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Abstracts

Abstracts 2011

55

SEB Main Meeting, Glasgow 2011

Bidder and Woolhouse Lectures

Bidder Lecture Morphodynamics: from genes to shape during flower development

Jan Traas (Laboratory of Plant Reproduction and Development, ENSLyon, France) During plant development the regulatory networks controlling growth and patterning must interfere with physical processes to generate specific shapes. How this is achieved, i.e. how molecules assemble into complex systems with a particular form is not known in any organism. We are addressing this central issue using the shoot apical meristem of the higher plant Arabidopsis. As a first step, we are correlating the activity of regulatory genes with specific morphogenetic events at the inflorescence meristem. To this end, we have developed a computational pipeline allowing us to express gene function in terms of quantified changes in tissue geometry. Using this tool, we are monitoring anisotropy and growth rates in gene expression domains and mutant backgrounds. In parallel we have started to analyse the molecular basis of shape control. Using a combination of physical, mathematical and biological approaches we have provided evidence for a model where molecular networks would impact on both growth rate and direction. In particular, we have identified a microtubule control of growth directions, which feeds back on local stress and strain pattern and which is coupled to the hormone-based control of overall growth rates. Models in the form of virtual flowers are now being developed to interpret the data and to propose precise hypotheses regarding gene function. Email address for correspondence: [email protected] 17:30 Friday 1st July 2011

Woolhouse Lecture Radical changes in plant biology research for better agricultural performance

Richard Flavell (Ceres) Harold Woolhouse was a `radical' thinker and enjoyed piecing together `radical' plans for the betterment of science. This is why I picked this title. We witnessed him enjoying such planning at the John Innes Centre and subsequently at the Waite Institute in Adelaide, Australia. Over the past few decades there have been many `radical' developments in plant science that have impacted plant breeding. These include use of model plants, discovering and deploying transgenes, whole genome sequencing, whole genome expression analyses and association mapping. I will discuss some current opportunities in plant breeding that, even if not radical, suggest that plant breeding in the future should not be as is the case now. Email address for correspondence: [email protected] 09:00 Saturday 2nd July 2011

56

Society for Experimental Biology

SEB Main Meeting, Glasgow 2011

Animal Section Young Scientist Award Session

YSAS.2 The heartbreak of migrating sockeye salmon at warm temperatures

Erika J Eliason (University of British Columbia, Canada), Timothy D Clark (University of British Columbia, Canada), Scott G Hinch (University of British Columbia, Canada) and Anthony P Farrell (University of British Columbia, Canada) It has long been established that temperature optima exist for growth and performance among different fish species. There are over 100 genetically distinct populations of sockeye salmon in the Fraser River watershed, each of which experiences unique environmental conditions during their upriver spawning migration. We hypothesized that cardiorespiratory performance and cardiac morphology would match the upriver migration challenges experienced by each population. To test this hypothesis, wild migrating adult sockeye salmon were instrumented to measure cardiorespiratory variables and swum at a single temperature (ranging from 8 to 26ºC) in a Brett-type respirometer. All populations maintained swimming and cardiorespiratory performance across the entire range of temperatures typically encountered during their upriver migration, with Chilko sockeye salmon emerging as the high temperature champions. In addition, populations with more challenging migrations had significantly greater aerobic scope, a larger relative ventricular mass and more compact myocardium compared to coastal populations traveling shorter distances. These results suggest that sockeye salmon populations have physiologically adapted to meet their upriver migration conditions on a very local scale. Next, we sought to determine how Chilko sockeye salmon have a higher and broader thermal tolerance compared to a co-migrating population (Nechako). Chilko sockeye salmon have a significantly higher density of adrenaline-binding ventricular -adrenoceptors, which may serve to protect the heart at temperature extremes and thereby expand their thermal tolerance. These findings suggest that some populations may be more susceptible to continued river warming, which has clear conservation concerns for biodiversity. Supported NSERC Canada, BC Pacific Salmon Forum. Email address for correspondence: [email protected] 09:00 Friday 1st July 2011

Sponsored by:

Ucrit and muscle mRNA levels. Although 3,3,5-triiodo-L-thyronine (T3) is widely recognized as the only active TH, recent work has shown that 3,5-diiodo-L-thyronine (T2) also stimulates metabolism. To determine whether T2 and/or T3 promote the metabolic effects of cold-acclimation, we supplemented hypothyroid fish with T2 or T3 during acclimation to 18ºC. Both T2 and T3 promoted the recovery of cold-acclimation (Ucrit and muscle mRNA levels). For the first time, we have identified central regulators underlying metabolic acclimation in ectotherms. Like T3, T2 also regulates the transcription of important metabolic genes such as PGC1. While T2 has been shown to stimulate metabolism, it has never, to our knowledge, been implicated in a physiological role until now. Because THs are conserved even back to the earliest invertebrates, these findings suggest that they may act universally to integrate environmental cues with acclimation response. Email address for correspondence: [email protected] 09:20 Friday 1st July 2011

YSAS.7 Revisiting oxygen supply and demand ­ oxygen availability, metabolic rate and thermal limits in aquatic ectotherms

Wilco Verberk (University of Plymouth, UK), David T Bilton (University of Plymouth, UK), Piero Calosi (University of Plymouth, UK) and John I Spicer (University of Plymouth, UK) Oxygen supply and demand are central to the hypothesis that oxygen limitation can set whole animal thermal tolerance limits. Yet there is no consensus as to what drives environmental oxygen availability. Here we resolve the question whether partial pressure or solubility limits oxygen supply in nature. Intriguingly, by returning to the first principles of gas diffusion, it becomes clear that more oxygen is actually available to an organism in warmer habitats, something which is exactly opposite to current wisdom. Observed oxygen shortages in warmer habitats are not caused by lower oxygen concentrations, but instead arise through animal oxygen demand exceeding supply. The role of both oxygen supply and oxygen demand in setting thermal limits was confirmed in an experiment on the aquatic nymphs of a stonefly: hypoxia lowered thermal maxima, while hyperoxia increased them. At the same time, individuals that strongly increased oxygen uptake at elevated temperatures had lower thermal maxima. These results on aquatic nymphs contrast with recent studies on terrestrial arthropods and suggest that many insects may be affected by oxygen limitation at some stage during their life cycle. Our discovery that environmental oxygen supply is actually higher in warmer habitats, represents a significant shift in our understanding of how oxygen shapes aquatic communities and makes physiological adaptations to escape oxygen limitation at thermal extremes more feasible. Email address for correspondence: [email protected] 09:40 Friday 1st July 2011

YSAS.5 Thyroid hormones 3,3,5-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2) are important thermoregulators in zebrafish (Danio rerio)

Alex G Little (University of Sydney, Australia) and Frank Seebacher (University of Sydney, Australia) In mammals, thyroid hormone (TH) is a principal regulator that stimulates metabolic heat production via brown adipose tissue and skeletal muscle. While regulators of metabolism in mammals are relatively well understood, those in ectotherms remain unknown. To determine whether TH promotes thermal acclimation in ectotherms, we induced hypothyroidism in zebrafish (Danio rerio) during three weeks acclimation to 18°C or 28°C using propylthuiouracil and iopanoic acid. The cold-acclimated control fish had significantly increased endurance swimming capacities and upregulated mRNA levels of metabolic genes (PGC1; PGC1; PPAR; COX VB; COX II; F0 F1 -ATP, ; ATPase 8/6) in liver and muscle compared to warm-acclimated controls. Hypothyroidism abolished the ability to cold-acclimate as measured by

Abstracts 2011

57

Plant Section Young Scientist Award Session

Sponsored by:

YSAS.3 Vacuolar Ca2+/ H+ transport activity is required for systemic phosphate homeostasis Involving shootto-root signalling in Arabidopsis

Tzu-Yin Liu (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan), Kyaw Aung (Plant Biology Department, Michigan State University, USA), Ching-Ying Tseng (School of Biological Sciences, University of Texas, USA), Tzu-Yun Chang (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan), Ying-Shin Chen (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan) and Tzyy-Jen Chiou (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan) Calcium ions (Ca2+) and Ca2+-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca2+/ H+ transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (Pi) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated Pi level in shoots as a result of increased Pi uptake in a miR399/PHO2independent signalling pathway. Microarray analysis of the cax1/cax3 mutant suggests the role of CAX1 and CAX3 as negative regulators to suppress the expression of one-fifth of Pi starvation responsive genes in the shoot, including genes encoding the PHT1;4 Pi transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not upregulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/ cax3 scion is responsible for high Pi accumulation in grafted plants, and that the pht1;1 rootstock is sufficient to moderately repress such Pi accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root Pi transporter system, partly via post-translational regulation of PHT1;1. Email address for correspondence: [email protected] 09:00 Friday 1st July 2011

A substantial increase in the activity of the citric acid cycle (TCA) during initiation of axillary meristem growth was observed. These changes were closely paralleled by the increased expression of the cell cycle S phase marker gene Histone H4. I will present data to show the changes of both gene expression and metabolism that occur during the early stages of axillary bud initiation growth in tomato. Email address for correspondence: [email protected] 09:20 Friday 1st July 2011

YSAS.8 Competition for light severely hampers defence signalling in Arabidopsis thaliana

Mieke De Wit (Utrecht University, The Netherlands), Ronald Pierik (Utrecht University, The Netherlands) and Laurentius Voesenek (Utrecht University, The Netherlands) Crops are typically grown in high densities where competition for light with weeds is intense and diseases can spread rapidly. It is, however, unknown how plants can deal with both these major stress factors simultaneously. Therefore, we investigate whether there is an interaction between the shade avoidance response to light competition and the defence response to pathogen attack, and how plant signalling is affected when both stress responses are induced. We show here that plants responding to a low red:far-red light ratio (low R:FR; the predominant neighbour detection signal) are more susceptible to the pathogen Botrytis cinerea than plants in control light. Notably, infected plants still exhibited low R:FR-induced petiole elongation, indicating that shade avoidance was prioritized over disease resistance. Indeed, induction of immune-related genes by the defence hormones salicylic acid (SA) and jasmonic acid (JA) was markedly reduced in low R:FR-treated plants, while shade avoidance and the associated marker gene expression were unaffected by the defence response. To elucidate the mechanisms underlying the suppression of defence during shade avoidance, microarray studies were performed on plants simultaneously treated with low R:FR and SA or JA. Low R:FR treatment resulted in massive downregulation of both defence responses; 88% and 43% of all differentially expressed genes in SA- and JA-treated plants, respectively, were suppressed in the combined treatment. Gene ontology analyses show which processes are affected by the combined induction of shade avoidance and defence and guide current studies on candidate regulators of light quality-mediated defence suppression. Email address for correspondence: [email protected] 09:40 Friday 1st July 2011

YSAS.6 Metabolic changes during axillary bud growth in tomato plants

Chloe Steels (University of Sheffield, UK), Steve Coates (Advance Technologies (Cambridge) Limited, UK), Andrew Fleming (University of Sheffield, UK) and Mike Burrell (University of Sheffield, UK) Axillary bud growth can affect the structure of the plant and can have a major outcome on agricultural practice and crop yield. Understanding the regulation of axillary bud development will improve our ability to manipulate plant form and, thus, increase food production. In comparison to molecular changes that occur during bud development very little is known about changes in metabolism. The aim of this research is to combine methods of spatial analysis of gene expression by in situ hybridization with advanced metabolite profiling techniques (nanospray-MS and MALDI-MS) to gain a comprehensive understanding of how axillary bud development is controlled. Axillary bud growth in tomato plants (Lycopersicon esculentum cv Alisa craig) was initiated by removing all aerial parts of the plant above the first leaf axil. At eight-hour intervals over the subsequent 48 hours of growth, the axillary meristem (100 µm in diameter), was dissected, the metabolites extracted and analysed using mass spectrometry. In parallel, the expression pattern of specific cell cycle genes was visualised by in situ hybridization, documenting the temporal progression of the initiation of axillary bud growth.

58

Society for Experimental Biology

Cell Section Young Scientist Award Session

Sponsored by:

YSAS.1 Identification of a plant nuclear envelope protein and its characterization

Ting Lu (Oxford Brookes University, UK), Katja Graumann (Oxford Brookes University, UK) and David E Evans (Oxford Brookes University, UK) In animal and yeast cells, a cross nuclear envelope complex termed LINC (The Linker of Nucleoskeleton and Cytoskeleton) complex is formed by interacting outer nuclear membrane (ONM) intrinsic KASH (Klaricht/ANC-1/Sync homology) proteins and inner nuclear membrane (INM) intrinsic SUN (Sun1/UNC-84) proteins. The LINC complex provides a physical signalling between nucleoskeleton and cytoskeleton and participates in nucleus positioning and nucleus movement in cell division. The identification of plant SUN proteins AtSUN1 and AtSUN2 in combination with the fact that no KASH DNA homologues have been identified suggested that there are some KASH-like proteins in plants, which do not share DNA homology with KASH proteins, but have similar functions to them. The newly identified protein, AtNE1, was shown to be localized to the plant nuclear envelope periphery. It is a small nucleoplasmic protein, which freely diffuses through the nuclear pore complex (NPC) and is anchored to the INM by interacting with an unknown INM-intrinsic protein. AtNE1 was predicted to have a nuclear localization signal (NLS), two coiled coil domains and one transmembrane (TM) domain. Domain deletion and truncation florescent protein constructs were observed by confocal microscopy. The subcellular localization of mutants implied the putative NLS is not essential for AtNE1 to diffuse through NPC but increases efficiency of targeting; both coiled coil domains participate in the interaction of AtNE1 with its INM interaction partner, and the putative TM domain appeared to be non-functional. The function of AtNE1 in plants will be studied by observing tDNA lines and BY-2 cell stable expression lines. Email address for correspondence: [email protected] 09:00 Friday 1st July 2011

growth and development were extremely sensitive to CK1 inhibitor treatment in a dose-dependent manner. Inhibitions of root hair growth were accompanied by the branching or cessation of new root hair formation. The microtubules in epidermal and cortex cells of the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts of the differentiation zone reoriented from transverse/ oblique to chaotic /longitudinal after D4476 treatment. Our studies provide evidence for the involvement of CK1 in the regulation of primary root development, especially root hair formation and growth, through the regulation of cortical microtubules organization, possibly through the tubulin phosphorylation in plant cell. Email address for correspondence: [email protected] 09:20 Friday 1st July 2011

YSAS.4 Casein kinase 1 is involved in Arabidopsis root hairs formation and growth via regulation of microtubules organization

Yarina Sheremet (Institute of Food Biotechnology and Genomics, NAS, Ukraine), Alla Yemets (Institute of Food Biotechnology and Genomics, NAS,Ukraine) and Yaroslav Blume (Institute of Food Biotechnology and Genomics, NAS, Ukraine) Our recent experiments on Arabidopsis thaliana line expressing chimeric gene gfp-map4 identified that various types of serine/threonine and tyrosine kinases are involved in regulation of microtubules organization in primary root cells (Yemets et al., Cell Biol. Int., 2008; 32:630­637; Sheremet et al., Cell Tissue Biol., 2010; 4(4): 399­409). Nevertheless the regulation of microtubules by direct phosphorylation of tubulins and its functional role in plant cell have not been widely observed. It was shown recently that A. thaliana casein kinase 1 (CK1) member CKL6 is associated with cortical microtubules in vivo and phosphorylates tubulin in vitro (Ben-Nissan et al., Plant Phys., 2008; 148:1897-1907). To examine a functional role of CK1 through its involvement in modulation of tubulin phosphorylation the effects of specific inhibitor D4476 on the microtubule organization in A. thaliana primary root cells were investigated. It was found that D4476 affects primary root growth and causes alterations of general root morphology. Root hair

Abstracts 2011

SEB Main Meeting, Glasgow 2011

59

PR - President's Medallists

PR.1 Beyond the lab: scientists as citizens in the 21st century

Dr Jennifer L Rohn (MRC Laboratory for Molecular Cell Biology, University College London, UK) It is human nature to assume that one person cannot make a difference. Many scientists, in particular, tend to be too busy with their research to be aware of social and political activities in the larger world that might threaten their profession. Yet the outside world, which has the power to deny our funding or regulate what experiments we do, is ignored at our peril. Despite science's increasing centrality to our way of life, many people are not interested in it, or choose not to believe the lessons it has to offer. This skepticism comes at a time when scientific solutions are seen to be increasingly crucial to overcome the various threats that our planet will encounter in the future. Although I have worked hard to be a good scientist over the years, I have also explored various ways that I ­ as one person ­ can have an positive effect on how society at large views science and scientists. From communicating about science for general audiences and setting up the LabLit.com website, to founding the Science Is Vital campaign in response to the UK government's funding threats last year, I have tried to be a good scientist citizen as well as a good researcher. In my talk, I will give more details about all the many things you might consider doing to make a difference, even if you have little time to spare, and why such activities are important now as never before. Email address for correspondence: [email protected] 09:00 Sunday 3rd July 2011

PR.3 From cell to tissue polarity: searching for the key that interlocks tip-growths

Veronica Grieneisen (John Innes Centre, UK) In both animal and plant systems, the coordination of cell polarities within tissues is fundamental for morphogenesis. Establishment of coordinated cell polarities over tissues is considered to involve intercellular interactions as well as a global orienting signal. In many cases, however, the global orienting signal emerges as a consequence of the individual cell polarities, giving rise to tightly regulated feedbacks between intracellular dynamics, intercellular interactions and global signals. Examples of this is the auxin gradient within the root, which is a consequence of the polar localisation of auxin efflux facilitators (PINs) along the cell membranes. A set of key players underlying cell polarity in plants, the so-called ROPs, are very similar to important polarity determinants in animals like Rac, Rho, and Cdc42, all belonging to the family of small G-proteins. Mathematical modelling has elucidated in how small G-protein biochemistry underlies intracellular polarity in both animals and plants. However, the mechanisms by which in plant cell polarities are coordinated are largely unknown. We address this question by experimentally focusing on the pavement cells (PCs) of leaves, which grow in complex forms that resemble pieces from a jigsaw puzzle, generating interdigitating patterns with the neighbouring cells. Our modelling efforts show how auxin can act as a go-between and trigger, through ROPs, the spontaneous emergence of intra-cellular polarity, independent of pre-patterns or localised polarising signals. Due to the generated intercellular auxin gradients, cell-cell communication arises enabling PCs to coordinate their polarities and interlock. Email address for correspondence: [email protected] 09:00 Monday 4th July 2011

PR.2 Genetic variability in soybean response to ozone: Identifying tolerance in a narrow base of germplasm

Elizabeth A Ainsworth (USDA ARS) In the past 100 years, tropospheric ozone concentrations have more than doubled as a result of industrial activities. Current ozone concentrations cause oxidative damage in plants, leading to yield losses that have been estimated to cost soybean growers $5.8billion per year worldwide. Models indicate that ozone could continue to rise another 25% by the year 2050, thus exacerbating the strain on agriculture to provide food at a time of rapid population growth. Genetic variation in soybean response to ozone determines the potential for breeding for increased tolerance and therefore productivity in the future. Over 20 soybean cultivars have been grown at elevated ozone in the field in central Illinois, USA under full open-air conditions, revealing significant variation in soybean tolerance to ozone. The exposure response of 7 cultivars (from 40 to 200 ppb) has recently been studied, revealing that any increase in ozone above ~38 ppb (today's ambient concentration) leads to a linear reduction in seed yield of 30 to 35 kg/ha. A recombinant inbred population has been developed from parents with different sensitivities to ozone to begin quantitative genetic analysis of tolerance. At the molecular level, elevated ozone causes widespread decreases in transcript abundance of genes related to the light reactions and Calvin cycle, and an increase in TCA cycle and respiration-related transcripts was seen. Therefore, it is likely that reductions in productivity are due in part to an overall decrease in photosynthetic capacity, which is exacerbated by increased respiration required to produce greater amounts of antioxidant compounds. Email address for correspondence: [email protected] 09:30 Sunday 3rd July 2011

PR.4 The evolution of periodic ventilation in insects

Craig R White (School of Biological Sciences, The University of Queensland, Australia) When resting, many species of insect exchange respiratory gases with the atmosphere intermittently and discontinuously. This discontinuous gas exchange cycle (DGC) has evolved independently in at least five lineages of insects and has been a cause of debate for decades, but no consensus on its evolutionary origin or adaptive significance has been achieved. Comparative studies show that DGCs are more common and longer in species from arid environments, and experimental studies show that water loss is lower during DGCs, that individuals acclimated to desiccating conditions open their spiracles for shorter periods than those acclimated to humid conditions, and that individuals that show DGCs live longer under water restriction than those that do not. These findings support the hygric hypothesis for the evolution of DGCs, which proposes that DGCs evolved or are maintained to reduce respiratory water loss. However, DGCs are not ubiquitous among arid insects and dehydrated insects often abandon DGCs, so the hygric hypothesis alone cannot explain the evolution of DGCs. Other hypotheses suggest that DGCs evolved to enhance gas exchange in subterranean environments or limit oxidative damage, but support for these hypotheses is similarly equivocal. Given that DGCs are exhibited by resting insects, and that developmental or artificial reductions in brain activity induce DGCs, we propose that this pattern results from the thoracic and abdominal ganglia regulating ventilation in the absence of control from higher neural centres. Subsequent adaptive modification of the discontinuous pattern could then act to reduce respiratory water loss or limit oxidative damage. Email address for correspondence: [email protected] 09:30 Monday 4th July 2011

60

Society for Experimental Biology

SEB Main Meeting, Glasgow 2011

A1 - Molecular physiology of epithelial transport in insects: a tribute to William R Harvey

A1.1

A1.3 Regulation of V-ATPase in blowfly salivary glands

Otto Baumann (University of Potsdam, Germany) Vacuolar-type proton pumps (V-ATPases) are multi-subunit heteromeric complexes composed of a H+ -translocating, membrane-bound V0 domain and a ATP-hydrolysing, cytosolic V1 domain. Located in endomembrane systems and in the plasma membrane of eukaryotic cells, V-ATPases fulfil a variety of functions, such as activation of acid hydrolases in acidic organelles, intracellular pH homeostasis and extracellular acidification. In some insect epithelia, V-ATPase molecules are highly enriched on the apical domain of the plasma membrane and energise trans-epithelial fluid secretion and reabsorption. Using the tubulous salivary glands of the blowfly Calliphora vicina as a model system, we examine the intracellular signalling cascade(s) that regulate V-ATPase activity in ion transporting insect epithelia. V-ATPase activity in blowfly salivary glands is under the control of the neurohormone serotonin (5-HT). 5-HT binds to two different 5-HT receptors, which are most likely members of the 5-HT2 and 5-HT7 receptor families. Binding of 5-HT to the former receptor leads to activation of the InsP3/Ca2+ signalling cascade and a rise in cytosolic [Ca2+]. Binding to the latter receptor causes an increase in intracellular cAMP level. The increase in [cAMP] induces a phosphorylation of V-ATPase subunit C via protein kinase A, and this event may trigger the reversible assembly of inactive V0 and V1 domains to functional V-ATPase holoenzymes on the apical membrane of the secretory cells. V-ATPase activity is, however, not independent of the InsP3/Ca2+ signalling cascade, since Ca2+ modulates the cAMP/PKA signalling, likely at the level of the adenylyl cyclase. Email address for correspondence: [email protected] 11:05 Friday 1st July 2011

Osmoregulation in insects: the evolutionary perspective

Timothy J Bradley (University of California Irvine, USA) Phylogenetic analyses indicate that the basal, i.e. primitive, osmoregulatory pattern in insects is one of terrestrial adaptation, involving the capacity to survive in dry air by producing hyperosmotic excreta. Some insect clades subsequently evolved the capacity to survive in fresh water, requiring powerful ion uptake mechanisms in the gut and external cuticle. Within these aquatic clades, a few species have evolved the capacity to survive in salt water. This requires the pumping of ions out of the insect. Each of these capacities will be discussed in the context of Bill Harvey's contributions to our understanding of the mechanisms involved, and in terms of the evolutionary history of the groups, using both macroevolutionary and microevolutionary perspectives. Email address for correspondence: [email protected] 10:35 Friday 1st July 2011

A1.2 Cellular sequestration and recycling of portasomestudded apical plasma membrane fragments (apm pouches) during the moulting cycle in the water vapour absorbing firebrat, Thermobia domestica (Packard)

John Noble-Nesbitt (University of East Anglia, UK) From its second instar, the firebrat is capable of extracting water vapour from the air down to relative humidities as low as 43%. This sustained active water vapour absorption (WVA) temporarily ceases during each moult as the cells believed to be responsible for WVA functionally switch to cuticle deposition and lose their distinctive apical mitochondria-apical plasma membrane (apm) complex. This process has been studied in ultrastructural detail. At apolysis (when the old cuticle first loses contact with the underlying cells) the apical complex begins to regress fully. The elongated mitochondria shorten and migrate perinuclearly. At the same time, the deep pleated apm infolds pinch-off into numerous small portasome-studded apm pouches, which remain sequestered within the cell cytoplasm. The uppermost portions of the apm remain at the cell apex and coalesce into a flat apical surface suitable for the deposition of a new cuticle. Towards the end of the moult, with the new cuticle deposited, the cells revert remarkably quickly to their absorptive role and structural complexity. This is facilitated by the rapid recoalescence of the sequestered apm pouches into deep pleated apm infolds, between which mitochondria migrate from their perinuclear position and elongate, reforming the distinctive, very regular hexagonal nature of the apical mitochondria-apm complex. WVA recommences immediately after ecdysis (see also Noble-Nesbitt, J. 2010. J Insect Physiol. 56, 488-491). Email address for correspondence: [email protected] 10:50 Friday 1st July 2011

A1.4 Ouabain sensitive Na/K ATPase and Ouabainresistant Na ATPase in the Malpighian tubules of Rhodnius and related insect tissues

Adriana D Gamez (Instituto Venezolano de Investigaciones Cientificas), Jesus DelCastillo (Instituto Venezolano de Investigaciones Cientificas, Venezuela), Guillermo Whittembury (Instituto Venezolano de Investigaciones Cientificas, Venezuela), Rafael Garcia (Instituto Venezolano de Investigaciones Cientificas, Venezuela) and Antonio M Gutierrez (Instituto Venezolano de Investigaciones Cientificas, Venezuela) We try to develop a model that explains fluid secretion in the UMT of Rhodnius prolixus. Among other features, it includes at its basolateral membrane a 5HT-stimulated K++2Cl-+Na+ cotransporter driving ions into the cell. This is balanced by the Na+/K+ ATPase (Na+ pump-I) which extrudes Na+ from the cytosol back into the basolateral space. The long postulated Na+ pump-II, a K+-independent, ouabain-insensitive Na+-ATPase (Biochim. Biophys. Acta 1975, 394: 281) has recently been isolated and cloned (Rocafull et al. Biochim. Biophys. Acta, 2011, doi:10.1016/j.bbamem.2011.02.010). To investigate whether it also plays a role in UMT secretion, we: (a) have studied the effect of ion transport inhibitors using perfusions of isolated UMT; (b) prepared microsomal fractions from Aedes albopictus C6/36 cell monolayers to obtain pellets to measure ATPase

Abstracts 2011 activities; (c) prepared SDS-PAGE and western blots for Na+/K+ and Na+ ATPases; (d) used immunofluorescence localization of Na+ATPases by means of anti-Na+/K+ ATPase and anti-Na+-ATPase IgY polyclonal antibodies; RNA isolations and RT-PCR; RT-PCR for P-type ATPases. The sequence analysis presents 100% identity with Rattus norvegicus Na+/K+ transport ATPase, and with alpha 1 polypeptide (Atp1a1). This demonstrates the presence of P-type ATPases, Na+ and Na+/K+-ATPases, in C6/36 cells. This finding indicates that pump II may be present in the UMT of Rhodnius, which would extrude Na+ from the cytosol back into the basolateral space to balance the huge rush of Na+ induced by the K++2Cl-+Na+ cotransporter after the latter is stimulated by 5HT. Email address for correspondence: adrianadinorahgamez @gmail.com 11:35 Friday 1st July 2011

61 basolateral membrane voltage. Intracellular recordings made with conventional microelectrodes show the PC basolateral membrane has a large K+ conductance and a smaller Cl- conductance. The intracellular activities of K+, Na+ and Cl- were measured with double-barrelled ionselective microelectrodes before and after kinin stimulation. From these data, the net electrochemical gradient of each ion across the basolateral and apical membranes was calculated, and the results incorporated into a model for the mode of action of diuretic kinins in A. domesticus. Email address for correspondence: [email protected] 12:15 Friday 1st July 2011

A1.7 Cloning and functional characterization of ion transporters in Aedes Malpighian tubules

Peter M Piermarini (Cornell University, USA) and Klaus W Beyenbach (Cornell University, USA) In the past few years, our laboratory has attempted to identify some of the major molecular players in the transepithelial secretion of fluid by Malpighian tubules of the mosquito Aedes aegypti. To guide our efforts, we tested the hypothesis that the basal membrane of principal cells, which are enriched with an apical V-type H+-ATPase, express: 1) a SLC4-like Cl/HCO3 anion exchanger (AE); and 2) a SLC12-like K, Cl cotransporter (KCC), as found in the alpha-intercalated cells of mammalian renal tubules, which are also enriched with an apical V-type H+-ATPase. First, RT-PCR on Aedes Malpighian tubules revealed the expression of the expected AE and KCC cDNAs. Second, functional characterizations of the encoded transporters in Xenopus oocytes demonstrated that: 1) the AE mediates DIDSsensitive Cl/HCO3 exchange; and 2) the KCC mediates swellingactivated, DIOA-sensitive K,Cl cotransport. Third, immunolabelling experiments in Malpighian tubules unexpectedly revealed that: 1) the AE does not localize to principal cells, but to the basal membrane of neighbouring stellate cells; and 2) the KCC localizes to the apical membrane of principal cells rather than the basal membrane. Fourth, assays of fluid secretion on isolated Malpighian tubules employing DIDS and DIOA indicate, respectively, that the AE only contributes to the diuretic rates of fluid secretion, whereas the KCC contributes to both the control of and diuretic rates of fluid secretion. We conclude that the AE in stellate cells supports the metabolic regulation of fluid secretion by principal cells, whereas the KCC plays an essential role in the transepithelial secretion of fluid by principal cells. Email address for correspondence: [email protected] 12:30 Friday 1st July 2011

A1.5 Biology of the pyrokinin-related peptides in the Chagas' disease vector Rhodnius prolixus

Jean-Paul V Paluzzi (McMaster University, Canada) and Michael J O'Donnell (McMaster University, Canada) Pyrokinin-related peptides are normally produced from multiple genes in insects. One such gene, which is often referred to as the capability or CAPA gene, encodes a single pyrokinin-related peptide that contains the consensus carboxy-terminal sequence of GM/LWFGPRL-amide. In addition, the CAPA gene produces two CAPA-related peptides that normally have a conserved carboxy-terminal sequence of FPRV-amide. The CAPA-related peptides have been shown to be important modulators of fluid secretion rates of insect Malpighian tubules. For example, CAPA peptides stimulate fluid secretion in dipteran insects, however in hemipteran insects CAPA peptides inhibit fluid secretion. Unfortunately, little information is known regarding the physiological effects of the pyrokinin-related peptides in insects. Thus, in order to elucidate the roles of the pyrokinin-related peptides in R. prolixus, we have isolated and functionally characterized receptors for these peptides. We demonstrate that receptor expression is associated with a number of tissues not previously recognized as targets of this peptide family. This research establishes novel possibilities for the physiological roles of pyrokinin-related peptides in this medically-relevant disease-vector. This research was funded by NSERC. Email address for correspondence: [email protected] 11:55 Friday 1st July 2011

A1.6 Intracellular ion measurements in Malpighian tubules of the house cricket: implications for the mode of action of diuretic kinins

Geoffrey M Coast (Birkbeck, UK) Kinins are potent diuretics, doubling the rate of secretion by the Malpighian tubules (MTs) of Acheta domesticus at sub-nanomolar concentrations. Their mode of action has been extensively studied in Drosophila melanogaster and Aedes aegypti. In both species, kinins target stellate cells (SCs), acting via a Ca2+-dependent mechanism to open a transepithelial Cl- selective conductance pathway, which increases anion movement into the tubule lumen. This results in a nonselective acceleration of KCl and NaCl secretion along with osmotically obliged water. The Cl- conductance pathway is described as being transcellular (through SCs) in D. melanogaster and paracellular (through intercellular junctions) in A. aegypti. Since SCs are only a few microns deep they are not amenable to microelectrode studies that might confirm the location of the conductance pathway. The MTs of A. domesticus lack SCs, and kinins act on principal cells (PCs) to open a transepithelial Cl- conductance, which depolarizes the apical membrane without impacting on the

A1.8 Cell signalling mechanisms in epithelial function

Shireen A Davies (University of Glasgow, UK), Gayle Overend (University of Glasgow, UK), Selim Terhzaz (University of Glasgow, UK), Sujith Sebastian (University of Glasgow, UK), Venkateswara Chintapalli (University of Glasgow, UK), Pablo Cabrero (University of Glasgow, UK), Lorraine Aitchison (University of Glasgow, UK), Andrew Finlayson (University of Glasgow, UK) and Julian AT Dow (University of Glasgow, UK) The Drosophila Malpighian tubule is an epithelial model for cellspecific, organotypic ion transport and cell signalling studies, functional genomics and gene discovery. The tubule is critical for osmoregulatory and detoxifying functions (Beyenbach, Skaer et al.) and fluid transport is modulated by neuroendocrine control and second messengers (Nassel and Winther). As barriers between the external and internal environment, epithelia play key roles in stress defence; and the Drosophila tubule is critical for organismal defence against salt and

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Society for Experimental Biology Stephanie M Graham (University of Glasgow, UK), Shireen-Anne Davies (University of Glasgow, UK) and Julian A T Dow (University of Glasgow, UK) FlyAtlas is an online resource that allows scientists to look at tissuespecific gene expression in the fruit fly Drosophila melanogaster. Unexpected expression patterns of previously characterized genes may hint at novel functions, thus helping to close the phenotype gap. To test this hypothesis we looked at the neuronal gene Fasciclin 2 (fas2), which has been exhaustively characterized (over 500 papers), with neural functions ranging from axonal growth in development to synapse stabilization in the adult. Surprisingly, FlyAtlas showed that fas2 is predominately expressed in the Malpighian tubule (a renal, rather than neural, tissue), hinting at a previously unreported function in this tissue. Results suggest fas2 may play an important role in apical microvilli development and stability in the principal cells of the tubules. We have also shown that fas2 may be involved in actin localization. Fas2 shows dynamic localization in response to cAMP and overexpression of the protein results in a significant increase in secretion when tubules are stimulated with cAMP. Therefore we can conclude that FlyAtlas offers scientists a unique starting point in order to determine novel functions for genes. Email address for correspondence: [email protected] 14:35 Friday 1st July 2011

oxidative stress (Terhzaz, Finlayson et al.; Stergiopoulos, Cabrero et al., 2009). Tubules are also immune tissues (Tzou, Ohresser et al., 2000; McGettigan, McLennan et al., 2005; Kaneko, Yano et al., 2006), expressing anti-microbial peptides via the IMD and Toll pathways. We now show that a tubule principal cell-specific cGMP-kinase (cGK) `switch' modulates NF-kB orthologue (Relish) and IMD pathway activation. This cGK `switch' modulates the survival of immunechallenged whole flies, the first evidence for cyclic nucleotide modulation of innate immunity. The tubule cGK switch also influences the response of the gut to bacteria; thus, response to infection may be dependent on tubule/gut communication. The high rates of metabolic activity in tubules and the associated production of reactive oxygen species results in specific adaptations to counter this, including the enriched expression of `antioxidant' genes. We show that the manipulation of specific genes in only tubule principal cells is sufficient to modulate organismal stress and immune responses. Thus, the tubule is a key stress-sensing tissue for the whole organism. Email address for correspondence: [email protected] 13:35 Friday 1st July 2011

A1.9 Mechanism and function of Capa receptor - activation by human NeuromedinU in vivo

Selim Terhzaz (Institute of Molecular Cell and Systems Biology, UK), Pablo Cabrero (Institute of Molecular Cell and Systems Biology, UK), Joris H Robben (Radboud University, Nijmegen Medical Centre, Netherlands), Jon C Radford (Institute of Molecular Cell and Systems Biology, UK), Brian D Hudson (Institute of Neuroscience and Psychology, UK), Graeme Milligan (Institute of Neuroscience and Psychology, UK), Julian A Dow (Institute of Molecular Cell and Systems Biology, UK) and Shireen A Davies (Institute of Molecular Cell and Systems Biology, UK) Insect control is extremely important and insect neuropeptides are a key research area for the potential novel routes of such control. The capa peptide receptor, capaR, is a G-protein-coupled receptor (GPCR) for the capa neuropeptides, Drm-capa-PVK 1 and -2 (capa1 and -2). capaR undergoes rapid desensitization, with internalization via a barrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal- FPRXmide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25, and the insect-PRLamides - Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and huging - on capaR, were investigated. NMU-25 but not huging nor Drm-PK-1 and -2, increases intracellular calcium ([Ca2+]i) levels via capaR. In vivo, NMU-25 increases [Ca2+] i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca2+]i and fluid transport. Finally, antiporcine NMU-8 staining of the larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. Taken together, these data suggest that NMU is a functional vertebrate orthologue of Drm-capa-PVK-1 and -2. Via gene expression analysis and the use of a capaR promoter-GAL4 line, we show that capaR is almost exclusively expressed in tubule principal cells in the fly. Targeted capaR RNAi in tubule principal cells significantly reduces capa-1 stimulated [Ca2+]i and fluid transport. Whole-organism survival studies reveal that adult capaR RNAi transgenic flies display a desiccation phenotype, where capaR RNAi increases survival under desiccation conditions. Email address for correspondence: [email protected] 14:15 Friday 1st July 2011

A1.11 Regulation of epithelial ion transport by tyramine: cell-to-cell communication in the Drosophila Malpighian tubule

Edward M Blumenthal (Marquette University, USA) and Haiying Zhang (Marquette University, USA) The biogenic amine tyramine (TA) is a potent diuretic agent in the Drosophila Malpighian tubule (MT). Application of nanomolar TA to isolated MTs causes a rapid depolarization of the transepithelial potential (TEP) associated with an increased transepithelial chloride conductance, resulting in diuresis. L-tyrosine, the precursor of TA, has similar effects, suggesting that MTs can both synthesize and respond to TA. Consistent with this hypothesis, mutation of the TA synthetic enzyme tyrosine decarboxylase 1 (Tdc1f03311, which reduces Tdc1 expression >100-fold) selectively eliminates tyrosine but not TA responses. Normal tyrosine sensitivity of mutant MTs can be rescued by overexpression of Tdc1 in the principal cells but not the stellate cells. To identify the TA receptor, we studied tubules mutant for CG7431, which was previously reported to encode a TA receptor (Cazzamali et al., BBRC 2005). The allele CG7431f05682, which lowers expression by >100-fold, reduces but does not consistently eliminate TA sensitivity. Deletion of both CG7431 and the adjacent homologous gene TyRII results in complete loss of TA sensitivity, while deletion of only TyRII has no effect. We conclude that CG7431 is the primary TA receptor in the MT with some degree of redundancy with TyRII. We have named CG7431 tarot (tro) (TA Receptor Of the Tubule). Finally, RNAi knockdown of tro expression in the stellate cells, but not in the principal cells, reduces TA sensitivity to the same extent as trof05682. Our data demonstrate the first example of cell-cell communication in an insect MT. Supported by NSF IOS-0744619 to EMB. Email address for correspondence: [email protected] edu 14:50 Friday 1st July 2011

A1.10 Using FlyAtlas to detect novel functions for wellknown genes in Drosophila melanogaster

A1.12 Control of salivary glands in black-legged tick Ixodes scapularis

Abstracts 2011 Ladislav Simo (Department of Entomology, Kansas State University, USA), Juraj Koci (Department of Entomology, Kansas State University, USA) and Yoonseong Park (Department of Entomology, Kansas State University, USA) Ticks that feed on vertebrate hosts use their salivary secretion, which contains various bioactive components, to manipulate the host's responses. The mechanisms controlling the tick salivary gland in this dynamic process are not well understood. We are interested in the mechanisms controlling tick salivation. Our studies searching the components that regulate the salivary glands yielded two neuropeptides: myoinhibitory peptide (MIP) and SIFamide. Both of them are produced in the two giant protocerebral neurons (PcSG) having an axonal projection to salivary glands acini II and III. Anatomical study suggests that these peptides control the basal cells of salivary gland acini, which produce dopamine, the potent activator of salivary secretion. We identified for the first time the tick D1 receptor activated by dopamine. Temporal and spatial expression patterns examined by immunohistochemistry and reverse transcription polymerase chain reaction suggest that the dopamine produced in the basal cells of salivary gland acini is secreted into the lumen and activates the D1 receptors on the luminal surface of the cells lining the acini. Therefore, we propose a paracrine function of dopamine that is mediated by the D1 receptor in the salivary gland at an early phase of feeding. The molecular and pharmacological characterization of the D1 receptor in this study provides the foundation for understanding the functions of dopamine in the blood-feeding of ticks. Email address for correspondence: [email protected] 15:40 Friday 1st July 2011

63 David F Moffett (Washington State University, USA), Zeping Wang (Washington State University, USA) and Stacia B Moffett (Washington State University, USA) Serotonin (5-HT) is a secretagogue for alkali secretion by the anterior midgut (AM) and acid secretion by the posterior midgut (PM) and stimulates diuresis by Malpighian tubules (Mt). The Aedes Vectorbase databank contains seven putative 5-HT receptors with vertebrate orthology. Of these, four (aael009573 and/or its paralogue aael015321, aael007644 and aael011844) were found by quantitative real-time PCR to be expressed in the AM and Mt. Immunocytochemistry showed aael015553 in epithelial cells, but not muscle cells, of AM and in stellate cells, but not principal cells, of Mt. aael015553 is a 5-HT6-like receptor and would be expected to transduce a cAMP second message, consistent with stimulatory effects of cAMP in both systems. A Ca2+ second message is apparently involved in transducing the stimulatory effect of 5-HT in all three tissues and cannot readily be accounted for by aael015553, or by aael009573, a 5-HT7-like receptor. Low expression of all predicted Aedes 5-HT receptors in the PM compared to the AM and Mt suggested that the PM is served by novel insect 5-HT receptors without significant vertebrate orthology. Email address for correspondence: [email protected] 16:15 Friday 1st July 2011

A1.15 Roles of Ca2+ and cAMP in serotonergic control of gut secretion in larvae of the yellow-fever mosquito Aedes aegypti

Urmila Jagdeshwaran (Washington State University, USA), David F Moffett (Washington State University, USA) and Greg G Goss (University of Alberta, Canada) In mosquito larvae, serotonin (5-HT) stimulates both extreme alkali secretion by the anterior midgut (AMG) and partial reversal (or reacidification) of this alkalinization in the posterior midgut (PMG). The effects are accompanied by characteristic changes in the transepithelial potential. We measured the intracellular Ca2+ activity (Ca2+i) in isolated gut tissues by intracellular fluorescent probe, Fura-2 AM. 5-HT increased Ca2+i in both the AMG and PMG, consistent with a second messenger function, although the patterns and time courses differed between the two regions. Ca2+-free solution abolished the secretory and electrical response to 5-HT in AMG, consistent with an extracellular source for the Ca2+. In the PMG the 5-HT response was not blocked in a Ca2+-free solution, but was blocked after subsequent addition of thapsigargan, an inhibitor of Ca2+ accumulation by the endoplasmic reticulum, suggesting that the source for the Ca2+ signal in PMG is intracellular. Although exogenous cAMP was a secretagogue for both tissues, it did not fully emulate the electrical responses characteristic of 5-HT, and did not overcome the Ca2+ requirement for serotonergic stimulation in either tissue. Email address for correspondence: [email protected] 16:30 Friday 1st July 2011

A1.13 A comparison of the perfused and unperfused Drosophila posterior midgut

Shubha R Shanbhag (Tata Institute of Fundamental Research, Colaba, Mumbi, India), Natalie M D'Silva (Tata Institute of Fundamental Research, India) and Subrata Tripathi (Tata Institute of Fundamental Research, India) Transport by the Drosophila midgut was studied in detail with microperfusion techniques in combination with SIET (Shanbhag and Tripathi, 2005; 2009a;b; 2010). The epithelium is so resistive that voltage-clamp of the epithelium is feasible despite the tubular geometry. The basal membrane can also be voltage-clamped. Since perfusion involves removal of the peritrophic membrane, the potential loss of GPI-anchored apical transporters was assessed in unperfused midguts and compared to the perfused state. SIET was used to scan gradients of ions on the basal side of the posterior midgut epithelium. When ion-selective microelectrodes were stepped in a flowing bath towards the gut wall of perfused midgut, there was an increase in H+, K+ and Cl- and a decrease in Na+. The extracellular pH decreased from 7.2 to 5.83 ±0.1 (n=45), K+ and Cl- increased from 5 to 7.3 ±0.2 mM (n=18) and 146 to 179 ±12.7 mM (n=6), respectively; Na+ decreased from 145 to 107 ±6.4 mM (n=8). In unperfused posterior midguts kept in a stationary bath, K+ and Cl- increased from 5 to 12.4 ±1.1 mM (n=8) and 141 to 159.3 ±4.2 mM (n=9) respectively and Na+ decreased from 141 to 95 ±3.6 mM (n=11). The posterior midgut of Drosophila secretes NaHCO3 and absorbs KCl and HCl, energized primarily by a V-type H+-ATPase, and secondarily by other transporters. Perfused and unperfused preparations are both viable and have comparable transport properties; the latter could be useful preparations for transport studies. Email address for correspondence: [email protected] 16:00 Friday 1st July 2011

A1.16 The role of adducin in the diuresis triggered by aedeskinin III in Malpighian tubules of the yellow fever mosquito

Jeremy T Miyauchi (Cornell University, USA), Peter M Piermarini (Cornell University, USA), Jason D Yang (Cornell University, USA), Diana M Gilligan (SUNY Upstate, USA) and Klaus W Beyenbach (Cornell University, USA) Adducin is a cytoskeletal protein that caps F-actin and promotes the association of actin with spectrin. A recent proteomic study by our laboratory implicated a potential role for adducin in the kinin-triggered diuresis mediated by the renal (Malpighian) tubules of the mosquito

A1.14 Differential expression of serotonin receptors in midgut and Malpighian tubules of mosquito (Aedes aegypti) larvae

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Society for Experimental Biology both Drosophila representatives of the newly-discovered CPA2 branch of the cation proton antiport family (also known as NHAs) are much better candidates for Wieczorek exchangers than the better known CPA1 branch that includes the classical Na+/H+ exchangers (or NHEs) (Day et al., 2008). Using Drosophila genetics, we analysed the physiological contributions of these exchangers to insect homeostasis. The exchangers are: specifically expressed in major transporting epithelia (Malpighian tubules, salivary glands, midgut and hindgut); coexpressed with other transport genes as distinct functional epithelial transcriptomic clusters; essential for survival; and regulated at the transcriptional level, being upregulated in response to salt loading. References: Day JP, Wan S, Allan AK, Kean L, Davies SA, Gray JV and Dow JAT (2008) Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa. Journal of Cell Science 121: 2612-2619. Harvey WR and Wieczorek H (1997) Animal plasma membrane energization by chemiosmotic H+ V-ATPases. Journal of Experimental Biology 200: 203-216. Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

Aedes aegypti. The goal of this study was to further characterize adducin's role in this diuretic response. Using RT-PCR on Malpighian tubules, we cloned two alternativelyspliced cDNAs that encode adducin-like proteins: AeAdd-A and AeAdd-B. Using an anti-adducin antibody, the immunoreactivity of adducin was localized to both principal and stellate cells of the Malpighian tubule epithelium. In principal cells, adducin immunoreactivity is prominent at the base of the brush border, and is also found throughout the cytoplasm. Immunoblotting experiments on isolated Malpighian tubules employing an anti-phosphoadducin antibody revealed that adducin is transiently phosphorylated when the tubules are stimulated with the diuretic peptide, aedeskinin III. Additional immunoblotting experiments using pharmacological modulators of protein kinases demonstrated that the kinin-induced phosphorylation of adducin is mediated by protein kinase C (PKC). Furthermore, physiological assays of isolated Malpighian tubules indicated that the known kinin-induced: 1) stimulation of transepithelial fluid secretion; 2) hyperpolarization of the basal membrane potential in principal cells; and 3) decrease in principal cell input resistance, are all blocked by an antagonist of PKC (bisindolylmaleimide I). Taken together, our results demonstrate that the phosphorylation of adducin by PKC plays a key role in the switch-like diuresis that is triggered by kinins in mosquito Malpighian tubules. Email address for correspondence: [email protected] 16:45 Friday 1st July 2011

A1.19 Characterization of AgNHA2, the second isoform of the cation/proton antiporter from Anopheles gambiae

Minghui Xiang (University of Florida-Jacksonville, USA), William R Harvey (University of Florida, USA), Charles W Heilig (University of Florida-Jacksonville, USA), Lanfang Li (University of FloridaJacksonville, USA), David A Price (University of Florida, USA) and Paul J Linser (University of Florida, USA) The newly identified metazoan Na+/H+ antiporter family has two isoforms that are postulated to be electrophoretic, i.e. voltage-driven. The first member, AgNHA1, was cloned from Anopheles gambiae larvae and immuno-localized with respect to the H+ V-ATPase by the Harvey laboratory. Attempts to characterize AgNHA1 cRNA that was expressed in NHA-minus mutant hamster or yeast cells or in frog oocytes were unsuccessful but it has been implicated in acidgenerating Na+/H+ antiport using Wieczorek's `acid quench of acridine orange' method (see abstract by Harvey, Okech and Sterling). Here we report studies on the second isoform, AgNHA2. It is predicted to have a relative molecular mass of ~60 kDa and shares 29.2% amino acid identity with AgNHA1. Immuno-localization with a specific antibody reveals that AgNHA2 is prominently expressed in stellate cells of Malpighian tubules of An. gambiae larvae, where it is mainly localized to the apical plasma membrane. AgNHA2 is expressed more ubiquitously in adults of An. gambiae. Immuno-localization study on the yellow fever mosquito Aedes aegypti produced very similar results. When heterologously expressed in the yeast Saccharomyces cerevisiae lacking endogenous cation/proton antiporters and pumps, AgNHA2 localizes to plasma membrane and enhances toxicity when yeast cells are cultured in growth media containing any one of the alkali metal cations: Li+, Na+ or K+. This property enables a functional characterization of AgNHA2 and provides a robust tool for the high-throughput screening of chemical libraries for AgNHA2 inhibitors that could be developed as mosquitocides. Email address for correspondence: [email protected] 11:50 Saturday 2nd July 2011

A1.17 High affinity solute transporters in membrane vesicles from whole Aedes aegypti larvae

William R Harvey (Whitney Laboratory, University of Florida, USA), Bernard A Okech (University of Florida, USA) and Kenneth M Sterling (University of Florida, USA) Phenylalanine uptake across the apical plasma membrane in the posterior midgut of Aedes aegypti larvae is mediated, in part, by a voltage-driven, Na+-coupled, nutrient amino acid transporter (AeNAT8). The voltage is generated by an H+ V-ATPase with secondary H+ and Na+ gradients. Both the H+ and Na+ are recycled by a postulated Na+/ H+ antiporter, AeNHA1. All three components, V-ATPase, antiporter and nutrient amino acid transporter, are functional in brush border membrane vesicles isolated from whole larvae of Ae. aegypti (AeBBMVw). The K0.5 for 3H-Phe uptake into AeBBMVw was 0.53 ±0.02 micromolar (mean ± SEM) compared to 0.19 ±0.02 millimolar for Phe-uptake into AgNAT8-cRNA-injected Xenopus laevis oocytes measured electrically. The affinity of Na+ for AeNAT8 in AeBBMVw was also low, at K0.5 10:40 Saturday 2nd July 2011

A1.18 Functional characterization of Drosophila melanogaster alkali metal (Na+ and K+) antiporters in the living whole animal

Venkat R Chintapalli (School of Medical Veterinary and Life Sciences, University of Glasgow, UK), Louise Henderson (School of Medical Veternary and Life Sciences, University of Glasgow, UK), Shireen-Anne Davies (School of Medical Veterinary and Life Sciences, University of Glasgow, UK) and Julian A Dow (School of Medical Veterinary and Life Sciences, University of Glasgow, UK) Alkali metal (Na and K ) antiports/exchangers - CPAs - play essential roles in the transport mechanisms of epithelial tissues. Insect epithelia are energized by an apical plasma-membrane V-ATPase (Harvey and Wieczorek, 1997). However, until recently, no plausible candidate for an apical antiport/exchanger partner for the V-ATPase (as required for the Wieczorek model) could be identified. Recently, we showed that

+ +

A1.20 Adaptive evolution and selective intervention of essential amino acid transport in vector biology

Dmitri Boudko (Rosalind Franklin University, Chicago Medical School, USA)

Abstracts 2011 Using bioinformatics, molecular cloning and reverse genetic approaches, we have identified, cloned and characterized a unique set of previously unknown secondary transporters that mediate thermodynamically active alimentary absorption and systemic redistribution of essential amino acids in model nematodes and dipteran insects. The characterized transporters form a large basal subfamily of the Neurotransmitter Sodium Symporter family, designated here as NAT-SLC6 (Nutrient Amino acid Transporters of the NSS a.k.a. solute carrier family 6, SLC6). In addition to B0 system-like broad spectra neutral amino acid transporters previously described in mammalian model organisms, we have identified a range of lineage-specific transporters with narrow selectivity and high affinity either for phenylalanine/phenolbranched, tryptophan/indole-branched, or methionine/cysteine/sulphur containing substrates, that together mediate absorption of the most underrepresented essential amino acids. NATs are highly expressed in the absorptive region of the metazoan alimentary canal and specific central and sensory neuronal cells. Transient RNAi of selected NATs in model mosquitos reduces growth and dramatically increases mortality in mosquito larvae. The consensus of identified biological traits across metazoan species implies that the NATs evolved to balance the acquisition of essential and conditionally essential amino acids relative to the specific metabolic needs of metazoan cells and organisms. The possibility of developing pharmacological and/or genetic tools for selective suppression of NAT functions in pathogenic, pest, and disease-transmitting invertebrates will be discussed. Supported by R56AI03464 NIH/NIAID. Email address for correspondence: [email protected] 12:05 Saturday 2nd July 2011

65 V-ATPase) all bring about neutralization of the pH gradient in the gut. Molecular cloning and immunochemical analyses of specific putative members of the bicarbonate regulatory system have demonstrated regionalized and even cell-specific expression of such proteins. Hence, compartmentalization of specific functionalities plays a central role in the generation and maintenance of the extreme pH gradients found in the lumen of the alimentary canal. Several carbonic anhydrases have been cloned and characterized from Anopheles gambiae and Aedes aegypti and regionalized patterns of mRNA expression and protein localization indicate a contribution to pH compartmentalization in the gut. Additionally, various members of the solute carrier families of proteins (SLCs) exhibit complex distributions that indicate anions including bicarbonate and chloride are regulated differentially at a very fine degree of resolution in the compartmentalized gut. For example, both apical and basal localizations for members of the SLC4a transporters are found at different locations along the contiguous epithelium of the larval alimentary canal. Microarray and RNAseq analyses show that members of the SLC26 family of anion transporters also exhibit cellspecific patterns of expression. Integration of data to date show that bicarbonate/anion trafficking in the gut is highly regionalized. A comprehensive model for this molecular physiology is slowly emerging but will in fact require details at the level of individual cells and small groups of cells. Email address for correspondence: [email protected] 12:35 Saturday 2nd July 2011

A1.23 A comparative investigation of organic anion transport in Halobiotus crispae (Tardigrada) and Schistocerca gregaria (Arthropoda, Insecta)

Kenneth A Halberg (University of Copenhagen, Denmark) and Nadja Møbjerg (University of Copenhagen, Denmark) We investigate transport of the organic anion chlorophenol red (CPR) in the tardigrade Halobiotus crispae compared to the Malpighian tubules of the desert locust Schistocerca gregaria, using a new method for quantifying non-flourescent dyes. CPR accumulates in the midgut lumen of H. crispae, indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial uncoupler DNP (1 mM; 83% reduction), the Na+/K+ATPase inhibitor Ouabain (10 mM; 23% reduction) and the vacuolar H+-ATPase inhibitor bafilomycin (5 µM; 21% reduction), and by the organic anions PAH (10 mM; 40% reduction) and probenecid (10 mM; 61% reduction, inhibition concentration-dependent). Transport by the two types of excretory organs exhibit a similar pharmacological profile; albeit markedly higher concentrations of CPR are reached in S. gregaria. Immunolocalization of the Na+/K+-ATPase -subunit in S. gregaria revealed that this transporter is abundantly expressed, and entirely localized to the basal cell membranes. Immunolocalization data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter-mediated with, likely candidates for the basolateral entry step being members of Oat and/or Oatp transporter families. Our results suggest that apical H+ and possibly basolateral Na+/K+ pumps provide the driving force for the transport; the coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.21 Nutrient signalling and transport in the fat body of Aedes agypti

Immo A Hansen (Department of Biology and Institute of Applied Biosciences, New Mexico State University, USA) Mosquitoes are major vectors of disease across the globe. Female mosquitoes obtain amino acids (AAs) from vertebrate blood and import them into their fat body, where they are used as building blocks for the synthesis of yolk proteins. This uptake process is facilitated by AA transporter proteins in the fat body plasma membrane. After a blood meal, the rise in AA levels in the mosquito blood triggers a conserved signal transduction pathway, the target of rapamycin (TOR) signalling pathway. TOR signalling activates yolk protein production in the fat body. Our focus is on seven AA transporters that are members of the SLC7 family and up regulated after blood meal in the fat body of adult females. We knocked down all seven SLC7 transporters independently using RNA interference (RNAi) and used a fat body organ culture system to determine the effect of the knockdown on TOR signalling. We also performed electrochemical characterization of one member of the SLC7 family, AaCAT1. Our results stress the importance of SLC7 transporters for nutrient signalling and transport in the mosquito fat body. Email address for correspondence: [email protected] 12:20 Saturday 2nd July 2011

A1.22 Molecular physiology of the bicarbonate system of mosquito larvae

Paul J Linser (Whitney Laboratory, University of Florida, USA) Mosquito larvae utilize an alkaline digestive strategy in the alimentary canal with a peak pH approaching 11 in the anterior midgut. Inhibitors of carbonic anhydrase activity, anion transport and proton pumping (via

A1.24 Ca2+ affects the InsP3/Ca2+ and the cAMP signalling pathways in blowfly salivary glands - from monologue to complex conversation

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Society for Experimental Biology

Kristoffer Heindorff (Institute of Biochemistry and Biology, University of Potsdam, Germany), Otto Baumann (Institute of Biochemistry and Biology, University of Potsdam, Germany) and Bernd Walz (Institute of Biochemistry and Biology, University of Potsdam, Germany) Fluid secretion in the salivary glands of the blowfly, Calliphora vicina, is induced by the neurohormone serotonin (5-HT). Binding of 5-HT to at least two distinct G-protein-coupled receptors leads to co-activation of the InsP3/Ca2+ and cAMP signalling pathways (Berridge and Heslop, 1981). Interestingly, there is mounting evidence for extensive crosstalk mechanisms between both pathways. Previous studies revealed a Ca2+-dependent increase in intracellular cAMP that somehow involves calcineurin (Voss et al., 2010). Possible targets of this interaction are enzymes that govern the intracellular cAMP concentration - adenylyl cyclases (AC) and phosphodiesterases (PDE). We show that Ca2+/calmodulin activates adenylyl cyclases in membrane preparations. In contrast, the activity of PDEs is largely independent of Ca2+. Calcineurin does not seem to be involved in the regulation of both enzymes, at least in vitro. We found that inhibition of calcineurin sensitizes the Ca2+-signalling pathway for 5-HT, suggesting a calcineurin-dependent negative feedback of Ca2+ on the InsP3/Ca2+ signalling pathway. We are currently characterizing this feedback mechanism in detail. In conclusion, Ca2+ has multiple and complex modulatory functions in intracellular signalling in the salivary gland. Funded by Deutsche Forschungsgemeinschaft (Wa 463/9-6) References: Berridge MJ and Heslop JP (1981) Br J Pharmacol 73: 729-738. Voss M et al., (2010) Am J Physiol Cell Physiol 298: C1047-C1056. Email address for correspondence: potsdam.de Poster Session Saturday 2nd July 2011 [email protected]

A1.26 Electrophysiology of the isolated and perfused posterior midgut of adult female yellow fever mosquitoes (Aedes aegypti)

Horst Onken (Wagner College, USA), Yolana Fuks (Wagner College, USA), Melanie Valencia (Wagner College, USA), Stacia B Moffett (Washington State University, USA) and David F Moffett (Washington State University, USA) The posterior midgut of female mosquitoes receives the blood meal. Salt and water are rapidly absorbed and secreted by the Malpighian tubules. Later, nutrients are digested and absorbed. To our knowledge, transport in the midgut of adult mosquitoes has never been studied with isolated preparations. Midguts were isolated and transferred into a bath with aerated mosquito saline (containing glucose, amino acids and dicarboxylic acids as nutrients). After mounting midguts on perfusion pipettes connected to a syringe pump, the transepithelial voltage (Vte) was measured. Mounting midguts with the posterior end onto the pipette allowed continuous perfusion, and a small, lumen negative Vte was detected. Addition of theophylline (10 mM, increasing intracellular cAMP concentrations) to the bathing solution resulted in Vte polarity changes and small, lumen positive values of Vte were recorded. As a valve separates midgut and hindgut, the posterior midgut inflated when the anterior midgut was connected to the perfusion pipette. After inflation with mosquito saline the initial, lumen negative Vte and the lumen positive Vte during exposure to theophylline were strikingly higher, suggesting that stretch stimulates the ion transport characteristics reflected in the voltage. Vte was inhibited with dinitrophenol, indicating that it reflects active ion transport. In the absence of amino acids the tissue did not generate a Vte, indicating that amino acids are involved in the electrogenic transport mechanisms and/or are required to generate ATP. Financial support by the NIH (1R01AI063463-01A2) and by a Wagner College Faculty Research Grant is gratefully acknowledged. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.25 To freeze or not to freeze: an energetic comparison of cold tolerance strategies in the Antarctic midge

Nicholas M Teets (Ohio State University, USA), Yuta Kawarasaki (Miami University, USA), Richard E Lee (Miami University, USA) and David L Denlinger (Ohio State University, USA) The Antarctic midge, Belgica antarctica, is the world's southernmost insect and the only true insect endemic to Antarctica. Larvae are tolerant of a number of environmental stresses, chief among them being low temperature. However, while previous studies have examined physiological responses to a single bout of cold, in nature the larvae of B. antarctica are exposed to multiple freeze-thaw cycles. Thus, in this study we examined the effects of repeated cold exposure (RCE) on the survival, energy content, and stress protein expression in larvae of B. antarctica. Additionally, we compared results between larvae that were frozen at -5°C in the presence of water and those that were supercooled at -5°C in a dry environment. Overall, RCE was deleterious for frozen larvae but not for their supercooled counterparts. By the end of five cycles of RCE, compared to supercooled larvae, frozen larvae had significantly higher mortality, higher tissue damage, depleted levels of key energy reserves (e.g. lipid, glycogen and trehalose) and higher expression of hsp70, a commonly used marker for protein damage and cellular stress. Thus, we conclude that it is preferable for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain supercooled during RCE. In addition, we are conducting similar experiments to compare cryoprotective dehydration and freezing as alternate strategies for long-term survival in the winter. Preliminary results will be presented from these experiments. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.27 The role of a K+/Cl- cotransporter in the transepithelial secretion of fluid by Malpighian tubules of the mosquito Aedes aegypti

Rebecca M Hine (Cornell University, USA), Peter M Piermarini (Cornell University, USA), Matthew Schepel (Cornell University, USA), Jeremy Miyauchi (Cornell University, USA) and Klaus W Beyenbach (Cornell University, USA) It is well known that electroneutral K+/Cl- cotransporters (KCCs) play important roles in cell volume regulation, in the maintenance of intracellular concentrations of Cl-, and in transepithelial transport. We have recently observed electroneutral transport mechanisms in the Malpighian tubules of mosquitoes, which gave us cause to look for KCC-mediated transport in this epithelium. Molecular cloning identified five partial alternatively-spliced cDNAs encoding putative KCCs in Malpighian tubules of adult female mosquitoes (Aedes aegypti). We chose the most abundant splice variant (AeKCC1-A1) for cloning the full-length cDNA. To functionally characterize the AeKCC1-A1, we expressed the protein in Xenopus oocytes and measured rates of 86 Rb+ uptake to trace K+ transport. These studies showed that AeKCC1-A1 mediates a Cl- dependent uptake of 86Rb+ that is stimulated by cell swelling, inhibited by dihydroindenyloxyalkanoic acid (DIOA), and dependent upon N-glycosylation of the transporter. Immunohistochemical studies localized AeKCC1-A1 to the brush border of principal cells. In studies of isolated Malpighian tubules we learned that DIOA significantly reduces the rate of transepithelial fluid secretion by ~60% without affecting

Abstracts 2011 the voltage and resistance of principal cells where AeKCC1-A1 is expressed. We conclude that AeKCC1-A1 in the apical membrane of principal cells is part of a transcellular electroneutral transport pathway for secreting KCl into the tubule lumen. Supported by NIH K01 DK080194-01 awarded to PMP and NSF IBN 0078058 awarded to KWB. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

67 physiological function of the enzyme, various regulation mechanisms have been proposed so far. Our aim is to reveal the role and regulatory mechanism of V-ATPase in the Malpighian tubules of Aedes aegypti using biochemical and electrophysiological approaches. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.30 What is regulating the reversible dissociation of V-ATPase?

Katharina Tabke (Animal Physiology, University of Osnabrueck, Germany), Andrea Albertmelcher (Animal Physiology, University of Osnabrueck, Germany) and Helmut Wieczorek (Animal Physiology, University of Osnabrueck, Germany) Vacuolar H+-ATPases (V-ATPases) are proton pumps found in every eukaryotic cell. During starvation conditions, they are regulated by reversible dissociation of the ATP-hydrolysing V1 complex from the membrane-bound, proton-translocating V0 complex. This type of regulation was first observed in the non-feeding, moulting tobacco hornworm (Manduca sexta). In the baker's yeast Saccharomyces cerevisiae, dissociation of the V-ATPase holoenzyme is induced by glucose deprivation. The shut-down of this energy consumptive pump appears to be an economic mode during diauxic shift. The re-addition of glucose induces the rapid and efficient reassembly of the holoenzyme without the need for biosynthesis of new subunits. By analysing localization of GFP-marked V-ATPase subunits in living yeast cells, we found that only the V1 subunit C and not the whole V1 complex dissociates from the membrane under starvation conditions. We were also able to show that a decreased intracellular cAMP concentration, the inhibition and/or deletion of protein kinase A subunits and the luminal pH are involved in regulating the dissociation of subunit C. Furthermore, dissociation is dependent on microtubules and the glycolytic enzyme aldolase. The interaction of subunit C with the cytoskeleton, especially with microtubules, seems to be a universal property. Finally, we found that the heterotrimeric complex RAVE may play a role not only in biosynthetic assembly of the V-ATPase but also in the re-association of subunit C to the holoenzyme. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.28 The Drosophila gut and tubule proteome

Maria Cundall (University of Glasgow, UK), Rujuta Shah (University of Glasgow, UK), Stephanie Graham (University of Glasgow, UK), Richard Burchmore (University of Glasgow, UK), Shireen A Davies (University of Glasgow, UK) and Julian A Dow (University of Glasgow, UK) Proteomic techniques are used for investigating global protein expression in a variety of organisms, tissues and cells. We have used several alternative approaches (1D LC-MS, 2D PAGE, Blue Native PAGE) to start to identify the adult Drosophila gut and tubule proteome. Over 600 proteins have been identified using the combination of these approaches, and using several techniques has led to the discovery of more proteins than using a single experimental approach. The merits of each technique will be discussed. We have attempted to enrich for plasma membrane proteins by density gradient centrifugation and fractionation and cell surface biotinylation, which has proved successful, with a significant enrichment of plasma membrane V-ATPase components and other transporter proteins in the datasets, which are known to be highly expressed in these tissues. Our proteomic data are compatible with existing gene expression data, with the vast majority of the proteins having been found to be expressed in adult Drosophila gut and tubules using expression microarray data (FlyAtlas). Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.29 The V-ATPase in the Malpighian tubules of Aedes aegypti

Felix Tiburcy (Animal Physiology, University of Osnabrueck, Germany), Klaus W Beyenbach (Department of Biomedical Sciences, Cornell University, USA), Helmut Wieczorek (Animal Physiology, University of Osnabrueck, Germany) The Malpighian tubules of the yellow fever mosquito Aedes aegypti are the main organs for the excretion of salts, water, metabolic waste products and xenobiotics, thereby regulating the homeostasis and the osmotic balance of the hemolymph. Especially in hematophagous insects that ingest huge amounts of blood during feeding, the Malpighian tubules fulfil a vital task, rapidly reducing the massive payload by diuresis. Since Malpighian tubules are not innervated, diuresis is under hormonal control. Several diuretic peptides have been found that are presumably released into the hemolymph by the central nervous system in order to regulate urine formation in the Malpighian tubules by the intracellular messengers Ca2+, cAMP and cGMP. One potential downstream target is the vacuolar H+-ATPase (V-ATPase) that is highly expressed in the principal cells of Malpighian tubules, where it is almost exclusively found at the apical membrane. The V-ATPase energizes the apical as well as the basolateral membrane and the paracellular pathway, thereby driving the transepithelial secretion of KCl, NaCl and probably other solutes. The highly diverse and critical role of the V-ATPase in cellular processes requires a tight physiological regulation of its expression and activity. Corresponding to the variability of the

A1.31 Molecular and pharmacological characterization of serotonin receptors in the salivary gland of Calliphora vicina

Claudia Roeser (Institute of Biochemistry and Biology, University of Potsdam, Germany), Wolfgang Blenau (Institute of Biochemistry and Biology, University of Potsdam, Germany), Arnd Baumann (Institute of Neurosciences and Biophysics, Research Centre, Jülich, Germany), Bernd Walz (Institute of Biochemistry and Biology, University of Potsdam, Germany) and Otto Baumann (Institute of Biochemistry and Biology, University of Potsdam, Germany) The salivary gland of the blowfly, Calliphora vicina, has been used extensively as a model system for the analysis of hormone-induced InsP3, Ca2+ and cAMP signalling as well as the regulation of V-ATPase activity. Salivation is hormonally regulated by the biogenic amine serotonin (5-HT), which is believed to activate at least two different membrane-bound G-protein-coupled receptors. Stimulation of these receptors with serotonin leads to an increase in the intracellular concentrations of the second messengers Ca2+ and cAMP. The mechanisms of intra- and intercellular Ca2+ signalling in blowfly salivary glands have been subject to numerous studies. Surprisingly, the molecular identities of the 5-HT receptor subtypes involved in the activation of saliva secretion have not been identified so far. A

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Society for Experimental Biology Lyudmila Popova (A. N. Belozersky Institute, Moscow State University, Moscow, Russia), Dmitri Boudko (Rosalind Franklin University, Chicago Medical School, USA) The recently identified subfamily of Nutrient Amino acid Transporters (NATs) of the Neurotransmitter Sodium Symporter Family (NSS a.k.a. SLC6) includes a large population of uncharacterized transporters that form lineage-specific clusters in the basal part of the SLC6 family tree. Here we report the cloning, functional heterologous expression and in situ hybridization of a novel insect NAT from the yellow fever vector mosquito, Aedes aegypti: AeNAT5 (NCBI accession, ABZ81822). In contrast to a previously characterized broad substrate spectra transporter, AeAAT1, from the same species, and tryptophan- and phenylalanine-substrate selective transporters from another culicid mosquito, Anopheles gambiae (AgNAT6 and AgNAT8, respectively), AeNAT5 performs selective absorption of L-methionine and, with 20 times reduced transport efficiency, L-cysteine, followed by other neutral amino acids. AeNAT5 mimics a general pattern of spatial expression of NATs in the insect gut. However, some substantial differences in the expression of AeNAT5 and AeAAT1 were identified in the anterior and posterior domains of the larval alimentary canal. These findings support our earlier hypothesis that the NAT subfamily evolved and acts in synergy as a principal molecular mechanism for intestinal absorption and somatic redistribution of essential amino acids. The narrow substrate spectra transporters AeNAT5, AgNAT6 and AgNAT8 represent specific duplications of insect NATs that amplify selective acquisition of the most underrepresented essential amino acids. Essential NATs are important for animal health and brain function, and the strong linage-specificity versus conserved neurotransmitter transporters reveals NATs as more rational targets in the management of medically and economically important invertebrates. Supported by R56AI03464 NIH/NIAID. Email Address for correspondence: [email protected] franklin.edu Poster Session Saturday 2nd July 2011

functional and pharmacological characterization, however, are crucial for the full comprehension of intracellular signalling pathways activated by serotonin in the salivary gland. Using different PCR-based strategies, we obtained full length cDNAs encoding for two putative 5-HT receptors of Calliphora (Cv5ht2 and Cv5-ht7). The deduced amino acid sequences display characteristics common to 5-HT receptors. To clarify the functional and pharmacological properties of the cloned receptors, we are currently analysing HEK 293 cell lines, stably expressing these receptors. The Cv5-HT7 receptor promotes the formation of cAMP with an EC50 of ~6 nM for 5-HT. Promising substances will be checked for their In vivo effect on salivary glands. In perspective, we also want to analyse the distribution of the receptors by immunohistochemistry. Supported by DFG (Grant Wa 463/9-6). Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.32 Unusual properties of a Na+-coupled broad specificity amino acid transporter from C. elegans

Ryan Metzler (Rosalind Franklin University, Chicago Medical School, USA), Jeffery Fox (Rosalind Franklin University, Chicago Medical School, USA), Ella Meleshkevitch (Rosalind Franklin University, Chicago Medical School, USA) and Dmitri Boudko (Rosalind Franklin University, Chicago Medical School, USA) Generic extinction of biosynthetic pathways for ten proteinogenic amino acids in metazoan organisms dictates a requirement for alimentary acquisition and cellular distribution of these substrates, which ensures reproductive viability and organism survival. Several members of the SLC6 family of transporters, resembling the Nutrient Amino acid Transporter (NAT) subfamily, have been associated with an essential amino acid uptake and distribution system in mosquitoes, flies, mice, caterpillars and humans. Our phylogenetic analysis of the Caenorhabditis elegans genome has revealed five potential NAT members (CeNATs) within the 14 total members of the CeSLC6 family. We have cloned and partially characterized a first representative of the identified CeNAT subfamily: CeNAT5 (snf-5). Similar to a few earlier reported broad spectra SLC6 transporters, CeNAT5 mediates sodium-coupled absorption of neutral amino acids. Surprisingly, and in contrast with earlier characterized metazoan NATs, heterologously expressed CeNAT5 has additional transport activity for acidic L-glutamate and aspartate, associated with a notable depolarization of cellular membranes. Expression profiling utilizing a transgenic strain expressing eGFP under a CeNAT5-specific promoter revealed broad and intensive gut expression as well as additional expression in the ASI sensory neurons responsible for mediating entry into the nutrient conserving dauer larval stage. The combined high expression in the putative nutrient condition detecting sensory neurons and CeNAT5/nutrient amino acid-induced depolarizing Na+ current suggest that CeNAT5 may play a role as a transporter/receptor (transceptor) in the regulation of metabolic rate in response to changing nutrient environment and, in particular, amino acid availability. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.34 Inward rectifier K+ (Kir) channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

Christin Kosse (Cornell University, USA), Matthew Schepel (Cornell University, USA), Peter M Piermarini (Cornell University, USA) and Klaus W Beyenbach (Cornell University, USA) Previous electrophysiological studies by our laboratory on Malpighian tubules of the mosquito Aedes aegypti have demonstrated that over 60% of the basal membrane conductance of principal cells is attributed to barium-sensitive K+ channels. The goal of the present study was to determine whether mosquito orthologs of the mammalian inward-rectifier K+ (Kir) channels contribute to the aforementioned conductance. Using RT-PCR on Malpighian tubules, we attempted to clone cDNAs encoding three putative Kir channels: AeKir1, AeKir2 and AeKir3. Fulllength cDNAs of AeKir1 and AeKir3 were cloned successfully. The AeKir1 cDNA encodes a protein of 486 amino acids that is an orthologue of the classical mammalian Kir2.x channels; the AeKir3 cDNA encodes a protein of 419 amino acids that has no clear mammalian orthologue. The AeKir1 and AeKir3 channels were expressed heterologously in Xenopus oocytes for their respective functional characterization via two-electrode voltage clamping. Compared to control (H2O-injected) oocytes, expression of the AeKir1 channel elicited a robust inwardrectifying conductance in the oocytes that is selective for K+ over Na+ and is blocked by barium. In contrast, expression of the AeKir3 channel did not result in any detectable changes to the electrophysiological parameters of the oocytes, relative to controls. In conclusion, the results of the present study indicate that at least the AeKir1 channel exhibits functional properties that are consistent with the known basal membrane conductance of principal cells in Aedes Malpighian tubules. Supported by NSF IBN 0078058 awarded to KWB.

A1.33 A novel methionine-selective transporter from the neurotransmitter sodium symporter family

Ella Meleshkevitch (Rosalind Franklin University, Chicago Medical School, USA), Dmitry Voronov (Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow, Russia), Jeffery Fox (Rosalind Franklin University, Chicago Medical School, USA),

Abstracts 2011 Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

69 signalling role annexin has in mediating the diuresis remains to be determined. Supported by NIH R21 AI072102 and NSF IOB-0542797 awarded to KWB. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.35 Expression and functional analyses of the kinin receptor in females of the Aedes aegypti (L.) mosquito

Cymon N Kersch (Texas AM University, USA), Patricia V Pietrantonio (Texas AM University, USA) and Hsiao-Ling Lu (Texas AM University, USA) The evolution of the blood feeding adaptation in arthropods includes the precise coordination of multiple physiological processes, such as digestion, rapid diuresis, changes in host-seeking behaviour and ultimately egg production. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known, including the Aedes kinins in female Aedes aegypti. Aedes kinins also stimulate hindgut contractions and leucokinin-like peptides have been discovered in the nervous and reproductive systems of other arthropod species. Given their potential to function in several physiological processes that are simultaneously stimulated by blood feeding, we investigated the global expression of the kinin receptor in the head, midgut, hindgut, ovaries and Malpighian tubules of non blood-fed and blood-fed females by Western blot and immunohistochemistry. Two specific antibodies unequivocally localized the Aedes kinin receptor in the stellate cell membrane in the Malpighian tubules, clarifying this much debated issue. Results also indicate widespread expression of the receptor protein in novel organs for haematophagous insects. These findings highlight the possibility that the Aedes kinins are integrative signalling peptides in Ae. aegypti that could coordinate multiple physiological systems after a blood meal, linking them to the evolutionary success of haematophagy in mosquitoes. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.37 The Drosophila Malpighian tubule: the beauty of a chance encounter between physiology and genetics in a transporting epithelium

Julian A Dow (University of Glasgow, UK) and Shireen A Davies (University of Glasgow, UK) A combination of painstaking physiology and pioneering biochemistry of the lepidopteran midgut led to a new paradigm for epithelial transport in which an apical plasma membrane H+ V-ATPase - rather than a basolateral Na+/K+ ATPase - plays a dominant role. This model has now been shown to apply to many epithelia, from insect to mammal. Despite its small size, the Drosophila Malpighian (renal) tubule has allowed a V-ATPase epithelium to be dissected genetically, with single genes knocked down (or overexpressed) in particular cell types in an otherwise normal epithelium, and in an otherwise normal organism. Such transgenic sophistication, combined with large postgenomic datasets, has allowed unparalleled insights into epithelial function. Armed with new information, it is now possible to make better sense of other transporting tissues, less fortunate in their transgenic technologies - even the lepidopteran midgut itself. References: Dow, JAT and Romero MF (2010) Drosophila provides rapid modeling of renal development, function, and disease. Am J Physiol Renal Physiol 299, F1237-1244. Dow JAT (2009) Insights into the Malpighian tubule from functional genomics. Journal of Experimental Biology 212, 435-445. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A1.36 Cloning and localization of an annexin from Malpighian (renal) tubules of the mosquito Aedes aegypti

Anish J Shah (Cornell University, USA), Peter M Piermarini (Cornell University, USA) and Klaus W Beyenbach (Cornell University, USA) The annexins are a family of calcium-dependent proteins involved in the gating of ion channels, trafficking of secretory vesicles and scaffolding of phospholipid membranes. In a recent proteomic study we observed a substantial reduction in the cytosolic abundance of annexin IX (AnxIX) after treating Malpighian tubules of the yellow-fever mosquito Aedes aegypti with the diuretic peptide aedeskinin-III (J. Exp. Biol. 2009; 212: 329). Thus, the goal of the present study was to clone and localize AnxIX in Aedes Malpighian tubules. Molecular cloning reveals that Aedes Malpighian tubules express six alternatively spliced transcripts derived from the Aedes AnxIX gene that encode a total of three distinct annexin-like proteins. Western blots locate AnxIX-immunoreactivity in the cytosolic and the total-membrane fraction of Malpighian tubules. Immunohistochemistry localizes AnxIXimmunoreactivity primarily in the brush border, but also in the cytoplasm and in the nuclear region of principal cells. Semi-quantitative Western blots demonstrate that treatment of tubules with the diuretic peptide aedeskinin-III or the calcium-ionophore A23187 results in a rapid and transient reduction to the cytosolic abundance of AnxIX, confirming our proteomic study. We are presently evaluating whether the cytosolic AnxIX relocates to the brush border (mitochondria) and/or nucleus upon stimulation with aedeskinin-III or A23187. In summary, annexin is a dynamic protein that transiently leaves the cytosol of Malpighian tubules during a kinin-induced diuresis. What

A1.38 Bicarbonate transporters in Drosophila and Anopheles

Michael F Romero (Physiology Biomedical Engineering and Mayo Clinic College of Medicine, USA), Lauren Brin (Physiology Biomedical Engineering, USA), Paul J Linser (Whitney Laboratory, University of Florida, USA), Pablo Cabrero (University of Glasgow, UK), Julian AT Dow (University of Glasgow, UK and King Saud University, Saudi Arabia) and Taku Hirata (Physiology Biomedical Engineering and Mayo Clinic College of Medicine, USA) Bicarbonate (HCO3) is the major physiological buffer, and pH homeostasis is essential for animal cells, tissues and whole organisms. Accordingly, both H+ transporters and HCO3 transporters mediate pH homeostasis, and we have focused on HCO3 as living in air requires both CO2 and HCO3 movements. HCO3 transporters are contained within two Solute-Leak Carrier (Slc) families: Slc4 and Slc26. Slc4 transporters are the `classic' HCO3transporters such as Cl--HCO3 exchangers (AEs), Na+/HCO3 cotransporters (NBCs) and Na+ driven Cl--HCO3 exchangers (NDCBE, NDAE). In the late 1990s we cloned, localized and characterized the first HCO3 transporter from Drosophila: NDAE1 [cg8177; Romero et al., JBC 2000;275:24552]. NDAE1 mRNA/proteins in Diptera are located in the basolateral membrane of midgut and Malpighian tubule epithelia. Slc26 proteins in mammals also can transport HCO3, but with varying stoichiometries of nCl--mHCO3 exchange. While Drosophila and Anopheles genomes contain seven to nine Slc26 genes and many are found in gut and Malpighian tubules, it appears that only the Slc26a5/

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Society for Experimental Biology Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

a6 paralogue (prestin) mediates HCO3 transport. While localization experiments are underway, we anticipate that this Slc26a5/a6 protein will be located apically in epithelia as mammalian prestin is apical. This placement would indicate that Slc26a5/a6 is part of the mechanism responsible for the alkaline midgut pH.

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Abstracts 2011

SEB Main Meeting, Glasgow 2011

71

A2 Conservation physiology

A2.1 More than just fine words the role for physiology in evidence-based conservation

Julian Metcalfe (Centre for Environment, Fisheries & Aquaculture Science, UK) and Craig E Franklin (University of Queensland, Australia) Conservation physiology has been proposed as a much-needed discipline that applies physiological tools to evaluate the ability of animals to respond to their environment and sustain their populations in response to natural and anthropogenic pressures. Yet sceptics might question that it is little more than a convenient `badge' that helps physiologists defend their research in the increasingly challenging world of science funding. Here we will focus on specific issues where physiological understanding has been used to support evidence-based conservation and management. Email address for correspondence: [email protected] 10:30 Sunday 3rd July 2011 William Cheung (School of Environmental Sciences, University of East Anglia, UK) and Daniel Pauly (Fisheries Centre, University of British Columbia, Canada) How changes in physical and biogeochemical conditions of the ocean affect marine organisms and fisheries is a critical question for modelling possible trajectories of marine ecosystems under global change and formulating mitigation measures. To address this issue, a framework to integrate responses of marine organisms to environmental changes at individual (physiological), population and community levels is needed. In this presentation, we suggest that, for fishes and invertebrates, oxygen is the key factor linking modelling components across these levels. We present the theoretical framework and empirical evidence illustrating the mechanism of such linkages. Using a recently developed dynamic bioclimatic envelope model, we demonstrate the application of such cross-level linkages in modelling ocean environmental changes on fish distributions, population dynamics and potential catch at the regional and global scales. Outputs from the model suggest that, under current trends of greenhouse gas emission, fish and invertebrate populations are expected to shift their distributions towards higher latitude and into deeper waters. In addition, their maximum body sizes are projected to change at individual, population and community levels. Ocean acidification and deoxygenation would increase the rate of such changes. This presentation highlights the importance in considering oxygen in modelling the effects of global change on marine ecosystems and the need for interdisciplinary collaborations between physiologists, ecologists and ecosystem modellers. Email address for correspondence: [email protected] 11:10 Sunday 3rd July 2011

A2.2 Linking physiology and ocean physics to manage marine fish...?

Myron A Peck (Institute of Hydrobiology and Fisheries Science, University of Hamburg, Germany) Populations of marine organisms are under increasing pressure from a variety of natural and anthropogenic sources. For example, notable climate-driven changes have occurred world-wide in the abundance and distribution of populations/stocks of marine fish species that are heavily exploited. Coupling long-term, retrospective analyses and novel, three-dimensional, biophysical, individual-based modelling shows great potential to reveal a `cause and effect' understanding of observed changes in key marine fish species. Case studies will be provided for some of the most ecologically- and commercially-important pelagic and demersal marine fishes including anchovy, sardine, herring and cod stocks in different large marine ecosystems. Individual-based models have become cutting edge tools that allow us to amalgamate organismal-level physiological responses and changes in ocean physics to better understand the processes affecting the productivity of marine fishes. However, translating these physiologicalbased model estimates into meaningful metrics (for management) is not always straightforward. Opportunities and challenges are discussed regarding the ability of these and other physiological-based tools to provide practical advice to management, such as projections of changes in marine fish species. The talk reveals how coupling physiology and ocean physics may contribute another key source of information required for evidencebased conservation of marine resources. Email address for correspondence: [email protected] 10:35 Sunday 3rd July 2011

A2.4 The role of conservation physiology in understanding and solving bycatch problems in inland commercial fisheries: insight from field studies on fish and turtles

Steven J Cooke (Carleton University, Canada), Graham Raby (Carleton University, Canada), Scott Hinch (UBC, Canada), Tony Farrell (UBC, Canada), Mike Donaldson (UBC, Canada), Alison Colotelo (Carleton University, Canada), Sarah Larocque (Carleton University, Canada) and Gabriel Blouin-Demers (University of Ottawa, Canada) Physiological tools have been widely used to understand and mitigate the consequences of capture and release in recreational fisheries, but comparatively little information exists for animals discarded in commercial fisheries. Our research has focused on bycatch in inland waters, a topic that has also received little attention. Using several case studies from Canada, we highlight examples of bycatch issues in two inland commercial fisheries with a focus on how physiological knowledge and tools have helped to identify problems and develop solutions. The first case study explores the condition and fate of coho and sockeye salmon encountered in commercial fishing gear in the Fraser River, British Columbia. We explore the potential of reflex impairment measures to serve as a proxy for condition and survival and test several gears that have the potential to facilitate the recovery of exhausted fish. The second case study examines bycatch of non-target fish and other vertebrates (e.g. mammals and turtles) in a commercial hoop net fishery in eastern Ontario. We use physiological tools to quantify sublethal disturbances and to test different management tools (e.g. temporal restrictions and turtle breathing spaces).

A2.3 Oxygen: a key factor in modelling responses of fish and invertebrate populations to global change

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Society for Experimental Biology increase in thyroid hormone, as well as the amount of metabolic heat produced during febrile mediation, is consistent and unaffected by ambient temperature, febrile body temperatures in ducks kept in hot environments become dangerously high. This differs from the physiology of the mammalian febrile mechanism and we think this variation makes birds' vulnerable to conditions of climate change. Email address for correspondence: [email protected] 12:25 Sunday 3rd July 2011

To summarize, we discuss where physiology has both provided important insight and where it has failed to inform conservation actions in an attempt to better understand the potential role of conservation physiology in addressing bycatch problems. Email address for correspondence: [email protected] 11:35 Sunday 3rd July 2011

A2.5 Using physiology to inform reserve design

Hamish Campbell (University of Queensland, Australia) and Craig Franklin (University of Queensland, Australia) Conservation of an animal species requires the protection of habitat where the rate of reproduction can achieve equal or greater rates than mortality. Physiologists have the methods and skills to identify what and where these habitats are, but how can that information be conveyed in a context that appeals to policymakers? In this talk, I will show how animal biological information can be incorporated within a spatial prioritization analysis to inform the placement of a protected area. Using modern telemetry techniques, detailed models of animal habitat utilization were created. Optimization algorithms were then used to weigh up the biological significance of a habitat against the monetary costs for purchasing, restoring and protecting it. This enabled an estimation of cost value at various levels of habitat protection, and showed conservation action scenarios - where the protection of preferred habitat could be maximized for the minimum total cost. This integration of physiological/behavioural data with economic information has the potential to improve the comprehensiveness, adequacy and efficiency of protected areas for the limited funds available for conservation. It may also be useful in setting target incentives to landowners and regulating land management in sensitive areas. This new field presents an exciting collaborative future between physiologists and spatial ecologists. Email address for correspondence: [email protected] 12:00 Sunday 3rd July 2011

A2.7 A novel osmoregulatory role for inositol in the eel may be disrupted by organophosphorus xenobiotics

Gordon Cramb (University of St Andrews, UK) Euryhaline teleosts exhibit a physiological flexibility essential for their successful acclimation to marine (salt water, SW) and freshwater (FW) environments. Although there have been numerous reports on the roles of osmolytes in marine `osmoconformers', such as elasmobranchs, there have been few studies into potential roles for organic osmolytes in `hypo-osmotic regulators' such as teleost fish. This is surprising, since organic osmolytes have an extensive evolutionary history and are known to have osmoregulatory functions from unicellular organisms to mammals. Myo-inositol is one major osmolyte that plays an important role in cytoprotection in many organisms exposed to a variety of environmental stresses. Myo-inositol monophosphatase (IMPA) is a cytosolic enzyme that de-phosphorylates any myo-inositol 1-phosphate recycled from the membrane phospholipid pool or synthesized de novo from glucose-6 phosphate to generate myo-inositol. Studies have identified increases in Impa1 expression in epithelial tissues exposed to the aquatic environment when eels move from FW to SW. IMPA1 mRNA and protein expression was upregulated throughout the basal/germinal cell layers within the stratified epithelia of the oesophagus, skin and fin, and also in chondrocytes within the cartilage of the fin rays, branchial arch and primary filaments. Preliminary studies have implicated a number of organophosphate pesticides as inhibitors of IMPA enzymatic activity. These results suggest that increases in Impa1 expression and intracellular inositol production protect epithelial/epidermal cells from the dehydrating effects of SW, and this protective function may be disrupted in SWmigratory fish previously exposed to certain pesticides in the FW environment. Email address for correspondence: [email protected] 13:30 Sunday 3rd July 2011

A2.6 Avian fever: a role for thyroid hormone in the susceptibility of birds to climate change

Manette Marais (School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, South Africa), Shane K Maloney (Physiology School of Biomedical Biomolecular and Chemical Sciences, University of Western Australia, Australia) and David A Gray (School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, South Africa) Introduction: Although fever is considered an evolutionary conserved immune reaction to pathogenic infection, there are differences in the characteristics of avian and mammalian febrile response. These differences might be due to variations in the physiological mechanism that underlies fever in these phyla. Methods: We studied: 1) the metabolic cost of the fever in Pekin ducks kept at different ambient temperatures; and 2) the role of thyroid hormone in activating metabolic heat production in ducks made febrile with bacterial endotoxin. Results: Pekin ducks do not adjust their metabolic heat production effort to mediate fever when they are kept in hot environments and because they have a limited capacity to dissipate heat, their core body temperatures become detrimentally high. Furthermore, we found a significant increase in the amount of free thyroxine in ducks, 60 minutes after treatment with bacterial endotoxin. The rise in plasma thyroxine was independent of ambient temperature. Conclusion: Activation of the innate immune response in Pekin ducks results in a release of thyroid hormone. We think this augments metabolic heat production which, in turn, mediates fever. As the

A2.8 The impact of diffuse pollutants on Atlantic salmon (Salmo salar L.) populations

Andy Moore (Cefas, UK) The declines in populations of Atlantic salmon throughout the northeast Atlantic region have been attributed to a range of factors operating both within the fresh water and marine environments. However, evidence from studies carried out in Europe and North America suggest that freshwater contaminants derived principally from intensive agriculture may have significant effects on Atlantic salmon at specific periods during the life cycle, often at concentrations frequently found in the environment. In particular, research has indicated that a range of pesticides may compromise reproduction, embryo development and the parr-smolt transformation and/or entry into saltwater. In this respect the research has highlighted that in terms of the life cycle of the Atlantic salmon, the freshwater and marine environments cannot be considered in isolation and that exposure to poor water quality in freshwater may be a key factor influencing survival of the fish once they migrate into the sea.

Abstracts 2011 An integrated programme of laboratory-based physiological and fieldbased behavioural studies has been undertaken to investigate the impact of a range of pesticides on Atlantic salmon. This approach, together with the development of life history models, has been used to assess the potential impact of diffuse pollution on salmon populations. The results of these studies form the basis of advice to Government and other evidence users on the relationship between contaminants and climate change and the potential regulation of salmon populations. Email address for correspondence: [email protected] 13:55 Sunday 3rd July 2011

73 months. Surprisingly, the experimental ocean acidification significantly increased the metabolic scope over the whole temperature range studied. Resting metabolic rate was kept constant from 10°C to 16°C, while the metabolic scope increased with temperature and was remained high at the highest temperatures (16°C and 18°C). This was in contrast with growth rate, which was optimal in the temperature range 10°C to 14°C and decreased at 16 and 18°C. Thus, it does not seem that metabolic scope limited growth performance in this experiment. The CO2-induced acidification lowered growth rate at the lowest temperature, but did not affect growth rate at the other temperatures. It is thus possible that by the end of this century ocean acidification may reduce growth rates in fish at the colder end of a species distribution range. Email address for correspondence: [email protected] 14:35 Sunday 3rd July 2011

A2.9 Coral reef fishes in a warmer and carbonated future

Göran E Nilsson (University of Oslo, Norway) Many coral reef fishes have long lived in a nearly stenothermic environment. For some of them, particularly cardinal fishes, relatively moderate increases in temperature (by 2 to 4°C) lead to large increases in their resting metabolic rate, resulting in drastic reductions in their aerobic scope that could threaten population survival, since less energy can be devoted to feeding and reproduction. In addition, their hypoxia tolerance is reduced, probably making them less able to avoid predators. Coral reef fish seem unable to acclimate to elevated temperatures as adults, but some hope lies in developmental plasticity and, of course, natural selection. Bad news for climate sceptics is that the predicted rise in the water CO2 level alone also has significant negative effects on the aerobic scope of coral reef fish. Moreover, elevated CO2 has drastic effects on neural functions in coral reef fish larvae, including changes in the olfactory preferences, possibly reducing their capacity to reach a proper habitat at the end of their planktonic phase and also exposing them to increased predation. Differences in the sensitivity of species and populations may lead to both geographical shifts, changes in fish community structure, and loss of diversity. Email address for correspondence: [email protected] 14:20 Sunday 3rd July 2011

A2.11 Combined effects of elevated pCO2 and nutrition on Mytilus edulis growth: comparison of field and laboratory studies

Jörn Thomsen (IFM-Geomar, Germany), Isabel Casties (IFM-Geomar, Germany), Christian Pansch (IFM-Geomar, Germany), Arne Körtzinger (IFM-Geomar, Germany) and Frank Melzner (IFM-Geomar, Germany) Highly elevated seawater pCO2 (>2000 µatm) reduces growth of the blue mussel Mytilus edulis. Nevertheless, juvenile M. edulis maintain their calcification rates under intermediate pCO2 levels (<1500 µatm) when food supply is sufficient. This is also observed in Kiel Fjord where, despite seasonally elevated pCO2 and permanent low [CO32-], mussels dominate benthic communities. In contrast, mussels that were transplanted to a low pCO2 site (average pH 7.93) accreted less shell CaCO3 compared to Kiel Fjord specimens (average pH 7.76) although higher pH should favour calcification. These results can be explained by twofold higher particulate organic carbon concentrations in the Fjord, indicating that calcification is a function of energy supply rather than carbonate chemistry. In a laboratory study, post settled larvae were exposed to different pCO2 and feeding treatments. Calcification was lowered by high pCO2 but its impact was much less pronounced than the effects of food supply. Better nutrition enables mussels to tolerate high pCO2 and overcome the negative impacts of seawater acidification. This is in line with increased energy turnover and NH4+ excretion rates in mussels acclimated to elevated pCO2 levels. Thus, today's dominance of mussels in Kiel Fjord is a consequence of the high CO2 tolerance of the earliest benthic stage and energy supply in an eutrophic habitat, which enables high growth rates. In conclusion, lowered rates of calcification might be a consequence of enhanced energy turnover under elevated pCO2 which is required for intracellular homeostasis. Under sufficient feeding conditions, mussel calcification should be relatively robust towards future ocean acidification. Email address for correspondence: [email protected] 14:50 Sunday 3rd July 2011

A2.10 Effects of temperature and ocean acidification on metabolic scope and growth rate in Atlantic halibut

Fredrik Jutfelt (Department of Zoology, University of Gothenburg, Sweden), Albin Gräns (Department of Zoology, University of Gothenburg, Sweden), Elisabeth Jönsson (Department of Zoology, University of Gothenburg, Sweden), Henrik Seth (Department of Zoology, University of Gothenburg, Sweden), Kerstin Wiklander (Department of Mathematical Sciences, Chalmers University of Technology, Sweden), Erik Sandblom (Department of Zoology, University of Gothenburg, Sweden), Sam Dupont (Department of Marine Ecology ­ Kristineberg, University of Gothenburg, Sweden), Kristina Sundell (Department of Zoology, University of Gothenburg, Sweden), Olga OrtegaMartinez (Department of Marine Ecology - Kristineberg, University of Gothenburg, Sweden) and Michael Axelsson (Department of Zoology, University of Gothenburg, Sweden) Due to the increasing atmospheric levels of greenhouse gases our oceans are getting warmer, and increasing CO2 dissolution leads to ocean acidification. It has been suggested that fish have an optimal performance temperature window that coincides with peak metabolic scope, and also that ocean acidification might suppress the scope. The aim here was to assess metabolic scope and growth performance as a function of temperature, and to investigate whether experimental ocean acidification would affect these parameters. We acclimated 500 juvenile Atlantic halibut to six temperatures (6 to 18°C), with or without CO2-acidified water (delta pH -0.4), for four

A2.12 Predator-prey interactions and changing thermal environments: determining the relationship between physiological and ecological performance

Veronica S Grigaltchik (University of Sydney, Australia), Frank Seebacher (University of Sydney, Australia) and Ashley Ward (University of Sydney, Australia) The abiotic environment can impact ecology and fitness by determining the physiological performance of organisms. Importantly, different species may respond differently to environmental change

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so that ecologically important interactions, such as predator-prey relationships, cannot be predicted from single-species performance curves. In this study, we tested the hypothesis that interspecific differences in the ability to thermally acclimate could impact and shift predator- prey interactions, thereby having important ecological consequences. We showed that the interaction between acclimation and acute test temperature resulted in differences in the locomotor thermal performance curves of a predator (Australian bass, Macquaria novemaculeata) and its prey (the Eastern mosquitofish, Gambusia holbrooki). During subsequent filmed interactions, thermal acclimation had a significant effect on measures of predatory behaviour such as duration of attack and eagerness to attack, as well as predator attack speeds and prey escape speeds. These findings suggest that ecological performance and predator success can be influenced by variable thermal conditions through intrinsic differences in the physiological responses of interacting species. This study looked at a predatory interaction comprehensively rather than just at the species involved and highlights the importance of interactions and community relationships when studying thermal impacts on organisms. Future studies in conservation and climate change should consider the importance of interactions between species, as well as the role of individual thermal sensitivities in determining fitness. Email address for correspondence: [email protected] 15:05 Sunday 3rd July 2011

A2.14 Predicting the impact of climate change upon a freshwater turtle

Mariana A Micheli-Campbell (University of Queensland, Australia), Craig E Franklin (University of Queensland, Australia) and David Booth (University of Queensland, Australia) Time-series measurements of ambient temperature in South-East Queensland, Australia, have shown a 0.4°C rise over the past decade. This region is home to many species of freshwater turtle, and this study aimed to quantify how rises in nest temperature during incubation may affect physiological and behavioural traits of the hatchling turtles. Freshly laid Elusor macrurus (Mary River turtle) eggs were collected and incubated at three constant thermal regimes (26, 29 and 32°C). Embryos incubated at the warmest regime developed faster, but post-hatch growth rate was significantly reduced compared to those incubated at lower temperatures. Swimming performance trials showed that hatchlings incubated at 32°C exhibited lower stroke force and spent the least amount of time swimming during eight minute trials, compared to the lower thermal groups. Importantly, these impairments upon the physiology constrained behavioural function because the turtles that exhibited inferior swimming performance (32ºC group) spent most of their time in shallow water, while turtles with improved swimming performance (incubated at 26ºC) preferred to inhabit deeper water-- where the hatchlings' food supply and refuges from predators exist. Considering that the average temperature of E. macrurus nests during the 2009 to 2010 season was 28.5°C and that climate models predict a 3°C rise in ambient temperature for this locality by 2070, our study results suggest that the survival of hatchling freshwater turtles may be compromised over the coming decades. Email address for correspondence: [email protected] 15:55 Sunday 3rd July 2011

A2.13 Is feeding under hypoxic conditions a good strategy? Insight from cardio-respiratory measurements on cod

Jane W Behrens (National Institute of Aquatic Resources, Technical University of Denmark, Denmark), Henrik Seth (Zoophysiology, Department of Zoology, University of Gothenburg, Sweden), Stefan Neuenfeldt (National Institute of Aquatic Resources, Technical University of Denmark, Denmark) and Michael Axelsson (Zoophysiology, Department of Zoology, University of Gothenburg, Sweden) Due to the absence of major intrusions of high-saline, oxygenrich North Sea water masses, the bottom waters in the vertically stratified Bornholm Basin of the Baltic Sea are almost permanently hypoxic. Knowledge on how hypoxic water volumes may restrict individual dispersion is crucial to predict the consequences on the distribution of fish populations under further deterioration of oxygen conditions. Employment of electronic archival tags on individual cod in the Bornholm Basin has shown that some fish undertake vertical migrations into the hypoxic waters, probably to feed (Neuenfeldt et al., 2009). An understanding of how the cardio-respiratory system responds to such behaviour will elucidate how this ultimately affects energy allocation for growth and maturation, being central life-history parameters that are highly relevant for the management of fish stocks. We made long-term (72 to 96 hour) pre- and postprandial measurements of oxygen uptake (MO2), cardiac output (Q) and heart rate (fH) and mesenteric and coeliac blood flow. In normoxia there was a pronounced SDA response and concomitant increase in Q and fH, with Q returning to routine values before MO2. Gut blood flow showed a clear temporal pattern with an initial increase in the celiac artery blood flow and a subsequent increase in mesenteric artery blood flow. The same parameters were next measured postprandially in cod exposed to a short hypoxic period corresponding to the time and oxygen level cod voluntarily endure when searching for food in the Bornholm Basin (35 to 40% oxygen saturation for 90 minutes) and subsequently returned to normoxia. Email address for correspondence: [email protected] 15:40 Sunday 3rd July 2011

A2.15 The heartbreak of migrating sockeye salmon at warm temperatures

Erika J Eliason (University of British Columbia, Canada), Timothy D Clark (University of British Columbia, Canada), Scott G Hinch (University of British Columbia, Canada) and Anthony P Farrell (University of British Columbia, Canada) It has long been established that temperature optima exist for growth and performance among different fish species. There are over 100 genetically distinct populations of sockeye salmon in the Fraser River watershed, each of which experiences unique environmental conditions during their upriver spawning migration. We hypothesized that cardiorespiratory performance and cardiac morphology would match the upriver migration challenges experienced by each population. To test this hypothesis, wild migrating adult sockeye salmon were instrumented to measure cardiorespiratory variables and swum at a single temperature (ranging from 8 to 26°C) in a Brett-type respirometer. All populations maintained swimming and cardiorespiratory performance across the entire range of temperatures typically encountered during their upriver migration, with Chilko sockeye salmon emerging as the high temperature champions. In addition, populations with more challenging migrations had significantly greater aerobic scope, a larger relative ventricular mass and more compact myocardium compared to coastal populations traveling shorter distances. These results suggest that sockeye salmon populations have physiologically adapted to meet their upriver migration conditions on a very local scale. Next, we sought to determine how Chilko sockeye salmon have a higher and broader thermal tolerance compared to a co-migrating population (Nechako). Chilko sockeye salmon have a significantly higher density of adrenaline-binding ventricular -adrenoceptors, which may serve to protect the heart at temperature extremes and thereby expand their thermal tolerance.

Abstracts 2011 These findings suggest that some populations may be more susceptible to continued river warming, which has clear conservation concerns for biodiversity. Supported NSERC Canada, BC Pacific Salmon Forum. Email address for correspondence: [email protected] 16:10 Sunday 3rd July 2011

75 of traits of a model amphibian species. The results from these studies reveal that multiple stressors can interact in ways that are not necessarily predictable from single-factor studies, and that the examination of one stressor in the absence of others potentially underestimates the impact of that stressor on amphibian populations in natural systems. Researchers in amphibian conservation biology should therefore continue to utilize a multi-factor experimental approach if we are to gain a comprehensive understanding of the stressors and mechanisms responsible for causing global amphibian declines. Email address for correspondence: [email protected] 16:40 Sunday 3rd July 2011

A2.16 Responses to thermal change are specific to populations not species

Frank Seebacher (University of Sydney, Australia) and Sebastian Holmes (University of Western Sydney, Australia) Pronounced regional climate changes can happen naturally or as a result of human forcing at rates that are too rapid for adaptive evolutionary responses to occur. Rapid climate change therefore causes organisms to migrate or become extinct, unless plastic responses of individuals can buffer the impact of chronic temperature change. Here we show that the capacity to counterbalance the impact of chronic temperature change differs significantly between populations of the mosquitofish Gambusia holbrooki. Populations differed in their absolute capacity for plastic responses and in their relative ability to respond to warming or cooling. Amplified fragment length polymorphism analysis showed that the genetic structure among populations explained a significant proportion of the variance in the scope for plastic compensation of temperature effects. Our results reveal a new dynamic in the response of animals to climate change. Current modelling of climate change impacts on biodiversity are most commonly averaged across species. For example, climate envelope modelling, which underlies most predictions of the impacts of global warming on biodiversity, assumes that species possess a homogenous and fixed physiological make-up. We show that while such models have been useful as initial estimates, their predictions are too simplistic. Future modelling and management of species under climate change will need to be resolved to a population level. Email address for correspondence: [email protected] 16:25 Sunday 3rd July 2011

A2.18 Digestion and oxygen consumption in Mexican axolotls

Karlina Ozolina (University of Manchester, UK), Heather Eyland (University of Manchester, UK) and Holly Shiels (University of Manchester, UK) Specific Dynamic Action (SDA) is the increase in an organism's metabolic rate after the intake of food. In this study we investigate the effect of feeding on long-term oxygen consumption in six wild-type axolotls (Ambystoma mexicanum) using stop-flow respirometry. Baseline oxygen consumption was established during the first 25 hours after which food was added and data were obtained for the next 100 hours. The results showed variation between individuals in terms of mean VO2 values, however VO2 followed the same general trend line with standard metabolic rate (SMR) averaging 0.57 ±0.28 SD VO2 mg/kg/h then increasing between five and 20 hours after feeding, where the mean SDA peak of 1.196 ±0.12 SD mg O2 /kg/h was reached. Afterwards, VO2 decreased at a much slower rate and did not return to the baseline SMR values within the time limit we had set for each experimental animal. These findings have various applications in animal husbandry and welfare as well as conservation physiology, because should an organism spend a significant proportion of its aerobic scope on food processing it will have limited energy resources left for other activities, such as locomotion, reproduction and growth. Therefore understanding SDA responses in environmentally endangered amphibians such as axolotls could be of importance to conservation strategies. Email address for correspondence: manchester.ac.uk Poster Session Sunday 3rd July 2011 [email protected]

A2.17 Understanding the causes of global amphibian declines: a lesson in complex interactions between multiple environmental stressors

Lesley A Alton (University of Queensland, Australia), Manuel Hernando Bernal (Universidad del Tolima, Columbia), Toby Mitchell (University of Queensland, Australia), Vincent O Van Uitregt (University of Queensland, Australia), Rebecca L Cramp (University of Queensland, Australia), Craig R White (University of Queensland, Australia), Robbie S Wilson (University of Queensland, Australia) and Craig E Franklin (University of Queensland, Australia) The phenomenon of global amphibian declines is a testament to the profound effects of human-induced global change on natural environments. With amphibians being the most threatened of all vertebrate taxa, and also important bioindicators of environmental health, understanding the causes of their declines is critical for their conservation, and possibly the conservation of other species. While research over the past two decades has identified a range of potential causative agents, it has become widely accepted that amphibian declines are likely to be a result of complex interactions between multiple environmental stressors. In a series of multi-factor experimental studies, we have examined the interactive effects of ultraviolet-B radiation combined with a number of natural stressors, including low temperature, predation stress, high conspecific density and hypoxia, on a suite

A2.19 Coupled analytical and numerical model of fish response to environmental changes

Paolo Domenici (CNR IAMC Oristano, Italy), Andrea Cucco (CNR IAMC Oristano, Italy), Matteo Sinerchia (CNR IAMC Oristano, Italy), Christel LeFrançois (University of La Rochelle, France), Paolo Magni (CNR IAMC Oristano, Italy), Angelo Perilli (CNR IAMC Oristano, Italy) Eco-physiology studies, performed in a laboratory under controlled conditions, provide an essential tool for assessing the potential impact of environmental changes on the metabolism and behaviour of individual fish. One way of quantifying such impacts is by measuring the metabolic scope (MS) of a given fish population. Laboratory experiments were performed to calculate the MS of Mugil cephalus from Cabras Lagoon (Italy) under different environmental conditions (temperature and oxygen). The equations derived were introduced into an ecological model, which was then coupled to a high-resolution hydrodynamic model. The model was calibrated for reproducing the environmental variability in the Cabras lagoon and Gulf of Oristano (Italy).

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Society for Experimental Biology genetic characteristics. These characteristics differentiate them from natural populations and help them to better adapt and perform in urbanized environment. The main aim of the investigation was to analyse genetic changes, DNA damage, as an effect of anthropogenic pollution. The oxidative damage of DNA, by assessment of abasic lesions, was measured with a DNA damage quantification kit (BioVision). For this purpose, we collected individuals of two species, Apodemus flavicollis and Apodemus agrarius inhabiting various green, urban and suburban areas of Warsaw city. The chosen species are from the same family, but are characterized by different periods of urbanization processes. We will present results and discus the differences in response and susceptibility of studied species. Email address for correspondence: [email protected] uj.edu.pl Poster Session Sunday 3rd July 2011

We used the model to reproduce the temporal and spatial variation in MS of the M. cephalus fish population, in order to investigate the relationship between changes in MS and the observed seasonal migration pattern between the gulf and the lagoon. Results show that, during the late spring-beginning of summer period, Cabras lagoon provides a higher MS for M. cephalus than the Gulf of Oristano. During the rest of the year, apart from some transitional phases, the Gulf provides more suitable conditions (higher MS) for M. cephalus. Results were compared to fisheries data, showing that M. cephalus catches are highest during the end of July to August period. This period is characterized by fish migrating from the lagoon into the Gulf and coincides with the reproduced drop of MS for M. cephalus in the lagoon. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

A2.20 Predicted long-term changes in ocean temperature and acidification affect growth rate, but not aerobic scope and in situ perfused heart performance in Atlantic halibut

Michael Axelsson (Department of Zoology, University of Gothenburg, Sweden), Sam Dupont (Marine Ecology, Sven Lovén Centre for Marine Sciences Kristineberg, University of Gothenburg, Sweden), Olga Ortega-Martinez (Marine Ecology, Sven Lovén Centre for Marine Sciences Kristineberg, University of Gothenburg, Sweden), Erik Sandblom (Department of Zoology, University of Gothenburg, Sweden), Albin Gräns (Department of Zoology, University of Gothenburg, Sweden), Fredrik Jutfelt (Department of Zoology, University of Gothenburg, Sweden), Kristina Sundell (Department of Zoology, University of Gothenburg, Sweden), Elisabeth Jönsson-Bergman (Department of Zoology, University of Gothenburg, Sweden) and Kerstin Wiklander (Department of Mathematical Sciences, Chalmers University of Technology, Sweden) On-going climate change leads to warmer ocean water and a drop in water pH (i.e. ocean acidification). Current theoretical models suggest that temperature and pH limitations on metabolic scope may compromise growth and other fitness-related traits in marine ectothermic animals. Here, growth rate, metabolic scope and in situ cardiac performance was assessed in Atlantic halibut acclimated for over four months to 6, 12 and 18°C at current ocean pH (8.1) and at a pH predicted for the year 2100 (pH 7.7). Growth rate was highest at 12°C and significantly lower at 6 and 18°C (both pH 7.7 and 8.1). While fish acclimated to 18°C had a significantly lower relative ventricular mass compared with 6 and 12°C acclimated fish, the 18°C fish had a higher aerobic scope, a higher maximum cardiac flow and a greater maximum cardiac power output compared with 6 and 12°C fish when tested at their respective acclimation temperatures. Low pH reduced growth rate at 6, but not at 12 and 18°C. If anything, metabolic scope and cardiac performance was elevated across temperatures in fish at pH 7.7. Thus, in contrast to established theories, growth rate did not seem to be limited by metabolic performance under the present experimental conditions. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

A2.21 Genetic effects of microevolution in populations of Apodemus spp. from urban areas

Renata Swiergosz-Kowalewska (Institute of Environmental Sciences, Jagiellonian University, Poland), Magdalena Mikowska (Institute of Environmental Sciences, Jagiellonian University, Poland) and Michal Kozakiewicz (Department of Ecology, University of Warsaw, Poland) In populations of animals that inhabit cities, changes have developed that have led to the creation of specific behavioural, physiological and

Abstracts 2011

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SEB Main Meeting, Glasgow 2011

A3 ­ The role of oxidative stress as a physiological mediator of life histories: contrasts between birds and mammals

A3.1 Does oxidative stress play a causative role in ageing?

Colin Selman (University of Aberdeen, UK) The idea that free radicals cause ageing via damage to various biomolecules was first proposed in the 1950s by scientists such as Denham Harman. Since the early 1990s, this idea (more commonly termed the oxidative stress theory of ageing) has become unquestionably the most dominant mechanistic theory as to why we age and ultimately die. Despite extensive correlative evidence supporting this theory from both laboratory and free-living organisms, several recent studies have raised reservations over its significance in causing ageing. These studies include several using genetically modified mice harbouring targeted deletions of the major antioxidant genes. While several of these mouse models display an increased level of oxidative damage, this was not coupled to a shortened lifespan. In addition, several long-lived novel animal models have recently been shown to have comparatively high levels of oxidative stress and be long-lived. I will present data from a range of studies that have attempted to modulate ageing by altering oxidative stress levels, and will discuss whether this idea still remains a useful mechanistic theory to understand the ageing process. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011 Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

A3.3 Oxidative stress and reproductive investment in house mice

Michael Garratt (University of New South Wales, Australia), Aphrodite Vasilaki (University of Liverpool, UK), Paula Stockley (University of Liverpool, UK), Francis McArdle (University of Liverpool, UK), Robert J Beynon (University of Liverpool, UK), Malcolm J Jackson (University of Liverpool, UK) and Jane L Hurst (University of Liverpool, UK) Investment in reproduction is costly and can decrease survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. We tested whether oxidative stress is a consequence of reproduction in house mice (Mus musculus domesticus), where males are required to invest heavily in scent signalling to acquire mates and females make substantial investments in offspring during gestation and lactation. After a long period of reproductive investment in the laboratory there was little evidence of increased oxidative stress among either males or females. Instead, in the liver, markers of oxidative damage indicated lower oxidative stress in reproducing individuals when compared with non-reproductive controls. Even during peak lactation in females, when the energetic costs of reproduction are extremely high, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur with reproductive investment in house mice, but suggest that individuals use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress. Email address for correspondence: [email protected] 12:00 Saturday 2nd July 2011

A3.2 Promotion of metabolic health and lifespan by transiently increasing oxidative stress

Michael Ristow (University of Jena, Germany) Recent evidence suggests that calorie restriction and specifically reduced glucose metabolism induces mitochondrial metabolism to extend life span in various model organisms, including S. cerevisiae, D. melanogaster, C. elegans and possibly mice. In conflict with Harman's free radical theory of ageing, these effects may be due to the increased formation of reactive oxygen species (ROS) within the mitochondria causing an adaptive response that culminates in subsequently increased stress resistance assumed to ultimately cause a long-term reduction in oxidative stress. This type of retrograde response has been named mitochondrial hormesis or mitohormesis, and may in addition be applicable to the health-promoting effects of physical exercise in humans and, hypothetically, impaired insulin/IGF1-signalling in model organisms. Consistently, abrogation of this mitochondrial ROS signal by antioxidant supplements impairs the lifespan-extending and healthpromoting capabilities of glucose restriction and physical exercise, respectively. In summary, the findings discussed in this review indicate that ROS are essential signalling molecules that are required to promote health and longevity. Hence, the concept of mitohormesis provides a common mechanistic denominator for the physiological effects of physical exercise, reduced calorie uptake, glucose restriction, and possibly beyond. Reference: Exp Gerontol, 2010;45:410-418. (PubMedID 20350594)

A3.4 Metabolically mediated trade-off between reproductive success and oxidative stress in a freeranging mammal

Quinn Fletcher (McGill University, Canada), Colin Selman (University of Aberdeen, UK), John R Speakman (University of Aberdeen, UK), Christiaan Leeuwenburgh (University of Florida, USA), Stan Boutin (University of Alberta, Canada), Andrew G McAdam (University of Guelph, Canada) and Murray M Humphries (McGill University, Canada) Cumulative physiological damage is the underlying cause of senescence and it results because of the allocation trade-off between reproduction and somatic maintenance. Levels of metabolic expenditure are one possible measure of allocation to reproduction that may mediate this trade-off. In mammals, high levels of metabolic expenditure are necessary to wean offspring, but a by-product of aerobic metabolism is the generation of reactive oxygen species that may lead to oxidative

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Society for Experimental Biology Here I present the results from a study linking oxidative stress and fatty acid composition in food, egg yolk, and plasma to life history variation in a wild population of great tits, Parus major. The oxidative stress is measured as total and oxidized glutathione (tGSH and GSSG), hydroperoxides (ROM) and total antioxidant capacity (OXY), and 14 different fatty acids were identified using gas chromatography. The study was conducted in a forest consisting of substantial smallscale environmental heterogeneity known to influence reproductive output and survival. The specific aims addressed were: (i) the extent of dietary variation in the wild; (ii) whether there is support for a functional link between oxidative stress and fatty acid composition, specifically, ROM and the peroxidizable polyunsaturated fatty acids that control membrane fluidity (docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA)); (iii) whether either or both are associated with fecundity, growth, and reproductive investment; and (iv) the relationship between fatty acids, oxidative stress, and survival. The results highlight the importance of incorporating nutrition and ecology to understand how oxidative stress may constrain life history variation. Email address for correspondence: [email protected] 15:40 Saturday 2nd July 2011

stress. The accumulation of oxidative stress is believed to mediate the rate of physiological ageing. Here, we examine the effect of metabolic expenditure on reproductive success and levels of oxidative stress accumulated during reproduction. Research was conducted on a marked natural population of red squirrels in Yukon, Canada. Levels of oxidative stress were determined from plasma samples paired with measures of field metabolic rate. Levels of oxidative stress were higher in lactating compared to nonbreeding females. We also detected a metabolically mediated trade-off whereby lactating females with higher field metabolic rates recruited more offspring into the population and experienced higher levels of oxidative stress. In conclusion, our results suggest that metabolic expenditure may be the underlying mediator of the trade-off between reproductive output and the somatic damage that underlies ageing. Email address for correspondence: [email protected] 12:30 Saturday 2nd July 2011

A3.5 Links between early growth, oxidative stress, metabolic rate and telomere shortening in zebra finches

Neil B Metcalfe (University of Glasgow, UK), Francois Criscuolo (CNRS Strasbourg, France) and Pat Monaghan (University of Glasgow, UK) There has been much speculation as to the role of oxidative stress in shaping life histories, since it is thought to play a leading role in determining the rates of cellular (and possibly organismal) senescence. We have examined the influence of early growth trajectories in this process, as previous work has suggested that catch-up growth (and a resulting elevation in oxidative stress) causes faster senescence through increasing the rate of telomere erosion. We measured withinindividual changes in growth rate, resting metabolic rate (RMR), antioxidant capacity, oxidative damage and telomere dynamics from early nestling life through to adulthood in the red blood cells of zebra finches Taeniopygia guttata subjected to different post-hatching dietary treatments that induced different growth trajectories. We found that although antioxidant capacity of the plasma increased with age, this was insufficient to prevent increasing oxidative damage (especially in females) as birds reached sexual maturity. Birds undergoing catch-up growth had the highest RMR, the greatest oxidative damage and the fastest rate of telomere loss, but these effects were not simultaneous and differences in telomere length among the growth treatment groups became progressively less marked as the birds grew older. Our results suggest that, while there are links between growth, metabolism, oxidative stress and telomere loss in birds, the reduced lifespan associated with catch-up growth is not due to permanent changes in telomere length induced during the period of growth compensation itself. We discuss potential reasons why this might be so, and suggest directions for future research. Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

A3.7 Oxidative stress as a link between life-history strategies and fitness in vertebrates

David Costantini (University of Glasgow, UK) It is increasingly recognized that the integration of ecological and behavioural sciences with oxidative stress physiology is important for understanding the proximate mechanisms underlying the evolution of life-history strategies, factors modulating phenotype development, and responses of natural populations to predictable and unpredictable environmental changes. In this talk, mostly relying on examples of avian studies, I will discuss which and how physiological and environmental agents can determine changes in redox state, stimulate antioxidant response or cause oxidative stress. I will also discuss how the degree of integration of the redox system may influence its flexibility and so, ultimately, the effectiveness of organism response to stressors. Email address for correspondence: [email protected] 16:20 Saturday 2nd July 2011

A3.8 Birds and bats: differences in enzymatic and nonenzymatic antioxidant defences

Mirella M Kanerva (University of Turku, Finland), Thomas Lilley (University of Turku, Finland), Janina Stauffer (University of Turku, Finland), Miia Koivula (University of Turku, Finland), Tapio Eeva (University of Turku, Finland) and Mikko Nikinmaa (University of Turku, Finland) Birds have a higher metabolic rate, body temperature and lifetime energy expenditure but lower ageing rate than mammals. Birds are also thought to have a less tight association between oxygen radical consumption and production and less oxidative damage than mammals. In this study we measured the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP,) glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), enzymes that are involved in regulating redox balance, the content of total glutathione (totGSH) and the ratio between reduced and oxidized glutathione (GSH/GSSG) from blood samples of great tits and blue tits (local omnivorous birds), and pied flycatcher (migratory insectivorous bird) and from Daubenton's bat (a flying insectivorous local mammal). Bats in general live much longer than expected on the basis of their body size. All the measured parameters except for GSH/GSSG differed significantly between the species. We were unable to detect GR and

A3.6 Mechanistic life-history biology in an ecological context: oxidative stress and fatty acids in great tits

Caroline Isaksson (University of Oxford, UK) Studying molecular and physiological systems that regulate biological functions is fundamental to understanding how variation in life histories arises. Constraints may be external, such as the reliance on specific micro- and macro-nutrients, or internal, such as the risk of oxidative stress or damage. Despite widespread acknowledgement that both may constrain life histories, they have rarely been explored simultaneously in an ecological context.

Abstracts 2011 G6PDH activities from bird blood. We found some differences between adults and juveniles in all of the species. In principal component analysis, the samples formed three distinctive groups: bats, flycatchers and tits. Bats had high SOD and low CAT activities, flycatchers had intermediate SOD and low CAT activities, and tits had low SOD and high CAT activities. Birds had higher totGSH level and GST activity and lower GP activity than the bat. The bats were closer to flycatchers than to tits, although the difference between birds and mammals was bigger than between insectivorous and omnivorous species. Email address for correspondence: [email protected] 16:40 Saturday 2nd July 2011

79 We measured oxidative damage to lipids and proteins in female bank vole (Myodes glareolus) after rearing one litter, two litters, and in non-breeding females. We used bank voles from lines selected for high maximum aerobic metabolic rates (which had high daily average metabolic rates) and non-selected control lines. The oxidative damage was determined in the heart, kidneys and skeletal muscles by measuring concentration of thiobarbituric acid-reactive substances as markers of lipid peroxidation, and concentration of carbonyl groups in proteins as markers of protein oxidation. Surprisingly, we found that the oxidative damage to lipids in the kidneys and muscles, and to proteins in the heart, was actually lower in breeding than in non-breeding voles, and did not differ between animals from the selected and control lines. Thus, contrary to our predictions, females that bred suffered a lower level of oxidative stress than those that did not reproduce. Elevated production of antioxidant enzymes and protective role of sex hormones may explain the results. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A3.9 Antioxidant capacity develops with maturation in the deep diving hooded seal

José Pablo Vázquez-Medina (University of California Merced, USA), José G Soñanez-Organis (University of California Merced, USA), Jennifer M Burns (University of Alaska Anchorage, USA), Tania Zenteno-Savín (Centro de Investigaciones Biologicas del Noroeste, Mexico) and Rudy M Ortiz (University of California Merced, USA) Maturation in hooded seals is characterized by the rapid development of their physiological diving capacity and is accompanied by increases in oxidant production but not with increases in oxidative damage. To test the hypothesis that the antioxidant system of hooded seals develops as they transition from a terrestrial to an aquatic environment, we obtained the complete cDNA sequence that encodes for the NF-E2related factor 2 (Nrf2), a central regulator of the antioxidant response, and compared Nrf2 mRNA and protein expression levels in muscle samples from neonate, weaned pups and adult hooded seals. We also compared glutathione (GSH) levels and the activity/protein content of the antioxidant enzymes catalase, glutathione peroxidase (GPx), peroxyredoxin VI (PrxVI), thioredoxin 1 (Trx1), thiorexoxin reductase (TrxR), glutaredoxin 1 (Glrx1), glutathione disulphide reductase, glutathione S-transferase and glutamate-cysteine ligase. The Nrf2 of the hooded seal is 1822 bp long and encodes for a protein of 606 amino acids with a leucine zipper domain and Keap1-mediated proteosomal degradation residues, which are key for Nrf2 function and regulation. Although neither Nrf2 mRNA nor Nrf2 nuclear protein content are higher in adults than in pups, GSH levels along with GPx, PrxVI, Trx1, TrxR and Glrx1 activity/protein content increase with maturation, suggesting that the potential for peroxide removal increases with development in hooded seals, and that these enzymes contribute to the regulation of the intracellular redox state and the prevention of oxidative damage in these diving mammals. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A3.11 Thyroid status and oxidative stress in birds: controlling mitochondrial oxidant generation in hypermetabolic states

Benjamin Rey (Wildlife Environmental Physiology, School of Physiology, Wits University, Johannesburg, South Africa), Damien Roussel (CNRS, Université Lyon 1, France), Jacques Bodennec (CNRS, Université Lyon 1, France), Jean-Louis Rouanet (CNRS, Université Lyon 1, France) and Claude Duchamp (CNRS, Université Lyon 1, France) Birds are challenging models to explore the relation between redox homeostasis and life history traits, particularly in the context of ageing. Indeed, as compared with mammals, birds present paradoxically extended longevity in regard to their high basal and field metabolic rates, elevated body temperature and high glycaemia. Such characteristics should increase endogenous reactive oxygen species (ROS) production, accelerate oxidative cell damage accumulation and stimulate senescence processes. In the present study, we investigated the effect of pharmacologically altered thyroid status on oxidative stress, mitochondrial oxidant generation, membrane susceptibility to ROS and antioxidant defences in duckling gastrocnemius muscle. We found that, contrary to mammals, the muscle tissue of hyperthyroid ducks presented no elevation in peroxidized lipid content (TBARS). Surprisingly, hyperthyroid ducks also exhibited: i) higher lipid unsaturation index (+13%) and lipid peroxidation index (+84%) in their mitochondrial membranes, suggesting higher susceptibility to ROS; and ii) no significant elevation of the main antioxidant enzyme activities (SOD and GPX). However, intermyofibrillar mitochondrial ROS generation was highly effected by thyroid status, being approximately 70% lower in mitochondria extracted from hyperthyroid as compared with those extracted from hypo- and euthyroid ducklings. Such endogenous ROS regulation may imply basal- and uncoupling protein-induced alteration of the mitochondrial inner membrane potential. We conclude that in birds, unlike mammals, controlling the rate of endogenous oxidant generation may be one of the first-line defences against oxidative stress in organisms subject to energetic constraints that involve large variations in thyroid status. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A3.10 Is reproduction costly? No increase of oxidative damage in breeding bank voles

Lukasz Oldakowski (Institute of Biology, University of Bialystok, Poland), Zaneta Piotrowska (Institute of Biology, University of Bialystok, Poland), Jan R Taylor (Institute of Biology, University of Bialystok, Poland), Katarzyna M Chrzascik (Institute of Environmental Sciences, Jagiellonian University, Poland) and Pawel Koteja (Institute of Environmental Sciences, Jagiellonian University, Poland) According to the life-history theory, investment in reproduction is associated with costs, which should appear as decreased survival to the next reproduction or lower future reproductive success. It has been suggested that oxidative stress may be the proximate mechanism of these trade-offs. Despite numerous studies of the defence against the reactive oxygen species (ROS) during reproduction, very little is known about the damage in the tissues caused by ROS.

A3.12 Anti-oxidant enzyme and complement activity in five wild-caught bat (Chiroptera) species from South-West Finland

Thomas M Lilley (University of Turku, Finland), Mirella Kanerva (University of Turku, Finland), Janina Stauffer (University of Turku,

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Finland), Veronika Laine (University of Turku, Finland) and Mikko Nikinmaa (University of Turku, Finland) The metabolic rates and consequent anti-oxidant enzyme and immune system activity vary between species as a function of genotype and environmental factors. We measured anti-oxidant enzyme activity (SOD, CAT, G6PDH, GR, GP and GST), total glutathione, GSH/GSSG-ratio and the response of the alternative complement pathway, a part of the innate immune system, in five wildcaught bat species: Daubenton's bat (Myotis daubentonii), Brandt's bat (M. brandtii), whiskered bat (M. mystacinus), brown long-eared bat (Plecotus auritus) and Northern bat (Eptesicus nilssonii) from the Finnish Archipelago in South-Western Finland with distinct diets and roosting preferences. The enzyme activities vary between species with apparent contrasts in GST, CAT and SOD activities, especially with Brandt's bat, which has high activities in comparison to the other bat species. Discussion and further results concerning complement pathway activity and the effect of diet and roosting preferences will be discussed in the poster. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

Abstracts 2011

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SEB Main Meeting, Glasgow 2011

A4 Under pressure: Costs and benefits of high systemic blood pressures

A4.1 Body size, dinosaurs and cardiovascular function

Roger S Seymour (University of Adelaide, Australia) To imagine cardiovascular function in dinosaurs it is necessary to understand how metabolic rate, blood flow rate, blood pressure and heart size are related to body size in living animals. High metabolic rates of endothermy are associated with high blood flow and high systemic blood pressure in birds and mammals. In turn, endotherms have more muscular hearts, according to the law of Laplace, and use more energy to pump the blood than ectotherms. Furthermore, larger mammals have greater vertical distances above the heart and compensate by having higher arterial blood pressures. Several lines of cardiovascular evidence, including vertical height, four-chambered hearts, and bone nutrient foramen size, indicate that dinosaurs were behaviourally active endotherms. The long-necked sauropod dinosaurs, however, pose severe cardiovascular problems that cannot be explained by reference to the giraffe. A vertically held neck of some species would have created systemic arterial pressures in the region of 700 mmHg, as compared to 200 mmHg in the giraffe. Such high pressure would have required left ventricles weighing about 15 times that of a similarly sized whale and more than doubled the animal's metabolic rate, both at rest and during activity. Potential solutions to this problem all fail, except for a horizontal posture or aquatic habits. Email address for correspondence: [email protected] 10:20 Sunday 3rd July 2011

A4.3 High blood pressures in ectotherms

James W Hicks (Ecological and Evolutionary Biology, University of California, Irvine, USA) In general, blood pressures (both systemic and pulmonary) in amphibians and non-crocodilian reptiles are considerably lower than equivalent sized mammals and birds. These reduced blood pressures are often correlated with 10x lower metabolic rates measured in these ectotherms. Additionally, the cardiac morphology of these `lower' vertebrates is correlated with the inability to effectively pressure separate the pulmonary and systemic circulations. Cardiac pressure separation is considered a hallmark of the fourchambered system found in endothermic vertebrates. However, the ventricular morphology of many ecotherms results in cardiac shunting. The direction and size of cardiac shunts are often accomplished through reflexive adjustments in the systemic and pulmonary vascular resistances that are correlated with physiological states. The resulting changes in cardiac shunting may convey some physiological advantages. Interestingly, there are three exceptions to these circulatory features of terrestrial ectotherms; the varanid lizards, pythons and crocodilians. In varanids and pythons, a large, partial ventricular septum results in mammalian-like pressure separations during the cardiac cycle. Crocodilians evolved a four-chambered heart similar to birds. The independent evolution of pressure separation anatomy may be associated with unusually high demands for oxygen during activity or digestion in these two species of squamate reptiles. However, the resulting trade-off is an inability to alter shunt patterns via changes in outflow vascular resistance, reducing the circulatory flexibility associated with low metabolic states. In crocodilians the factors that drove the evolution of the four-chambered heart are unknown, but can be tested via phenotypic manipulation of the outflow tracts. Email address for correspondence: [email protected] 11:40 Sunday 3rd July 2011

A4.2 Haemodynamic consequences of having a long neck: giraffes, ostriches and a snake

Tobias Wang (On behalf of the Danish Cardiovascular Giraffe Research Project (DaGiR), Aarhus University, Denmark) Animals with long necks, where the brain may be situated more than a meter above the heart level, have high arterial blood pressures to maintain adequate cerebral perfusion. In both giraffes and ostriches, the tallest living mammals and birds on Earth today, the pressure in the carotid arteries declines with the vertical distance above the heart according to gravity and results in a normal inflow pressure at the brain. In anaesthetised giraffes and ostriches, the pressure generated by the heart is reduced when the head is lowered below heart level, which may be an important mechanism protecting the brain. In both animals, a large volume of blood also pools within the jugular vein when the head is lowered, and this seems to reduce cardiac filling and hence ventricular pressure development on the giraffe heart, but a similar mechanism does not appear to apply to ostriches. In line with these observations, the arterial pressure of giraffes is exquisitely sensitive to volume depletion by bleeding, while the ostrich is less affected. Surprisingly, the relative mass of a giraffe heart is no bigger than other mammals, and we believe that its capacity to generate high pressures resides with relatively small end-diastolic and end-systolic volumes providing a normal mammalian wall tension. This may entail an evolutionary scenario where the ability to generate high blood pressures has evolved at the expense of a reduction in cardiac output. Email address for correspondence: [email protected] 11:10 Sunday 3rd July 2011

A4.4 Evolution of the vertebrate cardiac conduction system

Bjarke Jensen (Department of Biological Sciences, Zoophysiology, Århus University, Denmark), Tobias Wang (Department of Biological Sciences, Zoophysiology, Århus University, Denmark), Quinn D Gunst (Heart Failure Research Center, Academic Medical Center, Universiteit van Amsterdam, The Netherlands), Bastiaan J Boukens (Heart Failure Research Center, Academic Medical Center, Universiteit van Amsterdam, The Netherlands) and Antoon F Moorman (Heart Failure Research Center, Academic Medical Center, Universiteit van Amsterdam, The Netherlands) The endothermic mammals and birds evolved independently from reptile-like ancestors. Both groups nevertheless have similar hearts that beat several-fold more frequently to generate higher systemic blood pressures than in ectothermic vertebrates. Initiation and coordination of chamber contractions then becomes of even greater importance in fast beating hearts and we investigated whether a primordial version of their specialized cardiac conduction systems is present in ectothermic vertebrates. First, we studied the transcription of genes central to the development of the mammalian and avian cardiac conduction system in lizards

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Society for Experimental Biology brainstem oxygenation is within normal limits in the adult SHR, this is only the case when hypertensive; normalizing arterial pressure renders the brainstem severely hypoxic. The SHR brainstem vasculature exhibits up-regulation of genes controlling non-oxidative metabolism and there is depressed reactive hyperaemia when neuronal activity is increased. Moreover, the microvessels are inflamed and clogged with adhered leukocytes; the latter may cause focal ischaemia and oxidative stress. Thus, the SHR is highly vulnerable to hypoperfusion especially if arterial pressure is reduced as in sleep. Recent proof of principle studies demonstrate exaggerated sympathoexcitatory responses when vertebral artery flow is reduced in the PH-SHR but not normotensive rat. Chronic occlusion of both vertebral arteries and a carotid artery (with ipsilateral sinus baroreceptors denervated) produced long-term hypertension in conscious rats. Thus, brainstem blood flow contributes to the maintenance of arterial pressure set-point. Treatment of neurogenic hypertension could consider lowering cerebral vascular resistance. The British Heart Foundation and National Institute of Health funded this research. Email address for correspondence: [email protected] 14:20 Sunday 3rd July 2011

(Anolis carolinensis and A. sagrei) and frogs (Xenopus laevis); nppa, Bmp2, Tbx3, GJA5. The transcription domains in the mature hearts of these ectotherms were found to be comparable in form to those of embryonic mammalian and avian hearts. Second we optically mapped the spread of the activating action potentials over the adult cardiac ventricles with the voltage-sensitive fluorescent dye di-4-ANEPPS. The patterns of activation in fish (Danio rerio), frogs and lizards were found to resemble those of embryonic mammals and birds before the growth of compact walls, the interventricular septum and maturation of the specialized conduction system. We conclude that the conduction systems of adult mammalian and avian hearts are founded on the same morphological and genetic scaffold conserved in fish, amphibians and reptiles. Email address for correspondence: [email protected] 12:10 Sunday 3rd July 2011

A4.5 The role of kidney and blood pressure in the body fluid homeostasis of vertebrates

Hiroko Nishimura (Nephrology Institute, Niigata University, Japan, and University of Tennessee, HSC, USA) The kidney evolved as a device to excrete metabolic waste under low filtration pressure, but its structure and function changed in response to changing environments. The internal osmolality of marine cyclostomes largely depends on that of the external media. Freshwater cyclostomes excrete excess water volume as copious urine, while the distal nephron selectively reabsorbs NaCl to keep internal osmolality constant, making evolution of the kidney important in body fluid homeostasis. In lower vertebrates, renal arterial pressure is an important determinant of glomerular filtration rate (GFR) and urine flow. Accordingly, blood pressure-regulating hormones influence GFR, and intrarenal control of glomerular and renal function has subsequently evolved. For terrestrial vertebrates, water economy is a prerequisite for survival. Only birds and mammals, however, can form hyperosmotic urine to conserve body water. Although birds and mammals represent parallel lines of divergent evolution, the structure and function of avian kidneys have similarity to those of mammals, particularly new-born rodents. Countercurrent urine concentration in birds heavily depends on NaCl recycling that requires a source of energy, operated by a Na-K-2Cl cotransporter and Na/K pump on Henle's ascending limb. Also, the capillary network in avian glomeruli is simpler than that of mammals; thus, the filtration area per glomerulus would be smaller, and high glomerular perfusion pressure may help to maintain GFR. The remarkable features of mammalian kidneys include the development of the inner medulla and a mechanism to maintain a steep osmotic gradient for efficient urine concentration, and intrarenal mechanisms for precise control of glomerular and tubular function. Email address for correspondence: [email protected] 13:30 Sunday 3rd July 2011

A4.7 Blood pressure, resistance and arterial architecture

Michael J Mulvany (Department of Pharmacology, Aarhus University, Denmark) Normal systemic blood pressure (SBP) in humans is ca. 130/70 mmHg (systolic/diastolic). Those with higher SBP show structural changes in the resistance arteries, with decreased lumen diameters and increased wall to lumen ratios. It has long been a question whether the altered arterial structure is a cause or consequence of the high blood pressure. Given that in most organs capillary pressure is around 15 mmHg, it is not immediately clear why SBP of 130/70 mmHg is needed. However, renal glomerular capillary pressure is typically 40 to 50 mmHg, consistent with the filtration function of the kidney. These considerations suggest high SBP may be needed for kidney function. Thus narrowing of renal arteries, in particular of afferent arterioles, might raise blood pressure. This indeed is the case. Constriction of a. renalis results in hypertension. Likewise, in spontaneously hypertensive rats (SHR) the diameter of afferent arterioles is reduced. Moreover, in F2 SHR/WKY animals, those animals with narrowed arterioles when young later develop high blood pressure. In other words here altered structure appears to be a cause of high blood pressure. Further evidence that abnormal resistance artery structure is not the cause of hypertension also comes from animal and human studies, where correction of abnormal structure is not related to a reduction in blood pressure but to the ability of the therapy to dilate blood vessels. Thus abnormal resistance artery structure is more related to vasoactive tone than pressure, while in certain vascular beds, such as the kidney, the abnormal structure could play a role in the pathogenesis of hypertension. Email address for correspondence: [email protected] 14:50 Sunday 3rd July 2011

A4.6 The hypertensive brainstem

Julian F Paton (University of Bristol, UK) The mechanisms within the brainstem that generate excessive sympathetic nerve activity in conditions of neurogenic hypertension are unknown. We are exploring the hypothesis that elevated vertebrobasilar artery resistance drives hypertension. In drug-resistant hypertensive patients there is stenosis of the vertebral and/or carotid arteries. The causative association of this finding is being examined in the spontaneously hypertensive rat (SHR), which can be studied at a pre-hypertensive (PH) age. The vertebrobasilar vascular resistance is higher in the PH-SHR and exhibits increased wall thickness and narrower lumen. Although

A4.8 Cardio-respiratory interactions in vertebrates

Nigel West (University of Saskatchewan, Canada), Bruce Van Vliet (Division of Biomedical Sciences, University of Saskatchewan, Canada) The activity of the respiratory and cardiovascular systems in vertebrates is apparently coordinated to facilitate the conductance of the respiratory gases between the gas exchange medium and tissues. Populations of mechanoreceptors and chemoreceptors in both

Abstracts 2011 the cardiovascular system and respiratory organs provide feedback to the medulla to orchestrate this response. In bimodally breathing vertebrates these receptors additionally integrate the activity of two different gas exchangers. Evolution has resulted in homologies between receptors and their afferents in different vertebrate classes, even when the architecture of the respiratory organs and cardiovascular system is divergent. For example, a parallel, rather than a series organization of the systemic and gas exchanger circuits enables perfusion to be matched to ventilation on a temporal basis in vertebrates, such as amphibians and reptiles, in which ventilation is periodic. Nevertheless, important chemical variables such as arterial blood gas values and mechanical variables, such as arterial pressure, are monitored by receptors with general properties similar to those found in mammals and birds. For example, arterial baroreceptors have been identified in each class of jawed vertebrate, indicating a common need to control arterial perfusion pressure. However, baroreceptors capable of reporting arterial hypotension have only been identified in mammals, in which a high metabolic rate demands high systemic perfusion pressures and continuous ventilation. Historically, the role of feedback mechanisms in cardio-respiratory control has been emphasized, although it is evident that in some groups, such as chelonian reptiles, central feed forward is important in matching cardiovascular activity to ventilation. Email address for correspondence: [email protected] 15:40 Sunday 3rd July 2011

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A4.9 Embryonic development of cardiovascular function

Beerend P Hierck (Lieden University Medical Centre, The Netherlands) Shear stress is the frictional force exerted on vessel walls and the heart by the movement of blood. Endothelial cells (ECs) have the ability to sense these forces and respond with adaptations in protein activation and gene expression. Abnormal blood flow and concomitant shear stress during early embryonic stages leads to the development of congenital cardiovascular malformations. This has been studied in a chicken embryo model for heart development (Venous Clip Model). The differential response of ECs to unidirectional and bidirectional blood flow has been a particular area of focus, since bidirectional flow is usually associated with endothelial activation and the focal development of atherosclerosis in adult life. It induces the formation of primary cilia, which are involved in shear sensing and modulating the endothelial response to shear stress. ECs in predilection areas for atherosclerosis are also ciliated. Collaboration with the Laboratory for Aero- and Hydrodynamics at Delft University of Technology (Prof. J. Westerweel's group) facilitated the development of state of the art technology to visualize blood flow and measure wall shear stress in living chicken embryos, and provided a link between (altered) shear stress and cardiovascular function and development. Email address for correspondence: [email protected] 16:10 Sunday 3rd July 2011

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SEB Main Meeting, Glasgow 2011

A5 New frontiers in O2 homeostasis: NO, nitrite and H2S signalling in animal biology

A5.1 The role of hydrogen sulphide in cardiovascular oxygen sensing

Kenneth R Olson (Indiana University School of Medicine - South Bend, USA) Hydrogen sulphide (H2S) has been shown to have numerous effects on biological systems and it has been classified along with NO and CO as a `gasotransmitter'. Recent evidence suggests that H2S is the elusive oxygen `sensor' that directly couples tissue pO2 to appropriate physiological responses. In the cardiovascular system this enables blood vessels to directly match perfusion to either ventilation or metabolism, and it is the mechanism for chemoreceptor transduction of blood pO2. In this model, the tissue concentration of biologically active H2S endogenously produced in the cytoplasm is balanced by pO2dependent oxidation, and therefore inactivation, by the mitochondria. A number of observations support this hypothesis; the effects of hypoxia and H2S on a variety of tissues are virtually identical, H2S `donors' augment hypoxic responses and inhibition of H2S production inhibits these hypoxic responses. Furthermore, the recent development of amperometric sensors for measuring H2S in real-time and on living tissue, have shown that the transition from H2S consumption to H2S production occurs precisely over the physiological pO2 transition from normoxia to hypoxia. H2S-mediated O2 sensing is also supported by anecdotal evidence including the reciprocal relationship between H2S and O2 in the environment, the origin of eukaryotic cells from the combination of sulphide reducing Archaea and sulphide oxidizing bacteria and by studies that show H2S increases O2 consumption in many tissues. Support: NSF IBN0235223, IOS0641436 and IOS1051627. Email address for correspondence: [email protected] 10:30 Monday 4th July 2011

Sponsored by:

H2S might stimulate the mitochondrial production of superoxide, which is then converted to H2O2. Thus, H2O2 might act as the `down stream' signalling molecule in hypoxic vascular constriction. Email address for correspondence: [email protected] 11:00 Monday 4th July 2011

A5.3 Regulation of proteasome activity by hydrogen sulphide in H9c2 cardiomyoblasts

Daniele Mancardi (University of Turin, Italy), Annalisa Merlino (University of Turin, Italy), Claudia Penna (University of Turin, Italy) and Pasquale Pagliaro (University of Turin, Italy) In recent years hydrogen sulphide (H2S) has been demonstrated to exert several biological functions of pivotal importance. In fact, H2S has been implicated in the regulation of oxygen sensing, in maintaining normal blood pressure levels, in the response to ischaemic insult, and in the regulation of antioxidant defences, to name a few. With the increasing scientific interest, some molecular mechanisms responsible for H2S activity have been elucidated. It now appears clear that its high diffusion rate leads to the targeting of different mediators of the biological response, such as ion channels, enzymes and mitochondrial structural components. Ion channels have been shown to be regulated by exogenous H2S, while a large number of proteins undergo a S-sulfhydration posttranslational modification. Within this framework we wanted to verify whether the protein modification induced by H2S administration was able to regulate the proteolytic activity in a cardiac cell line. H9c2 cells were subjected to 100nM NaSH for 5', 15', 30' and 60' and proteasome activity was measured by fluorimetric assay. In this set-up we noticed a significant increase in the proteasome activation after both 30' and 60' NaSH treatment. These data confirm H2S as a new and important biologic signal molecule and as a new regulator of cell function. Email address for correspondence: [email protected] 11:25 Monday 4th July 2011

A5.2 The role of H2S in hypoxic vasoconstriction

Nini Skovgaard (University of Oslo, Norway) and Kenneth R Olson (Indiana University School of Medicine - South Bend, USA) Hypoxic pulmonary vasoconstriction (HPV) is an adaptive response that diverts pulmonary blood flow from poorly ventilated and hypoxic areas of the lung to more well-ventilated parts. This response is important for the local matching of blood perfusion to ventilation and improves pulmonary gas exchange efficiency. HPV is an ancient and highly conserved response, expressed in the respiratory organs of all vertebrates, including the lungs of mammals, birds and reptiles, amphibian skin and fish gills. The mechanism underlying HPV and how cells sense low pO2 remains elusive. In perfused trout gills (Oncorhynchus mykiss) acute hypoxia and H2S caused an initial constriction of the vasculature followed by dilation. Inhibition of the enzymes CSE and CBS, which block H2S production, abolished the hypoxic response. Blockers of all four complexes in the electron transport chain abolished both the hypoxic and the H2S-mediated constriction. Glutathione, an antioxidant and scavenger of superoxide, attenuated the vasoconstriction in response to hypoxia and H2S. Furthermore, an inhibitor of superoxide dismutase attenuated the hypoxic and H2S constriction. This strongly indicates that H2S mediates the hypoxic vasoconstriction in trout gills.

A5.4 The good and bad sides of nitrite and nitrite-derived NO

Frank B Jensen (Institute of Biology, University of Southern Denmark, Denmark) Nitrite can have both favourable and unfavourable consequences - depending on concentration - and can accordingly be considered a double-edged sword in physiology. Nitrite is endogenously produced

Abstracts 2011 as an oxidative metabolite of nitric oxide (NO), rendering it a marker of nitric oxide synthase (NOS) activity. Under hypoxic conditions, nitrite functions as a NO donor that is activated by various cellular proteins. Thus, nitrite can potentially fuel required NO production when NOS enzymes become compromised by a lack of oxygen. We are particularly interested in such relationships and their importance in hypoxia-tolerant fish. Our studies show that NOS activity is reduced during hypoxia and that nitrite is shifted from extracellular to intracellular compartments to defend intracellular NO homeostasis (Hansen and Jensen, 2010). This appears to be important for hypoxia tolerance and might contribute to metabolic depression and cytoprotection under severe hypoxia and anoxia. Freshwater fish have an additional nitrite supply route compared to mammals, namely the direct active uptake across the gills. This can lead to very high internal nitrite concentrations in nitrite-contaminated waters. The magnitude of nitrite uptake across the gills differs between normoxic and hypoxic fish, but nitrite accumulation inevitably causes excess NO production with extensive nitros(yl)ation of thiols, amines and heme groups. Nitrosative stress can therefore be considered an integral part of nitrite toxicity at high nitrite concentrations (Jensen and Hansen, 2011). References: Hansen M.N. and Jensen F.B. (2010) J Exp Biol 213, 3593-3602. Jensen F.B. and Hansen M.N. (2011) Aquat Toxicol 101, 318-325. Email address for correspondence: [email protected] 11:50 Monday 4th July 2011

85 Barometric pressure falls with increasing altitude while the fractional concentration of oxygen within the atmosphere remains constant. The consequent reduction in oxygen availability (hypobaric hypoxia) poses a formidable challenge to human physiology. Worldwide, more than 140million people live at altitudes >2500 m, and increasing numbers of workers and recreational visitors are exposed to environmental hypoxia. Pulmonary gas exchange and convective oxygen flux by the systemic circulation have long been thought to represent the decisive factors in determining an individual's ability to acclimatize and tolerate these challenges. However, this view has recently been challenged and changes in oxygen handling and utilization by the tissues are increasingly being implicated. Marked inter-individual differences exist in the ability to adapt to hypoxia, but little is known about the functional and molecular pathways underpinning these processes. Unravelling the underlying response mechanisms and identifying molecular signatures of beneficial or maladaptation is likely to be of relevance not only to high-altitude medicine but also to developmental biology and clinical situations associated with hypoxaemia. Recent integrative physiological studies performed during the 2007 Caudwell Xtreme Everest Research Expedition in combination with plasma biomarker studies have provided new insight into the regulatory pathways involved in the acclimatisation process. This presentation will provide an overview of the role of nitric oxide production and availability in the context of enhanced oxidative stress, highlighting some unexpected associations of NO-related biomarkers and physiological variables obtained at rest and during cardiopulmonary exercise testing at various altitudes during the ascent to Everest Base Camp. Email address for correspondence: [email protected] 13:30 Monday 4th July 2011

A5.5 Dramatic increase of nitrite in anoxic crucian carp hearts - possible role in cardioprotection

Guro K Sandvik (University of Oslo, Norway), Frank B Jensen (University of Southern Denmark, Denmark) and Gøran E Nilsson (University of Oslo, Norway) Nitric oxide (NO) has lately emerged as an important cytoprotective agent against anoxia/reoxygenation injury, e.g. via interaction with electron transport complexes in mitochondria. This is believed to prevent the generation of reactive oxygen species upon reoxygenation. Nitrite, once thought to be an inert metabolite of NO but later found to be converted to NO by many proteins, has also been shown to have cytoprotective effects in ischaemia. A close relative to goldfish, crucian carp (Carassius carassius), can survive several months without oxygen in an active state, relying on anaerobic glycolysis with the unusual end-product of ethanol. To investigate the role NO plays in the anoxia tolerance of this fish, we measured NO metabolites in crucian carp exposed to anoxia with chemiluminescence. We also cloned and sequenced crucian carp NO synthases variants and measured their mRNA levels in anoxia in several tissues. Interestingly, we found a massive increase in nitrite and other NO metabolites in anoxic hearts. Moreover, nitrite levels were maintained in brain, gill, liver, and to some degree in muscle and red blood cells. Nitrite levels were decreased in anoxic plasma, indicating nitrite transport from plasma into tissues. We speculate that nitrite in the tissues acts as a NO storage during anoxia, when NO synthases cannot produce NO from L-arginin and oxygen, and that NO and nitrite have cytoprotective effects under anoxia. This can be especially important in the crucian carp heart, which remains active in anoxia. Email address for correspondence: [email protected] 12:15 Monday 4th July 2011

A5.7 Nitric oxide metabolism in the anoxic turtle: insights into synthesis and excretion pathways

Angela Fago (Aarhus University, Denmark) Nitric oxide (NO) and its metabolites nitrite and S-nitrosothiols (SNO) play concerted roles in the control of blood flow and cellular oxygen consumption, particularly under severe hypoxic conditions, as shown in studies on humans native to high altitudes and on hypoxia-tolerant goldfish. The freshwater turtle Trachemys scripta is one of the very few vertebrates able to tolerate prolonged anoxia when submerged under water, which makes it possible to investigate the effect of such extreme adaptive behaviour on circulating NO metabolites. Using chemiluminescent and spectrophotometric assays, we found high basal levels of nitrite in the plasma of normoxic turtles, which suggests a high expression or activity of endothelial NO synthase. After anoxia, plasma nitrite decreased and SNO and iron-NO complexes markedly increased in the blood. These changes indicate that nitrite is consumed during anoxia to generate NO and SNO, consistent with the notion that when oxygen is limiting, nitrite acts as a substrate for NO-generating reactions. In anoxic turtles with no oxygen and low pH in the blood, deoxygenated haemoglobin and carbonic anhydrase are likely to be the main molecules responsible for the conversion of plasma nitrite to NO. Furthermore, we speculate that the high levels of SNO found in anoxia might reflect the lack of excretion of NO and of its gaseous products (NOx) due to lack of ventilation and that red blood cell carbonic anhydrase might be involved in the excretion of NOx in the lung. Email address for correspondence: [email protected] 14:00 Monday 4th July 2011

A5.6 Coping with thin air: the role of nitric oxide and nitrite

Martin Feelisch (University of Warwick, UK)

A5.8 New hypoxia-protective roles for myoglobin in fish

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Signe Helbo (Aarhus University, Denmark) and Angela Fago (Aarhus University, Denmark) For many years myoglobin (Mb) was believed to only play a role in oxygen (O2) storage/supply in oxidative muscle tissue. Recently, however, new Mb functions have been discovered including the regulation of nitric oxide (NO) homeostasis and scavenging of reactive oxygen species (ROS). Also Mb has now been identified in various non-muscle tissues in cyprinid fish and additionally a new Mb isoform was discovered in the carp and goldfish brain. Thus the versatility of Mb function and expression appears to range further than previously believed. In order to expand the understanding of Mb function we have explored the functional properties of Mb in various fish species with special emphasis on the roles in hypoxia-protection. Among other things, we have discovered that rainbow trout Mb has an unusually low O2 affinity that can be allosterically regulated by the covalent attachment of NO to reactive cysteines (S-nitrosation), a unique property that may contribute to O2-linked NO delivery in the trout heart during shortterm hypoxia. In carp, the brain-specific Mb isoform shows several distinct functional characteristics, in particular a fast reaction with H2O2, suggesting a specific role for this Mb in the protection of the brain against damaging ROS. This could be particularly important during reoxygenation after prolonged hypoxia, a condition frequently experienced by this species. These and other data reveal that, at least in fish, the roles of Mb in hypoxia protection are more numerous than previously recognized. Email address for correspondence: [email protected] 14:25 Monday 4th July 2011

A5.10 Effects of nitrite on metabolism and swimming performance of striped catfish

Sjannie Lefevre (Department of Biological Sciences, Aarhus University, Denmark), Frank B Jensen (Department of Biology, University of Southern Denmark, Denmark), Tobias Wang (Department of Biological Sciences, Aarhus University, Denmark) and Mark Bayley (Department of Biological Sciences, Aarhus University, Denmark) Nitrite occurs naturally and enters the blood of freshwater fish through the branchial chloride/bicarbonate exchanger. Once in the blood, nitrite diffuses into the red blood cells and creates methaemoglobin (metHb), which is unable to bind oxygen. Here we investigated the effects of nitrite-reduced oxygen carrying capacity on both the resting and active metabolism of the striped catfish Pangasianodon hypophthalmus. P. hypophthalmus is a facultative air-breather with a high capacity for both aquatic and aerial respiration (Lefevre et al., 2011) and airbreathing could be advantageous during nitrite exposure, since decreased gill ventilation should lower the uptake of nitrite. We therefore measured whether nitrite exposure altered the partitioning of oxygen uptake. We also investigated the effects of nitrite on swimming performance, measured as critical swimming speed (Ucrit) and maximum oxygen uptake (MO2max) and hypothesised that a high level of nitrite would reduce both, while a lower level would not, in accordance with Brauner et al. (1993). Nitrite did not induce air-breathing indicating that this air-breathing fish is insensitive to reductions in total blood O2 concentration. MO2max and factorial aerobic scope were only affected when the functional haemoglobin was reduced below 60% of normal levels. This indicates that P. hypophthalmus can compensate for significant reductions in oxygen-carrying capacity, but further studies are necessary to investigate the mechanism. References: Brauner, C.J., Val, A.L., and Randall, D.J. (1993) J Exp Biol 185, 121-135. Lefevre, S., Huong, D.T.T., Wang, T., Phuong, N.T. and Bayley, M. (2011) Comp Biochem Physiol A 158, 207-214. Email address for correspondence: [email protected] 15:40 Monday 4th July 2011

A5.9 Evolution of nitric oxide synthase in metazoans: a case study of chromatophore organs in Sepia officinalis

Anna Palumbo (Laboratory of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn, Naples, Italy) Nitric oxide (NO) is an important physiological messenger distributed throughout the phylogenetic scale from invertebrates to mammals. Important related issues in current NO biology concern: a) the origin and evolution of nitric oxide synthase (NOS) in metazoans; and b) the evolution of physiological roles of NO and NOS from invertebrates to mammals. As to issue a), we have recently characterized 181 NOS proteins obtained by genome data mining and direct cloning from the lamprey. Comparisons among protein and gene structures, combined with phylogenetic and syntenic studies, allowed for the formulation of a possible scenario of NOS evolution, resulting from multiple gene and genome duplication events together with changes in protein architecture. From the standpoint of comparative physiology (issue b), whereas in man and mammals NO is involved in few crucial functions that comprise neurotransmission, regulation of vasal tone and immune response, in invertebrates the situation becomes paradoxically more complex. A typical case in point is provided by the cuttlefish Sepia officinalis, in which NO plays apparently a much broader variety of functions related to inking and defence behaviour. Especially worthy of note is the regulatory role on chromatophores, the complex structures responsible of the variety of body colouration patterns. NO and NOS are detectable in pigment and muscle fibres in embryo, juvenile and adult chromatophore organs. Pharmacological bioassays and NO production monitoring revealed that NO induces chromatophore expansion through the cGMP/cADP-ribose/ryanodine receptor signalling pathway, acting as an important messenger in the long-term maintenance of body colouration patterns. Email address for correspondence: [email protected] 14:50 Monday 4th July 2011

A5.11 Rest- and submersion-associated apnoeas do not induce systemic oxidative damage in elephant seals

José Pablo Vázquez-Medina (University of California Merced, USA), Tania Zenteno-Savín (Centro de Investigaciones Biologicas del Noroeste, Mexico), Michael S Tift (Sonoma State University, USA), Henry J Forman (University of California Merced, USA), Daniel E Crocker (Sonoma State University, USA) and Rudy M Ortiz (University of California Merced, USA) Seals experience breath-hold (apnoea) bouts while diving and resting. Apnoea in seals is characterized by decreased cardiac output, bradycardia and peripheral vasoconstriction, resulting in ischaemia to peripheral tissues and in hypoxemia. At the end of an apnoea, an increased cardiac output restores blood flow to tissues and blood oxygen content, presenting seals with the potential for excess oxidant production and oxidative stress. As apnoea is a natural component of seals' behaviour, we hypothesize that it is not associated with increased oxidative damage. To test our hypothesis, we conducted rest- and submersion-associated apnoea experiments in six elephant seals. Seals were allowed to rest in a cage or freely submerge in a shallow water tank. Blood samples were collected before, at several time points during apnoeas and after the bouts. Levels of 4-hydroxynonenal, nitrotyrosine, protein carbonyls and 8-isoprostanes, xanthine and hypoxanthine (HX), were measured in plasma. Protein expression of xanthine oxidase (XO), Mn- and CuZn-superoxide dismutases (MnSOD, CuZnSOD), along with

Abstracts 2011 Hypoxia-Inducible Factor 1 (HIF-1) nuclear content, were measured in muscle biopsies collected before and after the apnoeas. None of the measured markers of oxidative damage changed among control, apnoea or recovery samples. Xanthine and HX increased at the end of the apnoeas and during recovery. Muscle XO, MnSOD, CuZnSOD and HIF-1 increased after apnoea. Results suggest that seals do not show adverse effects of apnoeainduced hypoxemia and ischaemia/reperfusion and that the upregulation of their antioxidant defences and HIF-1 are part of the cellular mechanisms that allow them to cope with this condition. Email address for correspondence: [email protected] 16:05 Monday 4th July 2011

87 prolonged fasting increases oxidant production, oxidative damage and inflammation. To test the hypothesis that prolonged fasting in elephant seals does not increase oxidative damage or inflammation, blood and muscle biopsies were collected from early (one to two week) and late (seven to eight week) post-weaned northern elephant seals. Muscle and plasma levels of oxidative damage and inflammation markers, plasma renin activity (PRA), red blood cell (RBC) levels of antioxidant enzymes and glutathione (GSH), and muscle activity/protein expression of NADPH oxidase 4 (Nox4), angiotensin receptor type 1 receptor (AT1) and antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase, glutathione-S transferase, 1-cys peroxiredoxin and glutamate-cysteine ligase) were compared between early and late fasting periods. Fasting induced a 2.5-fold increase in PRA, a 50% increase in AT1, a 70% increase in NADPH oxidase activity and a 2.0-fold increase in Nox4, suggesting that prolonged fasting increases pro-oxidant conditions in elephant seals. However, neither tissue nor systemic indices of oxidative damage or inflammation were increased. Furthermore, muscle and RBC antioxidant enzymes increased by 40 to 60% and GSH content increased 2.0- to 4.0-fold suggesting that the up-regulation of the antioxidant system contributes to the prevention of oxidative damage and inflammation in prolong-fasted seals. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

A5.12 Molecular adaptations to low temperature and high oxygen content in Antarctic fish and marine microorganisms

Cinzia Verde (National Research Council, Institute of Protein Biochemistry, Italy), Daniela Giordano (National Research Council, Institute of Protein Biochemistry, Italy), Roberta Russo (National Research Council, Institute of Protein Biochemistry, Italy) and Guido Di Prisco (National Research Council, Institute of Protein Biochemistry, Italy) The Antarctic marine environment is among the most extreme on Earth due to low and stable temperatures and high oxygen content. Polar marine organisms provide a great opportunity to learn more about the structure and function of haemoproteins and their physiological processes. Over evolutionary time, the globin phenotype undergoes dynamic changes in response to low temperature and high oxygen content. In our talk, the structure and function of neuroglobin of Antarctic notothenioid fish will be presented. We have cloned, over-expressed and purified neuroglobins isolated from the brain of the haemoglobinless icefish Chaenocephalus aceratus and from the retina of the closely related red-blooded fish Dissostichus mawsoni. A detailed structural analysis of the two Antarctic fish neuroglobins was carried out by Resonance Raman spectroscopy, molecular dynamic simulations and laser-flash photolysis to assess the ligand-migration pathways and docking sites. The overall structural view shows that Antarctic fish neuroglobins display several features that differentiate them from their mammalian counterparts, e.g. extensions at the N and C termini that can interact with the EF loop, a gap in the primary structure that could modify the CD-region structure/dynamics in such a way that the two Cys known to form the intramolecular disulphide bridge in human neuroglobin are less distant. Subtle differences between icefish and red-blooded fish neuroglobins were found, suggesting that the icefish protein could act more efficiently as an oxygen-storage protein than human neuroglobin, but also than the red-blooded fish protein. Email address for correspondence: [email protected] 16:30 Monday 4th July 2011

A5.14 Polyphenols ­ possible therapeutic agents against cardiovascular disease

Katarzyna Goszcz (The James Hutton Institute, UK), Ian Megson (Department of Diabetes and Cardiovascular Science, University of the Highlands Islands, Inverness, UK), Gordon McDougall (Plant Products and Food Quality Programme, The James Hutton Institute, Dundee, UK), Derek Stewart (Plant Products and Food Quality Programme, The James Hutton Institute, Dundee, UK) Many epidemiological studies have shown that daily consumption of fruit, including berries (an excellent source of polyphenols), significantly reduces the risk of incidence of numerous degenerative diseases including cardiovascular disease (CVD). Previously numerous studies have followed the paradigm that polyphenols exert their benefits by scavenging free radicals, thereby protecting epithelial cells and by association reducing the risk of CVD. However, more recent developments and the reported limited bioavailability of these phytochemicals have alluded to their role in several other protective mechanisms. These might include some or all of the following: metal chelation, enzyme modulation, cell signalling, pathway and gene expression modulation. The present study will assess whether any of these potential mechanisms of action can indeed reduce risk of cardiovascular disease. To this end, the polyphenol content and the composition of selected fruits [raspberry (Rubus ideaus cv. Glen Ample), blackcurrant (Ribes nigrum cv. Ben Dorain) and blackberry (Rubus fruiticosus cv. Loch Tay)] has been characterized. Extract and selected polyphenols will be subjected to in vitro metabolism and investigated for their bioavailability and bioefficiency in mammalian systems. Furthermore, mechanistic studies will focus on the impact of the polyphenol metabolites in model cellular studies, wherein their role in protecting against oxidative stress and the inhibition and/or retardation of degenerative disease progression will be elucidated. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

A5.13 Prolonged fasting up-regulates the antioxidant system of the northern elephant seal

José Pablo Vázquez-Medina (University of California Merced, USA), Tania Zenteno-Savín (Centro de Investigaciones Biologicas del Noroeste, Mexico), Henry J Forman (University of California Merced, USA), Daniel E Crocker (Sonoma State University, USA) and Rudy M Ortiz (University of California Merced, USA) Elephant seals experience up to three months of absolute food and water deprivation (fasting) while weaning, moulting or breeding without apparent detrimental effects. In terrestrial mammals

A5.15 Angiotensin II and cardiac plasticity in fish

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Society for Experimental Biology 30 days. Both groups showed similar Frank-Starling responses. However, in response to afterload increases, stroke volume rapidly decreased in the control hearts, while it was better maintained in the AngII-treated counterparts. Noteworthy, the AngII-elicited effect was abolished by pre-treatment with the AT2 receptor antagonist CGP42112. Recently, it was reported that cortisol, a major stress hormone in teleosts, induces compacta remodelling in the salmonid hearts (Johansen et al., 2011). Future studies will clarify whether the improved haemodynamic capacity of AngII-treated eel hearts is associated with structural remodelling of the ventricular wall and/or involves the NOS/NO pathway. References: Imbrogno et al. (2003) J Exp Biol 206, 2675-2684. Johansen et al. (2011) J Exp Biol 214, 1313-1321. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

Sandra Imbrogno (Department of Cell Biology, University of Calabria, Italy), B. Tota (Department. of Cell Biology, University of Calabria, Italy), D Amelio (Department of Cell Biology, University of Calabria, Italy) and F Garofalo (Department of Cell Biology, University of Calabria, Italy) The octapeptide angiontensin II (AngII), the principal effector of the renin-angiotensin system (RAS), is a pluripotential hormone whose biological actions include short-term modulation and long-term adaptations. In the eel, the short-term cardiotropic effects of AngII involve a nitric oxide (NO)-dependent pathway (Imbrogno et al., 2003). In contrast, information regarding a role of AngII in cardiac remodelling is lacking. This was explored in the present study using isolated heart preparations of freshwater eel (Anguilla anguilla L.), in which the mechanical performance was characterized on the basis of the Frank-Starling response. Myocardial changes were analysed in control animals and in their counterparts, which were intraperitoneally injected with AngII for

Physiological and Biochemical Zoology

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Physiological and Biochemical Zoology publishes current research in animal physiology and biochemistry at all levels of organization, from the molecular to the organismic, focusing on adaptations to the environment. PBZ covers a wide range of subdisciplines, with a specific emphasis on studies that investigate the ecological and/or evolutionary aspects of physiological and biochemical mechanisms. A valuable resource for teachers and researchers in zoology, physiology, and other biological sciences.

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Abstracts 2011

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SEB Main Meeting, Glasgow 2011

A6 General animal biology

A6.1 Common earthworms can separate the diffusion of oxygen and water vapour

Mark Bayley (Aarhus University, Denmark), Augusto S Abe (Sao Paulo University, Brazil), Martin Holmstrup (Aarhus University, Denmark) and Hans Malte (Aarhus University, Denmark) The common earthworm Aporrectodea caliginosa survives drought by constructing aestivation chambers formed from mucus and gut contents in the top-soil. These chambers are effective at slowing water loss during drought. Given that it has previously been shown that aestivating worms are metabolically depressed and that water can be expected to diffuse slightly faster than oxygen, it follows that the metabolic depression might be a result of hypoxia within the soil chamber. To investigate, we induced aestivation in a soil of a consistency allowing the removal of intact aestivation chambers containing a single worm. A hemisphere of the soil chamber was carefully removed and placed over a small humidity sensor and an oxygen optode embedded in a PVC surface. The edge of the hemisphere was sealed against the PVC with vacuum grease and the diffusion time constants for oxygen and water measured by filling the chamber with water-saturated nitrogen and allowing oxygen to diffuse in and water vapour to diffuse out. These measurements showed unequivocally that the cocoon material slows the diffusion of water while having virtually no effect on oxygen, giving rise to time constants for oxygen that are exceeded by those for water, by more than a factor of 100. The cause of this interesting separation in diffusion might be the presence of lipids or waxes in the soil chamber and data from gas chromatography analysis of the soil will be presented. We can further conclude that any metabolic depression during aestivation is not induced by hypoxia. Email address for correspondence: [email protected] 10:30 Friday 1st July 2011

A6.3 Effects of oxygen availability on maximum aerobic performance in mice selected for basal metabolic rate or aerobic capacity

Andrzej K Gebczynski (University of Bialystok, Poland) and Marek Konarzewski (University of Bialystok, Poland) Maximum aerobic metabolism in mammals is constrained by one (or many) steps of oxygen transport and utilization pathways. To elucidate these constraints we compared peak metabolic rate elicited by running (VO2run) in hypoxia (14% O2), normoxia (21% O2) and hyperoxia (30% O2) of house mice divergently selected for low and high basal metabolic rate (L-BMR and H-BMR, respectively), mice selected for maximum metabolic rate elicited by swimming (VO2swim) and unselected control lines. In all line types VO2run was lowest in hypoxia, intermediate in normoxia and highest in hyperoxia, which suggests a `central' limitation of oxygen uptake or delivery instead of a limit set by cellular oxidative capacity. However, the existence of a common central limitation is in disagreement with our earlier studies showing that selection on high VO (in contrast to selection on high BMR) resulted in considerably higher oxygen consumption during cold exposure in a He-O2 atmosphere than VO2run. While responses of VO2 to hypoxia were similar across different selection regimens, the selection lines showed contrasting responses under hyperoxic conditions. VO2run in the H-BMR line type was highest, suggesting that selection on H-BMR led to increased peripheral aerobic capacity. Overall, line type differences in the effect of pO2 on VO2run and in components of O2 flux pathways are incompatible with the notion of symmorphosis. Our results suggest that constraints on VO2max are context-dependent and determined by interactions between the central and peripheral organs and tissues comprising steps of O2 delivery. Email address for correspondence: [email protected] 11:00 Friday 1st July 2011

A6.2 Cardiorespiratory physiology of spiny rock lobster (Jasus edwardsii) in response to changes in ambient temperature and gas concentration

Leonard G Forgan (NZ Institute for Plant and Food Research, New Zealand) The spiny rock lobster (Jasus edwardsii) shows a remarkable ability to be immersed for extended periods despite having a fully aquatic life history. I present recent data from our laboratory on the development of a state of the art flow-through respirometry system for the measurement of metabolic rate in rock lobsters in both water and air, with simultaneous measurement of cardiovascular and respiratory parameters. Video tracking of the animal is also made possible with the use of an underwater camera. The apparatus allows the investigator to manipulate incurrent gas and water content and animal chamber temperature. Data demonstrating the validity of the preparation and exciting preliminary findings on the effects of anaesthesia, hypoxia, hypercapnia and temperature are presented. These data are placed in context within current research on rock lobster cardiorespiratory physiology. Email address for correspondence: [email protected] 10:45 Friday 1st July 2011

A6.4 Physiological adaptations of the hyperiid amphipod, Phronima sedentaria, to oxygen minimum zones

Leanne E Elder (University of Rhode Island, USA) and Brad A Seibel (University of Rhode Island, USA) Oxygen minimum zones (OMZs) are predicted to expand as global temperatures and CO2 levels increase, and oceanic oxygen levels decrease. OMZs are regions in the world's oceans where oxygen consumption is greater than oxygen supply. In such regions, the biomass of permanent deepwater species is limited. However, diel vertical migration is still prevalent, with many species spending daytime at oxygen levels below 5 µM. This study demonstrates that the vertically migrating hyperiid amphipod Phronima sedentaria is employing metabolic suppression and an increase in anaerobic glycolysis to survive the oxygen levels in the OMZ of the Eastern Tropical Pacific. As global warming continues, OMZs are expected to expand vertically, compressing the night-time habitat of Phronima and other migrating zooplankton, which has implications for both the ecology and biogeochemical cycling in the region. Email address for correspondence: [email protected] 11:15 Friday 1st July 2011

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A6.5 The effect of prolonged hypoxic exposure on the cardio-ventilatory response to acute hypoxia of bowfin (Amia calva) with and without access to air

Cosima S Porteus (University of British Columbia, Canada), Patricia A Wright (University of Guelph, Canada) and William K Milsom (University of British Columbia, Canada) We investigated the effect of acclimation to prolonged hypoxia of bowfin (Amia calva) with and without access to the surface to breathe air on the cardio-ventilatory response to acute hypoxia. Before exposure to prolonged hypoxia, fish without access to air during acute progressive hypoxia (140 torr, 112 torr, 84 torr, 63 torr, and 35 torr for 30 minutes at each level) showed a 2.5-fold increase in gill breathing frequency and a 30% reduction in heart rate; while fish with access to air increased air breathing 5.0-fold and no change in heart rate. After exposure to prolonged hypoxia (43 torr for seven days), fish without access to air during prolonged hypoxia had higher resting heart rates, increased heart rate sensitivity to acute hypoxia, and a decrease in gill breathing sensitivity to acute hypoxia compared to fish that had access to air during chronic hypoxia exposure. Fish with access to air during prolonged hypoxia also tended to have a decreased air breathing sensitivity compared to fish that did not have access to air during prolonged hypoxia, although this was not significant. Surprisingly haemoglobin oxygen affinity decreased after chronic exposure to hypoxia in both fish with and without access to air. Thus, acclimatization to hypoxia reduced respiratory sensitivity (with and without access to air) and increased heart rate sensitivity, indicating that A. calva utilizes different strategies for matching oxygen supply and demand following prolonged hypoxia. Email address for correspondence: [email protected] 11:30 Friday 1st July 2011

A6.7 Mechanisms of Hb adaptation to high-altitude hypoxia in Andean birds

Joana Projecto-Garcia (University of Nebraska Lincoln (UNL), USA), Zachary A Cheviron (UNL, USA), Chandrasekhar Natarajan (UNL, USA), Hideaki Moriyama (UNL, USA), Christopher C Witt (University of New Mexico, USA), Angela Fago (University of Arizona, USA), Roy E Weber (University of Arizona, USA) and Jay Storz (UNL, USA) Highland native species frequently exhibit long-term adaptations such as, for example, enhancement in haemoglobin (Hb) structure, usually leading to an increase in Hb oxygen (O2) affinity. Andean Passeriformes (sparrows) and Apodiformes (hummingbirds) can have similar altitudinal distributions, but occupy distant positions within the avian phylogeny. However, these bird groups might experience similar mechanisms of Hb adaptation to high-altitude hypoxia. To investigate this we are analysing Hb genes and Hb functional properties: a) within a species with continuous altitudinal distribution (Andean rufous-collared sparrow, ARCS); and b) between species with restricted altitudinal distribution (Andean hummingbirds, AHmb). The ARCS (Zonotrichia capensis) has a continuous distribution from sea-level to over 4600 m. Current research on Hb genes from this species shows strong allele frequency differences between low- and high-altitude populations. For these same populations, Hb functional data were generated through diffusion-chamber experiments. Preliminary results demonstrate a clear difference in Hb O2 affinity between low- and high-altitude populations, which agrees with the genetic data. We will present how these functional results can be integrated with the population genetic patterns. Hummingbirds are known to hover, which is a metabolic expensive behaviour, especially in hypoxia. AHmb have a restricted altitudinal distribution and haematological and flight performance data are available for at least nine species, high- and low-altitude combined. The integration of this information with Hb functional properties will help us to understand how high-altitude AHmb species cope with hypoxia. Preliminary results will be presented and compared to the ARCS study. Email address for correspondence: [email protected] 12:00 Friday 1st July 2011

A6.6 Estimating dive performance in early whales

Scott Mirceta (University of Liverpool, UK), Kevin L Campbell (University of Manitoba, Canada) and Michael Berenbrink (University of Liverpool, UK) Remarkable feats of breath-hold endurance have been observed among mammalian divers, with some species routinely diving for an hour or more. Although armed with adaptations to reduce the rate of oxygen consumption, mammalian divers must carry enough oxygen on-board to last the duration of a dive in order sustain aerobic metabolism. There are three main oxygen reservoirs - lung, blood and muscle - that might be drawn upon while submerged, and estimates of these stores are frequently used to predict dive time. As part of our on-going efforts to trace the evolution of breath-hold diving in endotherms, we aim to identify the oxygen pool that best predicts observed dive behaviour across seven lineages of mammalian divers. Data have been pooled from numerous literature sources and oxygen stores calculated from lung volume, blood volume, haemoglobin concentration, and muscle mass and myoglobin concentration. Dividing each store by measured basal rates of metabolism for each species and comparing the rate of oxygen consumption with observed maximal dive time, we find that muscle oxygen reserves are the best predictor of diving capability across all divers. Using phylogenetically informed analysis methods, we further show that maximal muscle myoglobin concentration strongly correlates with net surface charge of the protein, as calculated from myoglobin amino acid sequences. Ancestral myoglobin sequence reconstruction and fossil body mass estimates thus permit the evolution of maximal myoglobin oxygen stores and dive capacity to be reconstructed for early (Eocene) whales such as Pakicetus. Email address for correspondence: [email protected] 11:45 Friday 1st July 2011

A6.8 Reduced haemoglobin buffer values: novel mechanism for enhanced tissue oxygen supply in small endotherms?

Michael Berenbrink (Liverpool University, UK), Kevin Campbell (University of Manitoba, Winnipeg, Canada) and Pia Koldkjaer (Liverpool University, UK) Endotherm birds and mammals generally operate at several-fold higher metabolic rates than ectotherms of similar mass. Mass-specific metabolic rates in the smallest endotherms might furthermore exceed those in their larger relatives by orders of magnitude. It has long been recognized that this challenge to the maximal oxygen transport capacity of small and active endotherms may be met to varying degrees by an increased blood haemoglobin concentration, a stronger Bohr effect, increased tissue capillarization and a reduced haemoglobin oxygen affinity. Here we propose a novel mechanism for enhanced tissue oxygen supply in small endotherms. We have previously reported a distinct evolutionary reduction in physiological buffering amino acid residues in the haemoglobin sequences of passerine birds and shrews. Here we extend these findings to bats, another group of high metabolic rate mammals, and show by acid­base titration of purified bat, shrew and zebra passerine bird haemoglobins that they do indeed have much reduced specific buffer values compared to their larger-bodied relatives. Haemoglobin generally constitutes one of the largest non-bicarbonate buffer components of blood and we suggest that the parallel reduction

Abstracts 2011 of its specific buffering power in at least three groups of high metabolic rate endotherms enables larger arterial-venous pH shifts than would otherwise occur and enhances tissue oxygen supply by a greater exploitation of the Bohr effect. Email address for correspondence: [email protected] 12:15 Friday 1st July 2011

91 and fitness. The present study therefore includes the investigation of a relatively large number of physiological parameters including rates of oxygen consumption, activation energies of metabolism and thermal sensitivity. These parameters will be measured simultaneously over 18-month exposures to elevated CO2 and temperature conditions. This presentation discusses these physiological processes in terms of their possible effects on organism health (differential haemocyte counts and phagocytic activity), growth (growth rate and scope for growth) and reproduction (sperm motility and fertilization success) after three months of exposure. Email address for correspondence: [email protected] 13:30 Friday 1st July 201

A6.9 Woolly monkeys (Lagothrix lagotricha) can distinguish between human hunters and nonhunters in naturalistic conditions

Sarah K Papworth (Imperial College London, UK), E.J. Milner-Gulland (Imperial College London, UK) and Katie Slocombe (University of York, UK) Wild woolly monkeys (Lagothrix lagotricha) in Yasuni National Park, Ecuador, were presented with human models engaging in one of three behaviours `hunting', `gathering' or `researching'. These experiments were conducted at a hunted site and at a site that has been unhunted for at least 40 years. Differences in baseline visibility for individuals was observed between the hunted and unhunted site before model presentation, but no other differences in recorded behaviour were observed. After the presentation of the hunter model, no group increased their calling frequency, in contrast with the other two conditions were the most common response was no change in calling frequency. Groups at the hunted site made fewer large movements (over 5 m) after the presentation of the hunter model whereas groups at the unhunted site showed an increase in large movements. These results provide evidence of threat-sensitive predator avoidance, and suggest that elements of these threat-sensitive responses can be present even when the threat is not expected to have occurred within the lifetime of the prey species. Email address for correspondence: [email protected] 12:30 Friday 1st July 2011

A6.11 The blue mussel mantle transcriptome: identifying genes relevant for biomineralization

Frank Melzner (IFM-GEOMAR, Germany), Anne Huening (Alfred Wegener Institute for Polar and Marine Research, Germany), Eva Philipp (ICMB Kiel, Germany), Lars Krämer (ICMB Kiel, Germany), Philip Rosenstiel (ICMB Kiel, Gemany), Jörn Thomsen (IFM-GEOMAR, Germany), Magdalena Gutowska (Christian-Albrechts University, Germany) and Magnus Lucassen (Alfred Wegener Institute for Polar and Marine Research, Germany) Blue mussels (Mytilus edulis) in the Baltic Sea successfully compete in a challenging environment: low alkalinity leads to low calcium carbonate saturation states of the seawater and upwelling additionally brings CO2-rich water in contact with mussel beds. Yet, blue mussels can calcify at high rates and dominate communities in high pCO2 waters that are strongly undersaturated with calcium carbonate. This is especially interesting as the inner shell surfaces are in contact with an extracellular fluid that is characterized by even higher pCO2 values (as excretory CO2 diffusion gradients have to be maintained) and low pH that is not compensated during hypercapnia. In order to understand this remarkable biomineralization capacity under an adverse carbonate system speciation, we have sequenced transcriptomes from different tissues using 454 pyrosequencing. More than 2million reads from various tissues were assembled into ca. 75 000 contigs, of which 50 to 60% could be annotated. The comparison of transcriptomes from the central mantle and the mantle folds with other, non-calcifying tissues resulted in the identification of ca. 600 highly-expressed sequences that are at least tenfold up-regulated with respect to gill, muscle, foot and digestive gland tissues. Of these 600, ca. 250 are unique for the mantle. Of the unique sequences, 245 are located in the mantle margin, the primary shell formation zone. We are currently utilizing shell regeneration assays to study the function of these unique sequences. Constraining a biomineralization toolkit will enable us to better understand expression patterns in response to abiotic stressors in these ecologically important bivalves. Email address for correspondence: [email protected] 13:45 Friday 1st July 2011

A6.10 The effect of ocean acidification on the physiological responses and organism health, growth and reproduction of ecologically important benthic invertebrates

Samuel S Rastrick (University of Plymouth, UK), Chris Hauton (University of Southampton, UK), Helen Graham (Newcastle University, UK), Nichola Lacey (University of Plymouth, UK), Piero Calosi (University of Plymouth, UK), Helen Findlay (Plymouth Marine Laboratory, UK), Jasmin Godbold (University of Aberdeen, UK), Martin Solan (University of Aberdeen, UK), John Spicer (University of Plymouth, UK), Stephen Widdicombe (Plymouth Marine Laboratory, UK) and Nia Whiteley (Bangor University, UK) To assess the effect of future temperature increase and ocean acidification on intertidal benthic ecosystems, the physiological and immunological responses of three soft sediment and four hard substrate invertebrate species were investigated under different predicted scenarios. These species were selected due to their significant effects on the composition and functioning of coastal benthic ecosystems. As the survival of these organisms is to some extent determined by physiological acclimatization and/or adaptation, it is vital to understand the underlying physiological mechanisms in the face of environmental change, and their metabolic limitations. To date, many studies describing the impacts of global change drivers on organismal functions have focused on single physiological traits in isolation, investigated over relatively short-term exposures. Such studies risk missing long-term trade-offs between different physiological processes that might affect energy budgets and have an impact on performance

A6.12 Hypercapnia tolerance in cephalopod and fish during life history: a role of epidermal ionocytes in the regulation of acid-base disturbances

Marian Y Hu (Institute of Physiology, Christian-Albrechts University, Germany) Yung-Che Tseng (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan), Li-Yih Lin (Department of Life Science, National Taiwan Normal University, Taiwan), Frank Melzner (Leibniz-Institute of Marine Sciences, Germany) and Pung-Pung Hwang (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan) Adult fish (Oryzia latipes) and cephalopod (Sepia officinalis) were shown to tolerate high water pCO2 levels up to 0.6 kPa over long

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Society for Experimental Biology Among cellular signalling pathways, the MAPKs (mitogen-activated protein kinases), including ERK1/2 (extracellular-signal-regulated kinase 1/2), play a central role: they are activated by a wide range of environmental factors and can be involved in many cellular functions. In turbot hepatocytes, we have previously shown that a hypo-osmotic shock from 320 to 240 mOsm.kg-1 induced a rapid increase in ERK1/2 phosphorylation. However, these proteins do not play a role in the cell volume regulation process established to counteract the volume changes induced by the hypo-osmotic conditions tested. In this study, using Western-Blot analysis, some of the intracellular signalling pathways involved in the hypo-osmotic-induced ERK1/2 activation were investigated. Our results show that, in our cell type, ERK1/2 activation happens in two stages: a transient (from 0 to 5 minutes after the hypo-osmotic shock) and a sustained activation (10 minutes after the hypo-osmotic shock). This sustained activation is dependant of several intracellular signalling pathways, such as calcium, protein kinase C, phosphatidylinositol-3 kinase, tyrosine kinases, cytoskeleton or stretch-activated channels. Interestingly, the same signalling pathways are activated after the hypo-osmoticinduced cell swelling. At low osmotic levels, the transient activation of ERK1/2 is independent of all these signalling pathways. Further experiments are now needed: (i) to elucidate some elements involved in the transient activation; and (ii) to understand the role of the hypo-osmotic-induced ERK1/2 activation in these two stages. Email address for correspondence: [email protected] 14:30 Friday 1st July 2011

exposure times. Under the same conditions, cephalopod and fish early life stages show a slowed development accompanied with significant down regulation in the expression of ion-regulatory and metabolic genes. These similar responses towards elevated water pCO2 and increased sensitivity thresholds during early life history might be explained by extreme hypercapnic conditions inside the egg capsule and convergent acid-base regulatory features of fish and cephalopods. In adults of both taxa, the gill is the predominant site of acid-base relevant ion transport by the involvement of key transporters including Na+/K+-ATPase (NKA), Na+/HCO3--cotransporter (NBC), V-type H+ATPase (V-HA) and Na+/H+-exchanger (NHE). However, in early life stages, the skin and yolk membrane are the major sites of acid-base regulation. Using immunohistochemical and electrophysiological techniques, we show for the first time that cephalopod embryos exhibit epidermal ionocytes and that the skin is a mayor site for proton excretion. Similar to fish, ionocytes located on the skin and yolk of cephalopod embryos are characterized by high concentrations of mitochondria, NKA and NHE3. We propose that the presence of ionocytes on skin and yolk can be considered a key feature of high CO2 tolerance in cephalopod and fish early developmental stages. Email address for correspondence: [email protected] 14:00 Friday 1st July 2011

A6.13 Does salinity stress in Penaeus vannamei increase susceptibility to White Spot Syndrome virus?

Giffard M Ivone (Faculty of Marine Sciences, UABC, Mexico), Ramos C Santiago (Faculty of Marine Sciences, UABC, Mexico) and Giffard M Ivone (Faculty of Marine Sciences, UABC, Mexico) It is known that osmoregulation is one of the most demanding energy processes for an aquatic animal. Decapods, specifically Litopenaeus vannamei (or white shrimp), have adapted to live in a wide salinity range becoming the favourite for shrimp world aquaculture; it has been cultured from low salinity (2 ppt) to hyper-saline waters (>35 ppt) successfully. This industry has been severely damaged in past decade by the White Spot Syndrome virus (WSSV) disease and several contradictions have arisen about whether the shrimp is more or less susceptible to WSSV under different salinities. In order to address this question, we used real-time PCR to monitor the expression of a virus protein, WSSVVP664, following experimental infection with WSSV under different salinities. We found that the shrimp is more susceptible to WSSV under extreme salinities (5 to 54 ppt), where the virus induces changes in the shrimp haemolymph osmotic pressure in a way opposite to regular behaviour. Differences on WSSV-VP664 expression levels are related to salinity changes as a result of modifications in the pathogenicity of WSSV on L. vannamei mechanisms according to salinity. Email address for correspondence: [email protected] 14:15 Friday 1st July 2011

A6.15 Who cares if you have the ability, it's how you perform that's important

Gwendolyn K David (University of Queensland, Australia), Daniel Ortiz-Barrientos (University of Queensland, Australia), Michelle Smith (University of Queensland, Australia) and Robbie Wilson (University of Queensland, Australia) In nature, organisms routinely perform highly complex tasks that are critical to their fitness. Based on studies of animal performance, we know a great deal about how individual variation in complex performance is associated with maximal ability in whole-organism traits. However, this relationship may be less important in nature if individuals with poorer ability can behaviourally compensate for this decreased performance or only a sub-maximal ability is actually required to successfully complete complex tasks. Individuals may compensate by engaging in complex tasks with greater frequency or by specialising in specific complex tasks. The importance of such potential compensatory mechanisms is relatively unknown because only a handful of studies have simultaneously investigated three or more whole-organism traits when analysing performance in complex tasks, which is largely due to logistical constraints involved with quantifying non-human systems. In this study, we quantified individual ability across 14 whole-organism traits in humans and complex performance in football matches to test the following hypotheses: i) success in a complex task requires a minimum level of ability; ii) success in a complex task is negatively correlated with its frequency; and iii) individual ability is positively correlated with the highest performance in any one single complex task, but negatively correlated with average performance across multiple complex tasks. Our results reveal interesting relationships among abilities in wholeorganism traits, compensation strategies and performances in complex tasks. We will also discuss the application of this work for improving strategies for the development of young football players. Email address for correspondence: [email protected] 14:45 Friday 1st July 2011

A6.14 Intracellular signalling pathways involved in transient and sustained activation of ERK1/2 in turbot hepatocytes

Audrey Fouchs (Laboratoire ORPHY, Université Européenne de Bretagne, Université de Brest, France), Hélène Ollivier (Laboratoire ORPHY, Université Européenne de Bretagne, Université de Brest, France), Stella Roy (Laboratoire ORPHY, Université Européenne de Bretagne, Université de Brest, France), Patrick Calvès (Laboratoire ORPHY, Université Européenne de Bretagne, Université de Brest, France) and Karine Pichavant-Rafini (Laboratoire ORPHY, Université Européenne de Bretagne, Université de Brest, France)

A6.16 In vivo high-field magnetic resonance imaging of spiders

Abstracts 2011 Gavin D Merrifield (University of Edinburgh, UK), Jim Mullin (University of Glasgow, UK), Lindsey Gallagher (University of Glasgow, UK), Romain Pizzi (Royal Zoological Society of Scotland, UK) and William M Holmes (University of Glasgow, UK) Magnetic resonance imaging (MRI) has established itself as a powerful investigatory technique in both clinical and preclinical arenas, allowing the longitudinal and non-invasive study of a variety of subjects. Using MRI it is possible to obtain structural, functional and physiochemical information from scanned subjects. Recent years have seen the growth and development of highfield scanners optimized for working with small animals. Although increasingly popular in the biomedical field, there has been limited application of these scanners to general biological questions. We scanned several examples of large spiders (various Grammostola species) to test the translational applicability of established smallanimal MRI techniques to a novel type of subject. In terms of body size and anatomy, large spiders present many organs at comparative sizes with more typically scanned rodent subjects. We successfully obtained high-resolution 3D images showing excellent anatomical detail enabling identification of many internal structures as described in the literature. A modified angiography scanning sequence significantly highlighted the gut structure. Cinematic cardiac MRI scans of the beating tarantula heart allowed in vivo quantification of ventricular function using standard image analysis techniques. Our successes suggest that MRI is eminently suitable for studying a variety of biological subjects. Techniques and methods of analysis developed for the biomedical field can be immediately translated into more generic use. Equally, the versatility of MRI suggests that a wider variety of animal models could find use in biomedical research. Email address for correspondence: [email protected] 15:00 Friday 1st July 2011

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A6.18 Adjustment of metabolite composition in the haemolymph to seasonal variations in the land snail Helix pomatia

Annegret Nicolai (University of Rennes 1, France), Juliane Filser (University of Bremen, Germany), Valérie Briand (University of Rennes 1, France) and Maryvonne Charrier (University of Rennes 1, France) In temperate regions, land snails are subjected to subzero temperatures in winter and hot temperatures often associated with drought in summer. The response to these environmental factors is usually a state of inactivity, hibernation and aestivation, respectively, in a temperature and humidity buffered refuge, accompanied by physiological adjustments to resist cold or heat stress. We investigated how environmental factors in the microhabitat and body condition influence the metabolite composition of the haemolymph of the endangered species Helix pomatia. We used UPLC and GC­ MS techniques and analysed annual biochemical variations in a multivariate model. Hibernation and activity months had different metabolite composition. Snails used photoperiod as the cue for seasonal climatic variations to initiate a physiological state and were also highly sensitive to temperature variations, therefore constantly adjusting their physiological processes. Galactose levels gave evidence for the persistence of metabolic activity with energy expenditure during hibernation and for high reproductive activity in June. Triglycerides accumulated prior to hibernation might act as cryoprotectants or energy reserves. During the last month of hibernation, snails activated physiological processes related to arousal. During activity, protein metabolism was reflected by high amino acid level. An exceptional aestivation period was observed in April, providing evidence for heat stress responses, like the protection of cells from dehydration by polyols and saccharides, the membrane stabilization by cholesterol and enhanced metabolism using the anaerobic succinic acid pathway to sustain costly stress responses. In conclusion, physiological adjustments to environmental variations in Helix pomatia involve water loss regulation, cryoprotectant or heat protectant accumulation. Email address for correspondence: [email protected] 16:00 Friday 1st July 2011

A6.17 How does sexual dimorphism change along a latitudinal cline?

Skye F Cameron (University of Queensland, Australia) and Robbie S Wilson (University of Queensland, Australia) Sexual dimorphism arises from different selective pressures on the sexes, in association with competition for mates and resources. Latitude has the potential to modify the level of intrasexual competition due to associated latitudinal changes in population density, activity and resource availability. However, the role latitude plays in mediating the intensity of competition and sexual selection among populations is relatively unknown. In this study, we investigated sexual dimorphism among populations of the Asian house gecko (Hemidactylus frenatus) along a latitudinal cline. Asian house geckos are a highly invasive species that have recently dispersed across Australia, from the tropics to temperate regions. We quantified the influence of latitude on the levels of density, activity, sprint performance and bite performance of H. frenatus. We assessed the extent of sexual dimorphism in body and head size among populations and determined their functional consequences for sprint and bite performance. We predicted there would be higher levels of density and activity at lower latitudes and this would be associated with increased sexual dimorphism in both morphological and functional traits. As expected, changes in latitude resulted in clinal variation in both density and activity in conjunction with larger differences in wholeanimal performance between the sexes along this cline. Implications of this latitudinal cline in sexual dimorphism of both morphological and whole-organism performance traits will be discussed. Email address for correspondence: [email protected] 15:45 Friday 1st July 2011

A6.19 Cryopreservation of dried gemmules from freshwater sponges

Noboru Kaimori (Hachinohe Institute of Techonology, Japan) and Makoto Iijima (Koshigaya Middle School, Japan) Dried gemmules of the freshwater sponges Radiospongilla cerebellata and Ephydatia japonica were collected from a drained pond and preserved in liquid nitrogen vapour for periods of time ranging from one to 184 days (six months). After being stored in this way, they were removed from the liquid nitrogen vapour and incubated to hatch. The proportions of R. cerebellata and E. japonica gemmules that hatched after 184 days were approximately 90% and 70%, respectively. In both sponges, gemmule hatching rates were higher in experimental samples preserved in liquid nitrogen vapour compared to the control samples (non-preserved gemmules). In addition, higher hatching rates were obtained when longer preservation periods were used. Interestingly, the hatching rate of E. japonica gemmules cryopreserved for 184 days was approximately three times greater than that of the control. Sponges that hatched after being preserved for 184 days grew actively for ten weeks of culture in an aquarium, which was the same as that observed in the control specimens. Our results show that the gemmules of these species, and probably many other freshwater sponges, remain viable after extended periods of storage in liquid nitrogen vapour if they are dried at the time of storage. Email address for correspondence: [email protected] 16:15 Friday 1st July 2011

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Society for Experimental Biology Lewis Halsey (Roehampton University, UK), David Booth (University of Queensland, Australia), Manfred Enstipp (University of Strasbourg, France) and Todd Jones (Joint Institute for Marine and Atmospheric Research, USA) Accelerometry is a relatively new technique for estimating metabolic rate. While it shows promise with terrestrial species, its utility with airbreathing divers requires establishing. We undertook respirometry experiments to investigate whether rate of oxygen uptake (V'O2) at the surface in double-crested cormorants (Phalacrocorax auritus), green turtles (Chelonia mydas) and hatchling loggerhead turtles (Caretta caretta) correlates with overall dynamic body acceleration (ODBA), a derivative of acceleration commonly used as a proxy for energy expenditure. Cormorants dived in a 10 m-deep dive tank, green turtles submerged in a 1.5 m-deep tank, and loggerhead turtles swam while tethered in 16 cm-deep water such that they were always at the water surface. V'O2 was related to ODBA in both turtle species but not in cormorants. A likely explanation is that the cormorants, in contrast to the turtles, were alternating between the mediums of water and air during a dive cycle, affecting the V'O2-ODBA relationship. Due to the greater resistance of water, cormorant movement is damped during dives compared to during surface periods, with the result that activity at the surface has an unrepresentatively large effect on ODBA. Furthermore, cormorant body temperature decreases during dives, which may also mask a putative relationship between V'O2 and ODBA, whereas turtles rarely experience an increase in rate of heat loss when they undertake dives because they are almost constantly in water. Diving by air breathers is complex and these findings show that the validity of the accelerometry technique for estimating diving metabolism must be assessed case by case. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011

A6.20 Effect of mild hypothermia on cytokines and oxidative parameters during experimental sepsis

Karelle Leon (Université Européenne de Bretagne, France), Erwan L'Her (Université Européenne de Bretagne, France), Eric Quéméner (Université Européenne de Bretagne, France), Christine Moisan (Université Européenne de Bretagne, France), Aline Amérand (Université Européenne de Bretagne, France) and Karelle Léon (Université Européenne de Bretagne, France) Experimental studies have shown that pro-inflammatory cytokines and oxidative stress are involved in sepsis. Recently, a study performed in the lab has shown the beneficial effect of hypothermia on survival time in a septic rat. The objective of this study was to determine the effects of mild hypothermia on plasma levels of two cytokines (tumour necrosis factor (TNF)- and interleukin (IL)-6), radical production (hydroxyl radical, OH°), and heart tissue levels of superoxide dismutase (SOD) in septic rats. Thirty-six rats were randomly assigned to one of the six experimental groups. For each temperature, normothermia (38°C) or hypothermia (34°C), three experimental groups were considered: 1) the baseline group: the rats were operated on just a few minutes before sampling; 2) the sham group: the rats had a sham-operation four hours before sampling; and 3) the septic group: sepsis was induced four hours before sampling. Experimental sepsis was induced by cecal ligation and perforation. In each experimental group, plasma concentrations of TNF-, IL-6 and OH° were measured. Levels of SOD were determined in heart homogenates. At both temperatures, an increase in TNF- and IL-6 plasma levels was observed in the septic group compared to the sham group but was less pronounced at 34°C (+1095% for TNF- and +90% for IL-6). Email address for correspondence: [email protected] 16:30 Friday 1st July 2011

A6.23 A6.21 Tree-hugging as a thermoregulatory strategy in koalas

Natalie J Briscoe (University of Melbourne, Australia), Stephen Griffiths (University of Melbourne, Australia), Kathrine A Handasyde (University of Melbourne, Australia), Warren P Porter (University of Wisconsin, USA) and Michael R Kearney (University of Melbourne, Australia) Many animals thermoregulate by behaviourally exploiting spatial and temporal variation in microclimates. This is well quantified for ectotherms but remains poorly understood for many endotherms. We adapted a protocol for quantifying thermoregulation in ectotherms to measure the extent that koalas (Phascolarctos cinereus) select microclimates that reduce energetic and hydric costs of thermoregulation relative to a null model. We used a biophysical model (NicheMapper) to quantify the costs of thermoregulation under different microclimates and with different postures. Under hot weather conditions, koalas behaved non-randomly, altering their tree use and posture and hugging the trunks or large lower limbs of trees. We show that this behaviour facilitates heat loss via conduction to tree trunks, and might significantly reduce water loss when compared to alternative postures with minimal tree trunk contact. We also document variation in tree trunk temperatures between tree species, highlighting the potential importance of non-feeding trees for koalas during heat stress periods. Email address for correspondence: [email protected] 16:45 Friday 1st July 2011

Applying dynamic energy budget theory to kangaroo energetics

Jessica A Roberts (University of Melbourne, Australia) and Michael R Kearney (University of Melbourne, Australia) Metabolic theories formally quantify how animals acquire, use and allocate energy and other materials in a mechanistic and parameter-sparse manner. These models are constrained by the laws of thermodynamics and provide a framework for understanding ecological processes beginning at the level of the individual. Dynamic energy budget (DEB) theory is one such approach that links food availability with an organism's maintenance, development, growth and reproduction across its lifespan. We assess the capacity for Kooijman's DEB theory to predict energetics of eastern grey kangaroos (Macropus giganteus) in the context of varying nutritional and climatic environments. Email address for correspondence: [email protected] 10:45 Saturday 2nd July 2011

A6.24 Reproductive performance, growth rate and BMR: correlated after all?

Julita Sadowska (University of Bialystok, Poland), Marek Konarzewski (University of Bialystok, Poland) and Andrzej K Gebczynski (University of Bialystok, Poland) Only a few studies have demonstrated a correlation between basal metabolic rate (BMR) and reproductive performance. We used laboratory mice from the selection experiment in which animals are bred towards high (HBMR) and low (LBMR) BMR and found that

A6.22 Can we use accelerometry to estimate the metabolic rate of air-breathing divers?

Abstracts 2011 between line-type differences in the BMR of nursing females are preserved throughout the whole reproductive event. In a concurrent study, we showed than when given a cross-fostered litter of eight pups (four HBMR plus four LBMR), HBMR females are able to raise larger offspring at an ambient temperature (Ta) of 23°C as well as under conditions of higher metabolic load (17°C). We also collected milk at the eighth and fourteenth day of lactation from HBMR and LBMR females nursing at 17°C Ta. Milk produced by HBMR mothers was richer in protein and fat but not in lactose. We also determined the amount of milk produced by females from both lines at the end of the second week of lactation and found that HBMR females produced milk at higher rates. Our results show that there is a correlation between the level of BMR and reproductive performance and provide support for the assimilation capacity model of the evolution of endothermy. Email address for correspondence: [email protected] 11:00 Saturday 2nd July 2011

95 decreased thermogenic capacity (quantified as maximum Heloxinduced metabolic rate) significantly more in HBMR than LBMR mice. We conclude that large (>40%) intra-specific variation in BMR does not result in higher relative susceptibility to food shortage in the HBMR line, whereas the larger gastrointestinal tract in those mice might enable them to cope better with food shortages. Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

A6.27 Adaptations to exercise training in working and companion dog breeds

Nadine Gerth (Ludwig-Maximilians-University, Munich, Germany), Carolin Ruoss (Ludwig-Maximilians-University, Munich, Germany), Britta Dobenecker (Ludwig-Maximilians-University, Munich, Germany) and Matthias Starck (Ludwig-Maximilians-University, Munich, Germany) Exercise training enhances the oxidative capacity of muscles, increases muscle mass and improves capillary supply to the muscles in dogs. We measured the effects of exercise on oxygen consumption and muscle structure in a group of working Inuit sled dogs (ISDs) and foxhound-boxer-Ingelheim dogs (FBIDs), a companion dog breed. In contrast to the `standard performer' FBIDs, ISDs have been selected for high capacity for oxidative work, fatigue resistance and endurance. In ISDs living in Greenlandic husbandry conditions, we found enlarged muscle fibre diameters (M. adductor magnus) with a high maximum sustained energy expenditure (7.9x RMR) while hauling a sled for eight to ten hours daily in winter as compared to the summer resting condition. The capillary network of the muscle remained unchanged throughout the year. In FBIDs, seven weeks of tread mill training led to enhanced oxidative capacity, but no changes in muscle fibre diameter or capillary network. Peak metabolic rate was about 6.5x RMR. We suggest that the increase in oxidative capacity can root only in changes of the ultrastructural parameters and/or an increase in the mitochondrial volume. We are currently analysing the morphometric design and network of mitochondria in FBIDs and ISDs. We expect to provide explanations for the exceptionally high metabolic scope in ISDs by comparing mitochondrial ultrastructure of ISDs and FBIDs. Our current data strongly suggest that ISDs are exceptionally adapted to sustained oxidative work when compared to other dog breeds. Email address for correspondence: [email protected] 11:45 Saturday 2nd July 2011

A6.25 Fitness consequences of metabolic rate variation in the speckled cockroach, Nauphoeta cinerea

Natalie G Schimpf (University of Queensland, Australia), Philip Matthews (University of Queensland, Australia) and Craig R White (University of Queensland, Australia) It is well known that metabolism varies widely both within and between species. Mass is responsible for the majority of this variation, though even once this is taken into account, metabolic rate can still vary by up to several-fold among individuals within a species. The fact that such variation exists suggests that it is of evolutionary importance. There have been several studies in both endo- and ectotherms examining the consequences of this variation. The studies have looked at the relationship between metabolic rate and various life history traits that can be used as proxies for fitness, such as chick wing growth rates, juvenile and over-winter survival and reproductive performance. No clear relationships between metabolic rate and such traits have been revealed, with negative, positive and no correlations reported. Here we add to this body of research and present some interesting findings on the relationship between metabolic rate and a suite of fitness traits (reproductive output, dominance, cold tolerance and sprint speed) in the speckled cockroach Nauphoeta cinerea. Email address for correspondence: [email protected] 11:15 Saturday 2nd July 2011

A6.26 Does a high level of basal metabolic rate increase susceptibility to food shortage?

Pawel Brzek (University of Bialystok, Poland), Marek Konarzewski (University of Bialystok, Poland), Justyna Franczuk (University of Bialystok, Poland), Andrzej Gebczynski (University of Bialystok, Poland) and Aneta Ksiazek (University of Bialystok, Poland) Energy demands conferred by high basal metabolic rate (BMR) might represent significant ecological cost, particularly when food is scarce. Indeed, several studies have reported a positive link between environmental productivity and BMR on an inter- and intra-specific level. Here, we report the results of an experiment in which laboratory mice from lines artificially selected towards high (HBMR) or low (LBMR) BMR were subjected to eight weeks of dietary restriction (DR; 70% of normal food intake). Although higher food intake in HBMR mice is associated with larger energetically costly internal organs (small intestine, liver, heart and kidneys) and higher spontaneous locomotor activity, DR affected those traits similarly in both lines. However, DR reduced food digestibility significantly more in LBMR mice. DR also

A6.28 Mechanisms underlying individual variation in tolerance of prolonged fasting in European sea bass, Dicentrarchus labrax

D J McKenzie (CNRS Montpellier, France), B Chatain (Ifremer Palavasles-Flots, France), A Vergnet (Ifremer Palavas-les-Flots, France), M Vandeputte (INRA Palavas-les-Flots, France) and B Guinand (University of Montpellier 2, France) Fishes can experience prolonged periods of fasting as a result of seasonal or stochastic reductions in food supply. There is wide individual variation in the rate of mass loss during fasting in fishes such as the European sea bass. Although this reflects, at least in part, a trade-off against rates of mass gain (growth) when feed is available, its mechanistic bases are unknown. A population of sea bass was reared in captivity from broodstock that had been selected for either high or low relative tolerance of fasting. A sub-sample of 2000 individually tagged juveniles were submitted to two experimental cycles comprising three weeks' feed deprivation followed by three weeks' ad-libitum feeding, in a single common tank. Subsequently, 20 individuals from the opposite ends of the tolerance spectrum were identified on the basis of their relative rates of mass

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Society for Experimental Biology pretoriae), half of which were naturally infected with a cestode parasite. Males from both groups did not differ significantly in body mass, urinary testosterone and cortisol concentrations at the onset of the experiment. We observed significant reductions in food intake and body mass following the LPS administration that did not differ significantly between both groups. All males exhibited significant increases in cortisol and reductions in testosterone concentrations in response to LPS and the testosterone levels of infested males were significantly lower than those of uninfested males. Two weeks after the LPS challenge, infested males had significantly lower body weights than uninfested ones. In addition, infested males exhibited significantly reduced haematocrit levels and greater neutrophil to lymphocyte ratios. Our results suggest that male Highveld mole-rats can induce a stereotyped acute phase response irrespective of their infestation status. However, such a response appears to be more costly for infested individuals over the long-term, probably as a result of their depleted resources. Email address for correspondence: [email protected] 12:30 Saturday 2nd July 2011

loss during the periods of fasting. Methods of in vivo respirometry were used to investigate the following hypotheses: i) that tolerance of fasting related to the relative use of proteins as metabolic fuels, with intolerant individuals relying relatively more on proteins; and ii) that tolerance of fasting related to differences in standard and/or routine metabolic rate. These differences became increasingly significant as the period of fasting continued, and this reflected a gradual down-regulation of gut function in tolerant individuals. Methods of video tracking were used to investigate the hypothesis that relative tolerance related to differences in spontaneous activity levels, with intolerant individuals showing increased swimming, presumably reflecting increased foraging behaviour. Email address for correspondence: [email protected] 12:00 Saturday 2nd July 2011

A6.29 Ames dwarf mice: are membrane fatty acids the key to a long life?

Teresa G Valencak (Veterinary University, Vienna, Austria) and Thomas Ruf (Veterinary University, Vienna, Austria) Traditionally considered as beneficial dietary components, membrane polyunsaturated fatty acids (PUFAs) are suspected to shorten lifespan. The negative relationship between maximum lifespan and the proportion of certain fatty acids in tissue membranes has been explained by the biochemical properties of long-chained fatty acids. As PUFAs are particularly prone to peroxidation, they are considered a source of potent damagers of other cellular molecules. The `membrane pacemaker hypothesis of ageing' proposes that the amount of PUFAs in membranes is a determinant of lipid peroxidation and consequently of the rate of oxidative stress and ageing. We tested the hypothesis in a very long-lived mouse mutant, the Ames dwarf mouse. We sampled tissues from the animals at one, two and six months of age and repeatedly assessed tissue fatty acid profiles. We found that Ames dwarf mice in fact possess muscle phospholipids rich in PUFAs (53.4%) but significantly poorer in n-3 PUFAs than normalsized siblings. N-3 PUFAs are among those fatty acids with the highest susceptibility for peroxidation, yet n-6 PUFAs have been reported to cause the most detrimental effects on cells. Muscle tissues from Ames dwarf mice contain 16% more n-6 PUFAs relative to normal-sized, short-lived siblings and 25% more monounsaturated fatty acids. Thus, membrane composition in the Ames dwarf mouse indicates that, as suggested by interspecific comparisons, the ratio of n-3 and n-6 PUFAs affects longevity, yet the cause is unlikely to reside in peroxidation alone. Email address for correspondence: [email protected] 12:15 Saturday 2nd July 2011

A6.31 Pre- and post-natal corticosterone exposure in a precocial bird: investigating the short- and longterm effects on the individual phenotype

Valeria Marasco (Institute for Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, UK), Jane Robinson (Institute for Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, UK), Pawel Herzyk (Institute of Molecular Cell and Systems Biology, University of Glasgow, UK) and Karen A Spencer (School of Psychology, University of St Andrews, UK) Mammalian research shows that exposure to glucocorticoids during early development has permanent effects upon stress physiology in later life. The role of the mother is critical during both pre- and postnatal development. This makes it difficult to disentangle direct actions of the environment and indirect influences to which the developing phenotype is exposed through the mother. The use of a different model is required to overcome this confound. Bird systems are providing a good alternative in developmental stress research. Particularly, precocial birds, where maternal care is almost absent, allow a good bipartition between direct and indirect environmental components. Here, we used an experimental approach to independently manipulate both pre- and post-natal development with physiological doses of the stress hormone corticosterone in precocial Japanese quails. We aimed to determine how early life stress can influence the plasticity of stress physiology, which may alter growth, body homeostasis and behaviour. Preliminary investigations on growth revealed lower patterns only between chicks that experienced stress during both preand post-natal development and pre- and post-natal untreated chicks, but not among the other groups. Similarly, the analysis of glucose levels during restraint stress measured in early life and later during adulthood showed long-lasting sex-specific effects in quails that were treated during both pre- and post-natal development, and only in females. These results suggest that the combined actions of both pre- and postnatal corticosterone exposure may have an additive effect that might contribute to permanently alter the adult phenotype. Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

A6.30 Effects of parasite infection on the acute phase response, hormone levels and haematology of wild Highveld mole-rats

Heike Lutermann (Department of Zoology, University of Pretoria, South Africa) and Nigel C Bennett (Department of Zoology, University of Pretoria, South Africa) Parasites and pathogens divert resources from their hosts by initiating immune responses and directly competing for energy and nutrients. Consequently, host resources can be depleted by infection and this may impair a host's ability to respond to secondary infections. However, in nature multiple infections occur frequently and little is known about how hosts cope with multiple challenges. We simulated a bacterial infection by the administration of a lipopolysaccharide (LPS) to assess the physiological and behavioural responses of 20 wild male Highveld mole-rats (Cryptomys hottentotus

A6.32 Sneaky snails: variability and consistency in behavioural measurements of memory

Sarah Dalesman (University of Calgary, Canada) and Ken Lukowiak (University of Calgary, Canada)

Abstracts 2011 The ability to form memory is an important source of behavioural plasticity. Frequently studies of learning and memory measure a single focal behaviour; however it is likely that any learning paradigm will alter multiple behavioural traits in the same animal. We used video footage of the great pond snail (Lymnaea stagnalis), collected immediately prior to training (operant conditioning) and testing for memory to reduce the number of breathing attempts, to measure two additional `sneaky' behavioural traits: reducing the size of the pneumostome (breathing orifice) opening and shell tilt to cover the pneumostome. Typically the training regime used here, two half-hour sessions separated by an hour, results in memory to reduce the number of breathing attempts lasting 24 but not 72 hours. However, memory duration when measuring the two alternate behavioural traits differs significantly from that measured by the number of opening attempts; shell tilt is very short-lived, lasting only a few minutes after training; whereas the reduction in opening size is still apparent 72 hours after training, when the number of opening attempts has returned to the baseline level. Therefore, conclusions about the ability of L. stagnalis to form memory in response to a single type of training regime will differ significantly depending on the focal trait measured. In addition, we found individual consistency over time in all the behavioural traits measured following training, and significant correlations between the reduction in opening attempts and visible pneumostome area, indicating that these behavioural traits are co-specialized rather than compensatory. Email address for correspondence: [email protected] 13:45 Saturday 2nd July 2011

97 Daniel Hancox (University of Queensland, Australia), Robbie S Wilson (University of Queensland, Australia) and Craig R White (University of Queensland, Australia) Animal colour signalling is one of the most complex forms of intraspecific communication, involving not only the two or more individuals attempting to convey information but the entire visual context. Various aspects of the visual habitat, such as colour of the available light, the background against which signals are viewed, and signal transmission through the environment, can all have profound impacts on the efficacy of a given signal. A small change in any of these factors can often lead to local adaptation of phenotypic traits to the new visual environment, especially as these components often experience a simultaneous shift towards a dominant wavelength of light. Such cases could favour signals that match the dominant wavelength (to maximize signal strength) or those with the greatest contrast. We investigated how between-population variation in the visual habitat of the ornate rainbowfish, Rhadinocentrus ornatus, influences the relative frequency of red and blue colour morphs of this species. This colour-polymorphic freshwater fish inhabits streams and lakes of Fraser Island, Australia, that vary in water colour due to tannin content, which in turn effects the quality of available light and the visual background, creating `red-shifted' habitats. Using spectroradiometry and digital photography, we found that the relative frequency of red and blue individuals could not be predicted by any single habitat variable but relies on the chromatic differences between the available light and visual background. I will discuss the potential sources of variation between these visual habitat components and their implications for colour signalling in this system. Email address for correspondence: [email protected] 14:15 Saturday 2nd July 2011

A6.33 Is impaired memory and learning a cost of the crucian carp's ability to survive anoxia?

Jonathan Stecyk (University of Oslo, Norway), Kath Sloman (University of the West of Scotland, UK), Lisa Yuen (University of Oslo, UK), Christina Sørensen (University of Oslo, Norway) and Göran Nilsson (University of Oslo, Norway) For anoxia-intolerant vertebrates, death through lack of oxygen quickly arises from a failure of the heart and brain, the most oxygen-sensitive organs. In these tissues, cells die by either necrosis (uncontrolled cell death) or apoptosis (programmed cell death). It has long been assumed that anoxia-tolerant vertebrates like the crucian carp survive anoxia because they can effectively counteract deleterious effects of anoxia and do not suffer cell death in their vital organs. Surprisingly, however, we documented a 170% increase in the number of apoptotic cells in the telencephalon of crucian carp following seven days of anoxia and 24 hours of reoxygenation. The fish telencephalon is believed to be equivalent to the mammalian hippocampus, which is important for memory function and spatial learning. We thus examined whether crucian carp exposed to anoxia/ reoxygenation show impaired memory. Indeed, crucian carp trained to navigate a maze for a food reward, exposed to anoxia (5 days at 12°C), reoxygenated for 24 horus and retested took longer to navigate the maze and made a greater number of mistakes during navigation than control fish that were held under normoxic conditions between training and retesting. Thus, the increased prevalence of apoptosis in the brain following anoxia/reoxygenation was linked to actual behavioural impairments, namely reduced spatial memory retention. Research is ongoing to determine whether anoxia/reoxygenation also impairs the crucian carp's spatial learning ability and fear conditioning. Email address for correspondence: [email protected] 14:00 Saturday 2nd July 2011

A6.35 The physiological basis of unreliable signals of strength in males of the fiddler crab Uca vomeris

Candice L Bywater (University of Queensland, Australia), Craig White (University of Queensland, Australia) and Robbie S Wilson (University of Queensland, Australia) Unreliable signals of weapon strength (signals that are poor indicators of strength) are considered problematic for signalling theory and reliable signals are predicted to be the dominant form of signalling among conspecifics in nature. Despite theoretical assertions, the males of several species of crustaceans routinely utilize dishonest signals of strength during agonistic interactions. In this study, we examined the physiological basis of dishonest signals of strength in males of the fiddler cab Uca vomeris. Male fiddler crabs possess one enlarged and brightly coloured claw that is used both as a weapon during disputes with other males and to attract females during courtship. The males of many fiddler crab species can regrow a new claw after losing it during disputes, but this new claw has less mass, is less effective as a weapon and costs less to signal to opponents. Despite the weaker nature of these regenerated claws, they can operate as effective dishonest signals of strength and can deter potential opponents before they fight. We tested the claw strength and metabolic rate of claw tissue of U. vomeris original- and regenerated-claws to determine the consequences of possessing a putative dishonest signal of strength for this species. In addition, we examined the biochemical basis of muscle strength for both types of fiddler crab claw to discover whether individuals with regenerated claws regrow less effective muscle. We will discuss the importance of our results for understanding the evolution of signals of strength. Email address for correspondence: [email protected] 14:30 Saturday 2nd July 2011

A6.34 Visual habitat geometry predicts the relative abundance of red and blue colour morphs in the ornate rainbowfish A6.36 Animal personalities from a physiological perspective

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Uniza W Khan (Norwegian University of Life Sciences, Norway), Dag I Våge (Norwegian University of Life Sciences, Norway), Stig W Omholt (Norwegian University of Life Sciences, Norway), Ida B Johansen (Norwegian University of Life Sciences, Norway) and Øyvind Øverli (Norwegian University of Life Sciences, Norway) `Animal personality' is defined as consistent behaviour over time and in different situations. The evolutionary origins of consistent trait correlations are, however, poorly understood. This presentation will contend that consistency in behaviour is caused by physiological control mechanisms, some of which depend on genetic factors. This point is illustrated by studies of rainbow trout Oncorhynchus mykiss selected for divergent cortisol responsiveness. Focus is placed on the observation that stress-sensitive HR individuals stand out by largely lacking dermal eumelanin pigment, which forms the black skin spots typically seen on salmonid fish. Sequencing and expression analysis of melanocortin 1 and 2 receptors (MC1R, MC2R) and the endogenous MC1R antagonist Agouti signalling peptide (ASIP) reveal consistent gene expression patterns, which are also present in trout populations outside the selection programme. Specifically, stress-sensitive fish show enhanced MC2R expression in head kidney in combination with enhanced ASIP expression in skin, which nicely explains the link between coping style and pigmentation. This conserved molecular link between pigment patterns and stress physiology make salmonid fish a potentially valuable model for understanding the evolution and ecology of animal personalities, since the occurrence of different phenotypes in different environments and their response to variable selective pressures are easily recorded. Email address for correspondence: [email protected] 14:45 Saturday 2nd July 2011

A6.38 Effects of heavy metal accumulation on carbonic anhydrase activity in the mantle and gills of the pearl oyster, Pinctada radiata, from the Persian Gulf

Roghieh Talaei (UMR BOREA MNHN, France), Zohreh Hassan Abadi (Tarbiat Modares University, Iran), Asma Mohamad Karami (Tarbiat Modares University, Iran), Saber Khodabandeh (Tarbiat Modares University, Iran), Hossein Rameshi (Mollusc Studies Station, Langheh, Iran), Françoise Denis (UMR BOREA, National Museum of Natural History, France) and Stephanie Auzoux-Bordenave (UMR BOREA MNHN, France) In molluscs, carbonic anhydrase (CA) was identified in the gills and mantle and has long been linked with shell formation. Recently, a decrease in shell hardness has been reported in the Persian Gulf pearl oyster. Since heavy metals are known to inhibit shell formation and can cause significant changes in shell thickness and composition, the role of metallo-enzymes in this process is suggested. We investigated the relationship between Cu, Cd, and Zn accumulation and CA activity in the mantle and gills of Pinctada radiata originating from two stations in the Persian Gulf, Hendorabi and Langheh, exhibiting an increasing pollution level. Results showed that, whereas Zn accumulation in the mantle was not significantly different between the two stations, it was significantly higher in the gills from Langheh station. High levels of Cu and Cd were measured in both mantle and gills from the oysters of the two stations. CA activity was higher in the mantle than in the gills and for both tissues. Hendorabi samples showed a higher CA activity compared to the Langheh samples. To conclude, a high level of heavy metals in oyster tissues, particularly in the gills, is evidenced in Langheh station compared to Hendorabi. Moreover, the levels of Cu, Cd and Zn in oysters are associated with reduced CA activity. These preliminary results confirm that CA activity is sensitive to heavy metal accumulation and supports the idea that the bio-mineralization process of the pearl oyster can be affected by high Cu and Cd exposure in the marine environment. Email address for correspondence: [email protected] 15:45 Saturday 2nd July 2011

A6.37 Orb web spiders: a new behavioural model for studies on ageing

Mylene Anotaux (UMR, Nancy University, France), Julia Marchal (UMR, National Museum of Natural History, France), Guenael Cabanes (LIPN, University of Villetaneuse, France), Raymond Leborgne (Nancy University, France), Nicolas Châline (UFR, University of Villetaneuse, France), Caroline Gilbert (IPHC-DEPE, University of Strasbourg, France) and Alain Pasquet (UMR, Nancy University, France) Ageing is known to induce profound effects on physiological functions, but only few studies have addressed the associated behavioural modifications. In orb weaving spiders, the web is a complex geometrical structure that presents a visible regularity and varies with spider development. Its construction results of a succession of organized and repeatable activities classically considered stereotypical behaviours. We used the web of the Zygiella-x-notata spider as a model to test the hypothesis that ageing alters web geometry. Parameters taken into account to compare webs weaved by spiders at different ages were the number of anomalies (e.g. number of deviated radii, holes in the web, spiral threads stuck by two) and the regularity of the construction (angles between radii, number of spiral turns, parallelism between the spiral turns, and variability of distances between spiral turns). We showed that, at the inter-individual level, older spiders' webs presented more anomalies and were less regular. At the intra-individual level, these age-induced changes were highly significant when comparing the first web and last web weaved before death. These results suggest that web-building behaviour in adult spiders is age-specific and that the degree of changes induced by ageing is timeto-death dependant. Orb web spiders are therefore a pertinent and original behavioural model for the study of ageing. Email address for correspondence: [email protected] 15:00 Saturday 2nd July 2011

A6.39 `Toxic nectar': nicotine has both harmful and beneficial effects on honeybees

Angela Koehler (University of Pretoria, South Africa), Christian W Pirk (University of Pretoria, South Africa) and Susan W Nicolson (University of Pretoria, South Africa) The presence of secondary metabolites in floral nectar seems paradoxical, in view of the reward function of nectar, and little is known of their role in mediating plant-pollinator interactions. Nicotine, best known from the Solanaceae, is highly toxic to most herbivores and is also present in nectar and pollen. Nicotine and its synthetic analogues (neonicotinoids) are used as insecticides and have been suggested as contributing to the observed pollinator declines that are currently of great concern internationally. We were therefore interested in examining the effects of nectar nicotine on honeybee (Apis mellifera scutellata) foraging choices and worker longevity. Using preference tests on free-flying honeybees, we have shown that the dose-dependent deterrent effect of nicotine is stronger in more dilute nectars, consistent with secondary metabolites acting as partial deterrents that keep pollinators moving between plants and ensure cross-pollination. To assess the consequences of ingesting nicotine, newly emerged workers were caged in incubators and fed varying concentrations of nicotine in 0.63 M sucrose solution for 21 days. Honeybees tolerated naturally occurring nectar nicotine concentrations (30 µM), but nectar storage in the honey comb and survival decreased on high dietary nicotine (300 µM), thereby possibly having a detrimental effect on colony fitness. However, energetically challenged colonies that survived poorly on sugar-only diets demonstrated increased

Abstracts 2011 survival on nicotine diets. This suggests that low nectar nicotine concentrations could be beneficial to honeybees, possibly due to its antimicrobial properties. Email address for correspondence: [email protected] 16:00 Saturday 2nd July 2011

99 steroidogenic acute regulatory protein and cytochrome P450 sidechain cleavage in the brain and head kidney, were not altered by this pharmaceutical. However, venlafaxine exposure clearly attenuated the stressor-induced elevation in plasma glucose concentration. This may have important implications in the ability of the animal to regain homeostasis or in the display of adaptive behaviours in order to cope with secondary stressors. Email address for correspondence: [email protected] 16:30 Saturday 2nd July 2011

A6.40 Persistent organic pollutants interact with corticosteroid receptors in European flounder (Platichthys flesus)

Louise Colliar (University of Stirling, UK), Michael J Leaver (University of Stirling, UK) and Armin Sturm (University of Stirling, UK) It is well documented that certain pollutants can interfere with sex steroid signalling in fish. Less is known about the potential of environmental chemicals to disrupt other endocrine axes. In this study, the interaction of persistent organic pollutants with corticosteroid receptors (CRs) from European flounder (ef) was investigated. Using PCR techniques, we isolated cDNA encoding two glucocorticoid receptors, efGR1 and efGR2, and one mineralocorticoid receptor, efMR, from flounder gills. The mRNA expression of all receptors was detectable in all tissues examined. The ligand binding and transactivation properties of the ligand-binding domains of receptors were analysed by generating fusion proteins with the yeast transcription factor GAL4. GAL4GR1, GAL4GR2 and GAL4MR bound corticosteroids with high affinities. When constructs were transiently expressed in a fish cell line, transcriptional activities of GAL4GR1 and GAL4GR2 were induced by glucocorticoids, but not the mineralocorticoid aldosterone, and the GR antagonist RU486 blocked induction. With GAL4MR, aldosterone was a better agonist than glucocorticoids, and the MR antagonist spironolactone was a more potent than RU486. Thus, the ligand and antagonist selectivity of receptors appears to be retained in GAL4 fusion proteins. At concentrations of 100 nM and above, the persistent marine pollutant tris(tributyltin)oxide (TBTO) blocked the hormonal induction of transactivation activities of GAL4MR, GAL4GR1 and GAL4GR2. Bisphenol A and benzylbutylphthalate also showed antagonist activity on GR-derived constructs, but were less potent. In view of the widespread occurrence of these pollutants, further studies into their potential to cause endocrine disrupting effects in fish should be carried out. Email Address for correspondence: [email protected] 16:15 Saturday 2nd July 2011

A6.42 The interactive effects of ammonia exposure, nutritional status and exercise on the ecological fitness of gold fish (Carassius auratus L.)

Amit K Sinha (University of Antwerp, Belgium), Gudrun De Boeck (University of Antwerp, Belgium), Ronny Blust (University of Antwerp, Belgium), Marjan Diricx (University of Antwerp, Belgium) and HonJung Liew (University of Antwerp, Belgium) This study aimed to elucidate the combined physiological effects of high ambient ammonia following periods of feeding (2% body weight) and fasting (unfed for seven days prior to treatment) in gold fish (Carassius auratus). Both feeding groups of fish were exposed to elevated ammonia (1 mg/L) for 0 hours (control), three hours, 12 hours, one day, four days, ten days, 21days and 28 days. Measurements of weight gain (%), oxygen consumption, critical swimming speeds (Ucrit), plasma and muscle ammonia accumulation, ammonia excretion rate, glycogen level, lactate content in plasma and ammonia quotient during experimental periods were significantly affected by feed deprivation. This implies that unfed fish were more sensitive to external ammonia than fed fish. The toxic effect of ammonia exposure in both feeding treatments were exacerbated further when imposed to exercise (swum at 3/4th Ucrit), suggesting that fish become more vulnerable to external ammonia during exercise. Such effects were more pronounced in fasted fish as compared to fed fish. Therefore, it was evident from our study that feeding ameliorates ammonia toxicity during both non-exercise and exercise modes of swimming. Consequently, our findings recommend that nutritional status and swimming activities of fish must be considered when determining guidelines for the threshold concentration of waterborne ammonia toxicity. Email address for correspondence: [email protected] 16:45 Saturday 2nd July 2011

A6.41 Environmentally relevant levels of venlafaxine impact stress response in rainbow trout

Nataliya Melnyk-Lamont (University of Waterloo, Canada), Mathilakath M Vijayan (University of Waterloo, Canada) and Carol Best (University of Waterloo, Canada) Municipal wastewater treatment disposal is a primary route introducing pharmaceuticals into aquatic environments. Venlafaxine, a serotoninnorepinephrine reuptake inhibitor, is one of the most prescribed antidepressant drugs commonly found in aquatic systems of North America. The objective of this study was to investigate whether this pharmaceutical drug will affect stress performance in rainbow trout (Oncorhynchus mykiss). Fish were exposed to venlafaxine (0.2 ug/L and 1.0 ug/L) at concentrations detected in the municipal wastewater effluent around this region for seven days. These fish were then subjected to an acute handling stress for five minutes and sampled at one, four and 24 hours post-stressor exposure. Venlafaxine exposure did not modify the stress-induced cortisol response in rainbow trout. Also, the transcript abundance of glucocorticoid receptors or key steroidogenic genes, including

A6.43 Recombinant protein production of abundant larval transcript (ALT)

Karman Ashraf (Faculty of Pharmacy, Hamdard University, India) and Mujeeb Mohd (Faculty of Pharmacy, Hamdard University, India) Lymphatic filariasis is a major tropical disease caused by the mosquitoborne nematodes Brugia and Wuchereria. About 120million people are affected by this disease. Vaccine against filariasis must generate immunity to infective mosquito-derived L3 stage nematodes. Two highly expressed genes designated abundant larval transcript-1 and -2 (alt-1 and alt-2) produce the closely related proteins ALT-1 and ALT-2 (79%). The expression of the alt genes in E. coli BL21 plysS for the production of vaccine is major challenge as no vaccine is viable against this disease. Work has been carried out to express this protein at laboratory bioreactor scale. High cell density culture, maximum specific activity with good growth rate, etc., are some of the important factors that need to be maintained in order to get the maximum amount of recombinant protein. In shaken flask studies, after induction (maximum cell density and specific growth rate stage) a good expression of ALT protein was found. At laboratory-scale production done in bioreactor, however,

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Society for Experimental Biology Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

expression level drastically decreased. Plasmid stability analysis was carried out in a reactor and was found to be a cause of decreased productivity. The stability was improved by increasing the antibiotic concentration in the medium and also by pulsing antibiotics during induction. This led to better plasmid stability and increased expression in a reactor to expression levels similar to those in shake flask studies. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.46 Effects of selection for body size on early-life traits and gene expression in the skeletal muscle of zebrafish (Danio rerio)

Ian PG Amaral (University of St Andrews, UK) and Ian A Johnston (University of St Andrews, UK) This study investigated the effect of artificial selection for body size at 90 days post-fertilization (dpf) in the zebrafish (Danio rerio). After four rounds of artificial selection for divergent body size, three lines were produced: small (S-line), unselected (U-line) and large (L-line). The activation of the m-TOR signalling pathway regulating protein turnover was investigated in a fastingrefeeding experiment with the S- and L-lines. Larvae from the L-line were larger at six dpf, but survivorship at 30 dpf was not affected by the selection regime. Standard length and body mass of fish from the L-line was 5.1 and 15.7% higher than fish from the U-line and 12.3 and 41.9% higher than fish from the S-line. The general pattern of expression from the insulin-like growth factor pathway and other nutritionally-responsive genes in fast myotomal muscle was mostly similar in S- and L-lines. However, seven of the 27 genes studied showed significantly different patterns of expression with fasting (IGF2B, IGF1BR, IGF2R, IGFBP1A and IGFBP1B) or feeding (IGF1A and IGF1AR). These results contribute to a better understanding of the general effects of selection for body size on the life-history of the zebrafish and might help us to understand the genetic changes in fish subjected to selection pressures, with important implications in both aquaculture and fisheries ecology. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.44 Energy or specific nutrients: which one matters for metabolite storage in fruit flies?

Bohdana M Rovenko (Vassyl Stefanyk Precarpathian National University, Ukraine), Volodymyr I Lushchak (Vassyl Stefanyk Precarpathian National University, Ukraine), Oleh V Lushchak (Vassyl Stefanyk Precarpathian National University, Ukraine) and Dmytro V Gospodaryov (Vassyl Stefanyk Precarpathian National University, Ukraine) Many scientists suggest that diet composition and nutrient ratios affect metabolic processes to a greater extent than total energy intake. This work aims to assess the effects of dietary carbohydrate on energy balance, glycogen, total lipid and protein content, and body mass in fruit flies, Drosophila melanogaster. The media with glucose, fructose, sucrose and an equimolar mixture glucose and fructose in five concentrations from 0.25% to 20.00% were used. All diets contained yeast at a concentration of 4%, but had different carbohydrate to yeast ratios (ranging from 1/16 to 5/1) and different caloric content. Increasing the energy content in diets produced a gain in body mass, which accounted for the five- to sevenfold increase in glycogen and total lipids, except for sucrose-containing diets, which did not significantly increase body mass in flies of either sex. Regarding body protein content, contradictory results were obtained. The increase of carbohydrate content in diets enhanced body protein content with all dietary mixtures, except for males fed with sucrose. In contrast, the relative protein content (percentage protein weight relative to total weight) decreased in fruit flies of both sexes fed with glucose and fructose, but was not changed in flies fed with sucrosecontaining dietary mixtures. The observed differences in metabolite content in fruit flies can be explained by differences in food consumption of media with different carbohydrates. The results show that glucose, fructose and sucrose intakes have different effects on body composition and mass in fruit flies. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.47 External calcium availability alters memory formation in a freshwater snail

Sarah Dalesman (University of Calgary, Canada), Marvin Braun (University of Calgary, Canada), Vikram Karnik (University of Calgary, Canada) and Ken Lukowiak (University of Calgary, Canada) Dissolved calcium availability limits the distribution of many freshwater organisms, thought primarily due to limitations on growth and reproduction. For calciphiles, such as the great pond snail Lymnaea stagnalis, environmental calcium is considered to be the major abiotic limitation on their distribution, with a lower limit of 20 mg/l cited. Stress has been found to both enhance and block the ability of L. stagnalis to form long-term memory. We predicted that low calcium availability would act as a stressor for this species at a level they experience in natural populations; however, it was unknown whether this stress would block or enhance memory formation. L. stagnalis is a pulmonate, breathing via cutaneous respiration in eumoxia but switching to aerial respiration via a basic lung in hypoxic conditions. We can train the snails using operant conditioning to reduce aerial respiration attempts in hypoxia, normally resulting in long-term memory (> 24 hours) in our standard calcium environment (80 mg/l). Acute exposure to low environmental calcium (20 mg/l), however, blocked their ability to form long-term memory following this training regime. This was demonstrated both behaviourally and via a reduction in the electrophysiological response to training in a neuron of the central pattern generator that controls aerial respiration, RPeD1. We demonstrate that this is due to the snail's ability to directly sense calcium availability in their environment. Thus, despite this species surviving in low calcium environments, they exhibit less adaptability due to a decreased capability to form memory. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.45 Development of a model system for studying horse allergy

Sari S Sabban (University of Sheffield, USA), Hong Tu Yi (University of Sheffield, UK), Joy Zhao (National Institutes of Health, USA), Peter Schuck (National Institutes of Health, USA) and Birgit Helm (University of Sheffield, USA) The interaction of equine immunoglobulin E (IgE) with its high-affinity FcI receptor was investigated following the cloning and expression of equine IgE with specificity for NIP. Receptor recognition and effector functions were assessed in RBL-2H3.1 cells transfected with the -chain of the equine FcI. Receptor binding and stimulus secretion coupling were also tested for human, canine and mouse IgE. Results obtained showed that the equine FcI receptor recognizes both equine and canine IgEs and supports very similar -hexosaminidase release levels. By contrast, the equine FcI did not bind to human IgE and consequently no mediator release was observed.

Abstracts 2011

101 Shivayogeeshwar Neelgund (PG Department of Biochemistry, Kuvempu University, India), Gurumurthy D M Gurumurthy (PG Department of Biochemistry, Kuvempu University, India) and Sanna Durgappa (Indian Institute of Science, India) The extreme thermostable properties of enzymes from different thermophilic groups are of potential interest in industrial processing conditions. Many thermostable enzymes have placed a latent strategic position in extremist reactions like starch saccharification and in the production of high- glucose syrup. In this study, thermal springs were explored to isolate thermostable bacteria that could produce thermostable enzymes. The chemical profiling of thermal water revealed the elevated concentration of S, Fe and Ca. Other trace elements, such as Mg, Mn, Co, Cu, N and C, were present in lower concentrations. Ni, Hg, Ti and Sn were observed below the detection level. Thermostable alkali-resistant gram-positive, rod shaped Geobacillus sp Iso5 was isolated from the thermal water of Southern Indian springs. Identification of bacteria was done by conducting biochemical tests and 16s rRNA gene sequencing. The isolate comprises of 55% mol G+C content, iso-C15, iso-C16 and iso-C17 types of fatty acids. Phylogenetic constraint reveals that 98 to 99% sequence similarities was observed with the thermophilic members of the Geobacillus group: Geobacillus kaustophilus (98%), Geobacillus thermoleoverance (99%) and Geobacillus sp NT60. The sequence was deposited in NCBI under the accession number GQ140232. The bacterium was optimized for the production of hyper thermostable -amylase using starch as the sole carbon source. Further, thermostable - amylase was purified successively by using conventional biochemical techniques, such as ammonium sulphate precipitation, desalted using Sephadex G-150 gel filtration chromatography and DEAE-cellulose column. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.48 Long-term effects of ocean acidification on the Norway lobster, Nephrops norvegicus

Claudia SN Tavares (Heinrich-Heine Universität, Düsseldorf, Germany), Jim A Atkinson (University Marine Biological Station Millport, UK), Christopher R Bridges (Institut für Stoffwechselphysiologie Heinrich Heine Universität, UK), Alan C Taylor (Department of Ecology and Evolutionary Biology, University of Glasgow, UK) and Philip Smith (University Marine Biological Station Millport, UK) Ocean acidification is expected to have detrimental effects on commercially important crustaceans. This study investigates longterm changes in Norway lobster, Nephrops norvegicus, in response to reduced seawater pH resulting from increasing levels of atmospheric CO2. Norway lobsters sampled from depths of 80 to 100m in the Firth of Clyde, western Scotland, were maintained for five months at three different levels of dissolved CO2: 380 ppm (control, pH 8.2), 800 ppm (pH 7.75) and 3000 ppm (pH 7.25). Survival, activity and limb loss were monitored daily, and lobsters were weighed and haemolymph samples taken at monthly intervals. Early observations show that mortality and the incidence of cheliped loss were highest at 3000 ppm CO2. The first cheliped to be lost was more often the smaller, narrower (`cutter') claw. Responses in the 800 ppm group did not differ from the control group. Further investigation is planned to analyse changes in haemolymph chemistry and to establish whether limb loss is due to reduced calcification. To analyse structural changes in the exoskeleton, carapace and cheliped samples will be examined by scanning electron microscopy. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.49 Effects of corticosterone on reproductive decisions and parental behaviour in great tits, Parus major

Jenny Q Ouyang (Princeton University, USA), Marion Muturi (University of Konstanz, Germany) and Michaela Hau (Max Planck Institute for Ornithology, Germany) Hormones coordinate many aspects of life history and behavioural traits and are critically involved in the environmental adaptation of complex traits. However, our current knowledge of how the endocrine system regulates reproductive decision-making before the breeding season is limited. Glucocorticoid hormones maintain homeostasis and are candidates for regulation of the reproductive effort. In a correlative study on great tits, Parus major, we found that high levels of baseline corticosterone concentrations before egg-laying are indicative both of the proximity of an individual to lay and of high reproductive output over the entire breeding season. Therefore, in the current study, we experimentally elevated baseline corticosterone levels in free-living male and female great tits two weeks before egg-laying to test whether corticosterone causally regulates reproductive decisions. We assessed parental investment rules of manipulated and control individuals by determining the effects of corticosterone on lay-date, incubation behaviour and feeding rates. We will discuss the results in the context of the mechanisms that govern the expression of phenotypic variance. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.51 Strong effect of pH on the temperature sensitivity of red cell oxygen binding in rainbow trout

Samantha L Barlow (University of Liverpool, UK) and Michael Berenbrink (University of Liverpool, UK) It has long been believed that the physiology of fish is strongly affected by changes in environmental temperature. An increase in temperature increases metabolic rate, thus increasing a need for oxygen while decreasing haemoglobin oxygen binding affinity and oxygen solubility. As such, there has been much interest in species of tuna that are partially endothermic in nature, and produce and retain heat in some core muscles. It has been found that the oxygen-binding affinities of tuna haemoglobins have low or even reversed temperature sensitivity. A similar phenomenon has been observed in red cells of one genotype of Atlantic cod (Gadus morhua) and in the blood of Chub mackerel (Scomber japonicus). Here we analyse the oxygen affinity of rainbow trout (Oncorhynchus mykiss) red cells by determining the P50 of oxygen binding curves at 5, 15 and 25°C, and corrected to a constant pH of 7.5 and 7.9. The apparent heat of oxygenation (H) showed that at both pH values between temperatures of 15 and 25°C, haemoglobin was highly temperature sensitive (H ca. -20 kJ mol-1). This sensitivity was reduced between 5 and 15°C. This was particularly apparent at pH 7.5, where the apparent heat of oxygenation was reduced to close to zero. These data, together with other recent literature data call for a reevaluation of generalizations about the temperature sensitivity of haemoglobin oxygen binding in ectotherms. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.50 Refining of hyperthermostable -amylase from thermoalklophilic Geobacillus sp Iso5 from alkaline geothermal spring of Southern India

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Society for Experimental Biology

A6.52 Using biodynamics to predict waterborne accumulation of dissolved and nanosilver in the estuarine snail, Peringia ulvae

Farhan R Khan (Natural History Museum, UK), Eugenia ValsamiJones (Natural History Museum, UK), Superb K Misra (Natural History Museum, UK), Javier García-Alsonso (Natural History Museum, UK), Gabrielle M Kennaway (Natural History Museum, UK), Brian D Smith (Natural History Museum, UK), Philip S Rainbow (Natural History Museum, UK) and Samuel N Luoma (U.S. Geological Survey, USA) Engineered silver nanoparticles (AgNPs) are increasingly prevalent and are now used within a wide range of consumer products. An important first step in understanding their toxicity is characterizing their accumulation, from either the nanoparticle or dissociated silver, and in comparison with dissolved Ag. As accumulation is the result of the balance of uptake and efflux, we independently investigated these processes in the intertidal mud snail Peringia ulvae. Using a biodynamic approach, the uptake rates of citrate capped AgNPs (16.5 ±4.5 nm) and dissolved Ag (added as AgNO3) were measured over 24 hours and efflux was measured over a 16-day period post-exposure. Silver as AgNPs had a lower water uptake rate constant (KuAgNP =0.074 ±0.017 L g-1 d-1) compared to dissolved Ag (KuAg =0.15 ± 0.012 L g-1 d-1). Efflux rate constants for both nanosilver (KeAgNP =0.021 ±0.07 d-1) and dissolved silver (KeAg =0.026 ±0.02 d-1) were low and not significantly different, indicating that silver in both forms is readily accumulated post-uptake. Using these rate constants we modelled the accumulation of nano and dissolved Ag over 22 days and compared model output to measured concentrations following a 22-day exposure study. The newly developed model performed well in predicting accumulation with about 80% of modelled values within a factor of two of the observed values. As the primary site of uptake for waterborne uptake is perceived as the gill, AgNPs association to gill tissue was investigated, as was AgNP accumulation in the hepatopancreas which is a potential organ for AgNP deposition post-uptake. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.54 Influence of fragmented exogenous DNA preparation on the growth of experimental mouse tumours and activation of antigene-presenting dendritic cells

Ekaterina A Alyamkina (Institute of Cytology and Genetics, Russia), Evgenia V Dolgova (Institute of Cytology and Genetics, Russia), Anastasia S Proskurina (Institute of Cytology and Genetics, Russia) and Sergei S Bogachev (Institute of Cytology and Genetics, Russia) In the first studies it has been revealed that the combined influence of cytostatic cyclophosphan (CP) and fragmented exogenous DNA preparation results in a stable antitumor effect in mice. Moreover, the tumour growth is shown to be significantly down-regulated if the animals are pretreated with CP and DNA preparation and are immunized with tumour cells' homogenate. The results obtained suppose that the antitumor effect observed is mediated by the activation of antigene-presenting dendritic cells (DCs) resulting in the development of adaptive immunity. Further, ex vivo generated and in vivo derived mouse DCs activated for maturating with fragmented DNA preparation have shown a high level of surface maturity markers. Moreover, DC culture obtained also induces intense anticancer effect when used for animal vaccination. The combined cytostatic CP and doxorubicin (DOX) therapy has been used in our further experiments. DOX boosts the immunogenicity of pre-engrafted tumour due to the exposure of calreticulin protein at the cell surface, while CP actively destroys the tumour cells. Such tumour treatment with the subsequent stimulation of DCs of the organism with the exogenous DNA preparation leads to almost complete degradation of the growing tumour. Mononuclear fraction of the animal blood is shown to have high cytotoxic activity (86.5%). Not only did the maturation induction in the culture of human DCs generated extracorporally with the DNA preparation demonstrate high efficiency of cell maturating when compared with the standard LPS activator, but it also lead to highperformance proliferation of the perforin-containing CD8+ cytotoxic lymphocytes in mixed lymphocyte culture. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.53 Effect of diapause duration on queen weight loss and survival in the Bombus terrestris

Fehmi Gurel (Agricultural Faculty Animal Science Department, Akdeniz University, Turkey), Ayhan Gosterit (Cilimli Vocational School, Duzce University, Turkey) and Bahar A Karsli (Agricultural Faculty Animal Science Department, Akdeniz University, Turkey) A total of 150 Bombus terrestris queens were obtained from our laboratory-reared colonies. After successful mating and a transition period of one week at 13°C, the queens were randomly divided into three groups, weighed, numbered and subjected to one of three diapause regimes: stored for 30, 60 or 90 days at 4°C. At the end of the diapause duration, the surviving queens were weighed again. The highest mortality rate was found in the 90-day hibernated queens (66%), followed by 60-day hibernated queens (26%) and 30-day hibernated queens (12%). Mortality rate increased with longer storage times for queens at 4°C. The queens with a mean weight below 0.71g, 0.77 g and 0.85 g prior to diapause did not survive the 30-, 60- and 90day treatments, respectively. Results showed that queen weight prior to diapause and diapause duration were the key factors for diapause survival in B.tererstris. Email for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.55 Heavy metals' accumulation effects on CGRP-like molecules immunolocalization and concentration in the mantle and gills of the Persian Gulf pearl oyster, Pinctada radiata

Roghieh Talaei (UMR, BOREA, MNHN, France), Zohreh Hassan Abadi (Tarbiat Modares University, Iran), Asma Mohamad Karami (Tarbiat Modares University, Iran), Saber Khodabandeh (Tarbiat Modares University, Iran), Hossein Rameshi (Mollusc Studies Station, Langheh, Iran), Françoise Denis (UMR, BOREA, MNHN, France), Stephanie Auzoux-Bordenave (UMR, BOREA, MNHN-UPMC-CNRS, France) In marine molluscs, CGRP-like molecules were identified, but their role in shell formation still needs to be clarified. The pearl oyster, Pinctada radiata, is an important domestic species in the Persian Gulf whose populations have been severely depleted over the past 20 years. Here, we investigated the concentration of heavy metals (Cd, Cu, Zn and Pb), and their effects on CGRP-like molecules' immunolocalization and concentration in the oyster mantle and gills. The analyses were performed on oysters originating from two stations of the Persian Gulf, Hendorabi and Langheh, exhibiting an increasing pollution level. The results showed that the oysters from Langheh station exhibited an important decrease in shell hardness and size. Heavy metal measurements showed that significantly higher levels of Cu, Zn and Cd were detectable in oyster gills from Langheh station compared to those measured in Hendorabi. Pb accumulation in oyster mantle and

Abstracts 2011 gills from Hendorabi was significantly higher than that measured in Langheh samples. CGRP-like molecules were detected by immunohistochemistry in the mantle and gill tissues and although their concentration was not significantly different in the mantle of oysters from the two stations, its concentration was significantly lower in oyster gills from Langheh compared to Hendorabi samples. These results confirm a high level of heavy metal contamination (except for Pb) in oyster tissues in Langheh station compared to Hendorabi. We suggest that the decrease in CGRP-like molecule concentration observed in Langheh station could be linked to the disruption of shell formation process. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

103 following both in vivo injections and ex vivo co-cultivation without transfected factors. Quantitative analysis has shown that up to 1800 kbp of exogenous DNA in the form of fragments may be simultaneously presented in the nuclear space. Fragments of exogenous DNA are supposed to take part in the repair process, leading to destructive changes in the molecular cell systems. We found disturbances of the chromatin organization in the genome of mouse bone marrow cells, reflected in the amount of short retroposons (B1, B2) preserved. Under normal conditions, however, the number of repeats decreases within 18 to 24 hours after the CP injection. Also the level of bone marrow cell apoptosis becomes increased. Finally, the proportion of bone marrow cells relating to the lymphopoietic lineage precursors remains at a critically low level, which is caused by the destruction of the ability of pluripotent CD34+ cells to develop in the direction of the lymphopoietic lineage. Pathologoanatomic analysis of the organs derived from treated mice shows that the death of animals is due to multiple organ failure induced by accidental involution of the peripheral lymphoid organs and the inability to replace the worn-out spleen, thymus gland and lymph nodes with new immune lymphocytes. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.56 Effects of copper and parasitism on osmoregulation in the European eel Anguilla anguilla

Catherine Lorin-Nebel (Université Montpellier 2, France), Vincent Felten (Université de Metz, France), Eva Blondeau-Bidet (Université Montpellier 2, France), Evelyse Grousset (Université Montpellier 2, France) and Guy Charmantier (Université Montpellier 2, France) The European eel (Anguilla anguilla) is a catadromous species, breeding in the sea and migrating to estuarine, lagoon or freshwater habitats to grow. Silver eels, submitted to low or fluctuating salinities, are also exposed to multiple other stressors such as pollution, overfishing and parasitism that have contributed to the dramatic decrease of eel populations in several European countries. Our objective was to study the single and combined effects of waterborne copper and experimental infestation of eels with the nematode Anguillicola crassus after a salinity challenge from isotonic (15) to hypo- (5) and hypertonic (30) conditions in order to investigate the osmoregulatory capacity of eels exposed to those stressors. Blood osmolality measurements showed no significant effect of Cu2+ and A. crassus over the six weeks of contamination. At 5, a significant decrease in blood osmolality was detected in fish submitted to A. crassus infestation for two weeks with or without the addition of Cu2+. This decrease may originate from the lower Cl- levels measured in eels exposed to both stressors. Blood Na+ levels remained stable in all tested animals. No apparent branchial lesions were detected following the different treatments and immunolocalization of Na+/K+-ATPase (NKA) revealed welldifferentiated ionocytes. Gill NKA activities were measured in the different conditions and their relationships with osmoregulatory responses are discussed. Funded by ANR CES/CIEL 2008-12. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.58 Extremely high sound pressure level from a pygmy aquatic insect

James F Windmill (University of Strathclyde, UK), David Mackie (University of Strathclyde, UK) and Jerome Sueur (Museum of Natural History, France) To communicate at long range, animals have to produce intense but intelligible signals. This task might be difficult to achieve due to mechanical constraints; in particular relating to body size. While the acoustic behaviour of large marine and terrestrial animals has been thoroughly studied, very little is known about the sound produced by small arthropods living in freshwater habitats. Here, for the first time, we analyse the calling song produced by the male of a small insect, the water boatman Micronecta scholtzi. The song is made of three distinct parts, differing in their temporal and amplitude parameters, but not in their frequency content. Surprisingly, sound is produced at 78.9 (63.6 to 82.2) SPL rms re 2.10-5 Pa with a peak at 99.2 (85.7 to 104.6) SPL re 2.10-5 Pa, measured at a distance of one metre. This energy output is significant considering the small size of the insect. When scaled to body length and compared to 227 other acoustic species, M. scholtzi can be considered the loudest animal ever recorded, with the highest ratio of dB to body size, outperforming all marine and terrestrial mammal vocalizations. This water bug is clearly the exception that proves the rule that stipulates that the size and intensity of a source are positively related. Such an extreme display is interpreted as an exaggerated secondary sexual trait resulting from a runaway sexual selection without predation pressure. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.57 Toxic effect caused by the synergistic influence of cytostatic cyclophosphamide and exogenous DNA administered to adult mice

Evgenia V Dolgova (Institute of Cytology and Genetics, Russia), Sergey S Bogachev (Institute of Cytology and Genetics, Russia), Ekaterina A Alyamkina (Institute of Cytology and Genetics, Russia) and Anastasia V Prokopenko (Novosibirsk State University, Russia) We demonstrate here that injections of exogenous DNA in combination with the cross-linking cytostatic cyclophosphamide (CP) have resulted in the disease and death of experimental mice. It has been shown that fragments of exogenous DNA of various origins reach the nuclei of the bone marrow cells during reparation of double-strand breaks

A6.59 TRPV4 in Dicentrarchus labrax: a candidate protein for osmosensing?

Maryline Bossus (University of Montpellier 2, France), Guy Charmantier (University of Montpellier 2, France) and Catherine Lorin-Nebel (University of Montpellier 2, France) The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the Transient Receptor Potential ion channel superfamily that has been proposed as a potential fish osmosensor in previous

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Society for Experimental Biology Martin Boldsen (Aarhus University, Denmark) and Hans Malte (Aarhus University, Denmark) The cost of living has received much attention over the years as energy utilization plays a major role in shaping the behaviour, ecology and physiology of animals. Interestingly, intraspecific variation in metabolic rate still persists, even when ontogenetic processes are taken into consideration, i.e. development and total body mass adjustments. It is generally accepted that whole-animal metabolic rate is the sum of the metabolic rates of all the individual organs. These organs contribute to total metabolic rate by the product of mass and enzymatic intensity. Using intermittent-closed respirometry, we tested whether adult European eels (Anguilla anguilla L.) display intraspecific stability of mass-corrected metabolic rate. We also tested whether organ mass (heart, liver, intestine and spleen) or the activity of key enzymes of oxidative metabolism (cytochrome oxidase and citrate synthase) could explain some of the variation in individual's standard metabolic rates (SMRs). We found that the individual SMR of adult eels was stabile and repeatable over a two-month period. In addition, individuals with high SMR compared to conspecifics had a higher specific growth rate. Liver mass was found to correlate positively with SMR whereas we did not find any correlation between SMR and either liver cytochrome oxidase or citrate synthase activity. Our results are in agreement with the growing body of evidence that indicates that repeatability of metabolic rate is a universal phenomenon in vertebrates, and points to the importance of variation in the size of metabolically active organs for this variability. Email for correspondence: [email protected] Poster Session Saturday 2nd July 2011

studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 minutes to 30 days. TRPV4 mRNA expression was detected in organs involved in osmoregulation: the gills, kidney and brain. In gills, the expression increased significantly in FW from 24 hours to 30 days. In contrast, in the kidney the TRPV4 expression decreased from 10 minutes to seven days of exposure to FW and then it increased at 30 days. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membrane in branchial lamellae, chondrocytes in the cartilage of gill filaments, the posterior pituitary gland and in the apical region of the collecting ducts. Possible roles of TRPV4 in D. labrax are discussed. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.60 Influence of ammonia exposure, nutritional status and exercise on the expression pattern of potential biomarker genes in the common carp (Cyprinus carpio)

Lai Chan (University of Antwerp, Belgium), Marjan Diricx (University of Antwerp, Belgium), Hon-Jung Liew (University of Antwerp, Belgium), Amit K Sinha (University of Antwerp, Belgium) and Gudrun De Boeck (University of Antwerp, Belgium) High ammonia level and feed deprivation may occur simultaneously in natural water. Under these stress conditions, fish may be enforced to swim at a high speed (exercise) in order to catch prey, avoid predators and so on. Consequently, fish need to cope with these stressors by altering the enzymatic and hormonal activities, which are controlled by genes. In this present study, toxicogenomic analyses using real time PCR were used to characterize the expression levels of various genes in the common carp after exposure to high ambient ammonia following periods of feeding (2% body weight) and fasting (unfed for seven days prior to treatment). Both feeding group of fish were exposed to elevated ammonia (1 mg/L) for zero hours (control), three hours, 12 hours, one day, four days, ten days and 21 days. Gills and liver were sampled before and after exercise. The expression kinetics of growth, stress and other cellular toxicity representative genes such as those for Insulin-like growth factor 1(IGF-I), thyroid hormone receptor, prolactin receptor, cortisol receptor, heat shock protein 90 (HSP90), cytochrome oxidase subunit I (COI) and Na+/K+-ATPase were investigated. The transcript level of HSP90, COI, cortisol receptor and Na+/K+-ATPase showed a time-dependent increase in ammonia-exposed food-deprived fish. However, the expression levels of IGF-I, thyroid hormone receptor and prolactin receptor in same group of fish were considerably down-regulated. The mRNA transcript levels were amplified when ammonia-exposed fish were subjected to exercise. These results could enable the development of a `molecular biomarker system' to identify the combined effect of the stressors mentioned. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.62 Reduction of Salmonella enteritidis in the chicken model

Nabil A Ali (Plymouth University, YK) Introduction: In this study an in vitro model of the poultry digestive tract was used to determine the survival of a probiotic strain of Lactobacillus salivarius in drinking water and in fermented moist feed (FMF) and its effect on Salmonella enteritidis in the simulated digestive system of a chicken. Methods: in vitro model: Stage 1 crop: pH 4.6 (80 µl 37% HCl) for 45 minutes at 41.4°C. Stage 2 gizzard and proventriculus: pH 2.5 plus 0.320 g of pepsin for 90 minutes at 41°C. Stage 3 small intestine: pH 6.2 (with NaHCO3) plus 0.320 g of pancreatin and 0.220 g of bile salts for 120 minutes at 41°C. With the exception of L. salivarius for FMF, all bacteria were added at the beginning of stage 1. FMF was prepared by inoculating moist feed (1 feed: 1.2 water) with L. salivarius and incubating for 24 hours at 30°C. Lactobacilli and Salmonellae were enumerated at 0, 45, 135 and 285 minutes. All treatments were applied in triplicate. Results: At the end of stage 2 there was a 2.00 log reduction in L. salivarius in dry food and a 3.00 log reduction in FMF, after which numbers recovered. In the absence of L. salivarius, S. enteritidis fell from log 6.70 to 5.26 cfu/mL in stage 2 and then increased to log 6.80 cfu/mL in stage 3. In the presence of probiotics (L. salivarius) in drinking water, the numbers of S. enteritidis remained low. In the presence of FMF no S. enteritidis were detectable at the end of stages 2 or 3. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.63 A6.61 Temporal stability of the standard metabolic rate in the European eel and the effect of organ mass and enzymatic activity Bioenergetic and behavioural response of zebrafish to different dietary protein and lipid levels

Tim M O'Brine (Mars Petcare, UK), Jana Vrtelova (University of Saskatchewan, Canada), Kath Sloman (University of the West of

Abstracts 2011 Scotland, UK), Simon Davies (University of Plymouth, UK) and Donna Snellgrove (Mars Petcare, UK) The zebrafish, Danio rerio, is a popular aquarium species and a recognized model organism for scientific research, yet little is known about its nutritional requirements with possible implications for its welfare and use in research. Here, we investigated the effect of increasing dietary protein and lipid levels on the bioenergetics and behaviour of zebrafish. We found no significant effects of protein (32 to 75%) or lipid content (8 to 16%) on the specific growth rate (SGR) of zebrafish; only the 16% lipid diet elicited an increase in condition factor (fed at 5% body mass a day). Furthermore, there was no effect of diet observed on the standard metabolic rate of zebrafish. Interestingly, when the low (32%), medium (46%) and high (75%) protein diets were fed to zebrafish over the two-week behavioural observation period at a maintenance ration of 2% body mass, all diets showed a reduction in SGR, with the lowest protein fed fish showing the greatest reduction. Shoal cohesion and latency to feed were found not to be affected by dietary protein level. Currently there is no indication that changes in dietary protein or lipid content have any negative effects on zebrafish welfare. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

105 Simon J Uphill (University of Aarhus, Denmark), Tran Le Cam Tu (Can Tho university, Vietnam), Do Thi Thanh Huong (Can Tho university, Vietnam), Nina Iversen (Univeristy of Aarhus, Denmark), Tobias Wang (University of Aarhus, Denmark) and Mark Bayley (University of Aarhus, Denmark) The air-breathing Swamp eel Monopterus albus aestivates in mud during the Asian hot and dry season to escape unfavourable conditions. Previous reports are unclear concerning burrow construction and whether the animals are metabolically depressed, but it has been suggested that environmental hypoxia acts to lower metabolism. We fitted M. albus with interperitoneal heart rate (fH) transmitters and measured aerial oxygen uptake (VO2) and breathing frequency (fV) during stepwise increases in hypoxia. M. albus compensated by increasing fV but kept VO2 constant down to an ambient 5% O2. fH decreased gradually between 20% and 5% O2. At 2% O2, fH and VO2 dropped sharply to 33% and 19% of normoxic values, respectively, while fV increased more than 23 times. All parameters quickly returned to normoxic levels after two hours at 2% O2, and there was no apparent O2 debt. These animals subsequently burrowed into mud for estivation. VO2 measurements were no longer possible, but all fish maintained a small opening to the air. pO2 was measured in the fish chamber using an optode and found to be 15%, well within the range where M. albus's could maintain its standard metabolic rate. fH was slightly reduced during aestivation, possibly due to the state of torpor. We conclude that M. albus is able to cover its metabolic requirements with the O2 available during estivation and has no need of a significant metabolic depression. The effects of the prolonged fasting on metabolism and the fate of metabolically produced NH3 should be the focus of further experiments. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.64 Haemodynamic alterations due to ablation of control of the cardiac shunt in the South American rattlesnake, Crotalus durissus.

Cleo Leite (UNIFESP, INCT em Fisiologia Comparada, Brazil), Edwin W Taylor (University of Birmingham, UK), Tobias Wang (Aarhus University, Denmark), Denis Andrade (UNESP, INCT em Fisiologia Comparada, Brazil) and Augusto S Abe (UNESP - INCT em Fisiologia Comparada, Brazil) The South American rattlesnake, Crotalus durissus, has the sphincter on the pulmonary artery of its single lung innervated solely by the left vagus. Unilateral left vagotomy abolishes control of this sphincter, reducing peripheral resistance in the pulmonary circuit and causing a left-right (L-R) shunt. Long term comparisons between intact and left-vagotomised rattlesnakes provided no evidence of the importance of the cardiac shunt during several physiological challenges, such as digestion, growth, exercise and prolonged periods of food deprivation. These data question previous hypotheses, which have presented the undivided ventricle as an important adaptive advantage for reptiles. Such conflict leads us to question the relative contribution of the nervous control of such sphincter and the passive haemodynamic alterations caused by the adjustments of the peripheral resistance on guiding the cardiac shunt. We compared Qsys and Qpul in intact and left-vagotomized (LV) rattlesnakes at different temperatures (15, 20 and 30°C) at rest and during forced exercise. The intact snakes have a proportional reduction in both Qsys and Qpul with decreasing temperature (Qpul/Qsys~0.5). Activity caused an increase in blood flow in both circuits in intact rattlesnakes at 30°C, but had a relatively higher effect on Qpul at lower temperatures. Resting LV rattlesnakes have higher Qpul at 30°C but a lower Qpul/Qsys ratio. This ratio decreased further with temperature reduction, inverting the characteristic R-L shunt to L-R shunt at 15°C. Activity reinforced the reduction of the Qpul/ Qsys, and the R-L shunt became more prominent at 15°C. Supported by CNPq and FAPESP Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.66 Analysis of natural selection in a Drosophila circadian photoreceptor by experimental evolution

Shumaila Noreen (Department of Genetics, University of Leicester, UK), Mirko Pegoraro (Department of Genetics, University of Leicester, UK), Eran Tauber (Department of Genetics, University of Leicester, UK), Charalambos P Kyriacou (Department of Genetics and University of Leicester, UK) Genetic and biochemical experiments over the past decade have allowed the construction of a viable working model for the molecular mechanisms underlying circadian rhythm generation in Drosophila. Studies of genes like period and timeless not only reveal the molecular basis of the circadian clock, but also provide new insights into the evolution of this system. Genetic variation within these genes, and spatial patterns of allele frequencies observed along latitudinal gradients provide good evidence for natural selection at these clock loci. A single coding nucleotide polymorphism in cryptochrome (cry, the dedicated circadian photoreceptor in Drosophila) results in the isoforms cryH and cryL. A number of European populations from different latitudes were genotyped for this polymorphism but both alleles were common in all populations tested, with no evidence for a latitudinal cline. This suggests the polymorphism is actively maintained by balancing selection. In an attempt to test the role of selection underlying these allele frequencies, replicate population cages were initiated, having reversed frequencies of the two cry alleles (0.1:0.9). Somewhat remarkably, cryH and cryL allele frequencies converged to an approximately equal frequency after a period of about six months (~12 generations) recapitulating the situation in the wild. These results unusually show that a simple laboratory population experiment can sometimes simulate the more complex environment of the wild, and suggest that the underlying mechanism for this evident balancing selection may be experimentally tractable. Circadian photosensitivity and the general fitness of these genotypes will be analysed in an attempt to understand how this polymorphism is maintained.

A6.65 Lack of metabolic depression during aestivation in the Asian Swamp eel Monopterus albus

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Society for Experimental Biology The Roche 454 Genome Sequencer platform with titanium chemistry was used to generate ~75 000 sequence reads from the pooled subtracted cDNA libraries. Sequence assembler software was utilized to generate contig sequences that were then annotated using similarity searches against L. salmonis and other public databases. These sequences will provide templates for the generation of 60 mer oligonucleotide probes for inclusion on a 44K Agilent microarray, designed for the study of L. salmonis gene expression, including factors relevant to sensitivity to emamectin benzoate. We present the preliminary results of this study. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.67 Temperature but not intracellular 2,3-DPG underlies increased haemoglobin-O2 affinity in the hibernating golden mantled ground squirrel Spermophilus lateralis

Inge G Revsbech (Aarhus University, Denmark), Roy E Weber (Aarhus University, Denmark), Jay F Storz (University of Nebraska, USA) and Angela Fago (Aarhus University, Denmark) Hibernating mammals commonly exhibit drastic reductions in body temperature and in basal metabolic and ventilation rates with prolonged periods of apnoea, while predominantly remaining aerobic. Adaptive increases of the blood oxygen affinity during hibernation have long been attributed to the combined effects of lowered body temperature and reduced levels of the red cell intracellular anionic effector 2,3-DPG. To better understand these responses, we examined the isolated and combined effects of temperature, pH, DPG and chloride anions on purified haemoglobin (Hb) from a fossorial hibernator, the North American golden mantled ground squirrel, Spermophilus lateralis. We found the Bohr effect (pH induced change in O2 affinity) to be similar to whole blood and to be unaffected by the addition of DPG and/or chloride. The effect of temperature (heat of oxygenation, H) was lower than that of most mammalian haemoglobins, aligning with a trend seen in cold-adapted mammals, and similar to that previously reported for whole blood. Consistent with an observed low DPG sensitivity of Hb-O2 affinity, H was unchanged by the addition of DPG. These data suggest that the increase in blood O2 affinity during hibernation (that possibly contributes to the reduced metabolism) is not caused by decreased allosteric interaction with DPG, but solely by lowered temperature. This is possibly in combination with the indirect effects of decreased anion levels on the Donnan equilibrium of protons across the red cell membranes that might increase intracellular pH. These findings might be applicable to other mammalian hibernators. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.69 Functional morphology of the respiratory apparatus of a small gymnophthalmid lizard: Vanzosaura rubricauda (Boulenger, 1902)

Markus Lambertz (Institut für Zoologie Rheinische-Friedrich-WilhelmsUniversität, Bonn, Germany), Kristina Grommes (Institut für Zoologie Rheinische-Friedrich-Wilhelms-Universität, Bonn, Germany), Bianca Unger (Institut für Zoologie Rheinische-Friedrich-Wilhelms-Universität, Bonn, Germany), Steven F Perry (Institut für Zoologie RheinischeFriedrich-Wilhelms-Universität, Bonn, Germany) and Tiana Kohlsdorf (Departamento de Biologia, FFCLRP, Universidade de São Paulo, Brazil) The Gymnophthalmidae - or microteiids - are relatively smallbodied lizards that are closely related to teiids (Teiidae) and lacertids (Lacertidae). In order to gain some insight into the effects of miniaturization on the respiratory system, we focused on Vanzosaura rubricauda (Boulenger, 1902), which with a typical snout-vent-length of about 35 mm represents a small species even for a gymnophthalmid. The single-chambered lungs of this species have a homogeneous distribution of faveolar parenchyma similar to that of teiids but, with a total length of less than 7 mm, display miniaturization. We concur with Klein et al. (2005) in the presence of complete (left and right) dorsal and ventral mesopneumonia and the absence of a post-hepatic septum, the latter of which in general is characteristic for Teiioidea (Teiidae plus Gymnophthalmidae). In addition, however, we identified a very small post-pulmonary septum (PPS), which in squamates until now has only been reported for certain varanoid lizards and chameleons. Furthermore, our light and electron microscopical studies of the lungs revealed massive trabecular smoothmuscle cores, compared with those of other lizards. In addition, the capillaries bulge strongly into the air spaces and frequently display a single capillary net. The functional and evolutionary implications of a PPS and the pulmonary structures are discussed in this poster contribution, with particular emphasis on miniaturization effects. Reference: Klein et al. (2005) Org Divers Evol 5, 47-57. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.68 High-throughput analysis of genes constitutively expressed in two populations of Lepeophtheirus salmonis with differing susceptibilities to emamectin benzoate

Stephen N Carmichael (University of Stirling, UK), Alasdair Nisbet (Moredun Research Institute, UK), Andrew Tildesley (University of Stirling, UK), Karim Gharbi (The GenePool, UK), Philip Skuce (Moredun Research Institute, UK), James E Bron (University of Stirling,UK) and Armin Sturm (University of Stirling, UK) Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasitic copepod that parasitizes salmonid fishes and has become a serious problem for cultured and wild marine salmonid populations. One of the compounds currently employed to treat L. salmonis infections of Atlantic salmon is emamectin benzoate, used as an in-feed pre-mix (SLICE®), for which reduced treatment efficacy has been observed in major Atlantic salmon culturing countries. It has been possible to successfully cultivate multiple generations of two different L. salmonis populations at the Institute of Aquaculture's Marine Environmental Research Laboratory. One of these populations has been shown to have significantly reduced sensitivity to emamectin benzoate when tested using in vitro bioassay methods. Suppression subtractive hybridization was employed to generate two subtracted cDNA libraries that represent genes constitutively expressed in the respective L. salmonis populations.

A6.70 Effects of salinity on standard metabolic rate and critical oxygen tension in the giant freshwater prawn (Macrobrachium rosenbergii)

Rasmus Ern (Department of Biological Science, Aarhus University, Denmark), Do Thi Thanh Huong (College of Aquaculture and Fisheries, Can Tho University, Vietnam), Nguyen Van Cong (College of Environment and Natural Resources, Can Tho University, Vietnam), Tobias Wang (Department of Biological Science, Aarhus University, Denmark) and Mark Bayley (Department of Biological Science, Aarhus University, Denmark)

Abstracts 2011 The extensively farmed giant freshwater shrimp, Macrobrachium rosenbergii, can survive salinities up to 26 ppt, but the commercially important grow-out is done exclusively in freshwater areas. Recent studies suggest the shrimp equally capable of being grown in brackish water, and a better understanding of how this species responds to changing salinity could significantly impact freshwater prawn farming in deltas and coastal areas. Here, the effect of salinity (0 and 15 ppt) on post and intermoult standard metabolic rate (SMR) and critical oxygen tension (Pcrit) was measured in adult M. rosenbergii using intermittent closed respirometry. SMR was highest immediately after moulting (0.114 ±0.002 and 0.114 ±0.006 µmol g-1 min-1 in fresh and brackish water, respectively) and decreased by approximately 50% during the intermoult phase (0.053 ±0.004 and 0.045 ±0.003 µmol g-1 min-1), with no significant difference between the two salinities at any time in the moult cycle. During hypoxia M. rosenbergii kept oxygen uptake down to a Pcrit of 26.3 ±1.4 mmHg in fresh and 27.2 ±2.0 mmHg in brackish water (p=0.682), showing that salinity had no overall effect on oxygen conductance in the animals. These findings are in agreement with recent growth studies and provide further evidence that grow-out phase could be accomplished in brackish water areas. Thus, the predicted intrusions of brackish water in tropical deltas as a consequence of future global warming should not impact this important production. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

107 Most stingless bees (Apidae; Meliponini) inhabit tropical forests, visually complex environments that pose a sensory challenge for foraging individuals. In the present study, we investigated the ability of Melipona scutellaris to cope with complex foraging situations, testing their ability to use visual and olfactory cues for navigation through a maze. First, we trained 20 individuals to collect scentless sucrose solution at a feeder inside a maze composed of 25 cabins. The correct path to the food was highlighted with a visual cue (coloured circle or geometric pattern). Subsequently, the bees were tested on a different and longer path than during training. In the test, 15 bees (coloured circle), and 16 bees (geometric pattern), respectively, followed the visual cues to the feeder. These results demonstrate that the foragers had successfully learned the `visual-cuefood' context during training. To further evaluate which cue-modality (visual or olfactory cue) the bees prefer for their orientation through the maze, another 20 bees were first conditioned to associate a specific scent with food. Thereafter, the individuals were trained to collect the scented food in the maze. The path to the food was emphasized with yellow colour marks. In a subsequent choice between visual and olfactory cue, 11 bees chose the colour marks and nine the scent. These results allow two explanations: i) the foragers had preferentially learned one of the cuefood contexts during the training; or ii) the bees had learned the context `visual-cue+olfactory-cuefood' and chose one cue at random when offered separately from each other. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.71 The combined effects of ammonia exposure, nutrient status and exercise on the physiological, biochemical and genetic level in the common carp (Cyprinus carpio)

Marjan Diricx (University of Antwerp, Belgium) Ammonia is an environmental pollutant that is toxic to all organisms but most of all aquatic animals. In the present study, the impact of ammonia toxicity was tested on nutrient status (fed versus starved) and swimming performance activity (highest swimming speed versus resting). Fish were exposed to 1 mg/L of ammonia for a period of three hours, 12 hours, one day, four days and ten days, and were acclimated to fed (2% body weight) and starved (unfed for seven days prior to the experiment) conditions. Measurements of oxygen consumption, ammonia excretion, critical swimming speed (Ucrit), ammonia and lactate accumulation in the plasma was done for every experimental group. The changes in the energy budget of the fish (glycogen, lipid and protein) provide an indication of detoxification and/or the maintenance of normal body functional. The Na+/K+-ATPase activity in the gills was altered by ammonia exposure and thus disturbance in ion balance was obvious. Therefore the ion concentration was measured in order to understand the ion-osmo regulations during the exposure. Also ion-osmoregulation-controlling hormones like cortisol and thyroid hormones were measured. When fish were fed, it was observed that that they had different strategies to protect them from the toxic effects of ammonia, so they were less sensitive to external ammonia than starved fish. The toxic effects in both feeding treatments seemed to increase by exercise (3/4Ucrit). This suggests that fish become more vulnerable to external ammonia during exercise. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.73 Humoral regulation of heart rate during digestion in the Burmese python (Python molurus)

Sanne Enok (Aarhus University, Denmark), Lasse S Simonsen (Aarhus University, Denmark), Tobias Wang (Aarhus University, Denmark) and Nini Skovgaard (University of Oslo, Denmark) To sustain the increased metabolic demand associated with digestion, most vertebrates, including humans, experience a postprandial increase in heart rate. This increased heart rate is governed by nonadrenergicnoncholinergic (NANC) factors, in addition to sympathetic nervous regulation. Animals treated with plasma from digesting donor pythons experienced a threefold larger increase in double-blocked heart rate (treated with a combination of the -adrenergic antagonist propranolol and the cholinergic antagonist atropin) compared to animals treated with plasma from fasting pythons (13.4 ±2.8 min-1 versus 4.6 ±1.4 min-1). This shows that a humoral factor is responsible for up-regulating heart rate during digestion. Injections of the gastrin and cholecystokinin receptor antagonist proglumide had no effect on intrinsic heart rate or blood pressure. Histamine has been recognized as a NANC factor in the early postprandial period in pythons, but the mechanism of its release has not been identified. Mast cells represent the largest repository of histamine in vertebrates, and it has been speculated that mast cells release histamine during digestion. Treatment with the mast cell stabilizer cromolyn significantly reduced postprandial heart rate in pythons compared to an untreated group, but did not affect the doubleblocked heart rate. While histamine indeed induces postprandial tachycardia in pythons, its release during digestion is not stimulated by gastrin or cholecystokinin, nor is its release from mast cells a stimulant of postprandial tachycardia. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.72 Stingless bees use visual and olfactory cues for navigation through a maze

André Rodrigues (University of São Paulo, Brazil) and Michael Hrncir (Universidade Federal Rural do Semi-Árido, Brazil)

A6.74 Non-shivering thermogenesis in mice selected for high aerobic capacity

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Society for Experimental Biology Laboratory, Medical Department, Aarhus University Hospital, Denmark), Niels Jessen (Medical Research Laboratory, Medical Department, Aarhus University Hospital, Denmark), Gregers Wegener (Centre for Basic Psychiatric Research, Aarhus University Hospital, Denmark), Gudrun Winther (Centre for Basic Psychiatric Research, Aarhus University Hospital, Denmark), Jan Elnif (Animal Nutrition, Department of Basic Animal and Veterinary Sciences, University of Copenhagen, Denmark), Sebastian Frische (The Water and Salt Research Centre, Institute of Anatomy, Aarhus University, Denmark), Tobias Wang (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) and David Mayntz (Ecology and Genetics, Department of Biological Sciences, Aarhus University, Denmark) Dietary protein restriction in pregnant females reduces offspring birth weight and increases the risk of developing obesity, type 2 diabetes and cardiovascular disease. Despite these grave consequences, few studies have addressed the effects of preconception maternal malnutrition. Here we investigate how a preconception low protein (LP) diet affects offspring body mass and insulin-regulated glucose metabolism. Ten-week-old female mice (C57BL/6JBom) received either a LP or isocaloric control diet (8% and 22% crude protein, respectively) for ten weeks before conception, but were thereafter fed standard laboratory chow (22.5% crude protein) during pregnancy, lactation and offspring growth. When the offspring were ten weeks old, they were subjected to an intraperitoneal glucose tolerance test, and sacrificed after a five-day recovery period to determine visceral organ mass. The body mass of LP male offspring was significantly lower at weaning than controls, and a similar tendency was observed for LP female offspring. These differences in body mass disappeared within one week after weaning, a consequence of catch-up growth in LP offspring. Glucose tolerance tests of ten-week-old offspring revealed enhanced insulin sensitivity in LP offspring of both sexes. No differences were found in body mass, food intake or absolute size of visceral organs of adult offspring. Our results indicate that maternal protein restriction imposed prior to pregnancy produces effects similar to postconception malnutrition, namely low birth weight, catch-up growth and enhanced insulin sensitivity during young adulthood. This could imply an increased risk of offspring developing lifestyle-acquired diseases during adulthood. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

Julita Sadowska (University of Bialystok, Poland), Andrzej K Gebczynski (University of Bialystok, Poland) and Marek Konarzewski (University of Bialystok, Poland) Endotherms maintain elevated body temperature, which at rest stems exclusively from on-going metabolic processes. This heat production represents the basic maintenance costs of visceral organs such as the liver, kidneys and intestines and is called the obligatory nonshivering thermogenesis (NST). When exposed to cold, however, small mammals use so-called regulatory NST, which mostly involves heat generated by the brown adipose tissue (BAT). The relationship between obligatory and regulatory NST is unclear. Some models of endothermy evolution assume a positive genetic correlation between obligatory and regulatory NST. To verify this assumption we used laboratory mice from a replicated selection experiment, in which animals are bred towards high rates of VO2max elicited by swimming in 25°C water. We elicited NST in selected and control animals by norepinephrine injection and measured VO2 for 20 minutes at 23°C. We found that that NST was higher and BAT was larger in selected than control animals. However, the basal rate of metabolism (in other words obligatory NST) did not differ between selected and control line types. Thus, our results do not support the existence of the positive correlation between obligatory and regulatory NST. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.75 Time domains of the cardio-ventilatory response of bowfin (Amia calva) to hypoxia

Cosima S Porteus (University of British Columbia, Canada), Patricia A Wright (University of Guelph, Canada) and William K Milsom (University of British Columbia, Canada) We investigated the time domains of the hypoxic cardio-ventilatory response to seven days of hypoxia (43 torr) of bowfin (Amia calva) with and without access to the surface to breathe air. The immediate (acute) response to hypoxia of fish with access to air consisted of a threefold increase in air breathing frequency, no change in gill breathing frequency, and a tachycardia compared to resting values (normoxia). During prolonged exposure to hypoxia, the heart rate returned to resting values after 24 hours of hypoxia while air breathing frequency remained elevated. The immediate (acute) response to hypoxia of fish without access to air consisted of a twofold increase in gill breathing frequency, and a transient bradycardia compared to resting values (normoxia). During prolonged exposure to hypoxia, the heart rate returned to resting values after 48 hours of hypoxia while the twofold increase in gill breathing frequency was sustained. On return to normoxia after seven days of exposure to hypoxia, fish with access to air gradually reduced air breathing frequency. Interestingly, in fish without access to air the return of gill breathing frequency to normal levels was accompanied by an increase in heart rate that remained elevated for over 24 hours. The extent to which the differences in heart rate response of fish with and without exposure to air reflect differences in chemoreceptor input (water versus blood sensing), mechanical events (gill or air breathing) or other events, remains to be determined. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.77 Changes in blood flow patterns in crocodilians during digestion

Anders Findsen (Zoophysiology, Department of Biological, Sciences Aarhus University, Denmark) and Tobias Wang (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) Crocodilians have a complex cardiovascular anatomy, notable for the anatomical separation of the ventricle and maintenance of two aortae, which allows blood to bypass the lungs and be shunted to the systemic circulation. The adaptive role of this right-to-left (R-L) shunt is poorly understood, although recent studies have suggested that the R-L shunt might aid in acid-secretion during digestion. The haemodynamics of the R-L shunt during digestion are, however, largely unknown. We studied changes in shunt patterns, by measuring blood flow in conscious American alligators (Alligator mississippiensis) and Spectacled caimans (Caiman crocodylus). Recordings of blood flow in the right aorta (RAo), left aorta (LAo) and the celiac artery (CA) were continuously measured during fasting and digestion until 48 hours post-feeding. A complex pattern of blood flow was observed in the LAo during fasting, with both retrograde and anterograde flow, demonstrating that resting fasting animals are indeed utilizing the R-L shunt. Only anterograde flow was observed in the CA during fasting, with retrograde flow in the LAo matching the anterograde flow peak in the CA. This suggests that most of the flow in the CA during fasting originates from the RAo. This complex flow pattern was also observed during digestion, but the anterograde flow increased in both the LAo and CA, proportionally, indicating that the blood going to the CA during

A6.76 Maternal protein restriction before pregnancy reduces offspring early body mass and affects glucose metabolism in a gender-specific manner

Anete Dudele (Zoophysiology, Department of Biological Sciences, University of Aarhus, Denmark), Sten Lund (Medical Research

Abstracts 2011 digestion originates from the LAo. When digesting, the heart rate increased twofold and blood flow in the LAo and CA increased fivefold, pointing to an influence of R-L shunt during digestion. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

109 smooth muscle with no autonomic tone. This provides the possibility of mapping the function of adreno- and cholinoreceptor in vascular regulation in anurans. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.78 Go with the flow: Using non-invasive MRI to quantify blood flow to visceral organs in postprandial Yellow anaconda (Eunectes notaeus)

Jonas L Andersen (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark), Steffen Ringgaard (MRI-research Centre, Skejby University Hospital, Aarhus University, Denmark) and Tobias Wang (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) Using non-invasive, real-time magnetic resonance imaging (MRI), blood flow distribution to the stomach, along with that to the intestine and kidneys, was recorded during the massive digestive response in ten Yellow anacondas (Eunectes notaeus). Within the first 24 hours of digestion, blood flow to the stomach increased almost 18-fold (1.1 ±0.3 to 18.0 ±1.3 mL.min-1.kg-1), while flow to the intestine and kidney increased sevenfold (1.2 ±0.3 to 8.5 ±0.8 mL.min-1.kg-1). Both heart rate and stroke volume more than doubled, leading to an 8.5-fold increase in cardiac output (4.4 ±2.4 to 37.2 ±0.9 mL.min-1.kg-1), increasing throughout the 72-hour period after ingestion. The proportion of blood flow directed to the intestine remained statistically unchanged during digestion compared to fasting values (30 ±3 to 26 ±4%, respectively), whereas the proportional flow to the stomach increased twofold during digestion (31 ±7 to 64 ±4%). This may indicate that the stomach exhibits a large metabolic response to digestion. To verify the MRI flow measurements, a heart-phantom was set up with an open system of tubes, allowing for the MRI measurements to be compared directly to a known flow of water. In initial controls the MRI-measurements deviated less than 3% from the true flow, showing that MRI provides a novel method of non-invasive blood flow measurements. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.80 Molecular investigation of key components in ethanol production in the bitterling (Rhodeus amarus)

Cathrine E Fagernes (Department of Molecular Biosciences, University of Oslo, Norway), Stian Ellefsen (Centre for Medicine and Exercise Physiology, Lillehammer University College, Norway), Michael Berenbrink (School of Biological Sciences, University of Liverpool, UK) and Göran E Nilsson (Department of Molecular Biosciences, University of Oslo, Norway) When exposed to anoxia, the bitterling (Rhodeus amarus) has evolved the ability to produce ethanol as an anaerobic end-product, thereby avoiding lactic acidosis. This strategy is also utilized by its more distant relatives in the genus Carassius (goldfish and crucian carp). Apparently, this is made possible through adaptations in the pyruvate dehydrogenase (PDH) complex, which leaks acetaldehyde during anoxia in the ethanol-producing species. The acetaldehyde is further converted into ethanol by alcohol dehydrogenase (ADH). Ethanol diffuses from the blood into the ambient water over the gills. ADH is present in a broad range of organisms, and it is the primary enzyme responsible for the interconversion between alcohols and their corresponding aldehydes or ketones. Normally, its role is to metabolize alcohols that would otherwise be toxic to the organism. In organisms such as the Carassius species, however, ADH catalyses the opposite reaction (from acetaldehyde to ethanol) as part of anaerobic metabolism. While the anaerobic strategy seems to be restricted to skeletal muscle in Carassius, the bitterling has previously been shown to produce ethanol in both muscle as well as liver. In the present study, we have cloned and sequenced the PDH complex E1 subunits (alpha and beta) and the E2 enzyme, as well as ADH, in bitterling. Subsequently, we have analysed their expression levels in normoxic brain, heart, liver and muscle using real-time PCR. The function of these key components in the anaerobic metabolism of bitterling will be discussed. Email address for correspondence: c.e.fagern[email protected] Poster Session Saturday 2nd July 2011

A6.79 Mechanisms of adreno- and cholinoreceptors in isolated pulmonary and systemic vasculature of the cane toad (Rhinella marina)

Pil B Pedersen (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) and Tobias Wang (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) While the overall autonomic regulation of the heart and vasculature of anurans has been studied in some detail, little is known about the role of the adreno- and cholinoreceptors in smooth muscle when looking at resistance in the pulmonary and systemic vessels. Vascular smooth muscle is the primary site of regulation of resistance and therefore holds the capacity to control blood flow and pressure. This vascular control allows amphibians to regulate their shunt, i.e. partially bypassing either the pulmonary or systemic circuit. Shunting in amphibians has been studied due to their multiple respiratory modalities and their ontogeny including fundamental morphological changes during metamorphosis. Here we use wire myography to evaluate how the vascular tone of isolated blood vessels from the pulmocutaneous, pulmonary, cutaneous and systemic segments respond to sympathetic and parasympathetic stimulation. Hence, myography on vessels ranging from 0.2 to 2.0 mm allow for the investigation of isometric response to agonists and antagonists of

A6.81 Autonomic regulation of the heart during digestion in the bullfrog Rana catesbeiana

Débora Claësson (Aarhus University, Denmark), Tobias Wang (Aarhus University, Denmark) and Augusto S Abe (Universidade Estadual Paulista, Brazil) Digestion causes a substantial rise in oxygen uptake that must be attended by an increased cardiac output. To study the autonomic regulation of this postprandial tachycardia in an amphibian, we instrumented frogs with catheter in the femoral artery to record blood pressure and heart rate. Heart rate increased from a fasting value of 42.4 ±2.5 to 52.5 ±1.6 and 57.5 ±2.3 min-1 by 24 and 33 hours, respectively, after the frogs had been force-fed meals corresponding to 5% of their body mass. Using infusion of atropine and propanolol, we were able to demonstrate that part of the postprandial tachycardia can be ascribed to a progressive release of parasympathetic tone on the heart from 25.9 ±4.6% in fasting frogs to 6.3 ±5.1% by 33 hours into digestion. The sympathetic tone remained unaltered, at approximately 20%, throughout digestion. In addition to the withdrawal of vagal tone, digestion was also associated

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Society for Experimental Biology In this study, blood samples were collected from total of 504 Holstein breed cows randomly selected from populations raised in different parts of Antalya. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to identify individuals with these diseases. To cut the target regions TaqI restriction enzyme were used for BLAD alleles and AvaII restriction enzyme for BC alleles. However, a total 504 samples examined mutant (homozygous) BLAD and BC alleles could not be determined. As a result, although no BC carriers were found, the frequency of BLAD carriers (heterozygous) was found to be 1.5%. Email for correspondence: [email protected] Poster Session Saturday 2nd July 2011

with an increased intrinsic heart rate, revealed after the combination of atropine and propranolol, from a fasting value of 45.3 ±1.2 to 50.8 ±2.0 min-1 at 33 hours. This indicates that postprandial tachycardia in frogs, as has been previously shown in snakes, is governed partially by excitation of a non-adrenergic, non-cholinergic factor that might reflect the release of regulatory peptides from the gastrointestinal organs during digestion. Future studies are being designed to identify these factors. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.82 Cardiac function in the South American rattlesnake, Crotalus durissus terrificus

Gabrielle S De Paula (UFSCAR- INCT em Fisiologia Comparada, Brazil), Andre Guelli (UFSCAR - INCT em Fisiologia Comparada, Brazil), Lucas Ferro (UFSCAR - INCT Fisiologia Comparada, Brazil), Cleo A C Leite (UNIFESP - UFSCAR - INCT Fisiologia Comparada, Brazil), Augusto S Abe (UNESP - UFSCAR - INCT Fisiologia Comparada, Brazil), Francisco T Rantin (UFSCAR - UFSCAR - INCT Fisiologia Comparada, Brazil) and Ana L Kalinin (UFSCAR - UFSCAR - INCT Fisiologia Comparada, Brazil) The heart of reptiles, rather than being endothermic, undergoes wide changes in the extracellular content of gases, pH and temperature. This can result from alteration in environment or habit or be a consequence of the cardiac shunt connecting both pulmonary and systemic circuits. Even in the same reptile group, the Squamata, there is important variation on the cardiac myocyte ultrastructure and also on what the extracellular calcium depends on for contraction. Such variety is probably a reflex of the known diversity of the evolutionary pathways of the group. It is possible that species-specific peculiarities can reveal an even bigger plasticity of such contractile system. In order to understand this, we tested the importance of sodium-calcium exchanger (NCX), sarcoplasmic reticulum and adrenaline for the ventricular myocyte contraction of the South American rattlesnake, Crotalus durissus terrificus (753.38 g ±81.8 5g) at 30°C. The extracellular Ca2+ is important for the contraction and the blockade of NCX decreases speed of its relaxation. Ryanodine caused a 40% decrease in the force of contraction (Fc) and the adrenaline produced sustained an increase in Fc for a wide frequency of stimulation (0.2 to 0.8 Hz). Based on these data we can verify that the external Ca2+ is primary responsible for myocyte contraction and the NCX is the main Ca2+ pump working on the calcium output flow. Despite this, we could see that the reticular Ca2+ has an important role in the contraction. Furthermore, epinephrine is able to increase the affinity of contractile proteins to the Ca2+ and then further increases the Fc. Financial support: FAPESP, CNPq. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.84 Regeneration in the polyclad flatworm Boninia divae: a morphological study

Edna C Bonilla (Universidad de los Andes, Columbia), Diana M Bolaños (Universidad de los Andes and Universidad de Cartagena, Columbia) and Federico D Brown (Universidad de los Andes, Columbia) The Caribbean marine flatworm Boninia divae (Polycladida: Platyhelminthes) has the ability to regenerate from blastemal cells, as occurs in other taxa of the phylum. However, polyclad regeneration is dependent on the presence of the brain in the regenerating tissue. Using traditional histological stains and visualization by phase contrast and Nomarsky microscopy, we describe the polyclad blastema formation of anterior, posterior and lateral parts of the body after transverse and longitudinal sections of the brain. Information on polyclad regeneration was obtained at zero, two, five eight, 12 and 22 days after brain injury or amputation. Posterior brainless parts remain alive but do not regenerate a new head, even after 57 days of regeneration. Anterior sections including the brain regenerate in 14 days in a similar pattern and timing to freshwater flatworms, however anterior brainless sections are unable to survive more than eight days. Our results confirm that the presence of cephalic ganglia are necessary for complete regeneration in B. divae. We used serotonin and synapsin antibodies as neural markers to characterize neural populations and neuroregeneration. Our work is the first morphological study that describes regeneration in polyclad flatworms in detail, and therefore we are searching for differences in the regeneration or development of polyclads that may explain the brain dependence in these worms when compared to freshwater flatworms. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.85 Classification and molecular evolution of the sodium-phosphate co-transporter NaPi-II (SLC34)

Aslak Jørgensen (University of Copenhagen, Denmark), Kasper Andersen (University of Copenhagen, Denmark), Lea Prehn (University of Copenhagen, Denmark), Andreas Werner (University of Newcastle, UK) and Nadja Møbjerg (University of Copenhagen, Denmark) Sodium coupled-phosphate transporters (NaPi cotransporters) facilitate the transport of inorganic phosphate (Pi) across cell membranes. Traditionally three NaPi-II co-transporter (SLC34) isoforms - NaPiIIa (SLC34A1), NaPi-IIb (SLC34A2) and NaPi-IIc (SLC34A3) - have been identified from various vertebrates. However, recently numerous invertebrate NaPi-II sequences, together with additional vertebrate sequences, have become available primarily through genome sequencing projects. In the present study we investigate the classification and molecular evolution of the NaPi-II sequences using various phylogenetic inference methods. From our results it is clear that the current three-isoform

A6.83 Identification of bovine leukocyte adhesion deficiency (BLAD) and BC (bovine citrullinemia) alleles in Holstein cows reared in Antalya region

Emine Sahin (Institute of Natural Science), Taki Karsli (Institute of Natural Science), Murat S Balcioglu (Institute of Natural Science) and Askin Galic (Institute of Natural Science) Bovine leukocyte adhesion deficiency (BLAD) and Bovine citrullinaemia (BC) are autosomal recessive genetic diseases in Holstein cattle breeds and both result in the death of homozygous animals. The aim of this study was to investigate the presence of BLAD and BC alleles in Holstein cows reared in the Antalya region.

Abstracts 2011 classification can only be used for vertebrates. Within the invertebrates a number of distinct isoforms have evolved with NaPi-II isoforms from Cephalochordata and Echinodermata, inferred as the closest relatives to the vertebrates. Understanding the differential expression and evolution of traditional vertebrate NaPi-II isoforms (IIa, IIb and IIc) is dependent on increasing current knowledge of sequences from amphibians and reptiles. RTPCR amplification products of NaPi-II from selected amphibian and reptile species have been cloned and sequenced to increase their presence in the taxon sampling. Our results show that all three isoforms are present within Amphibia. Isoforms IIb and IIc have been found within anurans, isoforms IIa and IIb within salamanders, and isoform IIa within gymnophiones. Within reptiles, isoforms a and b are present. Some sequences previously identified as NaPi-IIb clearly belong to NaPi-IIa. The IIc isoform has previously been inferred to be unique to the mammalian lineage, however, as more data become available from genome sequence projects additional major taxonomic lineages are likely to reveal IIc isoforms. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

111 Germany), Philip Rosenstiel (Institute of Clinical Molecular Biology, Kiel University, Germany), Magnus Lucassen (Alfred Wegener Institute for Polar and Marine Research, Germany) and Frank Melzner (IFMGEOMAR, Germany) Increasing anthropogenic CO2-emissions are changing the oceans' carbonate system and leading to a decrease in water pH (ocean acidification), which may affect behaviour, homeostasis, calcification and reproduction of many marine species from diverse phyla. Here, the impact of ocean acidification on the mantle tissue transcriptome of the blue mussel Mytilus edulis was investigated to identify important genes that are differentially regulated during acid-base stress. Juvenile specimens from Kiel bight, Baltic Sea, were acclimated at two CO2 levels, 39 Pa (390 µatm, control) and 400 Pa (4000 µatm) for eight weeks under optimized feeding conditions. Following the incubation, outer (pallial and marginal zone) and inner (central zone) mantle tissue samples were dissected and analysed for gene expression patterns using 454 - pyrosequencing and quantitative real-time PCR. In total, about 40 000 unique transcripts have been identified in both tissues and 50% could be annotated and classified in gene ontology categories. Global analysis of the mantle transcriptome showed remarkable differences between inner and outer mantle as distinct matrix proteins, responsible for the organized formation of the mussel shell, could be identified exclusively in the outer mantle. Significant responses to elevated CO2 level were found in several biomineralization proteins and in transcripts coding for immuneresponsive genes. Detailed information on changes in gene expression in response to elevated seawater pCO2 was additionally gathered by quantitative real-time PCR. These observations indicate for the first time widespread changes in M. edulis gene expression patterns elicited by elevated seawater pCO2. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.86 The shell vasculature of Trachemys turtles investigated by modern 3D imaging techniques

Kasper Hansen (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark), Jesper Thygesen (Department of Clinical Engineering, Aarhus University Hospital, Denmark), Tobias Wang (Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark) and Michael Pedersen (MR Research Centre, Aarhus University Hospital, Denmark) Many freshwater turtles are extremely tolerant to a lack of oxygen and can survive the winter submerged in anoxic mud in ice-covered lakes. The pronounced anoxia-tolerance resides in a considerable depression of cellular metabolism and the ability to use the shell to buffer the acidosis arising from anaerobic metabolism. Infusion of microspheres has shown that the shell receives almost half of the cardiac output in turtles made anoxic at low temperatures. However, the vasculature of the turtle shell remains to be described. To visualize the vasculature within the carapace and plastron of the turtle Trachemys scripta, we perfused terminally anaesthetized turtles with different contrast-enhancing agents (Microfil [lead n/a]), barium sulphate [250 mg/kg], and iodine [15 to 250 mg/kg]), and the animals were then scanned by both single source as well as dual energy computed tomographic systems, to create three-dimensional representations. Dual energy computed tomography provided good visual contrast between blood vessels and bony tissue inside the turtle shell due to differences in energy level from the two simultaneously acquired X-ray sources. However, inadequate resolution of the clinical scanners prevented visualization of the smaller vessels with a diameter below approximately 600 µm. Thus, our technique clearly revealed the larger intracortical vessel embedded in the carapace and plastron of Trachemys, and future studies should seek to determine whether this vascularization is altered during prolonged anoxia. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.88 Baltic salmon (Salmo salar) sorted for early and late emergence show differential brain gene expression following confinement stress

Per-Ove Thörnqvist (Department of Neuroscience, Physiology Unit, Biomedical Centre (BMC), Uppsala University, Sweden), Erik Höglund (Technical University of Denmark, Denmark) and Svante Winberg (Department of Neuroscience, Physiology Unit, BMC, Uppsala University) Previous studies have demonstrated that early and late emerging Atlantic salmon fry differ in boldness behaviour, a trait that has been shown to be consistent over time. In rainbow trout, two strains divergent in their plasma cortisol response to stress, high responders (HR) and low responders (LR), has been established. Recently differences in gene expression between these lines were found after confinement stress. However, in this case boldness could not be linked to any strain or gene expression differences. In the present study we used six-month-old Baltic salmon previously sorted for early and late emergence. The fish were exposed to confinement stress and brain gene expression was analysed by qPCR in stressed and control fish that were both early and late emerging. Genes displaying divergent expression in the LR/HR strains, and genes believed to be involved in stress responses and aggression were selected. Blood cortisol levels and expression analysis could not link the early or late emerging salmon to a reactive or proactive coping style. However differences in expression between early and late emerging salmon were found. Genes linked to aggression such as GABA-RAP, Ependymin, and 5HT1A, point towards a reduction of aggression in late emerging salmon when exposed to confinement stress. These gene expression data, and previous studies showing a bolder behaviour in early emerging larvae, indicate that emergence time boldness and aggression are linked to each other, forming a behavioural syndrome in juvenile salmonid fish Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.87 Differential gene expression patterns of Mytilus edulis under elevated pCO2 a transcriptomic analysis

Anne Hüning (Alfred Wegener Institute for Polar and Marine Research, Germany), Jörn Thomsen (IFM-GEOMAR, Germany), Eva Philipp (Institute of Clinical Molecular Biology, Kiel University, Germany), Lars Krämer (Institute of Clinical Molecular Biology, Kiel University,

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Society for Experimental Biology Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.89 Heart mitochondria of the hypoxia-tolerant epaulette shark are more sensitive to hypoxia than those of a hypoxia-intolerant ray

Ania K Kutek (Griffith University, Australia), Fathima Iftikar (University of Auckland, New Zealand), Anthony J Hickey (University of Auckland, New Zealand) and Gillian M Renshaw (Griffith University, Australia) Current dogma states that extended hypoxic survival is improved by metabolic depression. However, this view has been based on responses to hypoxia at low temperatures1. The Australian epaulette shark is unusual among elasmobranchs as it is tolerant of hypoxia and anoxia at tropical temperatures2,3. Recent evidence demonstrates that epaulette sharks keep normal cardiac function down to oxygen concentrations less than half (~29 M) that of hypoxia-intolerant rays (77 M). This suggests that these sharks might only depress cardiac metabolism with extreme hypoxia and might have evolved systems that optimize cardiac oxygen delivery and/or oxygen uptake by mitochondria. We measured the affinity of heart mitochondria for oxygen in both the epaulette shark (Hemiscyllium ocellatum) and the pink whipray (Himantura fai) using differential spectroscopy and high-resolution respirometry. Surprisingly, mitochondria from epaulette sharks had a lower affinity for oxygen (P50 0.688 ±0.377 M) than those from pink whip rays (P50 0.044 ±0.005 M). Differential spectroscopy indicated that cytochrome c oxidase became more reduced at higher oxygen concentrations (5.8 M) in mitochondria from the epaulette shark than from the pink whipray, which still responded at extremely low oxygen tensions (0.028 M). Therefore while the epaulette heart is relatively tolerant to hypoxia, the mitochondria within the heart are acutely sensitive to progressive hypoxia and depress respiration at higher oxygen concentrations than the pink whipray. Respiratory depression may serve a cardio-protective function because it serves to limit free radical production. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.91 Endoscopic biopsy provides a possibility to investigate, in vivo, the integrity of the intestinal barrier in salmonids

Henrik Seth (University of Gothenburg, Sweden), Anders Edebo (Sahlgrenska University Hospital, Sweden), Michael Axelsson (University of Gothenburg, Sweden), Henrik Sundh (University of Gothenburg, Sweden), Kristina Sundell (University of Gothenburg, Sweden) and Anders Kiessling (The Swedish University of Agricultural Sciences, Sweden) There are several instances where there is a need to investigate the physical integrity of the intestinal epithelium. This includes the study of how the gastrointestinal canal is affected by stress, the presence of pathogens as well as how the composition of feed influences the integrity of the gut. The latter is important during the development of new alternative feeds aimed at reducing the level of fish meal and fish oil. Most of these analyses are performed post-mortem, however, due to the complexity of retrieving tissue samples from a live fish. This decreases the spatio-temporal resolution and impedes the analysis in itself. We therefore sought to use ocular examination and biopsies in anaesthetized salmonids in order to facilitate samples to be taken from the same fish at different time points and thus have the possibility to follow potential changes in the gastrointestinal barrier of the same individual. Using a flexible cystoscope and a specialized biopsy forceps, we were able to take several pointed biopsies at different locations in the gastrointestinal canal. This could be done rapidly with a negligible insult to the tissue, enabling a high throughput with a minimal impact on the physiology of the animal. We therefore believe that endoscopic biopsies will provide a significant advantage over the more traditional methods involving post-mortem sampling. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.90 Effect of body size on locomotion and metabolism in zebrafish, Danio rerio

Julie Lucas (LIENSs Laboratory, University of La Rochelle and Ifremer L'houmeau, France), Alicia Schouman (LIENSs Laboratory and Ifremer l'Houmeau, France), Fabrizio Atzori (LIENSs Laboratory and Ifremer l'Houmeau, France) and Christel Lefrançois (LIENSs laboratory, France) As a lot of other organisms, teleost fishes satisfy their energetic needs by oxidation of products originated from ingested organic matter. The capacity of an organism to sustain its energy-demanding activities can be assessed through its aerobic metabolic scope. Metabolism is known to be influenced by a set of parameters, such as environmental factors, but also intrinsic ones, such as body size. In this context, the aim of our study was to understand the effect of size on different physiological functions, and more especially on aerobic metabolism. The species employed for this study was the zebrafish Danio rerio, a freshwater species that is commonly used for experimental studies. The main objective was to measure standard (SMR) and active (AMR) metabolic rates in order to assess aerobic metabolic scope at different life stages in this species. In order to do so, oxygen consumption was measured by intermittent flow respirometry. Larvae were tested in a group, while adults were tested individually. Oxygen measurements were carried out during the resting period and after chasing in order to establish the SMR and the AMR, respectively. The results of this study will be discussed in order to establish the relationship between body size and metabolic capacity in D. rerio. This study will constitute the control for further experiments regarding the effects of organic pollutants on some physiological functions that will be assessed throughout the ontogeny of this species.

A6.92 How do we maintain the welfare of fish during slaughter? Investigating the physiological effects of cold shock and high levels of CO2 in the Arctic char (Salvelinus alpinus)

Henrik Seth (University of Gothenburg, Sweden), Erik Sandblom (University of Gothenburg, Sweden), Kristina Sundell (University of Gothenburg, Sweden), Michael Axelsson (University of Gothenburg, Sweden) and Anders Kiessling (The Swedish University of Agricultural Sciences (SLU), Sweden) With an increased awareness of welfare issues, there is a need for more ethically acceptable slaughter methods minimizing the stress and distress of fish while at the same maintaining the quality of the meat. Historically, one common method of immobilization/sedation prior to slaughter is to place the fish in ice cold water containing high levels of CO2. However, there is a limited amount of empirical data on the physiological impact of such treatment. We therefore measured several haemodynamic variables, such as blood pressure, heart rate, ventilation and haematocrit as well as plasma cortisol in order to investigate the physiological effects of cold shock and high levels of CO2. The results indicate that while cold shock alone had predictable effects on the physiology, with a decreased heart rate/ventilation and limited impact on the apparent behaviour. CO2 induced fundamental changes in both the physiology and behaviour of the fish. This was irrespective of whether high levels of CO2 were combined with cold shock or not.

Abstracts 2011 Aside from struggling behaviour during the initial 10 to 15 minutes, there was a large decrease in all cardiovascular variables, while the haematocrit of the deoxygenated blood increased. Therefore, CO2 s a method of immobilization does not meet the rational of minimizing the distress of the fish. The final clue as to how the fish experience the sharp increase in CO2 awaits the use of either an electroencephalogram or electrocortigram in order to establish the conscious state of the fish during the procedure. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

113 closer to that shown by rats under chronic ethanol exposure. In this study, we tested the hypothesis that the crucian carp has evolved gene expression profiles in relation to chronic ethanol exposure. To test this hypothesis, we exposed a non-anoxia-tolerant fish, the koi carp (Cyprinus carpio) - a close relative of the crucian carp -to chronic ethanol (10 mM, i.e. 0.05%) and compared the gene expression profiles of genes involved in GABAergic neurotransmission in these two species. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.93 Physiological and behavioural response in Arctic Hyas araneus larvae to elevated seawater pCO2

Melanie Schiffer (Alfred-Wegener Institute, Germany), Lars Harms (Alfred-Wegener Institute, Germany), Hans-Otto Pörtner (AlfredWegener Institute, Germany), Felix Mark (Alfred-Wegener Institute, Germany) and Daniela Storch (Alfred-Wegener Institute, Germany) On-going ocean acidification necessitates studies concerning the consequences of reduced seawater pH on marine organisms. Early life stages seem to be more sensitive to elevated seawater pCO2. We threfore investigated the effect of elevated seawater pCO2 on the development of Arctic Hyas araneus larvae. First, freshly hatched larvae of ovigerous females maintained at 390 ppm were reared at three different CO2 conditions (490, 1100 and 2400 ppm). Second, larvae of ovigerous females incubated at 390 ppm and 3000 ppm were reared in the same conditions as the mother females. Oxygen consumption, dry weight, mortality and developmental time of the zoea I stage were monitored. Additionally, the feeding rates and maximal survival time under starvation were investigated at four experimental temperatures (4°C, 9°C, 15°C and 21°C). The thermal tolerance of larvae was determined by measuring the heart rate and oxygen consumption during continuous warming. Elevated seawater pCO2 had no significant effect on larval mortality, developmental time and dry weight in the larvae of ovigerous females maintained at 390 ppm. However, we found high mortality and an extended larval development under high CO2 conditions in the larvae of females pre-incubated at 3000 ppm. Feeding rates were temperaturedependent and lower in larvae reared under elevated CO2. The maximal survival time of starved larvae was reduced under high CO2, except at 3°C where starved larvae kept at 3000 ppm survived longer than the control larvae. High seawater pCO2 seems to narrow the thermal tolerance of the larvae. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.95 Effects of food deprivation and exhaustive swimming impacts on swimming performance, metabolic responses and the energy budget of goldfish, Carassius auratus

Hon-Jung Liew (University of Antwerp, Belgium), Amit Kumar Sinha (University of Antwerp, Belgium) and Gudrun De Boeck (University of Antwerp, Belgium) Stress caused by rapid environmental changes can lead to chronic or acute food shortage in aquatic habitats that might depress the routine metabolism of aquatic animals. This study is designed to investigate the impact of short-term (seven days) starvation and feeding on the physiological responses of goldfish Carassius auratus challenged by respirometric approach to exhaustive swimming conditions. Starvation reduced biometric indexes, but did not depress Ucrit. Swimming significantly elevated MO2 by 38% in fed fish and 46% in starved fish compared to non-swimming fish. The NH4+ excretion rate was approximately twofold higher in swimming fish. Feeding itself also induced a twofold higher NH4+ excretion rate in fed fish as compared to starved fish. Swimming along with feeding resulted in a significant twofold higher ammonia quotient, suggesting that the percentage of protein used by fed swimming fish was approximately 96% compared to 74% in fed non-swimming fish. Plasma and muscle NH4+ levels increased significantly depending on feeding and swimming. Glycolysis increased inversely with swimming levels observed in both muscle and the liver. Swimming induced muscle lipolysis in both fed and starved fish, though changes in hepatic lipid reserves remained insignificant. Starvation significantly depleted the hepatic and muscle protein reserves, indicating that protein was used as a primary fuel during starvation. We conclude that protein catabolism was the main metabolic pathway used to sustain basal energy needs during starvation. Glycogen becomes as an important ready source of energy used to meet extra energy demand during exhaustive swimming. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.94 GABAergic gene expression in koi carp (Cyprinus carpio) brain after chronic ethanol exposure

Christine S Couturier (University of Oslo, Denmark), Göran E Nilsson (University of Oslo, Denmark) and Jonathan A Stecyk (University of Oslo, Denmark) The crucian carp (Carassius carassius) can survive for months in anoxia at low temperature. In these conditions, it relies on the anaerobic production of energy. The accumulation of acidic end-products is avoided by the transformation of pyruvate into ethanol, which is excreted by the gills. Nevertheless, blood ethanol concentration can rise up to 0.05%. Furthermore, in anoxia, brain activity is reduced, but the profiles of the relative expression of the six GABAA alpha subunits have shown to be almost unchanged in anoxia compared to normoxia. If they remain unchanged, they are drastically different from the one shown by another anoxia-tolerant species: the red eared slider (Chrysemis scripta) a freshwater turtle. The crucian carp profiles are

A6.96 Transcriptomic response of the spider crab Hyas araneus to ocean acidification and warming

Lars Harms (Alfred Wegener Institute, Germany), Melanie Schiffer (Alfred Wegener Institute, Germany), Felix Mark (Alfred Wegener Institute, Germany), Daniela Storch (Alfred Wegener Institute, Germany), Christoph Held (Alfred Wegener Institute, Germany) and Magnus Lucassen (Alfred Wegener Institute, Germany) Ocean acidification and warming, induced by elevated anthropogenic CO2 levels, seem to affect many marine organisms. For an understanding of species vulnerability it is necessary to examine the transcriptomic response, which forms the mechanistic basis of physiological plasticity at higher organisational levels. In this study the possible regulatory response to ocean acidification and warming was investigated in a potentially sensitive species, the

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Society for Experimental Biology The main purpose of the investigation was to study genetic diversity in wild populations of small rodents from isolated and metal polluted areas. Myodes glareolus, an abundant small mammal, was used as a good model for such an investigation. The animals were collected from nine populations (about 20 animals from each population). Three of the populations originated from areas near zinc and lead smelters (Katowice, Olkusz, and Miasteczko Slaskie), another three were island populations from Mazurian Lake District, which is a very clean area (Dejguny Lake, Dobskie Lake) and the Bieszczady Mountains (Skalista Island). We also collected animals from three clean open sites ­ Mikolajki (Mazurian Lake District), Niepolomice (Puszcza Niepolomicka) and Telelnica Oszwarowa (Bieszczady Mountains). Cadmium, lead, zinc and iron concentrations in the liver and kidney were measured to assess the animals' exposure to metals. To determine genetic diversity we used eight microsatellite markers. Based on these results, we will discus the influence of isolation and heavy metal pollution on genetic diversity in the bank vole populations studied. The levels of genetic diversity will be presented as Nei's genetic distance, expected heterozygosity, number of alleles and number of private alleles. Email address for correspondence: [email protected] uj.edu.pl Poster Session Saturday 2nd July 2011

osmoconforming spider crab Hyas araneus from Arctic waters. Adult males were incubated over a period of ten weeks at four different CO2 concentrations and two different temperatures (390, 1120, 1960 and 3000 ppm; 5°C and 10°C). To investigate the basis of a possible regulatory response, normalized cDNA libraries were constructed, and the transcriptome was determined by 454 pyrosequencing to serve as the backbone for in-deep expression studies. Furthermore, eight cDNA libraries were constructed for each treatment and sequenced with the Illumina high-throughput sequencing technology. About 60million reads were mapped on the backbone. The in-deep analyses of this high dataset are still in progress, but the first results already indicate the reasonable responsiveness of many biological processes in this osmoconforming species to elevated CO2 levels and increasing temperatures. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.97 Metabolic transition during long-term starvation and digestion in the tarantula, Acanthoscurria geniculata

Peter Skødt Knudsen (Department of Biological Sciences, Aarhus University, Denmark), Henrik Lauridsen (Department of Biological Sciences, Aarhus University and MR Research Centre Aarhus Un, Denmark), Kasper Hansen (Department of Biological Sciences, Aarhus University and MR Research Centre Aarhus Un, Denmark), Michael Pedersen (MR Research Centre, Aarhus University Hospital Skejby, Denmark), Tobias Wang (Department of Biological Sciences, Aarhus University, Denmark), Hans Malte (Department of Biological Sciences, Aarhus University, Denmark) and Johannes Overgaard (Department of Biological Sciences, Aarhus University, Denmark) Spiders are opportunistic feeders that often inhabit environments with fluctuating food availability. To thrive in such environments many species tolerate long fasting periods but retain the capacity to subdue and digest very large meals. In these ecological settings, spiders must be able to balance the use of endogenous energy stores with the need to maintain a digestive apparatus that can operate immediately due to the extra-oral digestion used by these animals. Here we have studied the effects of fasting on metabolic depression, the magnitude and duration of the SDA response and the content and preference of macronutrients (carbohydrates, lipids and proteins) of the Brazilian white knee tarantula (A. geniculata). Using magnetic resonance imaging (MRI) we also determine volume of the midgut mesenteron and diverticulas during progressive fasting and digestion. After two weeks of fasting, the spiders have metabolic rates of 40 to 55 ml O2/h/g at 28°C, but this is progressively depressed during starvation reaching 7 to12 ml O2/h/g after 150 to 200 days. Feeding in frequent-feeding spiders results in a nearly tenfold increase in metabolism and we are currently measuring the SDA response after prolonged starvation. Similarly, we will present estimates of organ atrophy/hypertrophy measured using MRI during progressive fasting and digestion. By measuring changes in the spiders' macronutrient content during digestion and following feeding we are finally assessing which energy sources are utilized during feeding and if/how these are replaced following feeding. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.99 Characterization of pattern recognition receptors in the blue mussel Mytilus edulis

Ulrike Findeisen (Institute of Clinical Molecular Biology, Christian Albrechts University, Germany), Lars Kraemer (Institute of Clinical Molecular Biology, Christian Albrechts University, Germany), Philip Rosenstiel (Institute of Clinical Molecular Biology, Christian Albrechts University, Germany) and Eva Philipp (Institute of Clinical Molecular Biology, Christian Albrechts University, Germany) Marine bivalves are constantly exposed to a multitude of potential pathogens through intense contact with the surrounding seawater at their epithelia. Germline-encoded receptors of the innate immune system are important for the recognition of foreign particles and danger signals and are present on cellular surfaces or intracellularly. In vertebrates the two major types of pathogen recognition-receptors (PRRs) in the innate immune system are the Toll-like receptors (TLRs), with an extracellular ligand binding domain, and Nod-like receptors (NLRs), which play a pivotal role for the recognition of pathogens in the cytosol. In the present study the TLR and NLR pathway of the bivalve Mytilus edulis was investigated to better understand the evolutionarily conserved mechanisms of pathogen detection and immune responses. Transcriptomes (454, Roche) of immune-challenged M. edulis individuals were searched for key constituents of both pathways. No indication of NLRs were found but 29 putative TLR transcripts could be identified within the M. edulis transcriptome, with eight of these transcripts showing the conserved domain structure of TLRs, i.e. LRRTM-TIR. Further, key components and target genes of the TLR pathway were found. in vivo and in vitro stimulation with lipopolysaccharide increased the expression levels of selected TLR pathway genes in Mytilus gills and haemocytes. Taken together, the results indicate a complex TLR system with a high number of TLRs that might play an important role in the Mytilus immune system. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.98 Isolation and heavy metal pollution as factors of genetic diversity in populations of the small rodent Myodes glareolus

Renata Swiergosz-Kowalewska (Jagiellonian University Institute of Environmental Sciences, Poland) and Magdalena Mikowska (Jagiellonian University Institute of Environmental Sciences, Poland)

A6.100 Comparison of the physiological responses of crucian carp (Carassius carassius) to high external ammonia and prolonged anoxia

Abstracts 2011 Jonathan Stecyk (University of Oslo, Norway), Mike Wilkie (Wilfrid Laurier University, Canada), Christine Couturier (University of Oslo, Norway) and Göran Nilsson (University of Oslo, Norway) Given the likelihood that fish experiencing regular episodes of oxygen deprivation also experience high ammonia levels, and the hypothesis that mechanisms underlying hypoxia/anoxia tolerance might also confer greater resistance to high external ammonia (HEA) (Walsh et al., 2007), we examined the tolerance and physiological responses of the crucian carp to HEA. Crucian carp were profoundly tolerant to HEA. No mortality was observed in fish acutely exposed to HEA (2 to 32 mM), although overturning and cessation of ventilation were noted at 16 and 32 mM ammonia (loss of equilibrium EC50 was 163 mmol/L for NH3 and 17.8 mmol/L for NH4+). Nevertheless, all fish completely recovered following a return to ammonia-free water. The physiological responses of crucian carp exposed to HEA (8 mM NH4Cl for up to 96 hours at 10ºC) differed markedly from crucian carp exposed to prolonged anoxia (96 to 168 hours at 8°C). First, unlike in anoxia-exposed fish, HEA caused brain swelling, as indicated by 25% increases in the brain water content of overturning fish. The increase strongly correlated with external and internal ammonia concentrations and was completely reversible. Thus, the crucian carp brain is not resistant to ammonia-induced brain swelling, as it is to anoxia-induced brain swelling. Second, crucian carp venous blood Na+, Cl-, pH, HCO3- and total CO2 were elevated with HEA, whereas these parameters decreased with anoxia exposure. Our findings suggest that, although crucian carp are extremely anoxia and ammonia tolerant, some of the physiological mechanisms of resistance to the two stressors differ. Reference: Walsh, P. J. et al. (2007) Comp Biochem Physiol 147A:332. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

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A6.102 Frictional values affect adhesion in insects: evidence for the influence of surface energies on the self-cleaning ability of insect adhesive devices

Mr Michael J Orchard (University of Hull, UK), Mika Kohonen (Australian National University, Australia) and Stuart Humphries (University of Hull, UK) The ability of insects to adhere to surfaces is enhanced by the use of adhesive organs found on the terminal leg segments. These organs must remain functional for the duration of the insect's life cycle; and so contamination from environmental detritus must be removed for them to function properly. Although grooming of antenna and body segments is a common behaviour in insects, any increase in time spent stationary can increase the potential risk from predation. Consequently, fouling can be an important problem, and it is likely that insect adhesive organs have self-cleaning abilities allowing them to minimise risk associated with grooming. To test this hypothesis we measured time taken to regain adhesive potential in Hymenopterans (Polyrachis dives and Myrmica scabrinodis) and Coccinellids (Harmonia axyridis and Adalia bipunctata) fouled with micro-spheres of differing sizes and physical properties on substrates of differing frictional values. The ability to adhere to surfaces was influenced by particle type and size in Hymenopterans with an interaction between the frictional values of the fouling particle and substrate, while in Coccinellids it was influenced by particle size and substrate properties. The adhesive organs of Coccinellids were found to possess superior self-cleaning abilities compared to those of Hymenopterans, although Hymenopterans were found to have better adhesion to both surface types. This work supports the hypothesis that although the pad forms may differ in ability to self-clean the interactions and influence of the physical properties of the fouling particles and substrates are common to both types. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

A6.101 Life without oxygen: a transcriptome analysis of the anoxic crucian carp (Carassius carassius) heart

Kåre-Olav Stensløkken (University of Oslo, Norway), Stian Ellefsen (Lillehammer University College, Norway), Olga Vasieva (University of Liverpool, UK), Yongxiang Fang (University of Liverpool, UK), Anthony Farrell (University of British Columbia, Canada), Lisa Olohan (University of Liverpool, UK), Jarle Vaage (University of Oslo, Norway), Göran Nilsson (University of Oslo, Norway) and Andrew Cossins (University of Liverpool, UK) Crucian carp can survive for extended periods in the complete absence of molecular oxygen, in part through maintained activity of the heart. Using a high-density cDNA microarray, we have explored the genomewide, gene expression responses to one and seven days of anoxia at either 8 or 13°C and to the subsequent restoration of normoxia for a further seven days. We show that at 8°C, anoxia and re-oxygenation elicited few changes in gene expression. In contrast, at 13°C we identified 844 unique genes that displayed mainly opposing responses to anoxia treatment and to the subsequent establishment of normoxia. There were few transcriptional changes between temperatures in the normoxic group, but 67 unique genes were regulated between 8 and 13°C in the anoxic group after seven days. The anoxia-affected genes were associated with cardiac muscle function, energy metabolism, calcium signalling, mitochondrial function and muscle morphogenesis. We also identified up-regulation of genes involved in protein turnover, pentose phosphate pathway, cell morphogenesis and angiogenesis. Down-regulated gene categories include RNA splicing and transcription. These patterns indicate divergent responses to anoxia at 8 and 13°C, with the latter but not the former driving a transcriptional reprogramming of cardiac function. Email address for correspondence: [email protected] Poster Session Saturday 2nd July 2011

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Society for Experimental Biology

SEB Main Meeting, Glasgow 2011

A7 Chemical messengers: neurotransmitters to pheromones

A7.1 Neurobehavioural innovations for vertebrate acoustic communication

Andrew H Bass (Cornell University, USA) The evolutionary origins of neurobehavioural innovations for social behaviours among vertebrates, such as vocalization, remain essentially unexplored. I will first present comparative developmental evidence that the evolution of vocal behaviours depends upon a highly conserved pattern-generating region of the central nervous system (CNS), a neuroectodermal compartment spanning the hindbrain-spinal junction. Next, I will present neurophysiological results showing how this CNS vocal compartment codes for frequency and duration, two of the most fundamental attributes of naturally occurring acoustic communication signals among all vertebrates. Further evidence shows how hormonal modulation of this CNS vocal network and vocal behaviour is also evolutionarily conserved. In summary, despite the wide range of variation in the spectral and temporal features of vocalizations across the major lineages of vocal vertebrates, the neural and hormonal mechanisms determining the production of those behaviours is remarkably conserved. Email address for correspondence: [email protected] 10:30 Monday 4th July 2011

A7.3 Maternal cortisol is a key chemical messenger for the developmental programming of growth in zebrafish

Matt Vijayan (University of Waterloo, Canada) and Dinushan Nesan (University of Waterloo, Canada) Maternal deposition of cortisol into fish eggs is well established, but the significance of this steroid accumulation on embryogenesis is less clear. In zebrafish (Danio rerio), we showed recently that maternallydeposited cortisol is depleted over time and the de novo synthesis of this hormone commences only after hatching. This provided a nice model to study the role of maternal cortisol on early developmental regulation in zebrafish. We have confirmed that glucocorticoid receptor (GR) signalling is essential for cortisol action during zebrafish development. This species is unique because it is the only teleost to have a single copy of GR in the genome, unlike other teleosts studied so far that have multiple copies. Knocking down GR in zebrafish revealed that cortisol signalling plays a critical role in mesoderm formation. Our results indicate for the first time that maternal cortisol modulates the expression of bone morphogenetic proteins, being a key morphogen involved in various aspects of growth and development, including embryo patterning. The talk will highlight the importance of GR signalling, as well as the maintenance of physiological cortisol levels in the embryos and during early development in zebrafish. Email address for correspondence: [email protected] 11:20 Monday 4th July 2011

A7.2 Corticotropin-releasing factor-related peptides regulate the stress response in fish through hypophysiotropic, neuromodulatory and paracrine actions

Nicholas J Bernier (University of Guelph, Canada) The neuropeptide corticotrophin-releasing factor (CRF) is best known for its role as the primary regulator of the hypothalamic-pituitaryinterrenal (HPI) stress axis in fish. Expressed at high levels in the preoptic area (POA) of the brain, a region known to project to the pituitary, CRF is considered the major regulator of adrenocorticotropic hormone (ACTH) secretion in teleosts. In fact, in vitro evidence suggests that both CRF and the related peptide urotensin I (UI) are potent hypophysiotropic regulators of ACTH, thyrotropin and alphamelanocyte-stimulating hormone. The close relationship observed in adult zebrafish between the dynamic changes in POA CRF gene expression and whole-body cortisol during exposure to a stressor also implicate CRF in the regulation of the HPI axis. While CRF-related peptides are important hypophysiotropins, the widespread central and peripheral tissue distribution of CRF peptides and receptors suggests a broader physiological role for the CRF system. For example, CRF-related peptides in fish play important neuromodulatory roles in the regulation of food intake, locomotion, ventilation and the cardiovascular system. Finally, while the abundant expression of CRF in the chromaffin cells of the head kidney in common carp may provide an opportunity for paracrine modulation of the HPI axis, the stimulatory effects of heat shock on CRF expression in early zebrafish embryos suggests that CRF can exert paracrine actions before it functions in the regulation of the endocrine stress response. Combined, the hypophysiotropic, neuromodulatory and paracrine actions of CRF-related peptides enable a coordinated neuroendocrine, behavioural and autonomic response to stressors. Email address for correspondence: [email protected] 11:00 Monday 4th July 2011

A7.4 Regulation of telencephalic cell proliferation in fish: a biphasic effect of cortisol?

Christina Sørensen (University of Oslo, Norway), Linda C Bohlin (University of Oslo, Norway), Göran E Nilsson (University of Oslo, Norway) and Øyvind Øverli (Norwegian University of Life Sciences, Norway) Fish have a very high rate and prevalence of brain cell proliferation, yet little is known about the regulation and functional implications of this phenomenon. We have shown that environmental enrichment stimulates and social stress suppresses cell proliferation in the fish telencephalon. Both treatments are associated with higher cortisol levels, though environmental enrichment elicits a moderate cortisol response while social stress leads to a marked elevation of plasma cortisol. In mammals, corticosteroid hormones have biphasic effects on many aspects of neurobiology, with moderately elevated hormone levels acting in a stimulatory manner while high levels are suppressive. This could also be the case for telencephalic cell proliferation in fish. To test this, rainbow trout were given cortisol in feed for six days at a low or high dose. Compared to controls receiving regular feed, this treatment caused moderately elevated plasma cortisol levels in the low-dose group and a considerable elevation of plasma cortisol in the high-dose group. Cell proliferation was investigated by immunohistochemistry for proliferating cell nuclear antigen (PCNA), a marker of actively cycling cells. Both cortisol-treated groups had a 50% reduction in the density of PCNA-positive cells in the telencephalon. Thus, under the current treatment regime, cortisol did not have a biphasic effect on telencephalic cell proliferation.

Abstracts 2011 Our results indicate that the suppressive effect of chronic social stress on telencephalic cell proliferation may be mediated by cortisol. In the case of environmental enrichment, the involvement of other factors counteracting the suppressive effect of cortisol appears likely. Email address for correspondence: [email protected] 11:40 Monday 4th July 2011

117 intense within the rostral periventricular area of the third ventricle (RP3V), septohypothalamic nucleus (SHy) and arcuate nucleus (Arc). Kisspeptin-ir cell bodies were mostly confined to the Arc. Kisspeptin-ir density in these regions was compared between breeding and nonbreeding seasons and between males and females. No differences in kisspeptin-ir density were observed between the sexes in any of the regions examined. In both sexes, the kisspeptin-ir density in the RP3V and Arc was significantly greater during the breeding season. In the SHy, there was no difference in kisspeptin-ir density between the seasons. These findings suggest a significant role for kisspeptin in regulating seasonal reproductive changes in the male and female E. myurus. It remains to be established whether this role involves relaying environmental signals concerning photoperiod and/or food availability to the central systems controlling reproduction. Email address for correspondence: [email protected] 12:20 Monday 4th July 2011

A7.5 Central influence of AFT and CRF on stress responses in rainbow trout (Oncorhynchus mykiss)

Tobias G Backström (Department of Neuroscience, Uppsala University, Sweden), Andreas Pettersson (Swedish University of Agricultural Sciences, Sweden), Markus Norén (Uppsala University, Sweden), Per-Ove Thörnqvist (Department of Neuroscience, Uppsala University, Sweden) and Svante Winberg (Department of Neuroscience, Uppsala University, Sweden) Arginine vasotocin (AVT) and corticotrophin-releasing factor (CRF) are two neuroendocrine factors of interest concerning both behavioural and physiological stress responses. We investigated the effects of AVT and CRF on agonistic interaction in rainbow trout. Fish were intracerobroventricullarly injected with peptides/antagonists at different doses and interacted with a control fish. We investigated the effects of social status on the expression of AVT, CRF and their receptors. In this case fish were allowed to interact for five days while behaviour was monitored and then brain was analysed for expression of mRNA. Further, fish were dietary supplemented with L-tryptophan (TRP), precursor to serotonin (5-hydroxytryptamine, 5-HT), for seven days and then brain was analysed for expression of AVT, CRF and their receptors. Both AVT and CRF injection induced subordination, and aggression affected components of the AVT and CRF system. These results suggest that AVT and CRF affect social behaviour through central effects. Dietary TRP also affected components of the AVT and CRF system. Funded by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). Email address for correspondence: [email protected] 12:00 Monday 4th July 2011

A7.7 Specific-specific information in hormonally-derived sex pheromones is conveyed by odour context in carp and goldfish

Peter W Sorensen (University of Minnesota, USA), Haude Levesque (University of Minnesota, USA) and Hangkyo Lim (University of Minnesota, USA) Several decades of study have demonstrated that many fishes detect gonadally-derived hormonal products with acute sensitivity and that these products serve as sex pheromones that drive powerful reproductive responses. This makes evolutionary sense, because the production of these cues naturally and precisely correlates with relevant events in the reproductive lives of fishes. On the other hand, their use is an enigma because by their very nature, endocrine products are highly conserved and poorly suited to conveying species-specific information. We explored this puzzle in the goldfish and common carp, two closely related species that rarely hybridize but produce, detect and use a common set of five hormonal products as sex pheromones. Initial behaviour studies confirmed that these species could distinguish each other using odour alone. Next, we measured the presence of known hormonal products in fish in different maturational conditions to discover the basis of odour specificity. To our surprise, we discovered evidence that while information on precise reproductive state was conveyed by hormonal products (steroids and F prostaglandins), species information was found in mixtures of polar and non-polar body metabolites that fish release continuously regardless of their endocrine state. Incorrect mixtures of body odours could even block responses to hormonal products. Recipient sensitivity to the presence of hormonal products within these pheromonal complexes varied with their reproductive state. In conclusion, fish appear to have evolved a remarkable ability to simultaneously perceive information about conspecific maturity and identity by discriminating changing arrays of hormonal and nonhormonal products which they release. Email address for correspondence: [email protected] 13:30 Monday 4th July 2011

A7.6 Changes in kisspeptin-immunoreactivity in the seasonally breeding eastern rock elephant-shrew (Elephantulus myurus) from South Africa

Katarina Medger (Mammal Research Institute, Dept. Zoology and Entomology, University of Pretoria, South Africa), Christian T Chimimba (Mammal Research Institute, Dept. Zoology and Entomology, University of Pretoria, South Africa), Jens D Mikkelsen (Department of Functional Neuroanatomy, NeuroSearch AS, Copenhagen University Hospital, Denmark), Nigel C Bennett (Mammal Research Institute Dept. Zoology and Entomology University of Pretoria, South Africa) and Clive W Coen (School of Medicine, King's College London, UK) Hypothalamic kisspeptin is considered important for the regulation of seasonal reproduction by actions that integrate environmental factors with the endocrine system. Whether kisspeptin plays a uniform role in seasonal breeding across species remains to be elucidated. Among the species that have been studied, there appears to be inconsistency in kisspeptin expression under different photoperiodic regimens and between breeding and non-breeding individuals. These findings have prompted further research, not only under laboratory conditions, but also in semi-natural and natural settings. The present study was designed to compare hypothalamic kisspeptinimmunoreactivity in wild-caught males and females of a small non-rodent mammalian species, the eastern rock elephant-shrew (Elephantulus myurus), between breeding (October) and non-breeding (April) seasons. Kisspeptin-immunoreactive (ir) fibre densities were

A7.8 The scent of dominance in crustaceans

Thomas Breithaupt (University of Hull, UK) Animals of all phyla use aggressive behaviour when competing over resources. Crustaceans are great models for the study of aggression, as the behaviour is conspicuous and involves potentially lethal weaponry. When individuals are placed in an aquarium, aggressive interactions gradually diminish over time, giving rise to stable dominance hierarchies

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Society for Experimental Biology The catadromous European eel spends part of its lifecycle in either the sea or freshwater. As a result, the eel requires sensory mechanisms that can detect changes in water salinity to regulate the osmoregulatory process. However, such sensory systems are poorly understood in fish. We hypothesized that eels use olfaction to detect changes in the aquatic composition of inorganic ions and to trigger rapid osmoregulatory responses. To test our hypothesis, we assessed olfactory sensitivity to Ca2+ and Na+, by olfactory nerve recording of sea- or freshwateradapted eels. All eels responded to increases in external [Na+]. Freshwater eels respond to increases in external [Ca2+] whereas seawater-adapted fish responded to reductions of external [Ca2+] in a concentrationdependent manner. We also tested whether olfactory responses to Ca2+ and Na+ might be mediated by sensing receptors or ionic channels by assessing olfactory responses to cations and mammalian Ca2+ agonists before and after exposure to nucleotide-gated ion channel blockers. We found that spermine was a Ca2+ agonist. Furthermore, we show by immunocytochemistry that Ca2+-sensing receptors in the nose were distributed throughout the olfactory lamellae in freshwater eels but confined to the interlamelae region in seawater eels. Finally, we evaluated the role of olfaction in adapting to salinity changes by measurement of osmoregulatory responses of anosmic eels after an acute transfer to either fresh- or seawater. We conclude that, in the eel, olfactory sensitivity to changes in environmental [Ca2+] and [Na+] could play a role in osmoregulation, possibly triggering necessary adaptation to new aquatic media during migration and reproduction. Email address for correspondence: [email protected] 14:40 Monday 4th July 2011

that are controlled by internal and/or external factors. In decapod crustaceans, urine-borne chemical signals play an important role in mediating dominance, particularly when competitors are sizematched. Lobsters and crayfish have been shown to release urinary signals during agonistic interactions and to be able to control the timing of signalling. If urine release is blocked, fights last longer, indicating that chemical signals are essential in the maintenance of dominance. Artificial introduction of urine from a dominant animal more than that of a subordinate reduces the duration of fights in Norway lobsters and crayfish. In crayfish, upon detection of introduced dominant urine subordinate individuals immediately reduce their level of aggression, even if the urine originates from an unfamiliar dominant. These subordinate responses enable bioassay-guided purification of dominance pheromones. Responses can be elicited with urine components smaller than 10 kDa indicating that proteins are not involved. During courtship, crayfish males do not advertise their status using chemical signals. In contrast to lobsters, female crayfish do not seem to use male dominance odours for mate choice. Further studies of crustacean urinary communication promise to decipher the information transmitted by these chemical signals and explore whether they can be used deceptively. Email address for correspondence: [email protected] 14:00 Monday 4th July 2011

A7.9 Glutamate mGluR1 and serotonin 5-HT1 type receptors act as odorant receptors in the goldfish (Carassius auratus)

Peter C Hubbard (Centro de Ciências do Mar, Portugal), Nikolay N Kolmakov (Centro de Ciências do Mar, Portugal) and Adelino V Canário (Centro de Ciências do Mar, Portugal) Odorant receptors and many neurotransmitter receptors belong to the G-protein coupled receptor super-family. Many aquatic organisms, including fish, have olfactory sensitivity to some neurotransmitters. The current study investigated whether neurotransmitter receptors could be responsible for this sensitivity in the goldfish olfactory epithelium. First, several putative neurotransmitter receptor (serotonin [5-HT1], L-glutamate [mGluR1] and -aminobutyric acid [GABAB]) mRNA sequences were identified in a goldfish olfactory epithelium EST library and compared with known sequences. Second, the olfactory sensitivity to the endogenous ligands (5-HT1, mGluR1 and GABAB) and specific agonists to the identified receptor types (1-(1-naphthyl) piperazine hydrochloride, quisqualic acid and baclofen, respectively) was assessed by electro-olfactogram (EOG) recording. Both 5- HT1 and mGluR1 and their respective agonists evoked robust EOG responses down to around 10-8 M. GABAB proved to be a less potent odorant and its agonist, baclofen, evoked virtually no EOG response. Third, cross-adaptation showed that the olfactory response to 5-HT1 could be specifically attenuated by prior exposure of the olfactory epithelium to its agonist 1-(1-naphthyl)piperazine hydrochloride and that the response to quisqualic acid could be nearly completely blocked by prior exposure to mGluR1. Together, these results suggest that both 5-HT1 and mGluR1 type, but not GABAB type, neurotransmitter receptors are used as odorant receptors in the goldfish olfactory epithelium. The role of GABAB within the olfactory epithelium receptor remains to be established. Email address for correspondence: [email protected] 14:20 Monday 4th July 2011

A7.11 Exposure to alarm substance alters physiological development in fish embryos

Katherine A Sloman (University of the West of Scotland, UK), Sulayman Mourabit (University of Exeter, UK), Samuel Voller (University of Plymouth, UK), Simon D Rundle (University of Plymouth, UK), John I Spicer (University of Plymouth, UK) and Ted Henry (University of Plymouth, UK) Most studies on the effects of alarm substance in fish have focussed on adults and juveniles, where exposure elicits characteristic antipredator behaviours. Recently, we demonstrated that exposure to alarm substance increased the rate of embryonic development in zebrafish, Danio rerio. Embryos exposed to alarm substance at the two-cell stage developed more rapidly, with time to first heart beat and muscle contraction occurring earlier than in unexposed embryos. Preliminary results indicate that the expression of a gene involved in heart development, (Nkx2.5 gene), is induced earlier in embryos exposed to alarm substance in a manner consistent with the early onset of heart activity. Thus, the release of alarm substance from injured adults has the potential to influence the development of their offspring and potentially increase their inclusive fitness. Email address for correspondence: [email protected] 15:40 Monday 4th July 2011

A7.12 Branchial and extrabranchial O2 chemoreceptors mediate behavioural responses to hypoxia in developing zebrafish, Danio rerio

Michael G Jonz (University of Ottawa, Canada) O2-chemoreceptive neuroepithelial cells (NECs) of the gills in zebrafish detect changes in arterial or water pO2 and initiate cardiorespiratory responses to hypoxia. Much remains to be discovered regarding

A7.10 Sniffing the water: is olfactory sensitivity to Ca2+ and Na+ related to osmoregulation in the eel?

Mar Huertas (Centre of Marine Sciences, Portugal), Peter C Hubbard (Centre of Marine Sciences, Portugal) and Adelino V Canario (Centre of Marine Sciences, Portugal)

Abstracts 2011 the chemical interactions of NECs with other cells and nerves, and the developmental processes that occur as zebrafish transition from anoxia-tolerant embryos to hypoxia-sensitive larvae during the first two to three days of life. Examination of zebrafish embryos using confocal immunofluorescence identified a transient population of innervated NEC-like cells of the skin at a time when embryos responded to hypoxia but gill chemoreceptors were absent. Chronic changes in water pO2 mediated growth and proliferation of embryonic NECs, and nerve degeneration studies correlated a loss of innervation with reduced behavioural responses to acute hypoxia. In larvae, serotonergic, cholinergic and dopaminergic pathways that mediated hypoxia-induced hyperventilation appeared in the gills at different developmental stages, and the timing of these events was consistent with the immunolocalization of serotonin and the vesicular acetylcholine transporter. In preliminary experiments in which wholegill specimens were removed from larvae and loaded with fura 2-AM, we recorded Ca2+ signals from gill regions containing NECs that were induced by acute hypoxia and quinidine, a blocker of O2 -sensitive K+ channels. These results were consistent with the Ca2+ responses of NECs isolated from the gills of adults. Thus, O2 sensing in embryos begins at an extrabranchial site and transitions to the gills, where NECs become functional and multiple neurochemical pathways develop during the larval stage. Email address for correspondence: [email protected] 16:00 Monday 4th July 2011

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A role of serotonin (5-HT) has been implicated in aggression, dominance, stress, behavioural inhibition and feeding as well as in several human affective disorders. Still, its effects appear elusive and it is far from clear which mechanisms 5-HT acts through. Numerous studies have reported that stress results in a rapid increase in brain 5-HT activity and that the 5-HT activity of subordinate animals remains elevated even after long-term interaction in an established dominance hierarchy. A positive correlation between brain 5-HT activity and plasma cortisol is often observed, and 5-HT has been suggested to stimulate HPA/ HPI axis activity. However, 5-HT is known to be important for stress coping and elevated dietary intake of L-tryptophan (TRP, a precursor of 5-HT) has been reported to elevate brain 5-HT activity along with a suppression of post-stress plasma cortisol. Similarly, stimulation of brain 5-HT activity by elevated dietary TRP results in an inhibition of aggressive behaviour. However, this as well as the effect of TRP on plasma cortisol only occur after feeding the fish elevated TRP for seven days, at a time when the direct effects on brain 5-HT levels are no longer apparent. Moreover, the behavioural inhibition observed in subordinate animals, which appear to be mediated by the 5-HT system, is only observed after long-term social interaction. Thus, in several cases the effects of 5-HT appear contradictory. Growing evidence suggests that 5-HT might act through effects on neuronal plasticity. Such mechanisms could explain the elusive and sometimes contradictory effects of elevated 5-HT release. Email address for correspondence: [email protected] 16:40 Monday 4th July 2011

A7.13 The pharmacodynamics and mRNA expression patterns of two toadfish serotonin receptors that are potentially involved in regulating toadfish chemical communication

M. Danielle McDonald (RSMAS, University of Miami, USA), Lea R Medeiros (RSMAS, University of Miami, USA) and Edward M Mager (RSMAS, University of Miami, USA) Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter with many functions in vertebrates. In the Gulf toadfish, Opsanus beta, 5-HT appears to play a role in the regulation of toadfish pulsatile urea excretion, which might serve as a possible mechanism of intraspecific chemical communication. We have recently cloned and obtained the full-length sequence of two 5-HT receptor subtypes, namely the 5-HT1A and 5-HT2A receptors, which are believed to regulate the teleost stress response and the toadfish branchial urea transporter, respectively. The toadfish 5-HT1A receptor (1271 bp) shows 67% amino acid sequence homology to mammalian 5-HT1A receptors and is expressed predominantly in the brain, namely within the pituitary and the midbrain/ diencephalon region that contains the hypothalamus, followed by the swim bladder and gonad. The toadfish 5-HT2A receptor (1490 bp) shows 70% amino acid sequence homology to mammalian 5-HT2A receptors and, unlike the 5-HT1A receptor, is expressed predominantly in the swim bladder, an organ used for vocal communication in toadfish, followed by the gonad, brain and gill. Within the brain, toadfish 5-HT2A receptor mRNA expression is highest in the hindbrain region. Using the Xenopus oocyte expression system, we have established toadfish 5-HT1A receptor pharmacodynamics that are similar to their mammalian counterparts and are presently investigating the pharmacodynamics of the toadfish 5-HT2A receptor. Email address for correspondence: [email protected] 16:20 Monday 4th July 2011

A7.15 HPI-axis activation and brain monoaminergic activity in Nile tilapia: comparing the acute stress response to the effect of conspecific alarm cues

Patricia I M Silva (UMB, Norwegian University of Life Sciences, Norway), Øyvind Øverli (Norwegian University of Life Sciences, Norway), Erik Höglund (Danish Institute for Fisheries Research, Institute of Aquatic Resources, Denmark), Hans M Gjøen (Norwegian University of Life Sciences, Norway) and Catarina I M Martins (Centro de Ciências do Mar, University of Algarve, Portugal) Many fish species exhibit threat-sensitive behavioural responses to avoid predation by freezing, hiding or moving away from chemical alarm cues emitted by injured conspecifics. Proximate neuroendocrine mechanisms translating the olfactory signal to a behavioural response, however, remain scarcely studied. In the present study, we investigated to what extent physiological responses to alarm cues are comparable to those induced by a known standardized stressor in fish: acute confinement in a small volume of water. Our model species, Nile tilapia (Oreochromis niloticus) respond to conspecific alarm cues by reducing bottom-grazing behaviour. Experimental fish were either exposed to acute stress, alarm cues or sampled as non-stressed socially isolated controls. Acute stress resulted in an elevation of whole body cortisol, as well as increased serotonin (5-hydroxytryptamine, 5-HT) metabolism in the dorsolateral area of the telencephalon, a brain region reported to show functional homology with the mammalian hippocampus. The same general pattern was seen after exposure to alarm cues, but this response was less pronounced than that seen in acutely stressed fish, and not statistically separate from non-stressed controls. In conclusion, a method for regional brain microdissection and neurochemical analysis was successfully established, but further studies are needed to pinpoint the signal systems involved in appetite suppression elicited by conspecific alarm cues in Nile tilapia. Email address for correspondence: [email protected] Poster Session Sunday 3rd July 2011

A7.14 Serotonin: an elusive neurochemical

Svante Winberg (Uppsala University, Sweden)

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Society for Experimental Biology

SEB Main Meeting, Glasgow 2011

A8 General biomechanics

A8.1 The SEM/FIB Workbench: nanorobotics system inside scanning electron or focused ion beam microscopes

Volker Klocke (Klocke Nanotechnik GmbH, Germany), Ivo Burkart (Klocke Nanotechnik GmbH, Gemrany) and Ralf Kaufmann (Klocke Nanotechnik GmbH, Germany) At the light microscopes it is natural for every expert to use tool sets like tweezers, knives, hooks, probes and several different measurement tools. Without such handling, manipulation and manufacturing tools, many present-day products and methods would not exist: no eye surgery, no wristwatch, no in vitro fertilization, just to mention a few.The operators of scanning electron microscopes (SEM), focused ion beam (FIB) or dual beam systems generally work without tool sets and call it natural, although the wave length limit of light is no physical boundary for using such tools. It can be imagined how technology would be pushed when the in situ SEM/FIB nanorobotics reach the same degree of usage as tool sets at light microscopes. The SEM/FIB Workbench developed by Klocke Nanotechnik offers, for the first time, an easy-to-use system for in situ nanomanipulation, object handling, material preparation and processing, assembly and manufacturing together with new methods for nano-characterization. Expanding the SEM/FIB to a material processing system and a nanoanalytical workbench opens the door to a world of new applications: from research to production, from material research over biology, pharmacy, pathology, forensic research, tribology, geology, and semiconductor technology up to nanofabrication. Several examples of these new interdisciplinary research and development fields will be described during the presentation - together with an invitation to participate in our research network. Email address for correspondence: [email protected] 10:30 Friday 1st July 2011 suck in prey at a maximum velocity of 1.7 ms-1 during food uptake. The whole gape cycle does not usually last over 150 ms, but the adduction of the hyobranchium to its initial position can take up to 10 seconds. We conclude that the rapid displacement of the jaws in A. davidianus represents the main mechanism that generates the powerful suction force and that the hyobranchial depression is mostly used for intraoral transport. Email address for correspondence: [email protected] 10:45 Friday 1st July 2011

A8.3 Force transmission in the suction feeding system of seahorses

Sam Van Wassenbergh (University of Antwerp, Belgium), Heleen Leysen (Ghent University, Belgium) and Peter Aerts (University of Antwerp, Belgium) Seahorses and other sygnathid fishes rely on a quick increase in the volume of the snout to suck prey into the mouth. This volume increase is observed as snout widening due to abduction of the suspensoria, the bones outlining the lateral sides of the snout. In contrast to other fishes, however, the start of suction is postponed until completion of the dorsal rotation of the neurocranium, a movement that brings the mouth close to the prey. In addition, the hyoid, which is generally considered the most important transmitter of force to the suspensoria in fish, rotates over more than 90° in the sagittal plane during feeding in seahorses. This is considerably higher than what is observed in all other suction feeders studied so far. To explain how seahorses abduct the suspensoria, we performed mathematical simulations with a model of force transmission via the hyoid and lower jaw. This analysis was based on three-dimensional reconstructions of the anatomy of the feeding system of the long-snout seahorse Hippocampus reidi, together with high-speed video data on hyoid and lower jaw rotation during prey capture in this species. Our results show a critical role for the inclined orientation of the hinge joint between the left and right ceratohyals, and suggest that force transmission also occurs via the lower jaw to produce suction in seahorses. The function of the peculiar arrangement of the three separate connections between the hyoid and the lower jaw in this process will also be discussed. Email address for correspondence: sam.vanwassen [email protected] 11:00 Friday 1st July 2011

A8.2 Analysis of the prey capture mechanism in the Chinese giant salamander Andrias davidianus

Egon Heiss (University of Vienna, Austria), Nikolay Natchev (University of Vienna, Austria), Anton Weissenbacher (Vienna Zoo, Austria) and Michaela Gumpenberger (University of Veterinary Medicine, Vienna, Austria) Being the largest recent amphibian species worldwide, Andrias davidianus may reach a maximum length of over 1.8 m and a weight of over 60 kg. The Chinese giant salamander shows a certain degree of pedomorphism, is a fully aquatic species, and lives in rivers and streams where it feeds on a variety of invertebrates, other amphibians, fish and occasionally on carrion. In the present study we show a movement analysis of the prey capture mechanism and discuss the role of the uniquely shaped hyobranchial apparatus. Our results show that A. davidianus approaches its prey very slowly to a minimum distance and engulfs it via a rapid strike. Analysis of digital high-speed recordings showed that the prey is sucked in by a rapid displacement of the jaws and after a clear delay (the mean delay is 70% of the total mouth opening duration) by hyobranchial depression. The jaw displacement is very fast (Vmean = 0.9 ms-1; Vmax =2.9 ms-1) and always faster than the hyobranchial depression (Vmean = 0.5 ms-1; Vmax = 1.5 ms-1). Using this performance, A. davidianus can

A8.4 Kinematics of the Atlantic mudskipper (Periophthalmus barbarus) feeding in aquatic and terrestrial environments

Krijn B Michel (University of Antwerp, Belgium), Sam Van Wassenbergh (University of Antwerp, Belgium) and Peter Aerts (University of Antwerp, Belgium) As part of the transition from water to land, the first tetrapods had to develop means of feeding in a terrestrial environment using aquatic feeding tools. The details of morphological changes occurring during the water-to-land transition are slowly becoming more evident from fossil records. Remarkably little work has been done on the morphology

Abstracts 2011 and function of the feeding apparatus of amphibious fish, however, particularly in a terrestrial setting. Here we describe the function and morphology of the feeding apparatus in Periophthalmus barbarus (the Atlantic mudskipper) in both a terrestrial and aquatic environment. P. barbarus feeds by protrusion of the premaxilla, positioning its oral cavity over the prey, engulfing it, while rapid closure of the dentary ensures prey capture. In a terrestrial setting, water is often carried in the oral cavity and deposited on the prey as it is imbibed. Kinematics show us that prey capture on land is very similar to that in water, where suction feeding is used. From high-speed digital X-ray videography we have learnt that the use of water is not required but can facilitate successful prey capture on land. Our results suggest that P. barbarus uses a modified form of aquatic suction feeding on land to facilitate swallowing and transport of prey inside the buccopharyngeal cavity. A versatile repertoire of volumetric expansion and jaw protrusion allows for terrestrial feeding with a primarily aquatic feeding apparatus. Email address for correspondence: [email protected] 11:15 Friday 1st July 2011

121 cause. Foot anatomy varies enormously among hoofed mammals, yet the functional consequences of foot anatomical variations are still a mystery. Elephants and rhinos, respectively, have five and three toes bound in a flexible pad of fibrous tissue (digital cushion), which is heavy and costly to swing, but seems to dampen vibrations when the foot hits the ground. Are the digital cushion and multi-toed foot structure thus marvellous solutions that control peak foot pressures in giant animals? How then do intermediate designs, such as cows and giraffes, work? We here study six species (pigs, horses, cows, rhinos, giraffes and elephants) to determine how foot anatomy and body mass influence the distribution of regional plantar pressures during walking. Our results show that peak pressure levels are maintained at near-constant relative levels across animal sizes by foot specializations, but that pressures on the distal ends of middle toes are significantly beyond those of other parts of the feet. Our study is the first attempt to measure plantar pressure distributions using spatially continuous descriptive statistical images and subsequent topological analysis, a set of techniques recently developed for human plantar pressure studies. Email address for correspondence: [email protected] 11:45 Friday 1st July 2011

A8.5 Sources of variance in temporal and spatial aspects of jaw kinematics in two species of primates

Jose Iriarte-Diaz (University of Chicago, USA), David A Reed (University of Chicago, USA) and Callum F Ross (University of Chicago, USA) Chewing is a strongly modulated behaviour, which responds to differences in material properties among different type of foods and to changes in the external physical properties of the food as the bolus is processed. Feeding, as with any complex biological behaviour, presents variation at multiple hierarchical levels, from among species to variation among chewing cycles within a single feeding sequence. To understand the mechanics and evolution of feeding systems requires an estimation of variability components at each of these hierarchical levels. In this study, we present data on the nature and sources of variation (from species to chewing cycle levels) in the kinematics of chewing in two species of primates, Cebus and Macaca, while feeding on foods of known material properties. Variation in chewing kinematics was not evenly distributed among hierarchical levels. Most of the variation was observed among chewing cycles, most likely in response to changes in the external properties of the food bolus. Species differences were found in duration and vertical displacement during slow close, suggesting that each species exhibits different power stroke dynamics. Cebus exhibited more variable gape cycles than Macaca, particularly when eating low-toughness foods. This increased ability to temporally and spatially modulate the gape cycle might reflect increased food processing efficiency because Cebus monkeys use fewer but longer cycles than Macaca when feeding on low-toughness foods. This is due to an increase in duration of the jaw opening phases of the gape cycle, when tongue manipulation repositions the food bolus in the oral cavity. Email address for correspondence: [email protected] 11:30 Friday 1st July 2011

A8.7 Frequency content of impact force signals in ungulates

Sharon E Warner (Royal Veterinary College, UK), Phillip Pickering (Royal Veterinary College, UK), Olga Panagiotopoulou (Royal Veterinary College, UK), Thilo Pfau (Royal Veterinary College, UK), Lei Ren (Manchester University, UK) and John R Hutchinson (Royal Veterinary College, UK) The causal link between impact vibration and injury remains controversial. The ability of the foot/limb to accommodate impact forces is partly dependent on the amplitude, frequency, duration and direction of oscillations experienced as a result of the foot-ground collision. At impact the foot must support, stabilize, brake and propel the body while providing necessary friction. The requirement to perform conflicting functions can leave the foot (and limb) vulnerable to mechanical insult. Force transmission demands high stiffness and low damping - features that reduce vibration attenuation. How do impact vibrations compare across ungulates and what is the influence of effective foot mass? We hypothesized that frequency of impact vibrations are kept at a constant level with increasing size and that effective foot mass and vibration frequency would have a significantly negative correlation. Using force platforms we measured the frequency content of impact forces in ten ungulate species (one to six individuals per species) spanning almost two orders of magnitude (ranging from an 18 kg blackbuck antelope to a 1058 kg giraffe) at normal walking and trotting speeds. Frequency of impact vibration appears to be invariant of body mass. In some species, the impact force experienced by the hindlimb contained a larger proportion of high frequency vibrations (50 to 100 Hz) than the forelimb, although this finding was not consistent across all species/all Froude ranges. Horizontal oscillations in the horse were consistently high at Fr > 0.5. Characterizing the mechanical consequences of foot impact has significant implications for animal welfare and further investigation might provide insight into mechanisms to prevent injury. Email address for correspondence: [email protected] 12:00 Friday 1st July 2011

A8.6 Regional plantar pressure distribution during walking in hoofed mammals

Olga Panagiotopoulou (Royal Veterinary College, UK), Todd C Pataky (Department of Bioengineering, Shinshu University, Ueda, Japan) and John R Hutchinson (Structure and Motion Laboratory, Royal Veterinary College, UK) Plantar pressures during locomotion have causal links with foot pathologies, e.g. osteoarthritis and foot abscesses. The causes of these pathologies are multifactorial but the foot-ground interaction is a major factor, particularly the frequencies and amplitudes of loading at foot impact, which can exacerbate pathology even if not the primary

A8.8 Gait transitions and interlimb coordination: pattern and process

Ludovic Maes (National Museum of Natural History, France) and Anick Abourachid (National Museum of Natural History, France)

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Society for Experimental Biology Alison P Wills (Royal Veterinary College, UK), Julia P Myatt (Royal Veterinary College, UK), Kyle Roskilly (Royal Veterinary College, UK), Stephen Hailes (University College London, UK) and Alan M Wilson (Royal Veterinary College, UK) Turning is a key requirement for terrestrial locomotion, as it enables animals to escape from predators and catch their prey. It is widely accepted that the majority of animals, including humans, horses and mice, have to slow down when turning at high speed. This is because it is necessary to increase the time each foot is in contact with the ground in order to withstand the increase in limb forces created by turning. It has, however, been shown that racing greyhounds do not slow down around bends. This has been attributed to the unique mechanisms this particular breed of dog uses to produce power and support its weight. Therefore, we aim to discover whether there is a force limit to turning at speed in other breeds of dog. GPS-IMU comprising units were attached to dogs via a standard dog harness. These dogs were then filmed chasing a lure that ran in a continuous loop including a 90° bend on a level grass playing field. Each dog was required to chase the lure in both directions. From the combination of GPS and video data, speed, radius of turn and duty factor were extracted for each trial. We explored whether dogs were able to maintain their speed on bends, and if limb force or grip limit appeared to limit speed in turning dogs. This study will enable further investigation into other limits of turning in quadrupeds, such as the coefficient of friction of the running surface. Email address for correspondence: [email protected] 13:30 Friday 1st July 2011

Gaits correspond to the stable coordination patterns of the four limbs resulting from complex biomechanical and neural interactions. In the wild, animals adapt their locomotion to environmental circumstances by changing their speed and thus changing gait. The steady speed coordination patterns are known but the transitions between gaits remain far less well studied. How do tetrapods adapt their coordination patterns when changing gait? Do gait transition patterns inform on the processes driving the interplay between biomechanics and the neural organization? In order to explore these questions, we studied the gait parameters of dogs during acceleration and deceleration-related gait transitions. We measured the spatio-temporal parameters of each limb and the coordination parameters using the APS method. Our results show that all transitions occur according to the same scheme: in all cases speed modifications are correlated to stance duration modifications. However, the swing duration was not correlated to speed. Changes in coordination were induced by punctual changes in swing duration. Changes in swing duration between anterior and posterior limb pairs allowed changes from one symmetrical gait to another; changes in the diagonal feet swing phases resulted in changes from symmetrical to asymmetrical gaits. As the mechanical exchanges between the animal and the environment occur during the stance phase, it is likely that the change in coordination is not driven by the mechanics of stance. However, the punctual changes in swing duration likely induce the change in coordination, and this change must be provoked by neuronal input. Email address for correspondence: [email protected] 12:15 Friday 1st July 2011

A8.9 Biomechanical determinants of transverse and rotary gallop in mammals

Carlo M Biancardi (Physiomechanics of Locomotion Laboratory, Department of Human Physiology, University of Milan, Italy), Alberto E Minetti (Physiomechanics of Locomotion Laboratory, Department of Human Physiology, University of Milan, Italy) In transverse gallop, the placement of the second hind foot is followed by that of the contralateral forefoot, while in rotary gallop it is followed by the ipsilateral forefoot, and the sequence of footfalls appears to rotate around the body. Three-hundred-and-fifty-one filmed sequences have been analysed to assess the gallop type of 89 investigated mammal species belonging to the Carnivora, Artiodactyla and Perissodactyla orders. Twenty-three biometrical, ecological and physiological parameters have been collected for each species, both from literature data and from experimental measures. Most of the species showed only one kind of gallop: transverse (40%) or rotary (39%). Some species performed rotary gallop only at high speed (17%), while a small number showed both kinds of gallop at any speed (4%). Two main principal components extracted by PCA accounted for size (PC1) and velocity (PC2), with the latter associated with the rotary galloper group. A canonical correlation analysis showed a significant separation of the two groups on the second canonical function (F = 14.2; d.f. = 1; p < 0.001), mainly based on the metacarpus/humerus and metatarsus/femur length ratio. The gait pattern analysis provided significant differences between transverse and rotary gallop in fore and hind duty factor (t-test; p < 0.001), and in duration of the fore contact (t-test; p < 0.001). Our results assessed the typical gallop gait in the investigated species and confirmed the correlation between cursoriality and rotary gallop and identified some morphological characters correlated with the gallop type. Email address for correspondence: [email protected] 12:30 Friday 1st July 2011

A8.11 Walking in children more bird-like than human?

Tatjana Y Hubel (Royal Veterinary College, UK) and James Usherwood (Royal Veterinary College, UK) Observations of walking in small children show that they move quite differently from what we would expect from downsized adults. Using a reductionist inverted pendulum model in order to explain limitations in walking speeds, we have to conclude that faster walking speeds require shorter steps. Walking requires that centrifugal forces due to the vaulting motion are compensated by the gravity component pointing along the leg in order to prevent toe drag or foot take-off situations. The further away the leg is from its vertical position the smaller the corresponding gravity component. With centrifugal force being a function of speed, leg angles have to be reduced with increasing walking speeds in order to be able to compensate for the centrifugal forces, leaving an increase in frequency as the only option to further increase walking speeds. Human adults are able to drive their legs actively with a frequency of about two to three times their passive swing frequency, walking at 4/3rds of the speed achievable with passive swing legs. Birds in contrast are limited to their passive swing leg frequency, walking considerably slower (for their size). We report findings from children (age one to five) walking and running over an array of force plates, and determine their resemblance to passive leg walkers such as birds and high-efficiency robots, and explore early indications that children may power walk with torques, contrasting with adults and current theoretical predictions of telescopepowering Email address for correspondence: [email protected] 13:45 Friday 1st July 2011

A8.12 Elastic energy storage in arboreal locomotion

A8.10 Kinematics of turning locomotion in the domestic dog

Samuel R Coward (University of Birmingham, UK), Robin H Crompton (University of Liverpool, UK), William I Sellers (University of Manchester, UK), Anthony R Ennos (University of Manchester, UK) and Susannah K Thorpe (University of Birmingham, UK)

Abstracts 2011 It is proposed that human bipedalism may have evolved within an arboreal environment similar to that in which orang-utans live. As orang-utans are also the great ape that most resemble us in the mechanics of their bipedal walking, they are an excellent living model through which we can study the formative stages of the evolution of our own bipedalism. In modern human bipedal walking, energy can be conserved within muscle tendon units, such as the triceps surae combined with the Achilles tendon, and released in a catapult like action. As non-human great apes do not have large Achilles tendons, theoretically they cannot substantially benefit from this form of elastic energy return. Orangutans do however use bipedalism on thin compliant supports, reacting to these supports like humans running on springy tracks, by increasing hip and knee extension, whereas other primates do the reverse. The benefit of straight legs for humans running on springy tracks is that energy can be stored and returned from the running surface. Here we investigate whether similar energy return can be obtained by orang-utans during bipedal walking on arboreal supports. Carbon fibre beams of varying compliance were used to replicate branches. The applied forces and displacements of the beams were recorded, allowing energy storage and return to be calculated. Results showed that energy was stored during foot strike and subsequently returned during lift off. This demonstrates a key adaptive benefit of using extended leg bipedalism on arboreal supports, which may have been instrumental in the adoption of this behaviour. Email address for correspondence: [email protected] 14:00 Friday 1st July 2011

123 Jeffery W Rankin (Royal Veterinary College, UK), Dimitrios E Tsaopoulos (University of Thessaly, Greece), Jonas Rubenson (University of Western Australia, Australia) and John R Hutchinson (Royal Veterinary College, UK) Due to their cursorial evolutionary background and bipedal nature, understanding how ostrich (Struthio camelus) morphology is adapted for movements such as walking can help elucidate the general biomechanical principles that govern movement, provide inspiration for biomimetic robots and facilitate the development of novel devices that assist in human movement, as well as infer aspects of avian locomotor evolution. However, little is currently known about the role that different lower limb muscles have in ostriches during walking. There are numerous challenges for empirically obtaining the data necessary to investigate muscle roles (e.g. electromyography, sonomicrometry and tendon buckles). In addition, ostrich limb musculoskeletal structure is complex, consisting of many multiarticular muscles and a large number of joints with three-dimensional movement. To date, these factors have obscured how ostriches successfully meet the biomechanical demands of walking. Recent advances in technology have, however, allowed for the development of musculoskeletal modelling and simulation techniques that complement and enhance established empirical methods by providing muscle-specific information during a movement (e.g. muscle force and musculotendon work), which can provide new insight into muscle roles and mechanical function during movements. This study uses a detailed musculoskeletal model, three-dimensional kinematic and kinetic data from experiments and a static optimization technique to determine individual lower limb muscle roles during ostrich walking. Email address for correspondence: [email protected] 14:30 Friday 1st July 2011

A8.13 A novel method for the quantification of the 3D orientation of the human femoral neck

Noémie Bonneau (National Museum of Natural History, France), Olivier Gagey (AP-HP Hôpital Bicêtre, France) and Christine Tardieu (National Museum of Natural History, France) Although numerous primates use bipedal locomotion, humans are characterized by a permanent bipedal posture and gait resulting in strong mechanical constraints on the femoral necks. An appropriate 3D orientation of the femoral neck is fundamental to ensure an efficient bipedal locomotion. A common problem in quantification of the orientation of the femoral neck is the difficulty in determining its true axis; however, the axis is generally only estimated visually. An accurate determination of the 3D orientation of the femoral neck requires us to reconsider the complex architecture of the proximal femur. The morphology of the neck results from: 1) the medial and arcuate trabecular systems that reflect the biomechanical constraints induced by bipedal locomotion; and 2) the strong asymmetry of the cortical bone. Given these considerations, we suggest modelling the femoral neck using two cylinders. Sixteen paired femora were used to evaluate this method. The surface geometry of the femoral neck was acquired and was subsequently used to fit one or two cylinders. The 3D orientation and standard deviation were extracted based on the mathematical parameters of each model. The model based on two cylinders provided a significantly smaller standard deviation than the one based on a single cylinder (p < 0.001) and reduced the intraobserver measurement error. Based on these results, a mathematic tool has been developed to propose a semi-automatic determination of the 3D orientation of the femoral neck. Both clinical (hip surgery and functional rehabilitation) and comparative evolutionary and biomechanical studies would be improved by this work. Email address for correspondence: [email protected] 14:15 Friday 1st July 2011

A8.15 Hindlimb-driven take-off and landing in small passerine birds (Taeniopygia guttata)

Pauline Provini (National Museum of Natural History, France), Anick Abourachid (National Museum of Natural History, France) and Anthony Herrel (National Museum of Natural History, France) Although take-off and landing are crucial components of avian flight, few studies have focused on these two phases. Zebra finches (Taeniopygia guttata) were filmed during short-distance free-flights between two perches situated at different heights. The lower perch was filmed by multiple synchronized high-speed video cameras and the reaction forces involved during take-off and landing on the perch were measured. The first and second derivatives of the centre of mass trajectory provided velocity and acceleration profiles, respectively. The kinematic analysis has revealed that Zebra finches are using hindlimbs and forelimbs successively in both take-off and landing. During take-off, maximum acceleration occurs while the bird is still on the perch and before the first wing down stroke. During landing, the maximum deceleration happens after the first touch, when the wings are folded. Our force recordings show that propulsive and dampening forces are similar in magnitude and can reach up to six times body weight. Most studies have focused on the importance of the wings in takeoff and landing, yet it has been demonstrated in this work that the hindlimbs are prominent in generating propulsion during take-off and dampen impact forces during landing in small passerine birds. Email address for correspondence: [email protected] 14:45 Friday 1st July 2011

A8.16 Decoding static-locomotion: ground reaction forces in caterpillar crawling and a simple mechanistic model of the environmental skeleton

Huai-Ti Lin (Tufts University, USA) and Barry Trimmer (Tufts University, USA)

A8.14 Biomechanical simulation of the mechanical roles of ostrich pelvic limb muscles during walking

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Society for Experimental Biology Manoeuvring during slow flight is important to the survival of almost all animals that fly, but relatively little is known about how animals achieve this. Alternative strategies include asymmetries in wing velocity observed in pigeons and asymmetries in wing posture and inertia in parrots. To initiate a comparative study of the relative importance of these alternatives, we studied steady hovering and low-speed manoeuvres in calliope hummingbirds (Stellula calliope, n = 4). We measured flight as the birds tracked a laterally-translating (0.5 m/s) and reversing (0.06 m/s/s) feeder. We obtained 3D wing and body kinematics (1000-Hz video) and flow dynamics in the wake (particle image velocimetry, PIV). Our results indicate that the birds initiate and arrest manoeuvres using within-wing beat, bilateral asymmetries in velocity and angle of attack. For example, a roll can be initiated with a velocity asymmetry in midupstroke, and then arrested without predictable asymmetry in the subsequent downstroke. Bilateral asymmetry in circulation shed into the wake was less during manoeuvres (30%) compared with steady hovering (40%). Wake tilt angle was correlated with lateral acceleration (27°, manoeuvre; 2°, hover). Thus, hummingbirds can laterally reorient their stroke-plane without consistent or prolonged asymmetry in wing velocity or angle of attack. Caution is necessary in interpreting our PIV data because of low temporal resolution (5 Hz) and the 2D aspect of our interrogation field. NSF grants IOS-0923606 and IOS-0919799. Email address for correspondence: [email protected] 16:00 Friday 1st July 2011

The caterpillar is a tractable model system for studying force transmission in soft-bodied animal locomotion. It has discrete attachment devices (the prolegs) that are either on or off, thereby facilitating gait analysis and biomechanical modelling. We devised a force beam system to measure the ground reaction forces in both the normal and fore-aft directions from a crawling caterpillar (M. sexta). Repeated measurements from each pair of prolegs revealed considerable step-by-step variation in the force profile but underlying patterns could be detected by comparing different segments. In the normal direction, all prolegs exhibited a simple sigmoid loading profile, with the most posterior and anterior prolegs bearing slightly more body weight during a crawl. Changes in the load-bearing capacity of different prolegs fitted a simple weight-shifting model. Forces in the fore-aft direction show a stereotypic tri-phasic pattern that shifts progressively with the most anterior prolegs, exerting the most thrust and the posterior prolegs producing net drag. The anteriograde wave of muscle contractions produces a forward propagating wave of intersegmental tension. Combining these ground reaction forces with the caterpillar crawling gait pattern suggests that caterpillar locomotion does not have any transverse mechanics. The forces in the direction of travel are used simply to stretch out the body segments and do not require active pressure changes to re-extend their bodies. This mechanism makes use of the substrate as a relatively stiff mechanical strut so we call this strategy an `environmental skeleton'. Email address for correspondence: [email protected] 15:30 Friday 1st July 2011

A8.17 Aerodynamics of tip-reversal upstroke in a revolving pigeon wing

Kristen E Crandell (University of Montana, USA) and Bret W Tobalske (University of Montana, USA) Bird species are classified into two distinct kinematic wing patterns during slow flight: the `flexed-wing' or distally supinated `tip-reversal' upstroke. Two hypotheses have been debated as to the functional significance of the tip-reversal upstroke. The first is that this behaviour is aerodynamically inactive and serves to minimize drag. The second is that the wing produces significant aerodynamic forces during the upstroke. Here, we combine 3D kinematics of a pigeon during slow flight with a well-established propeller model to explore the aerodynamic capabilities of the tip-reversal upstroke. Rock dove wings were spread and dried in postures characteristic of mid-upstroke or mid-downstroke and spun at in vivo Reynolds numbers to simulate forces experienced during slow flight. We compared 3D wing shape for the propeller and in vivo kinematics. We found that the wing in upstroke posture is capable of producing substantial aerodynamic forces. At in vivo angles of attack (66° at midupstroke, 46° at mid-downstroke), the upstroke wings averaged for three birds produced a lift-to-drag ratio of 0.91, and the downstroke wings produced a lift-to-drag ratio of 3.33. Peak lift-to-drag was 2.5 for upstroke and 6.3 for downstroke. Our estimates of total force production during each half-stroke suggest that downstroke produces a force that supports 115% of bodyweight, and during upstroke a forward-directed force (thrust) is produced at 36% of bodyweight. Supported by NSF grants IOS-0923606 and IOS-0919799. Email address for correspondence: [email protected] 15:45 Friday 1st July 2011

A8.19 Through the eyes of a bird: rapid flight path planning in a cluttered environment

Huai-Ti Lin (Harvard University, USA), Ivo Ros (Harvard University, USA) and Andrew Biewener (Harvard University, USA) Path planning at high speed imposes great neurological, biomechanical and physiological challenges to flying animals. Visual and vestibular systems must quickly integrate information about the bird's movement. Subsequent wing-body coordination has to generate accurate manoeuvres and support the power demands of flight. In this study, we challenged pigeons (C. livea) to fly through an artificial forest of 15 vertical poles (3.8 cm in diameter) spaced randomly over ~3x3 m area. The 3D kinematics data show that the birds typically altered their bearing <60° to avoid obstacles. Measurements of head saccades suggest that binocular vision is important for close-range collision avoidance. The pigeons' 300° panoramic view (lateral monocular plus frontal binocular) during flight trials was also assessed for given pole distributions, by combining body trajectory (bearing) and head orientation data. The resulting pattern of horizontal pole movements provides optical flow properties correlated to the future flight manoeuvres. Flights through a cluttered environment were significantly slower than unobstructed flights, with more than double the total estimated mechanical energy expenditure. While aerodynamic energy remained around 55% of this budget, the energy associated with breaking and reaccelerating increased from ~12% to almost 40%. Over repeated flight trials through the same obstacle distribution, pigeons tended to favour a learned fixed path. These preferred paths were often the straightest routes given the entry point. Interestingly, head orientation followed similar patterns for repeated paths within a given distribution. This result implies that head saccades are induced stereotypically by looming stimuli and/or head rotation is necessary to steer body bearing. Email address for correspondence: [email protected] 16:15 Friday 1st July 2011

A8.18 Kinematics and aerodynamics of low-speed manoeuvres in flying hummingbirds

Bret W Tobalske (University of Montana, USA), Douglas R Warrick (Oregon State University, USA), Ivo G Ros (Harvard University, USA)

A8.20 Desert locust aerodynamics: instantaneous wake volumes captured using tomographic particle image velocimetry

Abstracts 2011 Richard J Bomphrey (University of Oxford, UK), Per Henningsson (University of Oxford, UK), Dirk Michaelis (LaVision GmbH, UK) and David Hollis (LaVision UK Ltd, UK) Tomographic particle image velocimetry (Tomo-PIV) is becoming increasingly established in the experimental fluid mechanics community as a volumetric method for the analysis of complex flows, but is yet to be applied to animal flight despite the major advances in the field made possible by its planar predecessors. The technique captures a volume of instantaneous flow velocity vectors with equal resolution along each axis rather than the more limited experimental sampling plane of stereo-PIV. From the three-dimensional flow fields, a portrait of the flow features can be visualized and quantified. We recorded the wake of desert locusts flying tethered in a wind tunnel using broad sheet laser illumination operating at 1 kHz (pulse pairs) and a four-camera data acquisition system. This resulted in overlapping, near-wake volumes forming a time-resolved representation of the vortex wake. Here we present novel Tomo-PIV data revealing, for the first time, wake components of a flying animal that have been hitherto undetectable using existing techniques. The wake is complex, due primarily to the existence of out-of plane-vortex elements of variable strength. Email address for correspondence: [email protected] 16:30 Friday 1st July 2011

125 been unravelled, allowing us to explain the significant aerodynamic forces generated during flight as well as the underlying fluid dynamics. If major elements such as wings kinematics or leading edge vortex production are now partially understood for hovering flight as well as for forward flight, however, the aerodynamic mechanisms involved during the take-off phase remained unstudied. The aim of this study is therefore to understand the vortex production operating during an insect's take-off and how it could interact with the ground to take advantage of its proximity. To this end, fluid motions created by a butterfly (Pieris rapae) during its take-off flight were visualized by particle image velocimetry. Flow measurements were analysed during the first downstroke of the insect at four different spanwise positions: on the body, the wing root, mid-wing and wing tip. Compiling the dynamics of these data will allow us to propose hypotheses about the global mechanisms of vortices production and evolution and the role of the ground effect during take-off phase in this butterfly. This study should thus unveil aerodynamic mechanisms providing sufficient lift and power to perform the ground-to-air transition in this insect. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011

A8.23 Neuromuscular and biomechanical compensation for wing asymmetries in hovering flight

Maria Jose Fernandez (University of North Carolina, Chapel Hill, USA), Dwight Springthorpe (University of North Carolina, Chapel Hill, USA) and Tyson Hedrick (University of North Carolina, Chapel Hill, USA) In nature, wing damage is common in insects, resulting in wing asymmetries. Despite damaged wings, insects are able to maintain flight stability and perform complex manoeuvres. This response might be due to non-neural mechanisms, such as feedback between muscle and aerodynamic force or by neural modulation of muscle activity. Here we study the mechanisms by which insects compensate for damaged wings by simultaneously recording wing kinematics and neuromuscular activity in freely flying hawkmoths (Manduca sexta) with symmetric and asymmetric, artificially reduced wings. We recorded neuromuscular activity from four muscles: the left and right dorsal longitudinal muscles (DLM) and dorsal ventral muscles (DVM). These power the downstroke and upstroke, respectively. We examined differences in neural activation in clipped versus unclipped wings and their correlation to wingbeat kinematics. Our results show that hawkmoths altered wing kinematics and neuromuscular activation in response to wing damage. Overall wingbeat frequency increased, as did stroke amplitude on the clipped wing compared to the unclipped wing. While these findings partially match a non-neural feedback hypothesis, they were also associated with changes in neural activation found on the upstroke muscle (DVM), but not on the downstroke muscle (DLM). The modulation pattern was similar to that recorded previously from hawkmoths making a free-flight yaw turn; we suggest that hawkmoths continuously steer in the direction of the clipped wing to maintain hovering flight stability in the face of the wing asymmetry perturbation and that the flight power muscles are modulated to provide the response. Email address for correspondence: [email protected] unc.edu 10:45 Saturday 2nd July 2011

A8.21 Dynamic flight stability and stabilization control of a hovering insect

Mao Sun (Institute of Fluid Mechanics, Beijing University of Aeronautics and Astronautics, China) We made high-speed film of a hovering drone fly. Based on the observation, the equations of motion of the insect were derived and then simplified to that of a flying body using the assumption that the wing-beat frequency was much higher than that of the natural oscillatory modes of the body motion. A linear theory was developed and it showed that the flight was dynamically unstable, but the instability might not be a great problem to the insect because the time for the initial disturbances to double was more than eight times the wing-beat period and the insect had plenty of time to adjust its wing motion before the disturbances grew large. Approximate analytical expressions of the eigenvalues, which gave physical insight into the genesis of the instability, were derived. The expressions identified the moment produced by the forward/backward speed and the forward/backward tilting of the vertical force as the primary source of the longitudinal instability, and the moment produced by the side-speed and the side tilting of the vertical force as the primary sources of the lateral instability. Finally, the flight was numerically simulated by coupling the full equations of motion with the NavierStokes equations. The validity of the linear theory was tested using the solution of the full equations. Email address for correspondence: [email protected] 16:45 Friday 1st July 2011

A8.22 Mechanical principles of ground-to-air transition in a butterfly

Gaëlle Bimbard (Institut de Recherche sur la Biologie de l'Insecte, France), Thomas Steinmann (Institut de Recherche sur la Biologie de l'Insecte, France), Ramiro Godoy-Diana (Physique et Mécanique des Milieux Hétérogènes, ESPCI, Fance), Benjamin Thiria (Physique et Mécanique des Milieux Hétérogènes, ESPCI, France), Jérôme Casas (Institut de Recherche sur la Biologie de l'Insecte, France) Insects impress by their agility and their performances in flight. During the past few years, some of the characteristic kinematic patterns have

A8.24 Sensorimotor frequency response in the hawkmoth Hyles lineata

Shane P Windsor (University of Oxford, UK), Richard J Bomphrey (University of Oxford, UK) and Graham K Taylor (University of Oxford, UK)

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Society for Experimental Biology Measuring the flow around insects in flapping flight presents an intriguing challenge because insects are typically small, have high wingbeat frequencies, and the aerodynamics can be highly time variant and complex. Nevertheless, the flows they produce can be directly measured, and the efficiency of the process can be assessed using well-established aerodynamic theory. We used high-speed particle image velocimetry to calculate two efficiency factors by measuring the downwash distribution behind desert locusts (Schistocerca gregaria) flying tethered in a wind tunnel. Moreover, how the efficiency with which lift is generated varies throughout the course of the wingbeat cycle is described. The wings of the locusts were found to operate at a maximum span efficiency of 79% during downstroke (typically plateauing at about 60% for a large portion of the downstroke), but at low values during the upstroke, when lift forces are small. Furthermore, the highest span efficiencies plateau when large lift forces are being generated, suggesting that the combination of kinematics and morphology in locust flight is tuned to perform most efficiently when the wings are doing the most work. Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

The functional properties of an insect's flight control system can be explored experimentally by measuring the relationship between the stimuli that the insect experiences and the flight forces it produces in response. We measured the forces and moments produced by tethered white-lined sphinx hawkmoths (Hyles lineata) in response to oscillatory wide-field visual stimuli presented in a custom-built virtual reality flight simulator. We used constant frequency sinusoids and variable frequency sine sweeps to determine the frequency response of the moths in response to roll, pitch and yaw stimuli. The phasing of the measured frequency responses indicates that the flight motor responds to the position of the visual environment in roll. In contrast, the flight motor responds to both position and velocity in respect of pitch and yaw stimuli. These results may be explained by considering the head of the moth as a position tracking or gaze stabilization platform. The eyes detect the position and velocity of the visual world in the head's frame of reference, and the different responses to motion in the different axes might therefore be explained by differences in the range of motion of the head about each axis. Email address for correspondence: [email protected] 11:00 Saturday 2nd July 2011

A8.27 A8.25 The use of the alula in hoverflies

Simon M Walker (Oxford University, UK), Adrian Thomas (Oxford University, UK) and Graham K Taylor (Oxford University, UK) The alula is a large hinged flap near the base of the wings of most brachyceran flies. It is often flipped at right angles to the rest of the wing during flight. We used high-speed digital video of free-flying hoverflies (Eristalis tenax and Eristalis pertinax) to investigate how flipping of the alula was associated with changes in wing and body kinematics. We found that deployment of the alulae was significantly associated with body accelerations, consistent with reduction of the aerodynamic forces when the alula is flipped on one or both wings. Flipping of the alula was significantly related to several wing kinematic parameters, including stroke amplitude, stroke plane, mid-stroke stroke angle, the downstroke angle of incidence, the downstroke deviation angle, and the timing of supination at the end of the downstroke. Changes in state of the alulae were also significantly linked to changes in both the body and wing kinematics within the same wingbeat, indicating a mechanistic link with the alula. The stroke amplitude was lower and the timing of supination earlier when the alula was flipped, which are both central to the gear change mechanism in higher Diptera. The gear change mechanism comes into effect at mid-downstroke, which is also the time at which changes in the state of the alula first become apparent. Furthermore, the gearchange mechanism is linked to movements of the third axillary sclerite, which articulates directly with the alula. We therefore hypothesise that flipping of the alula is related to gear changes in flies. Email address for correspondence: [email protected] 11:15 Saturday 2nd July 2011

Analysis of guidance success rate in the migration of a flying swarm

Yoshinobu Inada (Department of Aeronautics and Astronautics, School of Engineering, Tokai University) and Yuta Morimoto (Department of Aeronautics and Astronautics School of Engineering Tokai University, Japan) When a swarm of organisms, regardless of whether flyers or swimmers, moves to its destination, a number of the individuals in a swarm are aware of the destination and take the leadership to guide the rest of the individuals. In this situation, the success of guidance, which means all individuals in a swarm can reach the destination successfully without dropping out, can be influenced by several factors, e.g. the percentage of leaders, the quality of mutual interaction among individuals, or the manoeuvring intensity, which means the degree of moving direction change in each turn of a swarm. We analysed the success rate of guidance in a flying swarm by using a computer simulation and clarified that the effect of leader-percentage on the success rate of guidance was non-linear, showing that the success rate tended to change quickly from a small value to 100% while the leader-percentage changes within a certain narrow range and tended to change gradually in other ranges. The success rate was small when the mutual interaction among individuals was slanted to either an approach or a parallel orientation, which is the tendency of individual to approach to its neighbours or to orient its body in parallel with the body direction of neighbours, respectively. The coupling of both tendencies yielded high success rate. The manoeuvring intensity also showed a non-linear effect on the success rate that suddenly decreased when the degree of moving direction change exceeded a certain threshold value, thus representing the complexity in the guidance of a flying swarm. Email address for correspondence: [email protected] 11:45 Saturday 2nd July 2011

A8.26 Time-varying span efficiency through the wingbeat of the desert locust

Per Henningsson (University of Oxford, UK) and Richard J Bomphrey (University of Oxford, UK) The efficiency with which an animal generates lift has wide ranging implications from the maximum range of flights between feeding, to the maximum load lifting capacity, to the length of time it is capable of staying aloft, the peak accelerations during manoeuvres, and other factors that are dependent on energy budget and flight performance.

A8.28 Why are climbing plants so invasive? Biomechanics, biomass and competition of invasive climbing plants growing on Sorghum under elevated CO2

Cloé Paul-Victor (AMAP University of Montpellier), Nick Rowe (AMAP University of Montpellier)

Abstracts 2011 Climbing plants are important though understudied elements of natural ecosystems. They have recently been shown to: (1) significantly increase in growth under CO2 enrichment; (2) represent serious invasive elements in both natural and cultivated areas; and (3) show potential changes in growth that might be changing vegetation dynamics in the tropics. These issues beg for detailed studies on the effects of CO2 enrichment on invasive climbing plants especially in terms of food production. The main aims of this study were to analyse the above ground effects of elevated CO2 on the development, biomass and mechanical properties of two selected invasive climbing species (Ipomoea triloba, Momordica charantia) and the agricultural C4 host plant (Sorghum bicolor) grown together but rooted in separate pots. We also investigated how elevated CO2 modified the effect of the climbers on the crop plant. Sorghum plants were grown with and without climbers under ambient (413 ppm) and elevated CO2 (755 ppm) in glasshouse conditions. Both climbing species showed changes in mechanical, morphological and photosynthetic traits under elevated CO2 resulting in enhanced growth (length + branching). The competition experiment demonstrated that under these glasshouse conditions sorghum plants are more negatively affected under elevated than ambient CO2 resulting in a weakened mechanical architecture, a decrease in panicle biomass and stem carbohydrate production. The results of this study suggest that under these experimental conditions, elevated CO2 can significantly influence the effect of climbing invasive weeds on an important stable crop via above ground interaction and climbing behaviour. Email address for correspondence: [email protected] 12:00 Saturday 2nd July 2011

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A8.30

Suckers and snappers ­ functional trap morphology and the biomechanics of fast underwater prey capture in aquatic carnivorous plants Simon Poppinga (Plant Biomechanics Group, Freiburg, Germany), Tom Masselter (Plant Biomechanics Group, Freiburg, Germany), Carmen Weißkopf (Plant Biomechanics Group, Freiburg, Germany), Olivier Vincent (Laboratoire Interdisciplinaire de Physique, Grenoble, France), Philippe Marmottant (Laboratoire Interdisciplinaire de Physique, Grenoble, France), Marc Joyeux (Laboratoire Interdisciplinaire de Physique, Grenoble, Framce), Catherine Quilliet (Laboratoire Interdisciplinaire de Physique, Grenoble, France), Simon Schleicher (Institute of Building Structures and Structural Design, Stuttgart, Germany), Julian Lienhard (Institute of Building Structures and Structural Design, Stuttgart, Germany), Jan Knippers (Institute of Building Structures and Structural Design, Stuttgart, Germany), Lubomir Adamec (Institute of Botany of the Academy of Sciences) and Thomas Speck (Plant Biomechanics Group, Freiburg, Germany) The biomechanics and functional morphology of traps of two aquatic carnivorous plant genera were investigated. The Utricularia (Lentibulariaceae) suction trap deflates by actively pumping out water, hence generating negative pressure and elastic deformation of the bladder walls. When firing, a trapdoor opens and closes very fast, and relaxation of the trap walls leads to suction of water. The door deformation comprises a morphologically predetermined buckling/ unbuckling process including a complete trapdoor curvature inversion. Suction can be triggered by prey touching the trigger hairs that protrude from the trapdoor, or occurs spontaneously when a critical negative pressure value is reached in the trap. Prey-triggered firings characterize the bladderwort's carnivorous habits, and prey animals are sucked into the trap in less than a millisecond with up to 600 times the gravitational acceleration. In contrast, spontaneous suctions are hypothesized to continuously accumulate biomass and provide additional nourishment for rootless Utricularia. We measured the development of negative pressure necessary for prey capture and spontaneous suctions. Aldrovanda vesiculosa (Droseraceae) catches its prey with snap-traps. In contrast to the snap-buckling instability mechanism in its closest relative, the terrestrial Venus Flytrap (Dionaea muscipula), we found that the closing movement in Aldrovanda is owing to deformation of the trap midrib and not of the two trap lobes. We present a mechanical model which explains how the midrib is kinematically coupled with the lobes, and show that this mechanism is more advantageous for underwater prey snap-trapping in comparison to the large-scale elastic deformation of the lobes in Dionaea muscipula. Email address for correspondence: [email protected] 12:30 Saturday 2nd July 2011

A8.29 Shrubs, climbers and stem brittleness ­ evolution of the mechanical architecture in cassava during domestication

Nick Rowe (AMAP University of Montpellier, France), Doyle McKey (CEFE University of Montpellier, France), Bruno Clair (LMGC University of Montpellier, France), Gilda Mühlen (University of Rondônia), Léa Ménard (AMAP University of Montpellier, France) Cassava (Manihot esculenta) is a staple crop of worldwide importance, grown in many parts of the tropics. Cultivation involves selection and planting of stem cuttings, growth as shrubs and harvest of the starchrich tuberous roots, which are processed for food. Recent phylogenies suggest that cassava was domesticated in southern Brazil and is derived from a geographically variable wild species currently referred to as Manihot esculenta subsp. flabellifolia in Brazil and as Manihot tristis in French Guiana. We compared growth forms and stem biomechanics of ancestral species with domesticated cassava from two regions of the Amazon basin in Brazil and French Guiana. Wild species show a high plasticity in growth form, developing robust shrubs in savannah and climbing plants in forest understory. We found that domesticated cassava will also grow as a climber if plantations are abandoned and overgrown. Field observations and flexure tests indicated that stems of domesticated cassava (both shrubs and climbers) are more brittle than those of the ancestral species. The results suggest that selection during domestication did not modify the overall ontogenetic trajectory or developmental plasticity ­ to grow as either shrub or climber ­ but drastically changed the material properties of the wood and hence survival as a climber. The results also provide evidence of a phenomenon that we have taken for granted though never adequately tested in the field: that certain types of climbing plant must develop stem properties that are flexible and resistant to brittle fracture. Email address for correspondence: [email protected] 12:15 Saturday 2nd July 2011

A8.31 Burrowing performance of Calyptommatus lizards from Brazilian Caatingas: sand granulometry plays a major role for differences among sister-species

Tiana Kohlsdorf (University of Sao Paulo, Brazil), Mariana Grizante (University of Sao Paulo, Brail), Vanessa Cunha (University of Sao Paulo, Brazil), Felipe Zampieri (University of Sao Paulo, Brazil) and Fabio Barros (University of Sao Paulo, Brazil) The locomotor performance exhibited by a given organism is often related to morphological features, and might be specific to the environment where the lineage evolved. However, some environmental conditions may enhance performance of a given activity in the organisms that evolved in such microhabitat as well as in those found in habitats that are slightly different. In the present study, we analyse burrowing performance of two fossorial sister-species of Calyptommatus lizards (Gymnophthalmidae)

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Society for Experimental Biology Understanding how temperature and habitat fragmentation affect the current and future presence of organisms is paramount given the ongoing anthropogenic disturbance of natural ecosystems. Understanding traits related to mobility is crucial in this respect given the importance of organismal mobility in allowing gene flow and migration to suitable habitats. Temperature is known to affect locomotor performance through its effects on physiology. However, effects of temperature may differentially affect different performance traits. For example, while muscle force generation is known to be temperature insensitive, ratedependent processes are highly influenced by temperature variation. Similarly, temperature may differentially affect cardiovascular and muscle contractile performance and as such drive speed-endurance trade-offs during locomotion. Here we explore the effect of temperature on locomotor performance traits (endurance and burst performance) at both the whole-organism as well as muscular levels. Our results show strong temperature dependence of both performance traits at the muscular level with higher temperatures leading to improved performance over a range from 15 to 32 degrees. Yet, these results are not reflected at the whole-organism performance level. Anatomical and physiological data suggest that in vivo performance is dependent on a whole suite of traits not limited to muscle contractile capacity. An integrative approach is thus essential in understanding effects of temperature changes on organismal performance and mobility. The role of trade-offs appears crucial in this respect and needs to be incorporated in models trying to predict effects of global change on the continued persistence of organisms. Email address for correspondence: [email protected] 14:00 Saturday 2nd July 2011

from microhabitats that differ in sand granulometry: particle distribution is much more heterogeneous in the sand from Cacimbas (locality of C. sinebrachiatus), in comparison to Ibiraba (locality of C. leiolepis). In this analysis, we first tested for differences between the two species in burrowing performance exhibited in their original microhabitat, and then tested the effects on locomotor performance of an experimental design where animals were stimulated to move on `swapped sands' (i.e. C. leiolepis, typical from Ibiraba, running on sand from Cacimbas, and C. sinebrachiatus, typical from Cacimbas, running on sand from Ibiraba). The two species differed in burrowing performance in their typical microhabitat, and burrowing speeds were also significantly affected in C. sinebrachiatus by the experimental setup of swapping sands: this species burrowed faster at the swapped sand, while the burrowing time of C. leiolepis was similar in both sands. Also, overall animals from both species burrowed faster in the homogeneous sand from Ibiraba. Physiological and morphological bases for possible biomechanical explanations involve variation in head morphology and muscle histochemistry. Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

A8.32 Estimation of the morphometric characteristics of two ecomorphs of anolis lizards

Pierre Legreneur (National Museum of Natural History, France), Vincent Bels ((National Museum of Natural History, France) and Leila Zghikh (University of Mons, France) Among some 400 anoles species, trunk-crown (Anolis carolinensis) and trunk-ground ecomorphs (Anolis sagrei) present variations in frequencies of walking and jumping in their locomotion behaviour to catch prey and to escape predators. For evaluating the relationship between phenotypic traits and mode of locomotion, only an inverse dynamics procedure can help characterize the dynamics of the lizard under the components of ground reaction data and kinematics (displacement, velocity, acceleration and angular) of the joints during the individual's displacement. This requires knowing their morphometric characteristics (lengths and weights of the segments, positions of the centers of gravity, moments of inertia) in advance. For this purpose, we propose to adapt the double pendulum procedure developed by Wells and DeMenthon (1987) to very low mass segments. Mass of the body segments were calculated as a function of whole body weight. All lengths were measured as percentage of the lizard SVL. Relative mass and length of hind limb segments were significantly greater in A. sagrei whereas the head-trunk-tail segment was heavier and the head was longer in A. carolinensis. For all other morphometric data, no significant difference was recorded. These results demonstrated that A. sagrei are able to develop more muscular power with their hind limb to move their body mass. Therefore, A. sagrei exhibits morphological properties more suited to escape performances such as running or jumping, unlike A. carolinensis that tends to adopt crypsis behaviour when adult.J. P. Wells, D. F. DeMenthon (1987) American Journal of Primatology 12, 299. Email address for correspondence: [email protected] 13:45 Saturday 2nd July 2011

A8.34 Coupling vibration of tympanic membranes into waves in the katydid ear (Orthoptera: Tettigoniidae)

Fernando Montealegre-Z (University of Bristol), Daniel Robert (University of Bristol) and Kate A Robson Brown (University of Bristol) Male katydids call to attract distant females for mating. In these insects, sounds are detected using an unconventional system of four eardrums, with one pair of tympana on each foreleg, which can vibrate in response to external and internal sound pressure. An air-filled tube, the acoustic trachea (AT), conveys sound from an opening (a spiracle) on the side of the thorax to the back of the eardrums. The AT extends forwards from the thorax through the fore femur, as a gradually narrowing pipe, until it reaches the knee joint where it enters the tibia and divides in two branches, each one backing a tympanic membrane. Dorsal to the AT division and adjacent to the mechanoreceptors, lies a fluid vesicle. Interestingly, the mechanoreceptors are not directly attached to the tympana, but lie on the external surface of the anterior tracheal division. Mechanoreception proper does not therefore occur near the tympanic membrane. In effect, it is unknown how the mechanoreceptive neurons are activated by incoming sound pressure. Using micro-scan laser vibrometry, micro-CT, and 3D computer generated models, we show that sound reaches the external and internal surfaces of the tympanum at different times. Our experiments suggest that tympanal vibrations and AT sound pressure are collectively transduced within the fluid vesicle first and subsequently passed to the mechanoreceptors. This vesicle seems to work as an impedance matching device, efficiently coupling the in-phase vibrations of both tympanic membranes to the mechanoreceptors; a role analogous to that of the mammalian ear ossicles. Email address for correspondence: [email protected] 14:15 Saturday 2nd July 2011

A8.33 Temperature, performance and physiology: does temperature drive performance trade-offs at different organizational levels?

Anthony Herrel (CNRS, National Museum of Natural History, France), Rob James (Coventry University, UK), Jason Tallis (Coventry University, UK) and Camille Bonneaud (CNRS, France)

A8.35 Biomechanical properties of the tympanic membrane

Abstracts 2011 Jef Aernouts (University of Antwerp, Belgium) and Joris Dirckx (University of Antwerp, Belgium) The mammalian ear is a complex and fascinating organ. The ear collects sound and turns it into electrical pulses sent to our brains. Our brains then use this information to build up a sound map of the world around us. The mammalian ear can be divided into three parts: the outer, middle and inner ear. The sound signal that reaches the ear in the form of air vibrations needs to be transmitted to the cochlea in the inner ear, which is a fluid-filled system. The impedance of the cochlear fluids is much greater than the impedance of air. As a result, most of the sound energy would be reflected back if airborne sound were to impinge directly on the cochlear fluids. The middle ear system overcomes this impedance mismatch by acting as a mechanical transformer that boosts the original signal so that energy can be efficiently transmitted to the cochlea. The tympanic membrane ­ or eardrum ­ initiates the mechanical transmission by converting the acoustic wave into vibrations of the middle ear ossicular bones. In the presentation, some interesting biomechanical features of the tympanic membrane are discussed: a) its conical-like shape and how this improves sound transmission; b) the elastic and viscoelastic properties of the membrane and the importance of these in modelling middle ear mechanics; and c) its interesting capability to handle both acoustic (low amplitude, high frequencies) and quasi-static (high amplitudes, very low frequencies) loadings. Email address for correspondence: [email protected] 14:30 Saturday 2nd July 2011

129 The ergogenic effects of caffeine in human exercise have been shown to improve endurance and anaerobic (activity 60 -to180 seconds) exercise performance. Some human studies have indicated that caffeine in blood plasma (70 µM maximum) may directly act at adenosine receptors on skeletal muscle causing enhanced force production. James et al (2005) subjected isolated mouse EDL (relatively fast muscle) to high frequency activation and demonstrated that 70 µM caffeine directly increased muscle power output (PO) by 3%. Our study used the work loop technique to investigate: 1) the direct effect of 70 µM caffeine on PO in maximally activated mouse EDL and soleus (relatively slow) muscle; 2) The effects of altered caffeine concentrations on PO of maximally activated mouse EDL and soleus muscle. 140, 70, and 50 µM caffeine treatments resulted in significant improvements in acute PO for both maximally activated EDL (3%) and soleus (6%) (P 0.420 in all cases). The ergogenic benefit was significantly greater in soleus compared to EDL (P 0.72 in both cases). Physiological concentrations of caffeine can directly improve maximal EDL and soleus muscle PO with greater benefit occurring in the relatively slower muscle. Reference: James, R.S., Kohlsdorf, T., Cox, V.M., and Navas, C.A. (2005) 70µM caffeine treatment enhances in vitro force and power output during cyclic activities in mouse extensor digitorum longus muscle. European Journal of Applied Physiology 95, 74-82. Email address for correspondence: [email protected] 15:30 Saturday 2nd July 2011

A8.36 High-resolution 3D ossicle displacements measured with point source X-ray stereoscopy

Wasil Salih (University of Antwerp, Belgium), Joris Soons (University of Antwerp, Belgium), Jan Buytaert (University of Antwerp, Belgium) and Joris Dirckx (University of Antwerp, Belgium) A combination of single micro point-source X-ray and stereoscopy is presented to measure high-resolution 3D coordinates and translations of small objects. The theory of the pinhole method is re-derived for a point-source X-ray projection setup using a conical beam. Stereo projections are obtained by rotating the object over 90º between subsequent recordings instead of using two sources in most of previous approaches. We used microscopic tungsten beads as marker points due to absorbance issue. The accuracy of the method is tested on a spherical calibration object, and found to be better than 10 m. Using a translation stage, the measurement uncertainty for translation measurements was found to be better than 5 m. The method is then used on gerbil middle ear to measure the displacement of the malleus when a pressure is applied to the tympanic membrane (eardrum). The amplitudes of malleus' motion at several frequencies are measured from pairs of images. The motion of the middle ear ossicles is an important input for testing of finite element models of middle ear biomechanics. The new method has the advantage over existing methods, such as laser vibrometry in that the structures under study do not need to be visually exposed. Due to the short measurement time and the high resolution, the method can be useful in several fields of biomechanics. Email address for correspondence: [email protected] 14:45 Saturday 2nd July 2011

A8.38 Is thermal sensitivity of skeletal muscle performance lower in ectotherms than endotherms?

Rob S James (Coventry University, UK), Jason Tallis (Coventry University,UK), Mike Angilletta (Arizona State University, USA), Anthony Herrell (C.N.R.S, National Museum of Natural History, France) and Camille Bonneaud (C.N.R.S., France) Unlike ectotherms, which experience a potentially wide variation in body temperature, endotherms usually regulate their core body temperature tightly to enhance physiological performance. Theory predicts that performance of an endotherm should proceed best at its modal body temperature and should be far more sensitive to temperature change than the performance of an ectotherm (Angilletta et al., 2010). The power output of any skeletal muscle increases with increasing temperature, mainly due to greater maximal shortening velocity and higher rates of activation and relaxation. Whilst many studies have examined the thermal dependence of skeletal muscle performance in ectotherms, none have yet quantified the thermal dependence of endothermic muscle using work loops. Here, we tested the hypothesis that thermal sensitivity of rate dependent mechanical processes in skeletal muscle would be higher in an endothermic species than in an ectothermic one. We isolated soleus and diaphragm muscles from eight mice (Mus musculus) and iliotibialis muscle from eight frogs (Xenopus tropicalis). Each muscle was subjected to isometric (constant length) studies to maximise twitch and tetanus performance before determining maximal power output via the work loop technique. Muscle performance was determined over a range of 15 to 40°C. Skeletal muscles of mice showed higher thermal sensitivity in tetanus activation and relaxation rates, but similar thermal sensitivity in power output to frog muscle. Sustained muscle performance improved with increased temperature in frogs, but tended to decrease at higher temperatures in mice. Reference: Angilletta Jr. MJ, Cooper BS, Schuler MS, Boyles JG (2010) Frontiers in Bioscience E2, 861-881. Email address for correspondence: [email protected] 15:45 Saturday 2nd July 2011

A8.37 Is there a dose dependant effect of physiological concentrations of caffeine on the power output of maximally stimulated mouse EDL (fast) and soleus (slow) muscle?

Jason Tallis (Coventry University, UK), Rob S James (Coventry University, UK), Val M Cox (Coventry University, UK) and Mike Duncan (Coventry University, UK)

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Society for Experimental Biology Email address for correspondence: [email protected] 16:15 Saturday 2nd July 2011

A8.39 Ontogeny of contractile behaviour in the flight muscles of birds

Brandon E Jackson (University of Montana, USA), Bret W Tobalske (University of Montana, USA) and Kenneth P Dial (University of Montana, USA) Flight is the defining characteristic of birds yet the mechanisms through which flight ability develops are virtually unknown. Recent efforts focused on pre-flight flapping behaviours (wing-assisted incline running and controlled flapping descent) in chukar (Alectoris chukar) have described the ontogenetic progression of kinematic and aerodynamic characteristics that permit very young (20 days) birds to achieve adult-like aerial locomotor performance. In this study we use these behaviours, which are common to all observed developing birds, as a stage to investigate the developmental trajectory of neuromuscular control and function in the pectoralis muscles of precocial chukar and semi-altricial pigeon (Columba livia). Using indwelling electromyography (EMG), sonomicrometry to measure muscle length, and surgically implanted strain gauges to measure muscle force (in the pigeon), we offer the first comparative data on the ontogeny of flight muscle function. Between 3 and 10 to 15 days after hatching, chukar chicks progress from using sporadic and variable timing of muscle activation to stereotypic and higher amplitude activation pulses. With increasing age, the muscle undergoes increasing strain at higher strain rates, and length trajectory becomes more asymmetrical and saw-toothed. At 20 to 25 days (12 to 15% adult mass), chukar pectoralis activity and locomotor performance approaches that of adults. Pigeon chicks demonstrate similar trends but at much older ages (5 to 8 weeks after hatching) and larger relative sizes (50 to 70% adult mass). These data demonstrate that development of muscle contractile behaviour coincides with aerodynamic force production by the wing and wholebody locomotor performance. NSF grants IOS-0923606 and IOS-0919799. Email address for correspondence: [email protected] 16:00 Saturday 2nd July 2011

A8.41 Using a muscle-robot interface to explore musculoskeletal dynamics during swimming

Christopher T Richards (Harvard University, uSA), and Christofer Clemente (Harvard University, USA) Engineers have used bio-robotic tools to illuminate how morphology and kinematics of propulsors contribute to rapid or efficient swimming. Separately, physiologists have used in vivo muscle approaches to characterize muscle dynamics. To further enrich our understanding of the neuromuscular control of limb motion, we introduce tools that integrate bio-robotics with in vitro muscle physiology. We used two FPGA-controlled servo motors to rotate and translate a robotic foot to mimic the kicking and forward swimming of Xenopus laevis frogs. Foot rotation was controlled either with force signals generated by: 1) a Hill-type muscle model in real-time control software; or 2) a living X. laevis plantaris muscle in vitro. To demonstrate the technique, we tested the robot's swimming performance while measuring fluid and muscle force in response to changes in foot morphology (keeping foot area constant). For the muscle model, as we increased foot aspect ratio (AR=0.5, 1, 2 and 4) muscle force increased from 0.95 to 1.1 to 1.3 to 1.5 N and peak power increased from 75 to 80 to 87 to 90 W/kg. Accordingly, hydrodynamic force and peak bio-robot swimming speeds also increased with AR. For the in vitro muscle, preliminary analysis reveals that although muscle shortening velocities increased from 4.0 to 4.2 to 5.4 to 6.2 ML/s, peak muscle force, power and swimming velocity only increased slightly as AR increased. Although preliminary, these findings suggest that our novel approach may be useful for integrating muscle force-length dynamics with limb morphology and swimming performance. Email address for correspondence: [email protected] 16:30 Saturday 2nd July 2011

A8.40 Tail beat frequency during swimming and power output of catshark muscle fibres

Roger C Woledge (Imperial College London, UK), Tim G West (Royal Veterinary College, UK) and Nancy A Curtin (Imperial College London, UK) Video records were made of catsharks swimming in a large tank at 12ºC. The fish were free to cruise at their chosen speed and direction. The field of view was approximately 5 m x 3.5 m and fish length ranged from 0.5 to 0.75 m. Video segments with the fish swimming in a straight path at a relatively constant speed were chosen for measurement. The forward velocity of the fish was found by tracking the forward movement of the front end of the fish. The frequency of the tail-beat was found from the side-to-side movements of the front end of the fish. In most observations the forward velocity was between 0.25 and 0.5 fish lengths/s, and the mean tail beat frequency was 0.76 Hz. We have previously measured the power output by muscle fibres isolated from catsharks during sinusoidal movement with intermittent simulation (Curtin, N.A. & Woledge, R.C. (1993) J. Exp. Biol. 185, 195206). These results show that during movement at 0.76 Hz the power output of the red fibres is close to 95% of maximum. So there is a good match between the performance of the red fibres and the whole fish during cruising. The power output of red fibres decreases sharply as frequency increases above 1 Hz. Thus white fibres, which produce their maximum power at about 3 Hz, will be recruited for faster swimming. We thank Dr Clem Wardle of the Aberdeen Marine Laboratory for making and giving us the video.

A8.42 Effect of foot webbing in frogs on hydrodynamics and muscular dynamics

Christofer J Clemente (Harvard University, USA) and Christopher Richards (Harvard University, USA) Aquatic animals apply power locomotion using paddles. Frogs use webbed feet for propulsion, while other animals use stiffer or inflexible fins. Flexible pads may simply reduce area and therefore resistive drag on the foot during the recovery stroke, while allowing for increased area, and therefore increased propulsive drag during the power stroke. If so then flexible webbed pads may have no effect or a deleterious effect on swimming performance. Alternatively, hydrodynamics and muscle dynamics may benefit from flexible webbing, leading to an increase in swimming performance. To test this we used servo motors to rotate and translate a foot to mimic the kicking and forward swimming velocity of Xenopus laevis frogs. Foot rotation was controlled with force signals generated by a Hill-type muscle model in real-time control software, such that hydrodynamic interactions of the foot interacted in real time with the muscle model. To test this we built a plastic frame based based on the bone structure of a Xenopus laevis hindfoot, and covered it in either hard inflexible plastic, or a thin (0.006 inches) flexible latex sheet. Results suggest that while muscle force produced was not significantly different between the trials, time-averaged hydrodynamic force was ~13% higher for webbed feet. Further, this caused a ~10% higher mean translational velocity. Preliminary data suggest that the change in timing of the muscular force with the activation nerve pulse, due to flexibility of the foot, results

Abstracts 2011 in a more favourable transmission of muscular force to hydrodynamic force, potentially explaining the observed differences. Email address for correspondence: [email protected] 16:45 Saturday 2nd July 2011

131 on individual toes showed the importance of a shear movement in the removal of particles. We demonstrate that a `flushing' effect of the secreted mucus plays an important role in shedding particles/ contaminants. Email address for correspondence: [email protected] 10:30 Sunday 3rd July 2011

A8.43 Sticking under wet conditions: a comparison of the attachment abilities of tree and torrent frogs

Thomas Endlein (University of Glasgow, UK), Jon Barnes (University of Glasgow, UK) and Ulmar Grafe (University of Brunei) Tree frogs climb surfaces by secreting a thin layer of mucus which spreads over the pads by means of a distinctive channel system. Adhesion is thought to be mainly due to capillary forces generated by the fluid­air interface around the edge of the pad. Torrent frogs, however, are able to adhere and move about even when their environment is completely flooded; i.e. in the absence of capillary forces. We measured the attachment of a tree frog and a torrent frog by challenging them to cling on to a rotatable platform which could be turned from an upright to an upside-down orientation. We tested their attachment to surfaces of different roughness and wetness. On smooth­dry surfaces, both frog species performed equally well. However, on flooded, rough surfaces, torrent frogs stayed attached significantly longer. In order to relate performance to contact area, we filmed the frogs on a rotatable transparent surface, using a special illumination technique that enabled us to quantify contact area. When we tilted the platform through 180°, the two frog species showed different strategies to remain attached. Torrent frogs increased the contact area over the course of the turn by bringing their ventral surface into contact with the surface, while tree frogs increasingly relied on their toe pads alone. Furthermore, we found that torrent frog pads possess a different cell shape which may facilitate the drainage of excess fluid underneath their pads in order to form a close contact with the substrate. Email address for correspondence: [email protected] 10:15 Sunday 3rd July 2011

A8.45 Influence of friction force reduction on feet grooming behaviour in beetles

Naoe Hosoda (National Institute for Materials Science) In insects, cleaning (grooming) of tarsal attachment devices is essential for maintaining their adhesive ability, necessary for walking on a complex terrain of plant surfaces. How insects obtain information on the degree of contamination of their feet has remained, until recently, unclear. In this study, influence of friction force reduction on feet grooming behaviour in beetles was investigated. Stiff polymer substrata with different degrees of nanoroughness (root mean square: 28-100 nm) were prepared for the experiment. Friction force by beetles Gastrophysa viridula (Coleoptera, Chrysomelidae) was measured on various substrata and occurrence of grooming on stiff polymer substrata was counted. Rough surfaces strongly reduced friction and adhesion without contaminating feet and increased occurrence of grooming movements. In this study, we were able to demonstrate that friction force between tarsal attachment pads and the substrate provides an insect with information on the degree of contamination of its attachment structures. We have shown that foot grooming occurrence correlates not only with the degree of contamination but also with the decrease of friction force. This result indicates that insects obtain information about the degree of contamination, not statically but rather dynamically and, presumably, use mechanoreceptors monitoring either tensile/compressive forces in the cuticle or tensile forces between leg segments. Email address for correspondence: [email protected] 10:45 Sunday 3rd July 2011

A8.44 The self-cleaning properties of the toe pads of tree frogs

Niall A Crawford (University of Glasgow, UK), Thomas Endlein (University of Glasgow, UK) and Jon Barnes (University of Glasgow, UK) Tree frogs use adhesive pads for climbing. They adhere by wet adhesion, aided by secretion of mucus into the space between pad and substrate. The pads get contaminated regularly through usage, but maintain their stickiness over time, and are able to recover adhesive and friction forces rapidly without grooming. Here we show in two experiments that the toe pads of White's tree frogs (Litoria caerulea) self-clean. We compared adhesive forces before and after contamination of frogs on a rotatable platform, where recovery of forces occurred quicker when the frog was encouraged to take a few steps as opposed to remaining stationary. We also measured the force recovery of individual toe pads in restrained frogs, mimicking individual steps using a motorised stage. The performance of individual toe pads was compared between `steps' with and without a shear drag, both for partial and full contamination. In both cases, the shear drag `steps' generated more force than the up-down `steps'. Videos of each form of step show faster contaminant removal for the steps with shear movement, which allow contaminant to be `smeared' off, thus increasing contact area. In both cases, adhesive forces recovered after a few steps, but took significantly longer in restrained frogs. The whole animal experiments show that use of the pads increases recovery, while the experiments

A8.46 Nubby versus smooth: Adhesive and frictional properties of tarsal attachment pads in two species of stick insects

Philipp Busshardt (Functional Morphology and Biomechanics, Christian-Albrecht-University, Germany), Harald Wolf (Institute for Neurobiology, University of Ulm, Germany) and Stanislav N Gorb (Functional Morphology and Biomechanics, Christian-AlbrechtUniversity, Germany) We measured adhesive and frictional properties of tarsal euplantulae in two stick insect species (Carausius morosus, Cuniculina impigra) on smooth and rough surfaces (roughness 3 µm). The surface of the euplantulae is smooth in C. impigra, while that of C. morosus bears small nubs. Adhesion experiments revealed that smooth pads produce higher adhesive forces on the smooth surface than on the rough one. The nubby euplantulae showed no difference in adhesion on both surfaces. A comparison between the species exhibited higher adhesion force of C. impigra on the smooth surface than that of C. morosus, while on the rough one we did not detect a difference. Friction experiments showed higher forces of the smooth pads on the smooth surface. The nubby pads generated stronger friction on the rough surface than on the smooth one. Inter-species comparison revealed higher friction force values of the smooth pads on the smooth surface than the nubby ones. On the rough surface, forces did not differ between species. In both species the euplantulae showed isotropic frictional behaviour in anterior and posterior direction.

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Society for Experimental Biology deposited on the surface. Hemispherical footprints of the fly had higher evaporation rate, suggesting a larger fraction of volatile compounds in the fly fluid. Within one hour, the droplet volume reduced to 21% of the initial volume for the fly, and to 65% for the beetle. Drop geometry for the fly footprints changes significantly during evaporation and shows pinning effects due to an assumed self organizing oil layer on top the water fraction of the micro-emulsion. This effect was not observed for the beetle, presumably due to the monophasic composition of the fluid. The data obtained let us suggest that the adhesion strength must be time-dependent due to the specific evaporation rate of the adhesive fluid. These results are important for understanding the functional mechanism of insect adhesive systems, but also for biomimetics of artificial capillarity-based switchable adhesive systems. Email address for correspondence: [email protected] 11:30 Sunday 3rd July 2011

The higher forces of smooth-on-smooth contact can be explained by the higher contact area compared to nubby-on-smooth and smoothon-rough ones. The nubby pads generate stronger friction on the rough than on the smooth surface, based on interlocking between nubs and surface asperities. We can therefore support the theory that microstructured pads have advantages in adapting to rough substrates, while smooth pads perform better on smooth substrates due to a large effective contact area. Email address for correspondence: [email protected] 11:00 Sunday 3rd July 2011

A8.47 Physical mechanisms of `wet' insect adhesion

Matthew J Anyon (University of Hull, UK), Mika M Kohonen (University of Hull, UK), Michael J Orchard (University of Hull, UK) and David M Buzza (University of Hull, UK) Many people take for granted the sight of an insect climbing a window or walking upside down on the ceiling, often forgetting that this is, in-fact, an advanced feat of biological engineering. The adhesive ability of insects, particularly on smooth substrates, requires specially adapted tarsal morphologies, and specialized physical mechanisms. For many insect species, it has been established that adhesive and frictional forces generated on smooth substrates are mediated by a liquid secretion ­ which the insect delivers to the contact-zone beneath the adhesive pads on their feet during locomotion. However, for most insects, the physico-chemical properties of these secretions are not well known. Taking an interdisciplinary approach, we study this `wet' adhesive system in Asian Weaver ants (Polyrhachis dives) and Harlequin ladybirds (Harmonia axyridis), possessing the smooth and hairy adhesive pad systems respectively.Using optical microscopy, interference reflection microscopy (IRM), scanning electron microscopy (SEM), dewetting experiments and gas chromatography-mass spectrometry (GC-MS) we characterized the adhesive pad and secretory `footprints' of these insects on a variety of smooth substrates. We determine properties including contact area, volume deposited, contact angle, viscosity and chemical composition. These parameters were fed into simple models of adhesion which were compared to experimental measurements of insect adhesive and friction forces obtained using different force measurement techniques in vivo. Results indicate that the standard `uniform thin-film' model does not adequately describe both the measured adhesive and frictional forces, and suggest that additional friction forces may be generated by dry contact between the adhesive pad cuticle and the substrate. Email address for correspondence: [email protected] 11:15 Sunday 3rd July 2011

A8.49 Underwater attachment to substrates of different surface roughness in the running water mayfly larvae Epeorus assimilis: The role of gill lamella surfaces

Petra Ditsche-Kuru (Zoological Institute, University of Kiel, Germany), Jochen H Koop (Federal Institute of Hydrology Koblenz, Germany) and Stanislav N Gorb (Zoological Institute, University of Kiel, Germany) The larvae of Epeorus assimilis (Heptageniidae, Ephemeroptera) can be exposed to strong currents while grazing algae from stones in running waters. To cling on the stone surface these larvae use specialized attachment structures. Beside their strong claws they bear special surface microstructures located ventrally on the part of each gill lamella tacked to the substrate. The aim of this study was to investigate the role of these setal structures (pads) in underwater attachment. We measured friction forces generated by the gill lamellae on solid substrates. Moreover, the influence of surface roughness on attachment force has been investigated for the first time. The setal structure has been visualized using scanning electron microscopy. Experiments show that the gill lamellae significantly contribute to friction force on smooth, slightly rough and some rough substrates. However, on an intermediate roughness the contribution of these structures to friction was rather low. The friction coefficient of the gill lamellae depended not only on the surface roughness of the substrate, but also on the pulling direction. The results indicate that interlocking between setae and substrate irregularities as well as molecular adhesion may contribute to different extent to friction on different substrates. Under natural conditions the contribution of the gill lamella structures to friction seems to be especially relevant on smooth and slightly rough substrates where claws cannot interlock with the surface irregularities. Email address for correspondence: [email protected] 11:45 Sunday 3rd July 2011

A8.48 Footprints of insects: Tarsal liquid evaporation dynamics in flies (Calliphora vicina) and beetles (Coccinella septempunctata)

Henrik Peisker (Christian Albrecht University, Germany) and Stanislav N Gorb (Christian Albrecht University, Germany) Insect adhesive structures deliver a thin layer of fluid into contact area. It has been repeatedly reported that the presence of the fluid significantly increases adhesion on various substrata. Previous data obtained on different insect groups suggested a difference not only in the chemical composition, but also physical properties between representatives of different taxa. In the present study, we have measured the evaporation rate of the fluid and changes in the droplet geometry over time in flies Calliphora vicina and beetles Coccinella septempunctata by the use of atomic force microscopy (AFM). Flattened droplets of the beetle showed oil like wetting tendency with continuous increase of droplet radius once

A8.50 From flow to fibres: using rheology to understand how silk is spun

Chris Holland (University of Oxford, UK), David Porter (University of Oxford, UK) and Fritz Vollrath (University of Oxford, UK) Silk production has evolved to be energetically efficient and functionally optimized, resulting in a material that outperforms most industrial fibres. However whilst we know much about the mechanical properties of the fibre we know little regarding the unspun silk dope. One novel and important way to approach this problem is through the application of rheology. Though analysis of the flow properties of the silk dope during processing we can understand how applied shear energy transforms a liquid gel into a final solid fibre with bespoke structurefunction properties.

Abstracts 2011 Our work has shown remarkably similar rheologies for native spiderdragline and silkworm-cocoon silk, despite their independent evolution and substantial differences in protein structure. In addition while native silks behave like typical molten polymers under flow, reconstituted silks do not. Therefore turning evolutionary constraints into design criteria will not only aid us in the creation of better materials but will also provide insight into the natural material along the way. Yet these are all bulk mechanical property measurements. The real excitement of this work surrounds the integration of rheology with techniques that allow us to visualize silk aggregation and fibril formation. As I shall discuss, we are now focusing on a series of projects at the life-sciences interface which are enabling us to understand, integrate and ultimately predict the kinetics, energetics and dynamics of silk protein aggregation. Website: www.oxfordsilkgroup.com Email address for correspondence: [email protected] 12:00 Sunday 3rd July 2011

133 mass. We investigated the development of the collagen network in 50 sheep divided over ten sample points between birth and maturity. We assessed three collagen network parameters: predominant collagen fibril orientation, collagen density, and collagen network anisotropy. Results were used in a numerical model to estimate the contributions of the development of each of these parameters to the development of the AC stiffness gradient. Predominant fibril orientation changed from parallel to the articular surface at birth, to a depth-dependent arcade-like structure. Collagen reorientation was finished before sexual maturity (36 weeks). Collagen density increased over the full tissue depth, increased most adjacent to the bone, and had not yet stabilised at 72 weeks. The numerical simulations show that collagen reorientation alone can result in a stiffness gradient that spans an order of magnitude. Based on the simulations, we hypothesize that reorientation of fibrils and increase in collagen density serve distinct functional roles: reorientation functions to establish the stiffness gradient, and collagen density increases to reduce fibril strains that tend to increase due to reorientation. Email address for correspondence: [email protected] 12:30 Sunday 3rd July 2011

A8.51 Comparative study of the epidermis architecture and material properties of the skin in four snake species

Marie-Christin G Klein (Functional Morphology and Biomechanics, Zoological Institute, University of Kiel, Germany) and Stanislav N Gorb (Functional Morphology and Biomechanics, Zoological Institute, University of Kiel, Germany) On the basis of structural and experimental data, it was previously demonstrated that the integument of all snakes consists of a hard, robust, inflexible outer surface (Oberhäutchen and -layer) and soft, flexible inner layers (alpha layers). The aim of this study was to compare the epidermal cross sections and the material properties of the outer scale layers (osl) and inner scale layers (isl) of the exuvium of four snake species specialized to different habitats: Lampropeltis getula californiae (terrestrial), Epicrates cenchria cenchria (all-rounder), Morelia viridis (arboreal), and Gongylophis colubrinus (burrowing), in order to relate the structure of the snake integument to its mechanical properties. Scanning electron microscopy (SEM) revealed a variation in epidermis structure and thickness between the four snake species. The nanoindentation experiments provided strong evidence for the presence of a gradient in the material properties in the snake integument within each species. The difference in both the effective elastic modulus and hardness of the osl and isl between the four species is not very large compared to the difference in epidermis thickness and architecture. The thickness of the epidermis and the presence of the gradient in material properties are discussed as possible adaptations to wear minimization in different habitats. Email address for correspondence: [email protected] 12:15 Sunday 3rd July 2011

A8.53 Inspired by snakes: Polymer surfaces with anisotropic frictional properties

Martina J Benz (Christian-Albrecht-Univerity, Germany) and Stansilav N Gorb (Christian-Albrecht-Univerity, Germany) Snakes keep their ventral body side in almost continuous contact with the substrate during locomotion. Therefore, the snake skin has to generate propulsion (high friction) and slide along the substrate (low friction) simultaneously, thereby anisotropic frictional properties of the surface can be assumed. The microstructure of ventral snake scales were analysed by scanning electron microscopy. The influence of skin microstructure on frictional properties is examined by microtribological techniques. Friction measurements were conducted with the following contact pair: ventral scales (exuvia) of the terrestrial snake Lampropeltis getula californiae and a smooth glass ball. To investigate the role of the stiffness of underlying epidermis layers on the anisotropy of frictional properties of the skin, two different types of sample cushioning (hard and soft) were tested in four different directions. The results showed strong anisotropic effect in frictional behaviour in both cases. The soft cushioned skin samples showed anisotropy between cranial to caudal, dextral and sinistral directions. The hard cushioned samples resulted in different frictional behaviour (anisotropy between caudal to cranial, dextral, sinistral directions). Inspired by the microornamentation of ventral snake scales in cooperation with our industrial partner, we developed microstructured polymer surfaces that generate anisotropic frictional properties. The tribological characterization of these artificial surfaces is currently under way by the use of similar experimental setup. As cooperation between the University of Kiel, the University Bonn and the company Leonhard Kurz Group Stiftung & Co KG, this work is funded by the Federal Ministry of Education and Research, Germany within the BIONA program. Email address for correspondence: [email protected] 12:45 Sunday 3rd July 2011

A8.52 Functional analysis of postnatal collagen network remodelling in articular cartilage

Mark C Van Turnhout (Eindhoven University of Technology, The Netherlands), Henk Schipper (Wageningen University, The Netherlands), Sander Kranenbarg (Wageningen University, The Netherlands) and Johan L Van Leeuwen (Wageningen University, the Netherlands) Healthy adult articular cartilage (AC) has a typical depth-dependent composition and structure that results in a tissue stiffness gradient: AC stiffness increases with increasing distance from the articular surface. This gradient is important for normal functioning of the mature joint, but is absent at birth and develops in early life. AC is a hydrated porous tissue in which the collagen network comprises ~75% of the solid

A8.54 Body mass specific muscle power output is maintained during daily torpor in the Djungarian hamster Phodopus sungorus

Rob S James (Coventry University, UK), Jason Tallis (Coventry University, UK), Frank Seebacher (University of Sydney, Australia) and Ken Storey (Carleton University, Canada)

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Skeletal muscle disuse causes atrophy, leading to reduction in absolute muscle force production, in both clinical and hibernation models of mammalian inactivity. No previous study has investigated the effects of daily torpor bouts on skeletal muscle size and mechanics. The Djungarian hamster undergoes daily torpor bouts in response to reduced photoperiod, causing a decrease in metabolism. In natural conditions torpor enables hamsters to survive the winter stresses of cold and reduced food availability. We hypothesized that prolonged cycles (14 weeks) of daily torpor in the Djungarian hamster would cause significant muscle atrophy, but limited changes in the mechanical properties of muscle. We isolated representative fast twitch (EDL) and slow twitch (soleus) muscles from eight hamsters subjected to short (torpor) photoperiod and eight hamsters subjected to long (control) photoperiod. Each muscle was subjected to isometric (constant length) studies to maximise twitch and tetanus performance, at 37°C, before the work loop technique was used to determine the power output-cycle frequency relationship. Torpor hamsters had significantly lower body mass and muscle mass than controls. Maximum soleus tetanic stress and work loop power output was higher in torpor hamsters than controls. Torpor decreased maximum EDL tetanic stress, but did not affect EDL power output. Rate of fatigue was not affected by torpor in either soleus or EDL muscles. If other locomotory muscles also respond in this way, with decreases in muscle size matched by reduced body mass, then daily torpor is unlikely to affect locomotor performance of the Djungarian hamster. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.56 Neuromechanical modelling of lizard locomotion: understanding morphology and behaviour

Kristiaan D'Août (University of Antwerp, Belgium and University Ghent, Netherlands), Kostas Karakasiliotis (EPFL Lausanne, Switzerland), Louis Flynn (Michigan State University, USA), Peter Aerts (University of Antwerp, Belgium and University Ghent, Netherlands) and Auke Ijspeert (EPFL Lausanne, Switzerland) The present study is part of the EU-funded project `LocoMorph' that aims to improve the application of morphing (changing form and/ or posture according the task) in robots, based on insights from ecological morphology, biomechanics, computational neuroscience and embedded artificial intelligence. Evolutionary adaptation to specific niches represents excellent biological paradigms for such technological morphing. The small long-tailed lacertid lizard (Takydromus sexlineatus), specialized for `swimming' through dense vegetation (grass), is used as the biological model for neuromechanical analysis. Dimensional and inertial properties of body segments (17 head-trunk-tail segments + 3 segments per limb) as well as dynamic coefficients of frictional with the substrate are determined. These are used as input for computer simulations (WEBOTS). `Body joints' have two (active or passive) rotational DOF's and limbs are modelled as single rotational units. In order to allow for the high cycling frequencies observed in vivo, damping has been introduced at the limb-trunk joints as well as at the limb ground interface. Optimizations (for speed) are run and compared with in vivo kinematics. Questions about the control of body-limb coordination, tail use and efficacy of movement are addressed. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.55 Comparing aerodynamic performance of 13 hummingbird species from Colombia and California

Jan Wouter Kruyt (Wageningen University, The Netherlands), Elsa M Quicazán-Rubio (University of California Riverside, USA), Sander W Gussekloo (Wageningen University, The Netherlands), Johan L Van Leeuwen (Wageningen University, The Netherlands), GertJan F Van Heijst (Technical University Eindhoven, The Netherlands), Douglas L Altshuler (University of California Riverside, USA) and David Lentink (Wageningen University, The Netherlands) Hovering is a key behaviour of hummingbirds that allows them to time-effectively extract nectar from flowers and catch arthropod prey, which is critical to their high-energy lifestyle. The aerodynamic power demands are high during hovering, but how these demands compare among different species is not fully understood. Here we compare the aerodynamic quasi-steady performance of wings from 13 species of hummingbirds from Colombia and California to determine how wing morphology mediates hovering performance. We attained lift and drag over a range of angles of attack for Reynolds numbers below 20 000. Whereas other spinner experiments recorded negative drag at low angles of attack, our spinner measured drag accurately across the full range of wing angles. The accurate drag and lift measurements combined allow us to compare aerodynamic lift and power factor. The power factor captures the efficacy of hummingbird wings to lift a unit weight with a minimum of aerodynamic power. To quantify the effect of wing morphology on the airflow we performed particle image velocimetry and visualized the leading edge vortex (LEV) for the upstroke and downstroke configuration of the wing. Even though wing camber is inverted for the upstroke, the structure of the leading edge vortex is still remarkably similar. The power factor on the other hand is much lower during the upstroke. Our findings suggest that precise drag measurements are important to evaluate the hover efficacy of spinning and flapping wings that generate leading edge vortices. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.57 How do animals with limited distal limb musculature use sensory feedback during locomotion?

Anna M Liedtke (TheRoyal Veterinary College, UK), Andrew Spence (Royal Veterinary College, UK), Thomas Witte (Royal Veterinary College, UK) and Sarah Moore (Royal Veterinary College, UK) Sensory feedback plays an important role in the control of locomotion. Understanding how the sensory system tunes locomotion can give fundamental insight into normal locomotion and shed light on the etiology of veterinary and human neurological disorders. Sensory feedback from the digits is known to be important for limb placement, touchdown posture, and compensation for environmental perturbations. Because limbs of different morphology are likely to have varying capability to exert control over the moving body, we postulate that animals with different limb musculature will use sensory feedback in distinct ways. Specifically, we hypothesize that animals with limited distal limb musculature will use limb touchdown position rather than axial leg actuation to control their locomotion. To test this, we measured the kinematics of horses with reduced levels of digital sensation. Optical motion capture was used to collect kinematic data from horses walking and trotting on a treadmill before and after an abaxial sesamoid nerve block was administered to remove digital sensation. Interestingly, preliminary results from three horses show that a lack of sensory input results in less variability in stance time. Future work will: 1) seek to determine whether the increased variability seen with sensory feedback supports the hypothesis of an overall limb control strategy; and 2) determine whether animals with more distal limb musculature choose a different control strategy. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

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A8.58 Biomechanics of adhesion in tree and torrent frogs

Diana Samuel (University of Glasgow, UK), Thomas Endlein (University of Glasgow, UK), Ulmar Grafe (Universiti Brunei Darussalam), Mathis Riehle (University of Glasgow, UK) and Jon Barnes (University of Glasgow, UK) Tree frogs adhere to surfaces using their toe pads. They utilize wet adhesion, which is dependent on two forces, capillarity and Stefan adhesion, and is facilitated by secreting fluid from their pads. Since locomotion is a dynamic process, the pads must be able to attach and detach from surfaces repeatedly, an ability that is lacking in man-made adhesives. Furthermore, detachment of the pads should be effortless, so that the frogs do not need to pull their pads from the substrate each time they take a step. We show that they achieve this by peeling, and exhibit other behavioural strategies that enable them to cling onto substrates in challenging conditions, for instance when adhering to overhanging substrates. Torrent frogs also possess expanded toe pads, but live in very wet environments, such as waterfalls. Such conditions result in their toe pads being submerged, thereby abolishing the air­fluid interface required for capillarity; yet they are still able to adhere to surfaces. This ability could potentially be utilized in the field of biomimetics for a number of applications, such as in the development of more efficient wet-weather car tyres. This poster aims to show that: a) detachment of tree frog toe pads is generally faster than their attachment and can occur in a number of ways; b) compared to torrent frogs, tree frog adhesive ability is drastically reduced on rough, flooded surfaces, thus showing the importance of capillarity; and c) torrent frogs have a more specialized toe pad morphology to cope with flooded conditions. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.60 Biomechanical of locomotion in Gymnophithalmini lizard species: snakelike x lizard-like body shapes

Vanessa Cunha (University of São Paulo, Brazil) and Tiana Kohlsdorf (University of São Paulo, Braizl) Locomotor performance is directly related to morphological traits and likely affects individual fitness in specific ecological contexts. In the present study we investigated possible associations between body shape and locomotor performance in five Gymnophthalmid lizard species: two snakelike forms (Calyptommatus leiolepis and C. sinebrachiatus) with short tail, no forelimbs and styliform hindlimbs; one intermediate morphology (Nothobachia ablephara) with long tail and trunk, styliform forelimbs and two-digits hindlimbs; and two lizardlike species (Vanzosaura rubricauda and Psilophthalmus paeminosus) with long tails and short trunks, four-digit forelimbs and pentadactyl hindlimbs. Lizards were placed in a racetrack having a thin sand layer from their natural habitats. Tests were performed in two sets of five races, in different days, and races were recorded using a high-speed video camera (500 frames/sec). Four anatomical points were marked along the body (scapular girdle, mid-trunk, pelvic girdle, mid-tail), and two to three linear runs, with at least three consecutive strides, were digitized frame-by-frame in Matlab. Number of consecutive strides and instantaneous velocity of each anatomical point were calculated. Overall, instantaneous velocity did not vary among anatomical points in each species, with exception of C. sinebrachiatus, where tails were faster than scapular girdles. In the comparative analyses, lizard-like lizards were faster than the snakelike species, although performing similar number of strides. Such variation can be also explained by behaviour: lizard-like species use limbs to help their propulsion when running away from predadors, while snakelike lizards are semifossorial and in nature burrow when threatened. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.59 Are different homing pigeon breeds morphologically different and are these differences consistent with aerodynamic theory?

Marinos Stavrou (Royal Veterinary College, UK) and James R Usherwood (Royal Veterinary College, UK) Birds come in different shapes and use different flying strategies. Is every strategy accompanied by specific adaptations and are these consistent with conventional aerodynamic theory? To investigate some aspects of this we used different homing pigeon breeds. These are used in various different races; some are better at short sprint races, others in long endurance races and some are good overall. Busschaerts were first bred for sprint races but over time they were developed to be good at all speeds. Roland Janssens do best at short sprint races while Aardens are best at endurance races. These breeds are under intense artificial selection in order to consistently win their respective races and hence we expect any morphological differences between them to indicate modifications that are suited to that particular flight strategy and are bred for in winning pigeons. The three different breeds were dissected to test for differences between selected morphological variables including wing span, wing area, aspect ratio, pectoralis and supracoracoideus muscle, percentage pectoralis and wing loading. Are all the combined effects of differences in the morphological parameters consistent with breeding for flight distance speciality? The measured parameters may change with each breed and flying strategy and it is usually the interaction between all of them that will contribute to the eventual aerodynamic capability. We developed a power curve model to find this combined effect by calculating flight energy costs for all the pigeons and comparing the three different breeds. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.61 Design and fabrication of a novel robotic flexible tool applicable in minimally invasive surgery for animals

Sanaz Mosafer (Amirkabir University of Technology, Tehran, Iran) and Siamak Najarian (Amirkabir University of Technology, Tehran, Iran) Minimally invasive surgery (MIS) is the surgical operation done through small incisions (ports) of 5 to 10 mm diameter with the help of specialized instruments and endoscopes. Unlike an open surgery in which large areas of animal's body have to be cut off, laparoscopic surgery requires only small incisions. In spite of all advantages such as reduction of trauma, risk of inflammation, disfigurement and pain, minimally invasive surgeries suffer from few negative limitations such as less tool placement flexibility compared to open surgeries. Non-intuitive effects on the tip movements caused by insertion point such as movement insertion and velocity scaling are the other important limitations. Since MIS instruments are rigid or only limitedly flexible, some anatomical regions are not accessible. Most current laparoscopic tools have rigid shafts, which make it difficult to approach the worksite through small incisions. In order to solve the mentioned problem, a novel hand-held surgical instrument with additional degrees of freedom at the instrument tip is presented. It can be used during specific phases of surgery when additional dexterity is required. The proposed surgical instrument has 5DOF with 8 mm diameter. This instrument is wire actuated using steel cables with the aim of size reduction. Its main advantages are its low weight and small size. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

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Society for Experimental Biology Masateru Maeda (Chiba University), Toshiyuki Nakata (Chiba University), Shuhei Ozawa (Chiba University), Hao Liu (Chiba University) Takeoff simulations of a butterfly have been performed. We captured a swallowtail butterfly Papilio xuthus by recording the takeoff sequences with high speed video cameras. From the images, three-dimensional wing shapes at each time step were reconstructed, as well as the body trajectory. Then, a geometric butterfly model and a takeoff kinematic model were constructed and an integrated simulation of 6 degrees of freedom (DoF) free flight was conducted. We utilized a coupled solver of fluid dynamics and rigid-body dynamics for the free-flight simulation. In the fluid dynamics solver, the flow field around flapping wings and a moving body are computed via threedimensional incompressible Navier-Stokes equations by means of finite volume method (FVM). There are two features unique in the takeoff flight. One is the aerodynamic effect termed "ground effect", where airflow produced by the wings is bent by the presence of the obstacle (ground). This effect is simulated by applying the no-slip boundary condition on the lower outer boundary. The other is kicking the ground, called "leg thrust." This not only augments vertical and horizontal forces but provides substantial additional torque(s) around the insect's centre of mass at the initial phase. Our results showed the leg thrust has a lot more impact on the takeoff than the aerodynamic ground effect. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.62 Neuromuscular control of stable locomotion through uneven terrain

Joanne C Gordon (Royal Veterinary College, UK), Monica A Daley (Royal Veterinary College, UK), Alan M Wilson (Royal Veterinary College, UK) and Jeffery W Rankin (Royal Veterinary College, UK) The control strategies that enable bipedal species to maintain stable locomotion over uneven or unpredictable ground surfaces remain uncertain. Locomotion involves a complex coordination of neural activation, muscle physiology and innate muscle mechanics that continuously adjust to variations in environmental terrain and behavioural context. Consequently, muscle activation patterns result from a multitude of control inputs. Here we investigate the role of visual feedback in the neuromuscular control of locomotion in uneven terrain. We measured in vivo myoelectric output of eight different limb muscles of the helmeted guinea fowl (Numida Meleagris) during speeds of 0.7 m/s and 1.3 m/s over both level and uneven terrain using a motorized treadmill. Two obstacle subsets were used, with low and high visual contrast to the treadmill surface. We hypothesize a proximo-distal gradient in joint neuromechanical function with greatest kinematic and myoelectric variability during obstacle strides in the more distal musculature. We expect increased visual information to result in more accurate anticipation of obstacles, augmenting feed forward mechanisms that facilitate context dependent neuromuscular control. We therefore expect highest swing phase myoelectric intensity in strides with high visibility obstacles, as compared to low visibility obstacles and level terrain. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.65 Effects of limb reduction on the trunk muscles of gymnophthalmid lizards

Kristina Grommes (Institut für Zoologie Rheinische, FriedrichWilhelms-Universität, Germany), Juliane Hammen (Institut für Zoologie Rheinische, Friedrich-Wilhelms-Universität, Germany), Markus Lambertz (Institut für Zoologie Rheinische, FriedrichWilhelms-Universität, Germany), Steven F Perry (Institut für Zoologie Rheinische, Friedrich-Wilhelms-Universität, Germany) and Tiana Kohlsdorf (Departamento de Biologia, FFCLRP, Universidade de São Paulo, Brazil) The loss of limbs and digits has repeated itself within several groups of squamates and is accompanied by a locomotor mode in which lateral undulation is most common. In the lizard family Gymnophthalmidae limb reduction and limblessness has occured independently several times.We compared the body wall muscles of two gymnophthalmid species, Calyptommatus leiolepis Rodrigues, 1991 and Vanzosaura rubricauda (Boulenger, 1902). Both are found in the Caatinga region of northeastern Brazil. C. leiolepis has a snakelike body shape, no forelimbs and vestigial hind limbs, and lives fossorially in sand dunes. In contrast, the habitus of V. rubricauda, which is found in the leaf-litter, is lizard-like with distinctive limbs. We focused on the comparison of the hypaxial and the epaxial muscles and the question, if the volumes and the forces that can be generated in these muscle groups differ between these species with respect to their different locomotor modes. The volumes were estimated using stereological methods. We found that volume and force of the entire body wall muscles are greater in C. leiolepis. In both species, epaxial muscles occupy more of the trunk than hypaxial muscles, but in C. leiolepis the epaxial muscles have a proportionally greater volume than in V. rubricauda. The reason must be the different locomotor mode: C. leiolepis moves by lateral undulation and uses the epaxial muscles for lateral bending. In addition, since C. leiolepis lacks limbs it cannot support its weight: according to the bow and string principle this would be accomplished by the hypaxial muscles. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.63 Are insect wings designed to be aerodynamically optimized?

Toshiyuki Nakata (Chiba University, Japan), Naoshi Nishihashi (Chiba University, Japan) and Hao Liu (Chiba University, Japan) Flexibility of insect wings often leads to passive and dynamic inflight wing deformations including bending, twist and camber. Our integrated model of insect flight with flexible wings revealed that wing shape changes due to elastic deformation are responsible for improving aerodynamic performance of flapping wings by enhancing lift production and aerodynamic efficiency. The deformed three-dimensional wing shape is usually determined by wing stiffness and its distribution. In this respect, the biological design of insect wing structures is of great importance in developing advanced bio-inspired MAVs (Micro Air Vehicles). In this study, the effects of wing shape and stiffness on wing deformation and aerodynamics are investigated by utilizing an integrated model of flying insect with flexible wings. The scaled structural model of insect wings is established on the basis of the integrated model of a hovering hawkmoth. A systematic study of aerodynamic performance of a flexible flapping wing is carried out over a wide range of Reynolds number of hovering insects including a fruit fly, a honeybee and a hawkmoth. Our results indicate that there exists an appropriate range of wing structure for optimized aerodynamic performance, which provides an essential guidance for developing flapping-wing MAVs. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.64 Estimating the impact of the ground in butterfly takeoff

Abstracts 2011

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A8.66 Force calculation in the uncinate processassociated muscles in zebra finch (Taeniopygia guttata castanotis) and budgerigar (Melopsittacus undulatus)

Kristina Grommes (Friedrich-Wilhelms-Universität, Germany), Jasmin Mühlisch (Friedrich-Wilhelms-Universität,Germany), Thomas Breuer (Friedrich-Wilhelms-Universität, Germany) and Steven F Perry (Friedrich-Wilhelms-Universität, Germany) Most birds have bony projections, uncinate processes (UP), on their ribs. Muscles associated with the UP are the appendicocostals (both inspiratory and locomotor/postural function), external intercostals (locomotion) and external oblique (expiration). We compared these muscles in two species with different UP morphology: zebra finches (Taeniopygia guttata castanotis), which have straight, slender UP, and budgerigars (Melopsittacus undulatus), which have UP with square bases. Zebra finches are perching/hopping birds that constantly vocalize, whereas budgerigars climb using their beak. Using stereological methods we estimated uncinate muscle volumes and calculated the putative generatable forces using trigonometrical formulas applied to the deviation from circular profile of the muscle fibres and the distance between the ribs. The putative force of appendicocostals is greater in zebra finches, correlating with their continuous vocal activity and need for rapid inspiration, whereas that of the postural/locomotor external intercostal muscles is greater in the budgerigars. Surprisingly, the force of the external oblique muscle was similar in both species in spite of different uncinate structure. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.68 Using bird flight modes to enhance overall flapping wing micro air vehicle performance

William Thielicke (Biomimetics-Innovation-Centre, Germany), Eize J Stamhuis (Biomimetics-Innovation-Centre, Germany) and Antonia B Kesel (Biomimetics-Innovation-Centre, Germany) Birds still outperform existing micro air vehicles (MAVs) in terms of manoeuverability and endurance. Recent studies on flapping flight in birds, bats and insects have shown that the ability to use unsteady, lift-enhancing mechanisms like the leading edge vortex is most likely responsible for increased manoeuverability and slow-flight capability. When flying at higher speeds, endurance and efficiency can be maximized using flapping or gliding flight modes with attached, steady flow. Whereas MAVs that incorporate the use of different aerodynamic mechanisms to increase overall performance are rare, birds combine the advantages of steady and unsteady aerodynamics. This study presents a flapping wing MAV design that creates lift and thrust with two different aerodynamic mechanisms: In slow-speed flapping flight, lift is created via unsteady aerodynamics and during faster flight, the MAV has the ability to intermittently glide efficiently. This combination of flight modes was possible using rigid wings and wing kinematics based on swifts and swiftlets. Wind tunnel measurements for both flight modes show that sufficient lift and thrust are created in flight. To prove the contribution of unsteady aerodynamics in slowspeed flapping flight, we modelled lift using a steady aerodynamics blade-element analysis. Lift observed in direct force measurements exceeds forces predicted by the model by a factor of two. Flapping wings hence offer more aerodynamic possibilities to create forces than fixed or rotary wings: Efficiency or magnitude of force can be maximized when desired; this feature is also promising for MAVs that need to complete missions with varying demands. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.67 Undulatory swimming: gradients in body stiffness passively affect swimming performance

René S Sonntag (Biomimetics-Innovation-Centre, University of Applied Sciences, Germany) and Antonia B Kesel (Biomimetics-InnovationCentre, University of Applied Sciences, Germany) Undulatory fish swimming yields maximal thrust and propulsive efficiency at Strouhal numbers of 0.25 < St < 0.4. Low undulatory frequencies additionally reduce muscle work, but require higher tail beat amplitudes, as St is proportional to tail beat frequency and amplitude. Large lateral deflections increase drag at high swimming speed. Thus fast swimming modes (subcarangiform to thunniform) show small anterior deflection and large posterior amplitudes. Previous studies showed that the undulatory body wave is induced by alternating lateral muscle contractions and modulated by body stiffness to match swimming speed. This depends passively on tissue characteristics (e.g. skin, tendons, skeleton) and is actively controlled by negative muscle work. As fish must adapt to different swimming speeds and turning manoeuvres, active control of body stiffness is essential. However, to what extent exclusively passive elements can affect swimming kinematics and propulsive efficiency still remained unclear. In the present study, simplified models from a carbon fibre/epoxy resin composite with different chordwise gradients in flexural stiffness were tested in a flow tank. Undulation was induced by an oscillating beam attached to the "head region". Keeping free flow velocity, beam deflection angle and driving frequency constant, trailing edge amplitudes and thus Strouhal numbers similar to those observed in rainbow trout were reproduced by applying a proper stiffness gradient. Our results imply that, kinematics associated with efficient propulsion can be controlled exclusively passive by appropriate material characteristics, for certain swimming speeds. These findings may help to simplify fish-like robots and propulsion systems. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.69 The biomechanics and the energetics of pedestrian locomotion in king penguins

Astrid S Willener (Roehampton University, UK), Yves Handrich (Strasbourg University, Strasbourg), Anick Abourachid (National Museum of Natural History, France) and Lewis G Halsey (Roehampton University, UK) The use of methods to measure energy expenditure considerably improves the knowledge of a specie by enabling the quantification of its energetic outcome and the state of its fitness leading to the improvement of its protection in applied biology. Measuring the energetic expenditures of king penguins while walking is important to know their fitness as they must sometimes walk kilometres to go back to the sea after an extended fasting period. Moreover, this subject is also interesting concerning the study of adaptation as the potential global warming may modify their environment and prolong their journey. I applied different known techniques used as proxy to determinate the energy expenditures while king penguins were walking (respirometre, accelerometer and heart rate frequencer). But I was also interested to measure the biomechanics of their gait to find the link of the energy expenditures depending of the gait via kinematic analyses, as, unlike humans, king penguins do not change their energy expenditures for the locomotion even they put on a third of their weight. By measuring the 3D kinetic movement as energy is expended, I hope to be able to discern if the capacity of king penguins to adapt their energy expenditure is due to a physiological or biomechanical adaptation while walking. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

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Society for Experimental Biology

A8.70 The function of the elytra in the flight of garden shafers (Melolontha melolontha)

Florian Hoffmann (Biomimetics-Innovation-Centre, Germany) and Antonia Kesel (Biomimetics-Innovation-Centre, Germany) Garden shafers (Melolontha melolontha) are flying with outspreaded elytra. The elytra move with the same frequency and in the same phase but with reduced amplitude than the wings (alae). Other beetles like flower chafers (Cetoniinae) are flying with closed elytra, so it seems that they are principally not necessary to fly. However, garden shafers can't fly without elytra, so that the elytra have not only a protective function for their wings. Using the particle image velocimetry (PIV) method on garden shafers flying tethered in a wind tunnel, the active and passive aerodynamic function of the flapping Elytra was investigated. The local flow structure around the elytra and wings are visualized in the plane parallel to the flow stream at different positions from proximal to distal. Primarily the elytra produce lift passively but only have low importance for the total lift. So that the elytra-wing and the wake-wing interactions seems to be more important for the flying capabilities of these beetles. Contrary to four winged insects the elytra and alae move in the same phase, so that the known wing-wake, wing-wing interference or wake capture should not work. Here we present a preliminary dataset which visualize the local flow conditions and wake-wing interactions and discuss their aerodynamic consequences. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.72 A simple model predicts walk/trot but not trot/gallop transitions in dogs: high speed gait transitions show signatures of additional constraints

Simon D Wilshin (Royal Veterinary College, UK), G C Haynes (University of Pennsylvania, USA), Jack R Porteous (Royal Veterinary College, UK) and Andrew J Spence (Royal Veterinary College, UK) A model for the relative leg phases of animals during gait transitions is compared to data obtained from five dogs on a treadmill. The model is derived by assuming that quadruped legs are approximately equivalent and that these legs may be varied independently. We also assume that small variations in relative leg phasing which do not change a specific measure of overall phase result in comparable deviations in metrics such as stability or energetics during a transition. In this sense we assume that all leg configurations are approximately equivalent. Combining these assumptions into the simplest possible action principle results in a set of differential equations. These equations describe the relative phases during a gait transition as a function of the normalized fraction of the transition completed. The model predictions and the observed relative leg phases are in good agreement during the walk/trot and trot/walk transitions. For the trot/gallop and gallop/trot transitions the model performs comparatively poorly. It is known that gait selection is driven by factors including energetics and stability, but it remains unclear what the determining factors are for the manner in which a gait transition is undertaken. We show how to add terms to our action which will allow us to test for different determining factors (energetics, stability, etc.) of body locomotion during a gait transition, since different determining factors lead to different additional terms. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.71 The effects of slipping on the kinematics of two cockroach species during high-speed running

Chris J Dallmann (Biomimetics-Innovation-Centre, University of Applied Sciences, Germany), Florian Hoffmann (BiomimeticsInnovation-Centre, University of Applied Sciences, Germany), Andrew Martin (Biomimetics-Innovation-Centre University of Applied Sciences, Germany) and Antonia B Kesel (Biomimetics-Innovation-Centre, University of Applied Sciences, Germany) Although insights into insect locomotion under various perturbations have already provided useful inspiration for robust and adaptive legged robots, stable locomotion in low-friction environments still remains a big challenge for technical designs. In order to determine how running insects are affected when their tarsi fail to make stable contact with the ground, we investigated the locomotion of two cockroach species, the long-legged American cockroach (Periplaneta americana) and the short-legged Dubia roach (Blaptica dubia), while encountering persistent and sudden artificial slipperiness. After triggering an escape response, animals traversed either a (i) high-friction (120-grid sandpaper), (ii) low-friction (plexiglass coated with paraffin oil), or (iii) half high- half low-friction substrate in random order while being videotaped dorsally and laterally with a high-speed camera. Kinematic analyses revealed that both species significantly reduce forward speed as soon as their tarsi slip during propulsive phases. The reduction of forward speed was correlated with significant decreases in stride length and stride frequency under both low-friction conditions, which points towards a fast adaptation of muscle activations and neural commands. In addition, P. americana was able to reduce the amount of slipping by increasing the touch-down angle of the propulsive hind legs, which indicates a beneficial change of ground reaction forces. We discuss the role of relative foot placements and ground contact times in regard to stability during locomotion and interspecific differences in limb and body morphology. Email for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A8.73 Strategies used to step over ground unavailable for foot placement during terrestrial locomotion

Jeremy Burn (University of Bristol, UK) and Katherine Daniels (University of Bristol, UK) In their natural environment, terrestrial animals regularly encounter areas of ground in the path of travel that are not viable for foot placement. Here we characterized the different strategies used by humans to traverse different sized unavailable areas in terms of patterns of mechanical energy change and kinematic parameters of gait. Subjects were asked to walk in a straight line along a 15-m long track without stepping in a rectangular area delimited by light projection from above. The width of the projected area was constant and equal to the width of the track but the length was varied randomly between 0.2 m and 1.5 m for each trial. Whole-body kinematics were recorded for each trial using optical motion capture and combined with published anthropometric data to calculate segmental mechanical energy together with basic kinematic parameters of gait. Subjects modulated the stepping kinematics of normal walking local to the obstacle. Obstacle traversal was generally associated with an increase in the mechanical cost of transport compared to normal walking. As obstacle length increased, subjects systematically changed stepping technique to include an aerial phase or `leap' step. Patterns of mechanical energy fluctuation for the body centre of mass during obstacle traversal were characteristic of walk at small lengths and run at large lengths. An additional strategy combining features of walk and run was observed at intermediate obstacle widths. Transitions between strategies may be made in order to maintain stability and maximize energy economy for the modulated strides. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

Abstracts 2011

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SEB Main Meeting, Glasgow 2011

A9 - Intraspecific variation in behaviour: functions and proximate explanations

A9.1 Individual variation in the costs of personality traits

Kathryn Arnold (University of York, UK), Katherine Herborn (University of Glasgow, UK), Lindsay Henderson (University of Glasgow, UK) and Lucille Alexander (WALTHAM Centre for Pet Nutrition) Behavioural differences between individuals that are stable within individuals across contexts are often referred to as `personality traits'. Individuals with different combinations of personality traits can differ in fitness but the mechanisms remain unclear. There is the suggestion, but few supporting data, that personality reflects underlying variation in physiology. Here, we tested whether wild birds differing in personality differed in their stress physiology, i.e. the glucocorticoid hormone corticosterone (Cort), and potential physiological costs specifically plasma indices of oxidative profile: antioxidant capacity (OXY), prooxidant status (reactive oxygen metabolites, ROMs) and oxidative stress. The personality traits neophobia (latency to approach food near novel objects), activity levels (during the exploration trial) and exploratory tendency (controlling for differences in activity) were repeatable within individuals but uncorrelated. ROMs and OXY were also uncorrelated with each other. Exploratory tendency and neophobia did not covary with baseline Cort. More exploratory birds had lower stress-induced Cort than less exploratory individuals. Oxidative stress was explained by a significant interaction between neophobia and exploratory tendency. To conclude: First, Personality types differed in both stress physiology and oxidative profile. Second, the physiological costs of different personality traits are not simply a product of the physiology, e.g. glucocorticoid response, of individuals. The combination of an individual's personality traits proved important in terms of the oxidative costs accrued. Finally, understanding how oxidative profile and thus physiological costs vary within and between personality traits may explain how personality can predict fitness. Email address for correspondence: [email protected] 10:30 Sunday 3rd July 2011 In general, our results challenge the view that personality traits can be predicted from constant associations with life-history trade-offs. Email address for correspondence: [email protected] 11:00 Sunday 3rd July 2011

A9.3 Consequences and causes of individual variation in fish

Katherine Sloman (University of the West of Scotland, UK) Individual variation in competitive ability often leads to the formation of dominance hierarchies, which have been well documented in juvenile salmonid fish. As a consequence of position in a social hierarchy, individual variation in physiology can be magnified with more subordinate individuals showing higher plasma cortisol levels, lower growth and other physiological compromises. What causes individual variation in competitive ability is less well researched. As environmental conditions under which embryos develop have the potential to affect adult phenotypes, two factors, maternal stress and conspecific presence, were investigated to determine whether they would alter levels of individual variation in behaviour and physiology later in life. Although both parameters affected overall behaviour and physiology of offspring, no changes in the level of between-individual variation occurred. Email address for correspondence: [email protected] 11:20 Sunday 3rd July 2011

A9.4 Behaviour and experience: how environmental factors shape intraspecific variation in the rainbow trout, Oncorhynchus mykiss

Jack S Thomson (University of Liverpool, UK), Phillip C Watts (University of Liverpool, UK), Tom G Pottinger (CEH Lancaster, UK) and Lynne U Sneddon (University of Chester, UK) Boldness describes a suite of correlated behaviours where bold animals are relatively more active, more exploratory and take more risks than shy animals. This intraspecific variation in behaviour may have adaptive consequences depending on the environmental pressures that individuals experience. Although predisposed towards boldness or shyness, previous experience is known to alter an individual's behaviour along that continuum. Bold and shy rainbow trout, Oncorhynchus mykiss, were classified according to their latency to approach a novel object in two investigations. In the first experiment, fish were held in groups of nine bold or nine shy trout before being behaviourally reassessed. In the second experiment, trout experienced variations in predator threat in addition to high or low food availability, and boldness was then reassessed. Plasma cortisol was also determined and brain mRNA was extracted for candidate gene analysis. In both experiments, bold fish significantly altered their behaviour, becoming shyer regardless of social group or threat, and their activity levels varied dependent upon threat and dietary regimes. Conversely, the behaviour of shy fish was more resistant to change and they remained cautious. High predation threat increased the expression of three candidate genes related to behaviour and stress responses.

A9.2 Personality and performance in the wild: exploring behavioural variation in brown trout

Jörgen I Johnsson (University of Gothenburg, Sweden) and Bart Adriaenssens (University of New South Wales, Sidney, Australia) The study of animal personality and behavioural syndromes is a fashionable and rapidly developing field of research. In many species individual differences in behaviour do show remarkable consistency across situations and contexts. Nevertheless, general assumptions and theories in the field are often inferred from studies in the laboratory and/or on captive and domesticated animals. In fish, for example, general consistency in behaviour is often assumed on the basis of short-term studies in the laboratory, whereas field studies are scarce. Thus, more long-term studies on wild populations in natural environments are needed to evaluate the current hypotheses aiming to explain consistent individual differences in behaviour. Using results from recent combined laboratory- and field studies on wild brown trout (Salmo trutta) we here discuss: (1) the repeatability of individual behaviour in the wild; (2) the strength and generality of behavioural syndromes; and (3) associations between personality and fitness-related traits. In light of this, recent adaptive explanations for variation in personality, e.g. the pace-of-life syndrome hypothesis, are discussed.

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Society for Experimental Biology an additional experiment was performed in order to evaluate the relative effect of visual and chemical stimuli from dominant individuals. In both cases external electrodes were used as a non-invasive method to study heart rate on swimming fish allowing behavioural and physiological responses to be measured simultaneously. Our results suggest that bystanders exposed to dominant individuals can rapidly and accurately assess the intrinsic fighting ability/quality of a potential opponent. This was reflected in an immediate behavioural response whereas the physiological stress response (increased heart rate) was delayed. The second study revealed that visual stimuli are more important as subordinate responded stronger towards visual cues compared with olfactory. Hence this ability may in nature allow bystanders to withdraw from costly contest with low probability of winning. Thus, escalated conflicts may be less common in nature than previously suggested by laboratory studies. Email address for correspondence: [email protected] 12:20 Sunday 3rd July 2011

The data suggest trout are able to alter their boldness as an adaptive response to environmental pressures, although the behaviour of bold fish is much more labile than that of shy conspecifics, a finding contrary to current theory on stress coping style and personality. Email address for correspondence: [email protected] 11:40 Sunday 3rd July 2011

A9.5 Neurogenesis and neural plasticity in fish brains: Gene regulation in the context of stress and individual stress coping styles

Øyvind Øverli (Norwegian University of Life Sciences, Norway), Ida B Johansen (Norwegian University of Life Sciences, Norway), Erik Höglund (Danish Technical University, Denmark) and Göran E Nilsson (University of Oslo, Norway) In birds and mammals post-embryonic neurogenesis is important in learning, memory, and regulation of mood and behaviour. Compared to endothermic vertebrates, fishes show a strikingly high rate of adult brain cell proliferation, with new cells being formed in all brain regions throughout life. The demonstration that social stress inhibits brain cell division in rainbow trout provided the first example of environmental regulation of neurogenesis in fishes. Stressful and dangerous situations however promote individual variability, and stress can have very different impact on different individuals. Low-responding/high-responding (LR, HR) rainbow trout lines have been established as a model to study heritable variation in stress coping style. LR fish show low flexibility in their foraging behaviour, high level of routine formation, and prolonged memory retention. HR fish are more sensitive to external cues and readily respond to environmental changes. We investigated brain mRNA expression of genes involved in postembryonic neurogenesis and neuronal plasticity in stressed and non-stressed LR and HR fish. Proliferating cell nuclear antigen (PCNA, a marker of actively cycling cells) expression was elevated in non-stressed HR compared to LR. Neurogenic differentiation factor (NeuroD) and doublecortin (DCX), both markers of young, differentiating neurons, were higher in HR compared to LR fish after long-term social stress. These observations suggest that neurogenesis and neural plasticity affect memory retention and responsiveness to environmental cues in fishes as well as mammals. Consequently, variability in these processes may underlie several differently termed phenomena such as stress coping style, behavioural syndromes, and animal personalities. Email address for correspondence: [email protected] 12:00 Sunday 3rd July 2011

A9.7 Natural variation in the stress and social molecular networks that underlie personality

Nadia Aubin-Horth (Laval University, Canada) Personality traits such as boldness and aggressiveness often covary at the population level to form a behavioural syndrome. Different mechanisms could underlie the presence of behavioural syndromes, including pleiotropy. Personality traits covary with stress reactivity in wild populations and selection lines, suggesting that the hormones involved in the stress response could affect personality traits and could have pleiotropic effects resulting in behavioural syndromes. To understand the molecular mechanisms underlying intraspecific variation in behaviour, it is thus necessary to determine which components of the stress molecular network differ among individuals with divergent personality. We first determined which components of the stress axis vary with personality in a wild population of threespine sticklebacks (Gasterosteus aculeatus). Our results show that natural variation in personality within a population is associated with differences in expression of specific components of the stress axis. Our results also show that the behavioural syndrome found in this population could be the result of a pleiotropic effect. We then studied the social and stress molecular networks in two populations of sticklebacks that differ in personality and that originate from ecologically-divergent habitats. Our results show that these population-level differences in personality are reflected in expression differences of genes known to be involved in social behaviours and in the stress response. The future experiments that will be necessary to uncover the genetic and developmental causes of these behavioural and molecular divergences and to confirm or rule out the pleiotropy hypothesis will be discussed. Email address for correspondence: [email protected] 13:30 Sunday 3rd July 2011

A9.6 Social status in salmonids; the importance of visual and chemical stimuli

Johan Höjesjö (Department of Zoology and Animal Ecology, University of Gothenburg, Sweden), Jörgen I Johnsson (Department of Zoology and Animal Ecology, University of Gothenburg, Sweden) and Michael Axelsson (Department of Zoology and Animal Ecology, University of Gothenburg, Sweden) The ability to adequately assess the fighting ability of a potential opponent will be important for all animals in order to avoid costly and escalated interactions with superior opponents. Generally, opponents obtain increasingly accurate information as the contest unfolds but recent studies also suggest that uninvolved animals can gather information of potential opponents. Here, two experiments using rainbow trout (Oncorhynchus mykiss) will be presented; first the ability of bystanders to assess the intrinsic fighting ability of potential opponents with and without contest information was examined. Then,

A9.8 Is boldness a fitness trait in rainbow trout?

Angela Sims (University of Liverpool, UK), Lynne Sneddon (University of Chester, UIK), Tom Pottinger (CEH Lancaster, UK), Steve Paterson (University of Liverpool, UK) and Phill Watts (University of Liverpool, UK) Behavioural syndromes comprise a suite of associated behaviours that indicate fitness. For example, aggression is often correlated with boldness, or exploration, in fish. Aggression has clear fitness benefits; aggressive fish tend to win agonistic contests with resultant advantages for survival and reproduction. However, fitness benefits of boldness are

Abstracts 2011 less clear; bold individuals have larger territories in some species, but lower fecundity in others. As heterozygosity can be used to indicate fitness, aggression and boldness should show clear links with heterozygosity if they are fitness traits in rainbow trout (Oncorhynchus mykiss). O. mykiss were tested for their aggressiveness by recording the frequency of aggressive interactions towards a conspecific. A second group of O. mykiss were tested for bold behaviour using a novel object test. DNA was extracted from muscle tissue and genotyped at 24 microsatellite loci for aggressive/less-aggressive trout and 82 loci for bold/shy trout. Aggressive trout had higher heterozygosity than less-aggressive individuals. In contrast, there was no difference in the level of heterozygosity between bold and shy trout. The association between heterozygosity and aggression in trout is consistent with the role of aggression in determining fitness. Conversely, the lack of correlation between boldness and heterozygosity demonstrates that boldness may not contribute to individual fitness. However, previous studies have shown that, under predation, boldness correlates with heterozygosity, which may mean that boldness in the context of predation does not correlate with exploration of a novel object. This suggests that the theoretical premise of behavioural syndromes may have to be reconsidered. Email Address for correspondence: [email protected] 14:00 Sunday 3rd July 2011

141 Individuals exposed to current-day or elevated CO2 were observed in a detour test where they were required to make repeated decisions about turning left or right. No bias for right or left turns was observed at the population level. However, individual fish were strongly lateralized and turned eitherleft or right with greater frequency than expected by chance. Exposure to elevated CO2 disrupted individual lateralization, i.e. the frequency of left and right turns of fish reared under elevated CO2 was not different from a random expectation. In contrast, the absolute lateralization of CO2 treated fish was not different from random but lower than the control fish, indicating that exposure to elevated CO2 disrupts lateralization. Given that lateralized fish were previously found to show higher performance in a number of anti-predator behaviours, it is possible that a loss of lateralization in a future high CO2 ocean could increased the vulnerability of larval fish to predation. Email address for correspondence: [email protected] 14.40 Sunday 3rd July

A9.11 Yolk reserves predicts social status in rainbow trout (Oncorhynchus mykiss) larvae

Madelene Åberg Andersson (Technical University of Denmark, Denmark) and Erik Höglund (Technical University of Denmark, Denmark) Establishing territories is important for future survival and fitness in salmonid larvae. It has been shown that the timing of emergence from the spawning nests in search of food can vary several weeks between offspring. Individuals emerging during the early stages have been shown to be more aggressive and to obtain more profitable territories compared to individuals that emerge later during the emergence period. Furthermore, individual variation in energy reserves preserved in the yolk-sac in emerging larvae has been demonstrated. In this study we examine how differences in energy reserves impact the chances of obtaining profitable territories. This was conducted by examining dominance and territory establishment in paired individuals of the same emerging time and total size but with different amounts of energy reserves. Dominance was determined by examining aggression and avoidance behaviours and the serotonergic activity in the larval brain. Moreover, energetic costs of social interaction were estimated by measuring yolk volume before and after social interaction. A higher probability to become social dominant was observed in larvae which had bigger yolk sacs before social interaction. Dominant larvae spent more yolk compared to subordinate individuals. Just as in more developed fish, subordinate yolk sac larvae were characterized by higher serotonergic activity in the brain. Email address for correspondence: [email protected] 15:00 Sunday 3rd July 2011

A9.9 Differences in repeatability between the laboratory and the field: Can temperature explain the difference?

Mark Briffa (Univesrity of Plymouth, UK) `Animal personality' means that individuals differ from one another in either single behaviours or suites of related behaviours in a way that is consistent over time, such that individual behaviour shows high repeatability. However, a trend seen across diverse study systems is for differences in repeatability between studies conducted in the field and the laboratory. It is usually assumed that personality is driven by variation in relatively stable `internal' factors, rather than by differences in external factors such as variation in microhabitat, which could influence metabolic rate. In marine invertebrates metabolic rate is strongly influenced by temperature, a factor that is subject to high temporal and spatial variation in intertidal habitats. Here we investigate the effect of temperature on consistent individual differences in two intertidal species; highly mobile hermit crabs (Crustacea), and sedentary beadlet anemones (Cnidaria). Email address for correspondence: [email protected] 14:20 Sunday 3rd July 2011

A9.10 Elevated CO2 affects lateralization in a coral reef fish

Paolo Domenici (CNR, IAMC, Oristano, Italy), Bridie Allan (JCU Townsville, Australia), Mark McCormick (JCU Townsville, Australia), Philip Munday (JCU Townsville, Australia) Elevated CO2 has recently been shown to affect chemosensory behaviourand activity levels of larval reef fishes, increasing their risk of predation. However, the effect of elevated CO2 on behavioural lateralization in fish, a phenomenon known to affect cognitive performance and fundamental traits such as escape responses, schooling and orientation, is unknown. Here, we tested the hypothesis that CO2 concentration that could occur in the shallow ocean by the end of this century (880 atm) may affect behavioural lateralization (i.e. turning bias in the left or right direction) in a coral reef fish, Neopomacentrus azysron.

A9.12 Personality, subjective well-being, and longevity in orang-utans (Pongo spp.).

Alexander Weiss (University of Edinburgh, UK), Mark J Adams (University of Edinburgh, UK) and James E King (University of Arizona, USA) Epidemiological studies have found that individual differences in cognitive abilities, personality, and subjective well-being are related to health outcomes, including mortality. Most impressive in this area of research are multiple studies showing that higher intelligence, conscientiousness, and happiness are linked to longer life. Similarly, studies of rhesus macaques have shown that personality traits in nonhuman primates are related to disease progression. We sought to determine whether personality or subjective well-being in 182 orang-

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Society for Experimental Biology At faster speeds, fish with lower MMR and AS swam near the rear of schools. These trailing fish required fewer tail beats to swim the same speed as individuals at the front of schools, indicating that posterior positions provide hydrodynamic benefits that reduce swimming costs. Conversely, fish with high aerobic capacity can withstand increased drag at the leading edge of schools, where they could maximize food intake while possibly retaining sufficient aerobic scope for other physiological functions. SMR was never related to position, suggesting that high maintenance costs do not necessarily motivate individuals to occupy frontal positions. In the wild, shifting of individuals to optimal spatial positions during changing conditions could influence the structure or movement of entire schools. Email address for correspondence: [email protected] 16:20 Sunday 3rd July 2011

utans (Pongo spp.) was related to longevity. Each orang-utan's personality was assessed via ratings on the Hominoid Personality Questionnaire; positive affect was assessed via four questions based on human measures of subjective well-being. We used discrete time survival analyses to test whether personality dimensions or subjective well-being are related to survival over periods ranging from three to seven years after ratings. Even after controlling for sex, age, species (Sumatran, Bornean, or hybrid), and number of life events, higher subjective well-being was significantly related reduced mortality risk. No similar effect was found for any of the orangutan personality dimensions. These findings indicate that ostensibly "subjective" measures may reflect objective health. These findings are suggestive with respect to the evolution of happiness. One possibility consistent with previous findings in humans, chimpanzees, and orang-utans is that happiness is a marker of genetic quality and evolved via sexual selection. The null findings with respect to personality may be explained by the absence of a conscientiousness dimension or the conditions of captivity, including health monitoring and prompt medical care. Email address for correspondence: [email protected] 15:40 Sunday 3rd July 2011

A9.15 Does personality affect individual thermal niches in fish?

Bart Adriaenssens (Evolution and Ecology Research Center, University of New South Wales, Australia), Albin Gräns (Department of Zoology, University of Gothenburg, Sweden), Michael Axelsson (Department of Zoology, University of Gothenburg, Sweden), Jörgen I Johnsson (Department of Zoology, University of Gothenburg Sweden) and Peter A Biro (Evolution and Ecology Research Center, University of New South Wales, Australia) The literature provides ample evidence for consistent individual differences in behaviour, often termed `personality'. The presence of personality implies that individuals perceive and react to changes in the environment in unique ways. Temperature is an important ecological variable known to vary both spatially and temporally in nature, causing fine-scale variation in suitable habitat within the home range of a species. If metabolism is linked to personality, we might expect that individuals will choose habitats that are optimized to their phenotype (niche specialization, or niche picking). Using results from recent studies on brown trout (Salmo trutta) and mosquitofish (Gambusia holbrooki) we explore links between repeated measures of behaviour, metabolic rate, and habitat choice along a temperature gradient. We discuss possibilities for personality-linked thermal niche specialization and its ecological implications. Email address for correspondence: [email protected] 16:40 Sunday 3rd July 2011

A9.13 Risk, attitudes to risk and learning in zebra finches

Sasha R Dall (University of Exeter, Cornwall Campus, UK) Risk pervades the lives of most animals. Broadly speaking there are two main strategies for adaptively managing the unexpected opportunities and dangers that individuals face day-to-day. Individuals can reduce their uncertainty in a particular context by gathering or providing information, or they can minimize the impact of uncertainty by buffering themselves against the bad times. Since the latter is always possible whenever information is available, the adaptive value of gathering information via learning should be influenced by how risky an individual perceives its environment to be. Here I present data from an experiment to test how well individual zebra finches, which vary in their stable attitudes to risk (tendencies to explore a novel environment), performed on an associative learning task under different levels of perceived danger (with or without protective cover). Both the likelihood of achieving the learning criterion and the speed at which those that learnt did so was influenced by both individual attitudes to risk and the manipulation of perceived risk. I go on to discuss the implications of these results for the evolution and maintenance of stable variation in attitudes to risk with populations, and for adaptive risk management strategies in general. Email address for correspondence: [email protected] 16:00 Sunday 3rd July 2011

A9.16 Intraspecific variation in aerobic metabolism: temporal repeatability and relations with organ size and enzyme activity in an ectothermic vertebrate, the brown trout

Tommy Norin (Aarhus University), Hans Malte (Aarhus University) Standard metabolic rate (SMR) and active metabolic rate (AMR) are two fundamental physiological parameters providing the floor and ceiling in aerobic energy metabolism. Previous studies on fish have found individual relative SMR to closely correlate with important life-history traits, and to be highly repeatable over time when fish are provided an ad libitum diet. In relation to this, we tested the temporal repeatability of individual body mass-corrected SMR, AMR, and absolute aerobic scope (AAS) in young brown trout (Salmo trutta L.) fed a moderately restricted diet (0.5 to 0.7% fish weight per day). A gradual loss of repeatability of all three metabolic parameters was observed over the 15-week experimental period. In other words, any existing hierarchy from relatively low to relatively high metabolic rate within the population gradually broke down. This phenomenon was interpreted as a plastic response to the apparent disadvantage of maintaining a high rate of metabolism during a period of low energy

A9.14 The effects of metabolic demand and aerobic capacity on the spatial position of individuals within fish schools

Shaun S Killen (University of Glasgow, UK), Stefano Marras (Université de Montpellier 2, France), John F Steffensen (Marine Biological Laboratory, University of Copenhagen, Denmark) and David J McKenzie (Université de Montpellier 2, France) The schooling behaviour of fish is of great biological importance, playing a crucial role in the foraging and predator-avoidance of numerous species. The extent to which physiological performance traits affect the spatial positioning of individual fish within schools is completely unknown. Schools of juvenile mullet Liza aurata were filmed at three swim speeds in a swim tunnel, with one focal fish from each school then also measured for standard and maximal metabolic rates (SMR; MMR), aerobic scope (AS), and maximum aerobic swim speed.

Abstracts 2011 availability. Since variations in metabolism may be explained by variations in the size of metabolically active organs, this was tested in the brown trout. No correlations between body mass, corrected organ weight and metabolism were found for either liver, heart, spleen, intestine or stomach, but instead, activity in the liver of two aerobic mitochondrial enzymes, citrate synthase and cytochrome c oxidase, was found to correlate with whole-animal metabolic rate. This finding offers potential insight into the source for the often observed intraspecific variation in metabolism. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

143 was made with thermoneutral ducklings of the same age. Our principal results showed that: (1) at ambient temperature (25°C), circulating serotonin was decreased in cold acclimated ducklings (-37%); and (2) in cold acclimated and thermoneutral ducklings, an intraperitoneal injection of glucagon (360 g.kg-1) was followed after 10 minutes by prominent lipolysis and a large increase in circulating serotonin. The elevation of circulating serotonin was less pronounced in cold acclimated ducklings. In the light of these results we suggest that the thermogenic action of glucagon in birds is probably indirect and involves at least the mobilization of lipids and the monoamine stimulation. The changes in peripheral serotoninergic activity during cold acclimation could be associated with adaptive changes leading to nonshivering thermogenesis. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.17 Influence of behavioural syndrome on cognitive bias in canaries (Serinus canaria)

Mathilde Lalot (Laboratoire d'Ethologie et Cognition Comparées, France), Davy Ung (Laboratoire d'Ethologie et Cognition Comparées, France), Florian Demangeon (Laboratoire d'Ethologie et Cognition Comparées, France), Patrizia D'Ettorre (Laboratoire d'Ethologie Expérimentale et Comparée, France) and Dalila Bovet (Laboratoire d'Ethologie et Cognition Comparées, France) Improving the quality of life of captive animals is dependent on developing relevant and efficient measures of animals' welfare. Until now, many researchers used indirect behavioural and physiological measures, indicating emotional arousal, but not emotional valence. Recent studies suggested that cognitive biases could constitute a novel measure of animal welfare. We trained 44 domestic canaries (22 males and 22 females) on a Go/No Go procedure to discriminate between two locations (the right and the left of the test cage). Each side is associated with a value: attractive food (mash and ground apple) or aversive food (some food with Bitrex®, a bitter tasting substance). During the test phase, an unreinforced dish is placed at three kind of intermediate positions, representing ambiguous information. An "optimistic" bird is supposed to fly to this location significantly faster than a "pessimistic" one and we assume that individuals living in the best conditions will be the most optimistic. We will look for a correlation between individuals' cognitive bias and their behavioural syndrome. We focus on some traits like: locomotion (using an aviary as an openfield), reaction to novelty (presenting a new objet, placed above the food source), sociality (measuring proximity to conspecifics for an individual placed in a long battery cage, with a side containing its cage mates) and aggression (putting an intruder bird in the cage of the tested individual). These results would show the existence of a cognitive bias in canaries and would highlight the influence of behavioural syndrome on these biases. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.19 Behavioural answers of male canaries (Serinus canaria) when they listen to different acoustic signals in various social contexts

Ophélie Boullet (Laboratory of Ethology and Compared Cognition), Eric Vallet (Laboratory of Ethology and Compared Cognition), Gérard Leboucher (Laboratory of Ethology and Compared Cognition) and Manfred Gajr (Max Planck Institute for ornithology) The social context and social status are very important for individuals because they determine behaviours and the adaptation to the environment. The study is about the social status and its influence on behaviours when different acoustic signals are played back. There are three different signals; a familiar ambience, an unfamiliar ambience and computer edited signals (songs with Sexy or non sexy phrases). In the study it was shown social status does not change during the year, the hierarchy is steady. The results demonstrate that dominants and subordinates do not behave in the same way. Concerning subordinates, they spend more time on feeding when the social context is familiar than in an unfamiliar social context. But there is no difference for dominants; whatever the social context they behave in the same way. Dominants and subordinates have different strategies for comprehending the social context; this can explain the results obtained. Concerning dominants, whatever the social context they behave in the same way because they need more informations for foraging. But it is not the same for subordinates because in familiar ambience they have sufficient information for foraging, they are sensitive to the social context. Males cannot free themselves from the social context. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.18 Involvement of the seretonin in cold-induced thermogenesis in Muscovy ducklings: A HPLC study

Taoufiq Fechtali (Hassan II University, Mohammedia-Casablanca, Morocco) Physiological studies have shown that glucagon is a potential mediator of nonshivering thermogenesis in birds. The present work was undertaken in order to investigate whether the observed thermogenesis results from a direct action of glucagon on avian thermoregulatory mechanisms or in fact requires the participation of other agents such as serotonin that is widely distributed within the brain of vertebrate animals and participates in the control of neural activity. Our experiments were performed using cold-acclimated ducklings which developed muscle nonshivering thermogenesis. A comparison

A9.20 Morphological plasticity based on environmental conditions: hearing in gregarious versus solitary locusts (Schistocerca gregaria)

James F Windmill (University of Strathclyde, UK) and Steve M Rogers (University of Cambridge, UK) Animals, based on differing capabilities to receive signals from the environment, are sometimes best suited to their habitat. For example, animals that see in dark conditions often have larger eyes or eyes with tapetum to reflect light. Animal ears too may be adapted to most efficiently receive predator or courtship signals. Often such morphological traits are under constant evolution; however, some animals show different phenotypes based on environmental conditions. For instance, genetically identical locusts exhibit two behavioural and morphological phases (solitary and gregarious) determined by their

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Society for Experimental Biology developed specific strategies to live in a zoo and this strategy my have a link with the morphology and the physiological characteristics of individuals living in this area. We carried out our observations in an enclosure housing capybaras, and we conducted observations before and after passage of caretakers. The pigeons visited this site more frequently after than before the passage of caretakers. Thus, the passage of caretakers seems to signal the avaibility of food. We found two behavioural profiles among the population of pigeons foraging in the capybaras enclosure. Regular visitor pigeons arrived just after the passage of the caretakers and remained the longest in the capybaras' enclosure. Occasional visitor pigeons were opportunist visitors of this food resource: they were seen less often, arrived later, and stayed a shorter time than regular visitor pigeons. Occasional visitor pigeons have more melanin than regular one, the melanin degrees of pigeons are correlated with level of parasites load; pigeons with less melanin have greater parasite load. We found no direct relationship between the behavioural profile and sanitary condition either in males or females. The behavioural profile of female is affected by body condition, the morph and the testosterone level, while in male the behavioural profile is affected by body condition and morph only. The GLM showed a link between behavioural profile and birds morph. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

environment. This form of epigenetic change is triggered within the locust's life cycle by several features of the animal's environment, centering primarily on social crowding conditions. The locusts in these phases have different colours, sizes and behaviours. Recent studies demonstrate differences in visual abilities and corresponding brain size between the phases suggesting the insect's capabilities match the needs of each phase. While much research focuses on hearing in locusts aimed at understanding basic tympanal ear principles; phase has never been taken into consideration as an element that may differentiate hearing abilities. This study focuses on the hearing of locusts in the different phases. Specifically, we measure frequency and amplitude sensitivity of the tympana as well as determine general morphology of the tympana of the solitary and gregarious locusts (Schistocerca gregaria). Understanding phase morphological and behavioural changes provides insight not only into how the environment can change the animal, but also to the evolution of which traits are better adapted to different environmental situations. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.21 Pigeons discriminate easily between human feeders

Ahmed Belguermi (Université Paris Ouest Nanterre La Défense, France), Dalila Bovet (Université Paris Ouest Nanterre La Défense, France) and Gérard Leboucher (Université Paris Ouest Nanterre La Défense, France) Considered a plague in many cities, pigeons in urban areas live close to human activities and exploit this proximity to find food which is often directly delivered by people. In this study, we explored the capacity of feral pigeons to take advantage of this human-based food resource and discriminate between friendly and hostile people. Our study was conducted in an urban park. Pigeons were fed by two experimenters of approximately the same age and skin colour but wearing coats of different colours. During the training sessions, the two human feeders displayed different attitudes: one of the feeders was neutral and the second was hostile and chased away the pigeons. During the two test phases subsequent to the training phase, both feeders became neutral. Two experiments were conducted, one with one male and one female feeder and the second with two female feeders. In both experiments, the pigeons learned to quickly (six to nine sessions) discriminate between the feeders and maintained this discrimination during the test phases. The pigeons avoided the hostile feeder even when the two feeders exchanged their coats, suggesting that they used stable individual characteristics to differentiate between the experimenter feeders. Thus, pigeons are able to learn quickly from their interactions with human feeders and use this knowledge to maximize the profitability of the urban environment. This study provides the first experimental evidence in feral pigeons for this level of human discrimination. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.23 Molecular characterization of boldness and the physiological stress response in rainbow trout, Oncorhynchus mykiss

Jack S Thomson (University of Liverpool, UK), Phillip C Watts (University of Liverpool, UK), Tom G Pottinger (CEH Lancaster, UK) and Lynne U Sneddon (University of Chester, UK) Boldness is an important behavioural syndrome primarily describing how individuals react in conditions of threat or novelty, where bold animals tend to be more active and exploratory than shy conspecifics. This intraspecific variation allows populations to adapt to a wide range of environmental conditions. Various studies have also identified a link between boldness and physiological stress responsiveness, termed stress coping style. Both behavioural and physiological responses to stimuli are hereditary traits, and two lines of rainbow trout, Oncorhynchus mykiss, bred for high (HR) and low (LR) cortisol responses to stress have previously been developed. The aim of this study was to correlate bold and shy behaviour with neuroendocrine stress responsiveness and the expression of related candidate genes in the selected rainbow trout lines. Boldness was determined over two using a standard novel object paradigm. Fish subsequently experienced a one-minute emersion to evoke a stress response, before a blood sample was taken for cortisol analysis. Brains were removed for qRT-PCR analysis of 16 candidate genes selected for their roles in behaviour and stress physiology. HR trout had a significantly higher post-stress cortisol profile than LR fish and boldness was highly correlated with activity levels, but no strong link was found between boldness and either stress response or gene expression. However, divergence in the stress response between the two lines was significantly linked with the expression of all but two candidate genes. This study demonstrated the complexity of relationships between behaviour and the stress axis. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.22 Foraging behaviour of feral pigeons in a zoological park `Regular versus occasional: two foraging strategies in feral pigeons?'

Ahmed Belguermi (Université Paris Ouest Nanterre La Défense, France), Dalila Bovet (Université Paris Ouest Nanterre La Défense, France) and Gérard Leboucher (Université Paris Ouest Nanterre La Défense, France) We studied the foraging behaviour of feral pigeons (Columba livia) in a zoological park in Paris. We tested the hypothesis that pigeons

A9.24 The two-choice system is a non-invasive method for identifying socially dominant individuals from a group of fish

Abstracts 2011 Danielle Caroline Laursen (Danish Technical University, Institute of Aquatic Resources, Denmark), Madelene Åberg-Andersson (Danish Technical University, Institute of Aquatic Resources, Denmark), Patricia M Silva (Norwegian University of Life Sciences, Norway) and Erik Höglund (Danish Technical University, Institute of Aquatic Resources, Denmark) The development of social hierarchies is commonly observed in several species of fish. Typically, a few individuals within a group become socially dominant while the majority will adopt a subordinate status. Dominants are responsible for most aggressive acts and tend to monopolize resources, resulting in heterogeneous growth rates and differing states of wellbeing between individuals. Currently, much research is focused on understanding the basis and function of social status in fish and the consequences of this. Therefore, providing a method to discriminate dominants from a group for further investigation may prove beneficial. With our study we present such a method. Rainbow trout weighing 300 to 440 g were stocked at 20, 40 and 80 kg/ m2 in three two-choice systems, consisting of two 700 l tanks attached to one another via a doorway. Individuals moved freely between the two tanks for three days, after which the number in each tank was determined. Generally, the distribution observed in each system was that one tank was occupied by a few dominant aggressive individuals ("dominant" tank) while the majority of the fish preferred to occupy the second tank ("crowded" tank). At the highest density (80 kg/m2), a higher proportion of the less aggressive fish entered the "dominant" tank, suggesting that at high densities the aversiveness of being crowded is similar to being reared together with highly aggressive individuals. However, at the lower densities (20 to 40 kg/m2) the twochoice system may provide a non-invasive method to identify dominant individuals in a group of fish to use for further investigation. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

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A9.26 The effect of elevated dietary intake of L-tryptophan (TRP) on the stress coping styles of rainbow trout (Oncorhynchus mykiss)

Hanna L Olsén (Uppsala University, Sweden), Kian Safarynejad (Uppsala University, Sweden), Tobias Backström (Uppsala University, Sweden), Per-Ove Thörnqvist (Uppsala University, Sweden) and Svante Winberg (Uppsala University, Sweden) Divergent stress coping styles have been identified in various vertebrates, including rainbow trout (Oncorhynchus mykiss). These coping styles, usually referred to as proactive and reactive, are characterized by different behavioural and physiological reactions when exposed to stressful situations. In short, reactive individuals are shy and respond to stress with a high elevation of plasma glucocorticoids, while proactive individuals are bold and display a lower rise in plasma glucocorticoid levels. The serotonin turnover in reactive individuals is low, while it is high in proactive individuals. Serotonin is synthesized from the amino acid TRP. The activity of the enzyme catalyzing the rate limiting step of serotonin synthesis, tryptophanhydroxylase, is restricted by the TRP availability. Therefore increasing the dietary intake of TRP stimulates brain serotonergic activity. Feeding rainbow trout TRP enriched feed decreases aggression and lowers the post stress plasma cortisol. In this study the effect of TRP enriched feed on the reactive and proactive stress coping styles was studied. The fish were characterized as reactive or proactive according to their post stress plasma cortisol. Fish were fed control feed or TRP supplemented feed for seven days. The boldness of the fish was tested in a novel object test before and after the treatment. Blood and brains were taken after the treatment period for analysis of post stress plasma cortisol and gene expression respectively. Results from the study will be presented. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.25 Regions of the rainbow trout (Oncorhynchus mykiss) genome under selection for the stress response

Angela Sims (University of Liverpool, UK), Lynne Sneddon (University of Chester, UK), Tom Pottinger (CEH Lancaster, UK), Steve Paterson (University of Liverpool, UK) and Phill Watts (University of Liverpool, UK) Genome scans are useful for detecting selection for a complex trait. Genetic mechanisms underlying variation in the stress response in rainbow trout (Oncorhynchus mykiss) are less well known than physiological mechanisms and, without a sequenced and annotated genome, it is difficult to assign function to one or many specific loci. Using O. mykiss lines that were selected for high (HR) or low (LR) cortisol response to a standard stressor, a genome scan was employed to search for signatures of selection. Eighty-two microsatellite markers were selected from known locations on a linkage map, spanning all chromosomes, to identify genome regions associated with the stress response. Two types of statistical method based on genetic variability and genetic differentiation were used to detect signatures of selection between lines. To validate candidate regions, a further seven microsatellites were chosen around regions with high evidence for selection. Two microsatellites possessed significantly reduced variability in LR trout compared with HR, whereas seven loci showed greater genetic differentiation between the lines. These outlier loci were located on several linkage groups, rather than a specific genomic region. Of these additional seven microsatellites, four were confirmed as providing strong evidence for selection. Selection for the stress response is evident at several genomic regions, likely reflecting the complex nature of the stress response and is confirmed at four major regions. These regions provide a starting point to pinpoint the positions of genes responsible for the stress response. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.27 Low-frequency urban noise erodes female domestic canaries, Serinus canaria, preferences for highly attractive low-frequency male songs

Guillaume Huet des Aunay (Laboratoire d'Ethologie et Cognition Comparées (LECC), France), Laurent Nagle (LECC, France), Hans Slabbekoorn (Behavioural Biology, Institute of Biology, Leiden University, The Netherlands) and Tudor Draganoiu (LECC, France) Female domestic canaries prefer male songs with a large frequency bandwidth and a high-repetition syllable rate called A-phrases. Moreover, low-pitched A phrases are preferred over higher pitched ones. We asked if low frequency urban noise affects female sexual responsiveness for these highly attractive phrases and if this effect is frequency dependent: are low frequency songs affected more than high frequency ones? We first measured female sexual responsiveness to low frequency (1 to 5 kHz) A phrases (A1-5) broadcasted at 65 dB level in a control (silence) condition and in three different urban noise levels (65, 71 and 77 dB). Copulation solicitation displays (CSD) were used as a behavioural index of female sexual responsiveness. Increasing noise level gradually affected female responses and females (n=13) displayed on average 32% less CSDs in the 77dB condition when compared to the control condition. A different group of females (n=15) was tested with the previous lowpitched ­ A1-5 and also high-pitched (3 to 7 kHz) A phrases ­ A3-7 both in a control (silence) condition and in a noisy (77dB) condition. Both the condition (silence versus noise) and the frequency level (low versus high) affected female preferences and there was a significant interaction between these two factors. Females displayed significantly more for low-frequency phrases in the control condition but not in the noisy one: noise significantly decreased

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the CSD response level for the low-frequency A phrases but did not affect the CSD response level for the high-frequency ones. Email address for correspondence: yahoo.fr Poster Session - Sunday 3rd July 2011 [email protected]

A9.30 Unexpected lack of food frustrates Atlantic salmon

A9.28 The costs of honesty: understanding why animals don't lie

Robbie S Wilson (University of Queensland, Australia), Candice L Bywater (University of Queensland, Australia), Alyssa Gibson (University of Queensland, Australia) and Vincent Van Uitregt (University of Queensland, Australia) Animals must be able to convey information about their inherent physical or reproductive quality to others of their species; this occurs via signals. Though many individuals could benefit by appearing of higher quality than they actually are, most signals are honest. The mechanisms that maintain signal honesty are hotly debated, but the dominant paradigm thought to maintain signal reliability is the Handicap Principle. However, recent theoretical models strongly challenge the Handicap paradigm and suggest handicap signals may play a lesser role than previously believed. We tested two key predictions associated with these recent theoretical models using studies of aggressive signalling among males of the slender crayfish (Cherax dispar). In this system, the signal is the size of the claw itself, but in actual fights the strength of the claw is what determines the winner. Our previous work has shown that claws are not always honest signals ­ in fact, individuals vary in the amount of muscle within the claw. This means that claws of the same size are not always the same strength; some are dishonest signals. Firstly, we investigated the metabolic costs associated with maintaining claws that have large amounts of muscle and strength (honest) with claws that have less muscle and strength (dishonest). Secondly, we tested whether the costs of possessing larger and stronger weapons on their locomotor performance were greater for low-quality than high-quality individuals. Our results are discussed within the framework of the maintenance of signal reliability and the debate over the importance of handicaps. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

Marco A Vindas (Norwegian University of Life Sciences, Norway), Tore S Kristiansen (Institute of Marine Research), Ole Folkedal (Institute of Marine Research), Lars H Stien (Institute of Marine Research), Uniza W Khan (Norwegian University of Life Sciences, Norway), and Øyvind Øverli (Norwegian University of Life Sciences, Norway) Currently there is substantial debate concerning the welfare of teleost fish in aquaculture, and whether fishes have capacities that merit such concern. Knowledge on learning capabilities and awareness in fish is however still lacking in many areas. Classical conditioning using cues signalling food reward have previously been demonstrated in Atlantic salmon (Salmo salar). The aim of this study was to investigate whether fishes are also capable of experiencing frustrative non-reward, i.e. whether sudden lack of reinforcement in a situation that was previously reinforced elicits behavioural responses indicative of stress and reduced well-being. Six tanks containing 200 fish each were conditioned over a period of trhee weeks to respond to light flashes as a conditioned stimulus (CS), presented 30 seconds before food (unconditioned stimulus, US) was distributed. Successful conditioning was demonstrated by congregation of fish in the feeding area in response to CS. Subsequently, three groups were subjected to a frustration paradigm, where the food reward was delayed for a period of 30 minutes. "Frustrated" fish showed enhanced levels of aggression during the period of delayed food distribution. Persistent CS-US uncoupling over a period of eight days also led to a significant increase in growth heterogeneity, an indication that feeding hierarchies were more pronounced in this treatment. These results indicate that fish respond to the omission of an expected reward by increased levels of aggression. Increased aggression in this context may represent a coping response, i.e. dominant individuals using displaced aggression towards subordinates as a stress reducing outlet. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.29 The outcome of fights for social dominance can be predicted by previous behavioural tests in zebrafish (Danio rerio)

Josefin S Dahlbom (Uppsala University), David Lagman (Uppsala University), Houner Ismail (Uppsala University), Katrin LundstedtEnkel (Uppsala University), Fredrik L Sundström (Uppsala University), Svante Winberg (Uppsala University) It has repeatedly been shown that animals that are bold towards a predator or in a novel environment also are more aggressive. In addition, the winner­loser effect has been studied and proved to be important. Even though the zebrafish is a schooling species it shows intense aggressive behaviour when kept in pairs. We performed a battery of behavioural tests to screen animals for boldness and then set up dyadic fights for social dominance. The subsequent social status of all fish could be correctly predicted from the behavioural tests.In addition, gene expressional analysis clustered upregulation of genes related to the dopamine and serotonin systems with being subordinate and upregulation of genes within the HPI-axis (corticotrophin, arginine vasotocin) with being dominant. The correlation betweenbrain levels of monoamine transmitters and the expression of their receptors and transporters will be analysed during the spring.

A9.31 Is there a behavioural syndrome in domestic canaries? Preliminary results

Davy Ung (Laboratoire d'éthologie et cognition comparées, France) and Gérard Leboucher (Laboratoire d'éthologie et cognition comparées, France) Behavioural syndromes have been defined as a suite of correlated behaviours across time and/or situations and are analogous to personality and temperament. Behavioural syndromes seem to be widespread among species and have received increasing attention from researchers in the last few years. Previous studies have shown that personality traits can have genetic and physiological basis and are correlated with essential aspects of animal's fitness such as survival rate and reproductive success. As the first step of a project aiming to study the relationships between personality and social behaviours, we investigated the existence of a behavioural syndrome in female domestic canaries (Serinus canaria). We measured six personality traits in 29 individuals using tests of activity, obstinacy, sociability, boldness, neophobia and aggressiveness. We also evaluated social dominance using competitions for food. All tests were performed twice at a 10-day interval. Repeatability analyses indicate that individuals behaved consistently across time for all personality traits. Correlations between personality traits showed the existence of a limited behavioural syndrome with obstinacy going hand in hand with activity. At last, social dominance

Abstracts 2011 was correlated with sociability only. The same protocols are being carried out at the moment on males. Results will be used in further experiments to study possible relationships between personality and social behaviours. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

147 differentially expressed. By examining the brains of fish which showed shifts in boldness, seven key genes were identified that also shifted towards the opposing phenotype of bold or shy. These results demonstrate that individual variation is more dynamic than previously thought and that bold and shy fish have divergent transcript profiles that correlate with these behavioural traits. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.32 Investigating the relationship between social rank and behavioural tendencies in crayfish

Jonathan Case (University of Hull), Thomas Breithaupt (University of Hull) Many studies have shown that animals from a variety of taxa display behavioural tendencies which differ between individuals. This may be somewhat surprising, since such tendencies bias individuals towards a specific behavioural response in any given situation, thereby limiting their capacity for behavioural plasticity. It has been suggested that social conflict is one of the main factors leading to the evolution of behavioural tendencies. Competition for social niches may favour the adoption of alternative social roles (such as different positions within in a social hierarchy) by individuals within a population, thus leading to differing behavioural tendencies. In crayfish, competition for resources leads to the establishment of social hierarchies, through agonistic encounters between conspecifics. In this study we investigate if crayfish (Pacifastacus leniusculus) of differing social ranks display different behavioural tendencies. To this end we test the responses of size-matched dominant and subordinate individuals to aversive stimuli. Crayfish, attracted by food odour receive an aversive stimulus mimicking a nearby predator. We analyse the nature of the response and in particular the latency individuals need to re-emerge from a shelter after this negative experience. We also test the responses of socially naïve crayfish to this stimulus and subsequently expose them to agonistic encounters in order to investigate whether pre-existing behavioural tendencies lead to adoption of subordinate or dominant roles or whether these roles and their associated tendencies only emerge as a result of agonistic encounters. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.34 Intraspecific variation and environmental context affect responses to pain in the rainbow trout

Lynne U Sneddon (University of Chester), Catherine R McCrohan (University of Manchester) Intraspecific variation in behavioural and physiological phenotype results in differing responses within a variety of contexts. This may be problematic for welfare assessment if behavioural responses to circumstances detrimental to welfare differ within species. Fish possess nociceptors similar to those that detect painful stimuli in mammals but their capacity for the central processing of nociception is unknown. In mammals and humans, the perception of pain can be reduced under stressful circumstances. Here, this approach was used to demonstrate that central processing of potentially painful stimuli takes place in a fish (rainbow trout, Oncorhynchus mykiss) and to determine whether candidate gene expression is altered in the brain. Fish given a noxious stimulus displayed fewer behavioural responses when placed in a more barren (stressful) environment, as opposed to one providing some shelter (enriched), suggesting that barren conditions reduces the perception of pain. Moreover, when given the choice of a barren or an enriched environment, noxiously-stimulated fish preferred the barren environment. Differences in plasma cortisol, delta opioid receptor and preopiomelanocortin (POMC) mRNA expression in the brain suggest a role for stress-induced analgesia in the observed reduction in nociceptive responses in barren conditions. Individual variation in stress coping style or boldness also affected responses to pain, with shy fish less likely to show evidence of nociception than their bold conspecifics. Individual variation coupled with stress-induced analgesia may mean assessment of pain is problematic. These findings provide the first evidence for central processing of nociception and possible stress-induced analgesia in a fish. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A9.33 Genes correlated withintraspecific variationin rainbow trout: transcriptomics of boldness

Lynne U Sneddon (University of Chester, UK) "Personality type" or boldness is thought to be an important influence in intra-specific behavioural variation and has been observed in many species including fish. The degree of boldness can have a profound effect upon the decisions made by individuals in different contexts. Studies have shown that the boldness of an individual is consistently expressed across a range of behavioural tests with bold individuals performing more risky behaviours compared with shy individuals. Little is known about how fixed these behavioural traits are or what the molecular basis of these divergent phenotypes are. The rainbow trout, Oncorhynchus mykiss, displays easily discernable bold or shy traits; therefore, the plasticity of boldness was investigated as well as brain transcript profiles using oligonucleotide microarrays. Rainbow trout were given positive or negative encounters by watching others respond to novel challenges or winning or losing fights to examine if prior experience affected boldness. Bold losers or bold observing shy demonstrators became shyer. Shy trout that watched bold demonstrators remained shy, however, shy winners and losers became bolder. The transcript profiles of the brains of bold and shy fish were compared and approximately 1000 genes were significantly

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SEB Main Meeting, Glasgow 2011

A10 - Physiological plasticity of thermal tolerance

A10.1 The yin and yang of thermal adaptation: physiological and behavioural plasticities in a warming world

Michael J Angilletta (Arizona State University, USA), Michael W Sears (Bryn Mawr College, USA) and Lauren B Buckley (University of North Carolina, Chapel Hill, USA) Human activities have triggered unprecedented changes in Earth's temperatures. Organisms can use physiological acclimation or behavioural thermoregulation to cope with their warming world, but we do not know if either of these strategies will enable species to persist in the coming decades. Some genotypes possess great capacity for thermal acclimation, but this capacity does not always enhance performance in novel environments. Other genotypes rely primarily on behavioural thermoregulation and possess limited capacities to acclimate; indeed, many species have already shifted their use of habitats in space and time instead of adjusting their tolerance of high temperatures. Ultimately, physiological and behavioural forms of plasticity should coevolve to minimize the loss of fitness during climate change. A mechanistic theory will be needed to understand the effects of physiological and behavioural plasticity on population persistence, as well as the coevolution of these plasticities in changing environments. Email address for correspondence: [email protected] 10:30 Monday 4th July 2011

Sponsored by:

A10.3 Co-gradient variation in physiological traits of a broadly distributed Australian butterfly (Heteronympha merope)

Madeleine G Barton (The University of Melbourne), Michael R Kearney (The University of Melbourne) Broadly distributed species often exhibit geographic variation in thermally sensitive traits in response to temperature gradients across the landscape. The processes underlying these patterns are often complex, difficult to identify and affect a species' overall capacity to adapt to environmental stress. An understanding of the mechanisms underlying such variation is therefore required to accurately predict a species likely response to climate change. This study focuses on a widely distributed Australian butterfly, the common brown (Heteronympha merope). Timing of adult emergence of this species varies across its range; populations from low latitudes emerge earlier in spring than those at high latitudes. Given this difference in development time, a latitudinal cline in body size is expected, however past and present datasets indicate a standard body size is maintained across the range. To explore the processes underlying this variation, or lack there-of, a common garden experiment was conducted. The impacts of temperature on larval growth, development and body size were measured on individuals collected from five populations throughout the species' range. A significant effect of population on growth and development, but not body size, was detected, suggesting low latitude populations grow and develop faster to attain the same body size as their southern counterparts. Selection pressures that may give rise to these patterns will be discussed. This study is one of few to show co-gradient variation in physiological traits, highlighting the importance of understanding the processes that give rise to variation in a species' response to temperature. Email address for correspondence: [email protected] 11:45 Monday 4th July 2011

A10.2 Costs and benefits of thermal acclimation in the field and in the laboratory

John Terblanche (University of Stellenbosch, South Africa) The relative costs and benefits of thermal acclimation are widely debated and have implications for forecasting responses of ectotherms to climate change at various scales. Here, we discuss the costs and benefits of thermal acclimation for laboratory and field responses of codling moth, Cydia pomonella. Using a component of field-fitness, we demonstrate that low temperature acclimated, laboratory-reared moths are recaptured significantly more (~2-4x) under cooler conditions in the wild relative to warm-acclimated or control moths. However, improvements in low temperature performance in cold-acclimated moths came at a cost to performance under warmer conditions. At high ambient temperatures, warm-acclimation improved field performance relative to control or cold-acclimated moths. Laboratory assessments of thermal activity and their limits matched the field results, indicating that these laboratory assays may be transferable to field performance. This study demonstrates clear costs and benefits of thermal acclimation on laboratory and field performance and the potential utility of thermal pre-treatments for offsetting negative efficacy in pest control programmes under adverse thermal conditions. Thus, the present work shows that evolutionary principles of phenotypic plasticity can be used to improve field performance and thus possibly enhance pest control programmes seeking increased efficacy. Email address for correspondence: [email protected] 11:15 Monday 4th July 2011

A10.4 Cold acclimation response in Drosophila melanogaster: molecular correlates and metabolomic fingerprints

Abstracts 2011 Hervé Colinet (University of Louvain and University of Rennes 1, France), David Renault (University of Rennes 1, France) and Ary Hoffmann (Melbourne University, Australia) To cope with stressful environmental temperatures, organisms can enhance thermotolerance when exposed to sub-lethal temperatures before thermal stress, a phenomenon referred to as thermal acclimation. Acclimation includes different forms: developmental, gradual or rapid. Here we completed a comprehensive assessment of how these interact and affect cold tolerance in Drosophila melanogaster adults. We also investigated whether the distinct forms of acclimation promoted differential molecular responses to cold stress (acute and chronic). Finally we investigated the metabolite profiles of cold-acclimated versus non-acclimated populations in response to acute and chronic cold stresses. Acclimation treatments had very large effects on cold tolerance and resulted phenotypes ranging from sensitive to tolerant individuals within the specific stress applied. Cold acclimation also influenced level of expression several responsive genes (Hsp23, Hsp70, Hsp40, Hsp68, Starvin, and Frost) during recovery cold stress and effects depended on the nature of the acclimation treatment. Complementary interactions occurred between different acclimation forms, and these as well as the different molecular responses point to different underlying mechanisms. Cold acclimation resulted in phenotypes that had very distinct metabolite profiles. The cold-acclimated population was able to maintain metabolite homeostasis after both types of cold stresses, while severe (and irrecoverable) perturbations of metabolite homeostasis were observed in the non-acclimated populations. A number of altered metabolites (e.g. fructose, saccharose and sorbitol) make good candidates for cold-acclimation responses. The results show major differences in Hsps expression and metabolites profiles between different acclimation-related phenotypes and highlight that acclimation may strongly impact field stress resistance. Email address for correspondence: [email protected] 12:00 Monday 4th July 2011 Email address for correspondence: [email protected] 12:15 Monday 4th July 2011

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A10.6 Thermal tolerance, cardiac function and niche utilizationin tunas

Barbara A Block (Stanford University) Bluefin tunas have unique attributes that allow occupation of a wide thermal niche including endothermy and a robust cardiac physiology that is reliant on excitation-contraction coupling. In bluefins, elevated metabolic rates are coupled with heat exchangers that enable heat conservation in muscle, viscera, eye and brain tissues. However, the hearts of all tunas operate at ambient temperatures. This unusual physiological arrangement is unique amongst vertebrates and can result in a cold heart supplying the metabolic demands of warm tissues. To better understand tuna physiology we examined the thermal niche of two closely related species, the bluefin and yellowfin tunas and the cardiac physiology of both species in the lab. Mean daily surface temperatures from 376 electronically tagged juveniles revealed a thermal preference for Pacific bluefin of 17.36°C ±0.05 (s.e.) and for yellowfin tuna of 21.55°C ±0.16. Results from molecular, structural and physiological data indicate a significant role of Ca2+-induced Ca2+ release in the cardiac myocytes of tunas from internal SR stores. We maintain that increasing reliance on SR function is crucial to the performance of the cold-tolerant bluefin heart and that the SR Ca2+ ATPase is the malleable unit of cellular Ca2+ flux, vital for increasing the capacity to operate at both low and high temperatures. Gene expression studies also indicate increased reliance in the bluefin on internal Ca2+ stores. These findings have implications beyond endothermic fish and may help to delineate the key steps required to protect vertebrate cardiac function at extreme temperatures. Email address for correspondence: [email protected] 13:30 Monday 4th July 2011

A10.5 Environmental stress adaptation: multi'omic and network approaches to the underpinning molecular mechanisms

Andrew R Cossins (University of Liverpool) Living organisms often display powerful tolerance adaptions to seasonal cold. The underpinning mechanisms of this in animals were linked to changes in lipid saturation, though this was not involved in acquired cold resistance in C. elegans is not linked to the regulation of lipid saturation by desaturases. We have adopted large-scale screening approaches of transcripts and metabolites to seek alternative mechanisms of adaptations and to identify regulatory genes that initiate and control responses. Using Affymetrix arrays we have identified ~8K genes displaying significantly altered transcript abundances with very conservative statistical criteria. The overall pattern indicates a very large-scale down-regulation of intermediary metabolism, and long-term changes to chromatin. We have also identified many new candidate genes, including a novel RNA-binding protein that shows a 60-fold upregulation within three hours. Second, a metabolomic screen of 117 compounds link the concentration of several compounds with the gains and loss of cold tolerance, and their involvement was confirmed by the cold protective effects of these compounds when supplemented into the growth medium at 20°C. Third, we have used the RNAi technique to screen ~400 regulatory and other candidate genes for a role in inducible cold protection, and the positive genes have been directed the analysis of the emergent networked picture linking upstream regulatory interactions with the downstream effector responses. Finally, we are collaborating with European labs in the use of expression QTL to identify regulatory gene loci. These loci that are serve as foci for the network-based analysis of regulatory pathways.

A10.7 Tolerance to sustained high temperatures in two coral-dwelling gobies adapted to high daytime temperatures

Christina Sørensen (University of Oslo, Norway), Philip L Munday (James Cook University, Australia) and Göran E Nilsson (University of Oslo, Norway) Tropical reef fishes might be sensitive to rising ocean temperatures from global warming because they have evolved within a relatively narrow and stable temperature range. However, some coral reef fish inhabiting shallow waters are already exposed to high daytime temperatures during extreme low tides. Two such species are the closely related coral-dwelling gobies Gobiodon histrio and Gobiodon erythrospilus. These sister species have identical habitat preference, yet they have different latitudinal distributions with G. histrio more prevalent in warmer, low latitude waters. We used closed respirometry to investigate the thermal reaction norms of both species at the overlapping mid-point of their distributions. Resting oxygen consumption (MO2) and hypoxia tolerance ([O2]crit) increased with temperature in a similar way for both species, suggesting that their aerobic capacities are similar. [O2]crit increased less than observed in other coral reef fishes, and they thus perform relatively well at elevated temperatures. The species differed in anaerobic capacity: At 33°C (4°C above the current average summer temperature) a significant increase in [O2]end (oxygen saturation when ventilation stopped) and decrease in anaerobic survival time (time from reaching [O2]crit to termination of ventilation) were seen in G. erythrospilus, but not G. histrio. Mortality was significantly increased at 33°C for G. erythrospilus, but not for G. histrio.

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Society for Experimental Biology Under constant temperature, the magnitude of hsp induction was positively correlated with acclimation temperature, whereas under variable temperature, the magnitude of hsp induction was negatively correlated with acclimation temperature variability. Thus, there could be distinct energetic consequences of increasing mean temperature versus thermal variability. The most strongly induced hsp genes varied with acclimation conditions. There was large variation of thermal induction within HSP gene families (e.g., hsp70), highlighting the different cellular roles of these chaperonins. Email address for correspondence: [email protected] 14:45 Monday 4th July 2011

These results suggest that coral reef fishes already routinely exposed to elevated water temperatures may perform better in a global warming scenario, and that anaerobic performance can explain species differences in distribution along the temperature gradient. Email address for correspondence: [email protected] 14:15 Monday 4th July 2011

A10.8 Control of ion transport by mitochondrion-rich chloride cells of eurythermic teleost fish: Cold shock versus cold acclimation

William S Marshall (St Francis Xavier University, Canada) Seawater acclimated eurythermic mummichogs (Fundulus heteroclitus L.) were acclimated to 5 and 20ºC for four weeks. From warm-adapted fish, opercular epithelia containing numerous mitochondrion-rich chloride cells were removed, mounted in Ussing-style membrane chambers and cooled to 16, 13, 10, 5 and 2.5ºC, then subjected to hypotonic shock that normally inhibits Cl- secretion (as Isc). Initially, cold exposure slowed Cl- secretion with a Q10 of 1.24-1.78 and membranes responded rapidly and reversibly to hypotonic shock, but below 10ºC a step decrease (Q10 = 6.98 from 10 to 5ºC ) occurred and membranes were refractory to hypotonic shock. By immunocytochemistry, Focal Adhesion Kinase (FAK) phosphorylated at tyrosine 407 (pY407) colocalized with CFTR in apical membrane and dephosphorylated with hypotonic shock at 20ºC but failed to dephosphorylate at 5ºC. Opercular epithelia from cold-adapted fish at 5ºC responded normally to hypotonic shock and to warming to 20ºC with a large overshoot in Isc above the levels for 20ºC acclimated tissues. Cold and warm adapted membranes had the same densities of MR cells (by DASPEI fluorescence), but cold shock of warm-adapted membranes reduced density of exposed apical crypts (by SEM), indicating cold shock causes paving over of MR cells and concomitant reductions in Isc. Cold-adapted mummichogs had increased polyunsaturated and decreased saturated fatty acids, indicating homeoviscous adaptation. In conclusion, eurythermic fish adapt osmoregulatory systems to cold by maintaining membrane fluidity and the complex transport regulation pathways. Supported by NSERC, CFI, UCR Email address for correspondence: [email protected] 14:30 Monday 4th July 2011

A10.10 Seasonal adaptations in high arctic Svalbard rock ptarmigan

Jonathan R Codd (University of Manchester) The Svalbard rock ptarmigan, Lagopus muta hyperborea experiences extreme photoperiodic and climatic conditions on the Arctic archipelago of Svalbard. This species, however, is highly adapted to live in this harsh environment. One of the most striking adaptations found in these birds is the deposition, prior to onset of winter, of fat stores which may comprise up to 32% of body mass and are located primarily around the sternum and abdominal region. This fat, while crucial to the birds' survival, also presents a challenge in that the bird must maintain normal physiological function with this additional mass. In particular these stores are likely to constrain the respiratory system, as the sternum and pelvic region must be moved during ventilation and carrying this extra load may also impact upon the energetic cost of locomotion. Here we demonstrate that winter birds have a reduced cost of locomotion when compared to summer birds. A remarkable finding given that during winter these birds have almost twice the body mass of those in summer. Svalbard ptarmigan are also extremely efficient at terrestrial locomotion. As energy conservation is paramount to these birds, minimizing the costs of moving around when resources are limited would appear to be a key adaptation crucial for their survival in the barren Arctic environment. Email address for correspondence: [email protected] 15:30 Monday 4th July 2011

A10.11 A10.9 Heat shock protein gene expression in porcelain crab, Petrolisthes cinctipes, following acclimation to a range of constant and fluctuating thermal regimes

Jonathon H Stillman (San Francisco State University, USA) At critical temperatures organisms expend increased energy to induce the expression of heat shock proteins (HSPs), and to use those proteins to chaperone unfolded proteins. Increased expression of HSPs is known to be associated with increasing thermal tolerance at a tradeoff of reducing fitness under benign conditions. Organisms living in highly thermally variable and unpredictable environments, such as the marine intertidal zone, must engage in an energetic tradeoff between increasing expression of HSPs to protect against possible thermal extremes that may occur during low tide versus decreasing expression of HSPs to increase fitness under the benign thermal conditions during high tide. Porcelain crabs living in the marine intertidal zone experience seasonal and latitudinal variation in seawater temperature during high tide and body temperatures can increase 20°C during low tide. Using a 25K unigene cDNA microarray built from a 98 K EST database, the expression of 92 different hsp genes was profiled in heat stressed and control crabs following acclimation to a 14°C range of constant temperatures, or daily thermal variability of up to 20°C.

Body temperature variation of free ranging South African antelopes across a climatic gradient

Anil K Shrestha (Resource Ecology Group, Wageningen University, The Netherlands), Sip E Wieren (Resource Ecology Group, Wageningen University, The Netherlands), Frank V Langevelde (Resource Ecology Group, Wageningen University, The Netherlands), Andrea J Fuller (Brain Function Research Group, University of Witwatersrand, South Africa), Robyn S Hetem (Brain Function Research Group, University of Witwatersrand, South Africa), Leith Meyer (Brain Function Research Group, University of Witwatersrand, South Africa), Steven D Bie (Resource Ecology Group ,Wageningen University, The Netherlands) and Herbert H Prins (Resource Ecology Group, Wageningen University, The Netherlands) To understand adaptive capacity of species in response to global change, we investigated body temperature (Tb) variation of three African antelopes: eland, wildebeest and impala along a climatic gradient of less seasonal/mild winter (Mapungubwe National Park) and more seasonal/long cold winter (Asante Sana Game Reserve) in South Africa using abdominally implanted temperature data loggers. We hypothesized that habitat with long cold winters are suboptimal for these African antelopes and therefore, antelopes in Asante Sana are expected to exhibit a higher variation (amplitude) in Tb during winter because of higher energetic cost to maintain homoeothermic status. We also predict that antelopes in Asante Sana will reduce their

Abstracts 2011 minimum body temperature (MinTb) during winter to reduce Tb and ambient temperature gradient to save energy. Neither eland nor impala showed significant variation in body temperature between study areas or a reduction of MinTb, irrespective of season. However, in wildebeest, Tb variation did differ significantly during winter and spring. Unlike our expectation, wildebeest in Mapungubwe showed a higher variability in Tb as much as 4ºC and also a lower MinTb during winter (36ºC ±0.8) and spring (36.5ºC ±0.5). This variation in Tb and reduced MinTb in Mapungubwe wildebeest was influenced by amplitude of ambient temperature (positive) and cumulative rainfall (negative). We interpreted this response of wildebeest in Mapungubwe was to cope with nutritional and water stress during winter and spring. With an expected increase of drought frequency in Southern Africa, wildebeest and other grazers likely will experience greater nutritional and water stress in future. Email address for correspondence: [email protected] 16:00 Monday 4th July 2011

151 potential (RP) and excitatory junction potential (EJP) do not predict the responses in seasonal acclimatized crabs. The possibility of a photoperiod influence on these adaptive responses to acclimation arises. No consistent effect of either short or long day photoperiod was found on muscle membrane RP in 8ºC or 22ºC acclimated crabs. However, in 8ºC acclimated crabs short day exposure resulted in consistently higher EJP amplitudes than following long day exposure. In 22º C acclimated crabs day length had a less marked effect, but short day appears to alter the pattern of response to temperature towards that for 8ºC acclimated crabs. Finally, photoperiod was shown to have an effect on muscle tension, particularly in 8ºC acclimated crabs. Long day as compared to short day exposure resulted in a higher force generated at temperatures below about 17ºC with a marked temperature dependency. In 22ºC acclimated crabs the difference between short and long day exposure was less marked but in this case the force generated was consistently greater following short day exposure. Thus photoperiodic effects may play a significant role in the acquisition of acclimatization. Email address for correspondence: [email protected] 16:30 Monday 4th July 2011

A10.12 Revisiting oxygen supply and demand ­ oxygen availability, metabolic rate and thermal limits in aquatic ectotherms

Wilco Verberk (University of Plymouth, UK), David T Bilton (University of Plymouth, UK), Piero Calosi (University of Plymouth, UK) and John I Spicer (University of Plymouth, UK) Oxygen supply and demand are central to the hypothesis that oxygen limitation can set whole animal thermal tolerance limits. Yet there is no consensus as to what drives environmental oxygen availability. Here we resolve the question whether partial pressure or solubility limits oxygen supply in nature. Intriguingly, by returning to the first principles of gas diffusion, it becomes clear that more oxygen is actually available to an organism in warmer habitats, something which is exactly opposite to current wisdom. Observed oxygen shortages in warmer habitats are not caused by lower oxygen concentrations, but instead arise through animal oxygen demand exceeding supply. The role of both oxygen supply and oxygen demand in setting thermal limits was confirmed in an experiment on the aquatic nymphs of a stonefly: hypoxia lowered thermal maxima, whilst hyperoxia increased them. At the same time, individuals that strongly increased oxygen uptake at elevated temperatures had lower thermal maxima. These results on aquatic nymphs contrast with recent studies on terrestrial arthropods and suggest that many insects may be affected by oxygen limitation at some stage during their life cycle. Our discovery that environmental oxygen supply is actually higher in warmer habitats, represents a significant shift in our understanding of how oxygen shapes aquatic communities and makes physiological adaptations to escape oxygen limitation at thermal extremes more feasible. Email address for correspondence: [email protected] 16:15 Monday 4th July 2011

A10.14 Thyroid hormones 3,3,5-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2) are important thermoregulators in zebrafish (Danio rerio)

Alex G Little (University of Sydney, Australia) and Frank Seebacher (University of Sydney, Australia) In mammals, thyroid hormone (TH) is a principal regulator that stimulates metabolic heat production via brown adipose tissue and skeletal muscle. While regulators of metabolism in mammals are relatively well understood, those in ectotherms remain unknown. To determine whether TH promotes thermal acclimation in ectotherms, we induced hypothyroidism in zebrafish (Danio rerio) during three weeks' acclimation to 18ºC or 28ºC using propylthuiouracil and iopanoic acid. The cold-acclimated control fish had significantly increased endurance swimming capacities and upregulated mRNA levels of metabolic genes (PGC1; PGC1; PPAR; COX VB; COX II; F0 F1-ATP, ; ATPase 8/6) in liver and muscle compared to warm-acclimated controls. Hypothyroidism abolished the ability to cold-acclimate as measured by Ucrit and muscle mRNA levels. Although 3,3,5-triiodo-L-thyronine (T3) is widely recognized as the only active TH, recent work has shown that 3,5-diiodo-L-thyronine (T2) also stimulates metabolism. To determine whether T2 and/or T3 promote the metabolic effects of cold-acclimation, we supplemented hypothyroid fish with T2 or T3 during acclimation to 18ºC. Both T2 and T3 promoted the recovery of cold-acclimation (Ucrit and muscle mRNA levels). For the first time, we have identified central regulators underlying metabolic acclimation in ectotherms. Like T3, T2 also regulates the transcription of important metabolic genes such as PGC1. While T2 has been shown to stimulate metabolism, it has never, to our knowledge, been implicated in a physiological role until now. Because THs are conserved even back to the earliest invertebrates, these findings suggest that they may act universally to integrate environmental cues with acclimation response. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.13 Do temperature and photoperiod interact in the adaptive responses to temperature in the neuromuscular system of crabs?

Ken Bowler (University of Durham, UK), David Hyde (University of Durham, UK), Suhaila Qari (University of Durham, UK) and Richard Hopkin (University of Durham, UK) Evidence is reviewed demonstrating that the adaptive responses to temperature made by the walking leg neuromuscular system of crabs (Carcinus maenas and Cancer pagurus) are in response to local temperature and not in response to hierarchical influences by the CNS and hormonal systems. Evidence is presented showing that the laboratory acclimation responses in muscle membrane resting

A10.15 Autonomic control of the heart in the bullfrog at three temperatures: a comparison with the African clawed toad

Edwin W Taylor (University of Birmingham, UK), Nini Skovgaard (Aarhus University, Denmark), Cleo Leite (UNESP, Brazil), Marina

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Society for Experimental Biology Zuzana Starostova (Charles University, Prague, Czech Republic), Michael J Angilletta Jr. (School of Life Sciences, Arizona State University, USA), Lukas Kubicka (Charles University, Prague, Czech Republic) and Lukas Kratochvil (Charles University, Prague, Czech Republic) In ectotherms, environmental temperature is the most prominent abiotic factor that modulates life-history traits. Here, we explore influence of environmental temperature on maternal investment to reproduction. We kept females of the Madagascar ground gecko (Paroedura picta) at three constant temperatures (24, 27, 30°C) and recorded their reproductive traits. As with all geckos, females of this species lay clutches of only one or two eggs within short intervals. For one clutch per female, we measured the wet mass and dry mass of eggs directly, and estimated energy content through bomb calorimetry. Increasing temperature positively influenced reproductive rate. The mean intervals between clutches were 9, 10 and 15 days at 30, 27 and 24°C, respectively. Although females did not differ in body mass or body length, they laid different sizes of eggs. Females at 30°C laid significantly smaller eggs than did females at 24 or 27°C. Eggs from different thermal treatments also differed in water content, but the scaling of relative water content did not differ among treatments. Females at all temperatures produced eggs with the same energetic density. Females at 24°C allocated fewer calories per day to reproduction than did females from higher temperatures; however, females at 24 or 27°C allocated significantly more energy per egg than did females at 30°C. Our results accord with previous studies that revealed strong thermal effects on egg size and reproductive output in ectothermic animals. Email Address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

Sartori (UNESP, Brazil), Gabrielle De Paula (UNESP, Brazil) and Augusto Abe (UNESP, Brazil) In heterothermic animals heart rate and degree of autonomic tone on the heart typically vary with temperature. A predominant inhibitory tone is exerted by the parasympathetic system via the vagus nerve that is supplied by vagal preganglionic neurones (VPN) in the brainstem. This study measured autonomic tone on the heart of the bullfrog (Rana catesbeiana) at 10, 20 or 30°C and correlated the results with a neuranatomical study of the distribution of VPN in the brainstem. The results are compared with an earlier study on the toad, Xenopus laevis (Taylor and Ihmied, 1995). Although heart rate increased with temperature, cardiac vagal tone in Rana was below 10% at all 3 temperatures whereas, in Xenopus it varied from 75% at 5ºC to 329% at 25ºC. In Rana VPN were located in a single medial nucleus within the central grey, whereas Xenopus had an additional distinct lateral group of smaller VPN located in the white matter. It seems possible that high levels of cardiac vagal tone are associated with dual origins for VPN in these amphibians. Supported by CNPq and FAPESP. Reference: Taylor, E.W. & Ihmied, Y.M. (1995). J. therm. Biol. 20, 55-59. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.16 Multigenerational analysis of synergistic effects of temperature and salinity variability on metabolic rate and acute thermal and salinity tolerance in Daphnia pulex

Xi Chen (San Francisco State University, USA) and Jonathon H Stillman (San Francisco State University, USA) Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. We investigated the synergistic effects of multi-generational exposure of n=5 replicate isofemale lines of Daphnia pulex in n=9 treatments of daily fluctuations in temperature (15, 15-25, 15-30°C) and salinity (0, 0-2, 0-5). Metabolic rates and developmental timing were measured at the 1st, 5th and 6th generations. After five generations, newborns of the 6th generation were cultivated in either each of the nine experimental treatments, or at the control condition (15°C, salinity 0). No statistically significant differences in metabolic rates were found among all treatments at the 1st generation. However, Daphnia exposed to both higher temperatures and salinities showed significantly slower metabolic rates at both the 5th and 6th generations. Metabolic suppression was maintained in Daphnia reared at the control condition after six generations. Exposure to lower temperature may slow growth due to Q10 effects, whereas exposure to higher temperatures may slow growth due to energetic demands of constitutively upregulating stress responses. Acute tolerance to temperature (LT50 ) and salinity (LC50) was measured after the 14th generation. LT50 and LC50 were both highest in cultures raised at 15-30°C but 0 salinity. Both temperature and salinity tolerance decreased in Daphnia reared under the most extreme conditions. Our results indicate that increasing temperature confers cross tolerance to salinity stress, but that exposure to multiple stressors does not lead to greater overall tolerance. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.18 Will climate changes affect the ability of fish to process and utilise food? Effects of temperature and pH on gut motility in Atlantic halibut

Catharina Olsson (Zoology), Kerstin Wiklander (Mathematical sciences), Henrik Seth (Zoology), Fredrik Jutfelt (Zoology), Michael Axelsson (Zoology), Erik Sandblom (Zoology), Albin Gräns (Zoology) Since the beginning of the industrial era, the extensive use of fossil fuels has lead to an increase in atmospheric CO2. It has been predicted that the oceans not only get warmer due to global climate changes, but also more acidic. This will likely affect many physiological parameters in fish, not least the ability to process and utilize food. Here we focus on gut motility. As part of a large scale study where we attempted to mimic possible future scenarios combining changes in pH with different temperatures, Atlantic halibut were divided into 12 groups, each subjected to a different combination of temperature and pH, and acclimated for approximately four months. The stomach was dissected out and longitudinal strip preparations were mounted in organ baths, at the acclimation temperature. Cumulative concentration-response curves for carbachol (10-8 to 10-4 M) were generated as well as single dose exposures to 50 mM KCl. While the maximal response to both KCl and carbachol showed clear temperature-dependence, pH did not show any general effect on the contractility. At 6°C as well as 18°C, the amplitude of contractions was lower than at intermediate temperatures. The EC50 values for carbachol however, showed no differences between the groups. The relation between temperature and maximal contraction follows a similar curve as the relation between temperature and growth in these fish. Hence, it might be suggested that the lower growth rate at 6°C and 18°C is related to impaired processing of the food. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.17 Temperature influences energy allocation to reproduction in a tropical gecko, Paroedura picta

Abstracts 2011

153 Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.19 Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the Marine toad (Rhinella marina)

Johannes Overgaard (Aarhus University, Denmark), Jonas L Andersen (Aarhus University, Denmark), Anders Findsen (Aarhus University, Denmark), Pil B Pedersen (Aarhus University, Denmark), Kasper Hansen (Aarhus University, Denmark), Karlina Ozolina (University of Manchester, UK) and Tobias Wang (Aarhus University, Denmark) A number of recent studies have convincingly correlated reductions in aerobic scope to the upper thermal tolerance limit of aquatic ectothermic animals. The reduction in aerobic scope has been associated with a progressive failure of capacity of the cardiovascular system to transport oxygen with increased temperature. Here we investigated whether a similar conceptual model can be applied to an air-breathing and semi-aquatic vertebrate ­ the toad Rhinella marina. The potential effects of acclimation on thermal tolerance were investigated by chronic acclimation to 20, 25 and 30°C, respectively, but we found no differences in the thermal responses between acclimation groups. The absolute difference between resting and maximal oxygen consumption increased progressively from 10 to 40°C, such that there was no decrease in aerobic scope even at temperatures approaching the lethal limit (41°C). Haematological and cardio-respiratory measurements were performed at rest and immediately after strenuous activity at benign (30°C) and critically high (40°C) temperatures. Within this temperature interval, both resting and active heart rate increased proportional to the increase in oxygen consumption and there was no indication of respiratory failure as evident from the high arterial oxygen saturation, pO2 and [HbO2]. In conclusion, we found no evidence of cardio-respiratory failure at temperatures immediately below lethal temperatures, indicating that maintenance of aerobic scope and oxygen transport is not related to the upper thermal limit of this air-breating semi-aquatic vertebrate. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.21 Is the resting metabolic rate of juvenile Atlantic halibut independent of acclimation temperature?

Henrik Seth (University of Gothenburg, Sweden), Albin Gräns (University of Gothenburg, Sweden), Erik Sandblom (University of Gothenburg, Sweden), Fredrik Jutfelt (University of Gothenburg, Sweden), Elisabeth Jönsson-Bergman (University of Gothenburg, Sweden), Kerstin Wiklander (Chalmers University of Technology, Sweden), Sam Dupont (University of Gothenburg, Sweden), Olga Ortega-Martinez (University of Gothenburg, Sweden), Kristina Sundell (University of Gothenburg, Sweden) and Michael Axelsson (University of Gothenburg, Sweden) When the temperature is increased within a relevant interval, most physiological processes expedite and this is reflected in an animals resting metabolic rate. In the current study juvenile Atlantic halibut were acclimated for four months to sox different temperatures (6, 10, 12, 14, 16 and 18°C). Using intermittent flow-through respirometry oxygen consumption was measured for 24 hours. Resting metabolic rate was calculated from the three lowest recordings. In comparison with all other groups, the resting metabolic rate was significantly lower in fish acclimated to 6°C and higher when acclimated to 18°C. However, the resting metabolic rate of fish acclimated to 10, 12, 14 and 16°C did not differ but showed a distinct metabolic plateau at these temperatures. Importantly the growth rate in these acclimation groups was the same except for a significantly decreased growth in the 16°C group. In contrast to this Atlantic halibut acclimated to 10°C show a linear increase in resting metabolic rate when subjected to an acute stepwise temperature increase from 10 to 16°C (2°C h-1). Our results show that juvenile Atlantic halibut have a stable resting metabolic rate over a wide range of acclimation temperatures and that they seem to prioritize this level of metabolic rate even at 16°C when growth is suppressed. The benefits of maintaining a fixed level of resting metabolic rate over a range of acclimation temperatures will be discussed. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A10.20 Thermal fluctuations in insects: how much does it improve survival in chill susceptible species?

Hervé Colinet (Catholic University of Louvain, Belgiium), Lisa Lalouette (University of Paris 6, France) and David Renault (University of Rennes 1, France) To date, the effects of thermal fluctuations on insects' survival have usually been assessed using two temperatures cycling over 24 hours. Therefore, important combinations `recovery temperature' x `duration of the recovery' may have been overlooked and that the subsequent picture drawn on the survival under FTR remains incomplete. In this study, we aimed to investigate the thermal sensitivity in adults of the tropical beetle Alphitobius diaperinus and hypothesized that fluctuating thermal regimes enhance the cold survival of this species compared to constantly cold-exposed adults. We examined: (i) survival at constant low temperatures (0, 5, 10 and 15°C); and (ii) at 25 fluctuating thermal regimes using 0°C as the low temperature and combined with recovery temperatures ranging from 5 to 20°C and durations of recovery periods ranging from 0.5 to 4.0 hours. All combinations improved the duration of survival in adult A. diaperinus compared to constantly cold exposed insects. However, the fluctuating thermal regimes moderately increased the duration of survival when the recovery temperature was below 15°C or when the duration of the recovery was less than 2.0 hours. Above this thermal threshold (15°C), the beneficial effect of the warm pulses arise from both reduced amount of accumulated cold injuries combined with the repair of the chilling injuries. Based on our data, mathematical models suggested that the duration of survival should increase in an exponential manner, concomitantly with the repair of chill injuries when the duration of the recovery period and the recovery temperature increase.

A10.22 Cold hypertrophy and connexin remodelling in fish heart

Andrew J Fenna (University of Manchester, UK) and Holly A Shiels (University of Manchester, UK) Cardiac hypertrophy in humans can lead to sudden death and one reason for this is remodelling of the gap junction protein connexin 43 (Cx43). Indeed, altered expression, phosphorylation and cellular distribution of this conductance channel has been observed in maladaptive mammalian cardiac hypertrophy. In cold acclimated fish, cardiac hypertrophy has been observed but its effect on gap junctions are unknown. We cold acclimated (4°C at eight weeks) rainbow trout and found significant increases in cardiomyocyte area, paralleled with extensive remodelling of the connective tissue and muscle layers of the heart, compared with warm acclimated (17°C) fish. We also found a novel gap junction protein in trout heart at the mRNA and protein level which varied between thermal acclimation groups. Associated with these changes we report temperature dependent differences in signalling molecules (ERK ½, p38 MAP Kinase, GSK - 3 and Smad 3), all of which are of potential importance in both hypertrophic growth and connexin remodelling. These results will be discussed in relation to the effects of chronic temperature on electrical conductance in the trout heart.

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Email address for correspondence: manchester.ac.uk Poster Session - Sunday 3rd July 2011

A10.23 Do thermal windows vary between reproducing females, their eggs and males of the king crab Lithodes centolla?

Daniela Storch (AWI), Raphaela Kathöver (AWI), Hans Otto Pörtner (AWI), Gustavo Lovrich (CADIC-CONICET), Carola Romero (CADICCONICET) and Natasha Schvezov (CADIC-CONICET) Species distribution can be influenced by temperature and might vary between life stages. The most sensitive stages are thought to be reproducing adults and the early life stages including eggs. Therefore, experiments to determine the thermal tolerance windows of ovigerous females, their eggs and males of the king crab Lithodes centolla were conducted. Threshold temperatures (TcI, TpI, TpII, and TcII) defining the thermal tolerance window were identified by break points in ventilation rate and corresponding changes in pO2 and heart rate of females and males and in oxygen consumption of eggs. The implications of our findings for the observed distribution of the king crab will be discussed. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

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SEB Main Meeting, Glasgow 2011

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A11 - New transport systems in vertebrate tissues

A11.1 Intercellular networks in the epididymis: Regulation of V-ATPase-dependent luminal acidification

Sylvie Breton (Massachusetts General Hospital Harvard Medical School, USA) Male infertility is often caused by sperm that have low motility and interact poorly with the oocyte. These functions are acquired in the epididymis. A low bicarbonate concentration and low pH maintain sperm dormant during their transit in the epididymal lumen. HCO3- is secreted from principal cells following stimulation, a process that "primes" sperm before ejaculation. Using a multidisciplinary approach that combines 3D microscopy imaging of intact organs from WT and transgenic animals with real-time ion flux measurements, we showed that clear cells acidify the lumen via an apical vacuolar H+ ATPase (V-ATPase). Luminal HCO3- induces V-ATPase apical membrane accumulation via the bicarbonate-activated adenylyl cyclase, sAC, leading to cAMP increase and PKA activation. The HCO3--induced V-ATPase apical insertion in clear cells allows for the re-establishment of luminal acidic pH. Luminal angiotensin II also regulates V-ATPase via activation of basal cells, which extend narrow body projections that cross the tight-junction barrier to scan the luminal environment. Basal cells then secrete NO, which diffuses out to stimulate proton secretion in clear cells following cGMP elevation. In addition, luminal ATP and adenosine induce V-ATPase apical accumulation in clear cells. ATP is released from sperm and principal cells and is then metabolized into adenosine by ecto-nucleotidases.In conclusion, epithelial cells and sperm in the epididymis have established a complex intercellular communication network for the control of luminal pH. Monitoring and decoding these "intercellular conversations" will provide new frameworks to promote diagnosis and treatment of male infertility related to post-testicular malfunction. Email address for correspondence: [email protected] 13:35 Saturday 2nd July 2011 columnar cell membrane, sequestration in acidified vesicles, vesicle transport via microtubules and exocytosis at the basal membrane. It is further assumed that excess ammonia that is not utilized for amino acid anabolism in the fast growing larva is secreted into the Malpighian tubules for further excretion via the hindgut where extremely high ammonia concentrations were detected. The study was supported by the DFG (WE 2868/1-1-3001961) and NSERC (Discovery Grant). Email address for correspondence: [email protected] 14:25 Saturday 2nd July 2011

A11.3 In vivo Drosophila model for calcium oxalate nephrolithiasis (kidney stones)

Taku Hirata (Physiology Biomedical Engineering and Mayo Clinic College of Medicine), Pablo Cabrero (University of Glasgow, UK), Donald S Berkholz (Physiology Biomedical Engineering), James R. Thompson (Physiology Biomedical Engineering and Mayo Clinic College of Medicine), Julian A. T. Dow (University of Glasgow, UK), Michael F. Romero (Physiology Biomedical Engineering and Mayo Clinic College of Medicine) Nephrolithiasis (kidney stone formation) is a major public health problem with a complex and varied aetiology. Most stones are composed of calcium oxalate (CaOx), with dietary excess a risk factor. The fruit fly, Drosophila melanogaster, reliably develops CaOx stones upon dietary supplementation, and the nucleation and growth of microliths can be viewed in real-time in vitro. The Slc26 anion transporter, dPrestin, is strongly expressed in Drosophilakidney, and biophysical analysis shows that it is a potent oxalate transporter. When dPrestin is knocked down by RNAi in fly kidney, formation of microliths is reduced, identifying the Slc26 family as key players in oxalate excretion. These results thus demonstrate that stone formation is an ancient conserved process, which can be modelled informatively across >400 My of divergent evolution. Email Address for correspondence: [email protected] 14:50 Saturday 2nd July 2011

A11.2 Ammonia transport in the tobacco hornworm Manduca sexta

Dirk Weihrauch (University of Manitoba, Canada), Anne-Kathrin Blaesse (University of Veterinary Medicine, Hannover, Germany), Gunnar Broehan (University of Osnabrueck, Germany) and Heiko Meyer (University of Osnabrueck, Germany) As documented in numerous publications by Bill Harvey and other investigators, the midgut of the tobacco hornworm, M. sexta represents a very unique epithelium that is energized only by the V-type H+ATPase and consists mainly of two cell types: the goblet cells, which promote a massive K+ secretion, and the neighbouring columnar cells, where nutrition uptake takes place. We found that the midgut of M. sexta exhibits an active uptake of ammonia, a molecule usually toxic to animals. This uptake persisted even against a 100 fold PNH3 gradient and was sensitive to the inhibition of the H+-ATPase, apical (not basal) H+/cation-exchangers and the microtubules network. We further identified an Rh-like ammonia transporter in the midgut of the caterpillar which showed, however, only low mRNA expression levels when compared to expression levels found in the Malpighian tubules and hindgut, respectively. It is presumed that ammonia from the midgut lumen is taken up by a mechanism that features a pH-dependent uptake across the apical

A11.4 Identification of the first animal sucrose transporter

Heiko Meyer (University of Osnabrueck, Germany), Olga Vitavska (University of Osnabrueck, Germany) and Helmut Wieczorek (University of Osnabrueck, Germany) According to a classical tenet, sugar transport across animal membranes is restricted to monosaccharides. Here we report on the first animal sucrose transporter, SCRT (SuCRose Transporter), which we detected in Drosophila melanogaster at each developmental stage. We localized the protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells. The fact that knockdown of SCRT expression results in significantly increased lethality demonstrates an essential function for the protein. Experiments with Saccharomyces cerevisiae as a heterologous expression system revealed that sucrose is a transported substrate. Since the knock out of SLC45A2, a highly similar protein belonging to the mammalian Solute Carrier Family 45 (SLC45) causes oculocutaneous albinism and since the vesicular structures in which SCRT is located appear to contain melanin, we propose that these organelles are melanosomelike structures and that the transporter is necessary for balancing the

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Society for Experimental Biology genes involved in transport are altered. For example, UNC93 and Synaptic vesicle-2 (SV2) are highly induced during arboviral infection. In mammals these genes are involved in intracellular transport in mammals. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant SINV resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Similarly toxin exposure alters not only transport processes but also a number of regulatory genes. A discussion of these genes will be presented Email Address for correspondence: [email protected] 16:40 Saturday 2nd July 2011

osmotic equilibrium during the polymerization process of melanin by the import of a compatible osmolyte. In the hindgut epithelial cells sucrose may also serve as a compatible osmolyte, but we cannot exclude that transport of this disaccharide also serves nutritional adequacy. Email address for correspondence: [email protected] de 15:40 Saturday 2nd July 2011

A11.5 Quantitative phosphoproteomics reveals major vasopressin signalling pathways

Ming-Jiun Yu (Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taiwan), Markus M Rinschen (National Heart Lung and Blood Institute, National Institutes of Health, USA), Trairak Pisitkun (National Heart Lung and Blood Institute, National Institutes of Health, USA), Jason D Hoffert (National Heart Lung and Blood Institute, National Institutes of Health, USA) and Mark A Knepper (National Heart Lung and Blood Institute, National Institutes of Health, USA) Protein phosphorylation is one of the major regulatory processes by which vasopressin mediates renal water transport. Using stable isotope labelling by amino acids in Cell culture (SILAC) coupled with LC-MS/ MS based quantitative protein mass spectrometry, we quantified 2,884 phosphopeptides in a mouse cortical collecting duct cell line mpkCCD in response to vasopressin analogue dDAVP (0.1 nM, 30 minutes) versus vehicle. While the majority (82%) of the quantified phosphopeptides did not show changes in abundance, 273 showed increases and 254 showed decreases in abundance in response to dDAVP. Immunoblotting confirmed the quantitative phosphoproteomic results. Phosphorylation motif analysis revealed dominance of basophilic motifs in the increased phosphopeptides and dominance of proline-directed motifs in the decreased phosphopeptides, suggesting that vasopressin activates basophilic kinases and inactivates prolinedirected kinases. Aquaporin-2 is one of the major targets regulated by vasopressin. Vasopressin increases aquaporin-2 phosphorylation of 2 basophilic sites (serine 256 and 269), and decreases phosphorylation of 1 proline-directedsite (serine 261). In vitro kinase assay showed that basophilic protein kinase A can directly phosphorylate serine 256 while proline-directed kinase ERK and JNK can directly phosphorylate serine 261. In summary, these data show that the vasopressin actions are mediated largely via increases in basophilic phosphorylation and decreases in proline-directed phosphorylation. Email address for correspondence: [email protected] 16:10 Saturday 2nd July 2011

A11.6 Transport processes in the midgut and Malpighian tubules in response to pathogen and toxin exposure

Sarjeet S Gill (University of California, USA), Amy M Evans (University of California, USA), Supaporn Likitvivatanavong (University of California, USA), Jianwu Chen (University of California, USA), Subum Lee (University of California, USA) and Corey Campbell (Colorado State University, USA) The mosquito midgut and Malpighian tubules play critical roles not only for nutrient uptake and excretion but also in the transport of pathogens and are portals for toxin action. However, we know little of how these transport process are affected by pathogen infection and upon intoxication by bacterial pathogens that have been used for the control of larval populations. To examine gene expression on a global level in response to pathogens and toxins we used whole genome microarrays. We show that many

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SEB Main Meeting, Glasgow 2011

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A12 - Multi-scale mechanics of biological and bio-inspired hierarchical materials and surfaces

Sponsored by:

A12.1 Cellular materials in nature

Lorna J Gibson (MIT, USA) and Michael F Ashby (Cambridge University, UK) Cellular materials are widespread in nature. Wood and cork have a honeycomb-like structure with cells that are roughly hexagonal prisms. Trabecular bone, plant parenchyma, adipose tissue, coral and sponge all have a foam-like structure, with polyhedral cells. Natural structures often have a cellular component: skulls and leaves of monocotyledon plants are sandwich structures, with dense outer skins separated by a foam-like core; animal quills and plant stems are nearly fully dense cylindrical shells supported by a foam-like core; and palm and bamboo stems are cylinders with radial density gradients. This talk provides an overview of cellular materials in nature and illustrates how the cellular structure gives rise to increased mechanical performance. Email address for correspondence: [email protected] 14:10 Sunday 3rd July 2011

of a thin closed-pored foam (flavedo), a thick open-pored foam (albedo) and two filmy layers represented by the outer and the inner epidermis. These layers are additionally permeated by fibre bundles, which continuously branch from the inner epidermis towards the flavedo (bundle diameter inside to outside: Ø = 0.50 mm ± 0.10 mm to Ø = 0.30 mm ± 0.04 mm). On the next lower hierarchical level the albedo emerges as three-layered graded foam consisting of foam types with coarser and narrower pores. Biomechanical tests carried out on cylindrical peel samples proved that the fibre bundles stabilize the surrounding foamy tissue and strongly influence its compaction behaviour during quasi-static and dynamic compression. These and other structural findings on different hierarchical levels are currently transferred to casted metal foams, which will be tested under the same experimental conditions as their biological role models. Email address for correspondence: freiburg.de 14:55 Sunday 3rd July 2011 [email protected]

A12.3 Glass sponge skeleton design - lessons in multiscale mechanics

Joanna Aizenberg (Harvard University) Structural materials in nature exhibit remarkable designs with building blocks, often hierarchically arranged from the nanometer to the macroscopic length scales. Skeleton of the hexactinellid sponge Euplectella sp. represents a fascinating example of such elaborate structures. Consolidated, nanometer-scaled silica spheres are arranged in well-defined microscopic concentric rings glued together by organic matrix to form laminated spicules. The assembly of these spicules into bundles, affected by the laminated silica-based cement, results in the formation of a macroscopic cylindrical square-lattice cagelike structure reinforced by diagonal ridges. The ensuing design overcomes the brittleness of its constituent material, glass, and shows outstanding mechanical rigidity and stability. The mechanical benefits of each of eight identified hierarchical levels and their comparison with common mechanical engineering strategies will be discussed. Email address for correspondence: [email protected] 15:40 Sunday 3rd July 2011

A12.2 The influence of hierarchical structuring on the impact behaviour of the pomelo (C. maxima) peel

Marc Thielen (Plant Biomechanics Group, Botanic Garden Faculty of Biology, University of Freiburg, Germany), Thomas Speck (Plant Biomechanics Group, Botanic Garden Faculty of Biology, University of Freiburg, Germany) and Robin Seidel (Plant Biomechanics Group, Botanic Garden Faculty of Biology, University of Freiburg, Germany) Pomelos (C. maxima) weigh up to 1.5 kg and grow on tall trees in heights of approximately 10 m to 15 m. Thereby they have high potential energies and reach impact velocities after being shed of up to 17 m/s. Impact tests with whole fruits proved their peel to be highly suitable as role model for the development of innovative bio-inspired impact resistive structures. To allow the implementation of ideas from biological role models into biomimetic products, various hierarchical levels of the pomelo peel have been analysed in detail according to their structural composition. In technical terms the peel consists of a sandwich structure composed

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A12.4 Self-assembled 3D synthetic calcite spicules with wave-guiding properties

Filipe Natalio (Institut für Anorganische Chemie und Analytische Chemie. Johannes Gutenberg-Universität, Germany), Tomas Corrales (Max Plank Institute fuer Polymer Forschung, Germany), Martin Panthöfer (Institut für Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universität, Germany), Michael Kappl (Max Plank Institute fuer Polymer Forschung), Hans-Jürgen Butt (Max Plank Institute fuer Polymer Forschung, Germany), Werner EG Müller (Institut für Physiologische Chemie Abteilung Angewandte Molekularbiologie, Johannes Gutenberg-Unive, Germany) and Wolfgang Tremel (Institut für Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universität, Germany) Mesoscale hierarchical structures with diverse functions are abundant in Nature. Here we use the self-assembly properties of CaCO3silicatein-system - a protein involved in silica biomineralization ­ to fabricate microsized structures reminiscent those of the calcareous spicules (monoaxon) of Sycon spp. The formation of these synthetic 3D structures starts from CaCO3 crystalline nanoclusters with specific binding of silicatein-a to one specific crystal surface and proceeds via filamentous structures by nanocrystallite cross-linking in a "brick-by-brick" fashion through an protein-protein hydrophobic interactions to microsized 3D structures needle-like structures. Good crystallinity, hierarchical organization, self-assembly, structural homogeneity and alignment of the calcite building blocks impart excellent wave-guiding properties to the synthetic inorganic-organic hybrid materials. Email address for correspondence: [email protected] 16:25 Sunday 3rd July 2011

A12.6 Learning from the past for the future: Fossil plants as concept generators for biomimetic materials and structures

Tom Masselter (Plant Biomechanics Group, Freiburg, Faculty of Biology Botanic Garden, University of Freiburg, Germany) Living organisms represent only a tiny fraction of the total biodiversity of plants and animals that have lived on this planet. The presented idea of searching for biomimetic applications inspired by the fossil record is interesting for several reasons: (1) A by far greater diversity of organisms for studying form and function becomes available. (2) It allows to analyse how, when and under what conditions significant biological structures have evolved and established successfully. Early `prototypes' or `intermediates' might have become extinct but may have played important roles in evolving complex structures for the long term survival of descendents. (3) Structural and functional studies of fossil organisms using theoretical as well as analytical and numerical approaches can be augmented by studies on comparable living species on which relevant biomechanical experiments and studies of the form-structure-function-relationships as well as physiological analyses can be carried out. A focus of this concept is the analysis of a selection of extinct plants that possessed structures and growth forms with a high potential for a transfer into innovative bio-inspired technical products. Of special interest are structures and growth forms that can no longer be found in extant plants. In several extinct groups of tree-like and semi self-supporting fossil plants such `evolutionary lost' structures can be found and offer new concepts for a transfer into biomimetic structures, making these groups particularly suitable for this kind of study. Email address for correspondence: [email protected] 17:05 Sunday 3rd July 2011

A12.5 Enhancing mechanical properties of calcite by Mg substitutions: A quantum-mechanical study

Pavlina Elstnerova (Max-Planck-Institut for Iron Research, Duesseldorf, Germany), Martin Friak (Max-Planck-Institut for Iron Research, Duesseldorf, Germany), Tilmann Hickel (Max-Planck-Institut for Iron Research, Duesseldorf, Germany), Helge Otto Fabritius (Max-PlanckInstitut for Iron Research, Duesseldorf, Germany), Dierk Raabe (MaxPlanck-Institut for Iron Research, Duesseldorf, Germany), Andreas Ziegler (University of Ulm, Germany) and Joerg Neugebauer (MaxPlanck-Institut for Iron Research, Duesseldorf, Germany) Nearly 90% of all animal species in nature protect themselves by a cuticle that represents a hierarchical biocomposite often containing calcite as a mineral stiffening component. Calcite crystals rarely occur in their stoichiometric state and contain impurities. Common impurities in these systems are Mg or P, their role however is still the topic of intense debates. We present results of a parameterfree quantum mechanical study of thermodynamic, structural, and elastic properties of calcite single crystals containing Mg atoms. Density functional theory calculations were performed employing 30 atomic supercells within the generalized gradient approximation (GGA). Based on the calculated thermodynamical results, the site preference of Mg atoms was determined. Examining the structural characteristics, the behaviour of the carbonate group is shown to be nearly independent on either the volume or concentration of Mg atoms. Based on the computed elastic values, the Mg atoms are predicted to stiffen the calcite crystals, specifically to increase the bulk modulus, but also to increase local strains due to the large size mismatch when substituting Ca atoms by Mg ones. Reference; Elstnerova et al., Acta Biomaterialia 6 (2010) 4506-4512 Email address for correspondence: [email protected] 16:45 Sunday 3rd July 2011

A12.7 Silks

Fritz Vollrath (University of Oxford, UK) In silks, proteins are the structural components and water is the solvent. Protein and water combine and separate ­ under ambient pressures and temperatures ­ to make silk threads with a wide range of tunable properties. Spider silk is a case in point for outstanding mechanical performance and hence an excellent starting material in the quest to unravel animal silks and their many trade-secrets. The tools we use to study silks are all based on comprehensive biological understanding and include the `mining' for natural silks with interesting properties in combination with state-of-the-art experimental analysis and polymer modeling. Such studies provide us with novel insights into the behaviour of silk proteins and their interaction with water, the great modifier of material properties. So far, these studies have lead us to a number of important discoveries ranging fromtunable nano-scale composite structures (that absorb energy hydro-electrically) to complex self-assembling micro-machines (that absorb energy mechanically) all the way to the building of complex webs cleverly engineered to absorb energy aerodynamically. Silks are not only interesting as highly evolved natural materials but seem to have a bright future both as models to guide our understanding of energy efficient bio-polymers but also as prototype models to guide the design of totally novel polymer systems be it for medicine or engineering. Email address for correspondence: [email protected] 10:30 Monday 4th July 2011

Abstracts 2011

159 is currently developed, based on mechanical properties of five hulls, representing the different tissue layers. Email address for correspondence: [email protected] de 11:35 Monday 4th July 2011

A12.8 The coagulation of plant latex as a fast self-repair mechanism

Georg Bauer (Plant Biomechanics Group, Faculty of Biology, University of Freiburg, Germany), Anke Nellesen (Fraunhofer Institute, UMSICHT, Oberhausen, Germany) and Thomas Speck (Plant Biomechanics Group, University of Freiburg, Germany, Competence Network Biomimetics and BIOKON) Many discussions on the function of plant latex can be found in the literature. Thereby, a defence mechanism is the most established opinion. Although further functions, such as a nutrition or waste storage and transport system, are proposed by other scientists, a self-healing mechanism was not mentioned. In this study we examined the latex coagulation mechanism of plant species from several genera and quantified the self-healing effect of Ficus benjamina latex. We compared our results with the well-known coagulation theory for Hevea brasiliensis latex, which reports the sealing of lesions after injury by cross linking rubber particles via the protein hevein. In the intact laticifer this protein is stored in vacuolar structures that burst due to the pressure drop upon injury and thus release the protein hevein. We could prove that the mechanical properties of Ficus benjamina stems were partly restored after a short period of time after injury due to the coagulation of latex. So, in addition to a fast sealing of lesions in the bark, a fast self-healing effect can be attributed to the coagulation of Ficus benjamina latex. Studies on the mechanisms of latex coagulation of plant species from the genera Ficus, Euphorbia and Campanula comprised IR-spectroscopy, duration of latex coagulation, pressure and oxygen dependency as well as particle size analyses. Our results suggest different mechanisms of latex coagulation for different taxonomic groups, ranging from simple physical effects like evaporation to coagulation via chemical bonds comparable to the above mentioned theory for Hevea brasiliensis. Email address for correspondence: [email protected] de 11:15 Monday 4th July 2011

A12.10 Hydro-actuated movements of plants

Ingo Burgert (Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Germany), Matthew J Harrington (Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Germany), Michaela Eder (Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Germany), John W Dunlop (Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Germany) and Peter Fratzl (Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Germany) Plants are able to actuate movements of their organs for various functions. Impressive examples of sophisticated actuation devices include leaves for trapping of prey (Venus flytrap) or folding for protection (Mimosa), as well as seed and spore dispersal systems (moss peristomes, pine cones etc.). Those organs that perform rapid movements are mainly based on changes of the internal water pressure in living cells. Slower movements are often hydration-based and are conducted by non-living organs. Such directed movements in the absence of a metabolism typically originate at the molecular level from water-swellable polymers in the cell walls and consequently, require a sophisticated hierarchical organization of the plant organs. In this talk various plant structures exhibiting hydro-responsive movements will be presented. A specific focus will be laid on two systems which are both actuated by a swellable cellulose layer filling the lumen of cells, but possessing completely different deformation patterns. The first example is tension wood which generates tensile stresses that bend stems and branches in dicot trees or shorten contractile roots. The other is a honeycomb-like tissue found in seed capsules of ice plants which deforms almost unidirectionally upon swelling and thereby facilitates a hydro-responsive opening and closure of the capsule. The general principle is based on directing the volume increase of active gels by a specific orientation of stiff fibres. This concept could be particularly interesting for biomimetic materials research in terms of self-sustaining convertible structures. Email address for correspondence: [email protected] 13:30 Monday 4th July 2011

A12.9 Self-healing processes in plants as a concept generator for biomimetic self-repairing materials

Olga Speck (University of Freiburg, Germany) Self-sealing and self-healing are very common in nature. As one can assume a high evolutionary pressure on the development of abilities for self-repair in nature, independent evolution of these properties including various mechanisms and structures in different plant groups and plant species is probable. Over the last decade plants have proved to be an important source of inspiration for biomimetic materials and structures with self-repairing effects. Studies revealed self-sealing of the sclerenchymatous outer ring of the twining liana Aristolochia macrophylla mediated by turgescent parenchymatous cortex cells. Due to their internal pressure these cells expand into the (micro)fissures and seal them. This functional principle has already been successfully transferred into the development of a biomimetic patent-registered PU-foam coating polymerized under pressure for pneumatic structures. Within a current R&D-project we selected Delosperma cooperi, a member of the Aizoaceae family with succulent leaves, as an especially promising role model. Growing in arid environments external wounds lead to an exceptional drought stress. Rapid self-repair protects the plant from dehydration and is therefore of eminent selective advantage. After an artificial injury, wound-sealing in leaves of Delosperma cooperi takes place by deformation and movement of the leaf. Two principles are involved: (1) rolling in of the fringes of the lesion within a few minutes; and (2) curvature of the entire leaf within a time span of 20 to 30 minutes. An analytical model describing the self-sealing process

A12.11 Biomechanics and functional morphology of the ramifications of arborescent monoctoyledons

Tobias Haushahn (Plant Biomechnics Group Botanic Garden University of Freiburg), Tom Masselter (Plant Biomechnics Group Botanic Garden University of Freiburg), Thomas Speck (Plant Biomechnics Group Botanic Garden University of Freiburg) The anatomy of arborescent monocotyledons (e.g. Dracaenaceae and Pandanaceae) and their ramifications differ considerably from that observed in softwood and hardwood trees. Their hierarchical stem organisation consists of isolated lignified vascular bundles (fibres) in a partly lignified parenchyma (matrix), representing a typical fibre-reinforced composite. Whilst these fibres run in a regular spiral manner in the main stem and in the branches, they form a densely packed, irregular and intertwined network within stem-branch-transition zone. Stem and branch tissue can be easily distinguished, since the side branches are attached to the main stem by clasping of the main stem. This results in a `flangemounted' structure of the ramification, often showing a distinct notch between stem and branch. Among the different investigated species the ramifications vary considerably in their geometric

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Society for Experimental Biology films of bacteria, micro algae, and other organic material are found on any surface in humid environments. In very humid environments, so called macrofoulers (e.g. barnacles, mussels, tunicates) additionally start to colonize these surfaces. Drifting seeds reach their growing area by drifting with ocean currents, thus they have to be buoyant. However, macrofoulers increase the specific weight of the seeds and decrease their mobility. Therefore, drifting seeds would benefit from surface properties inhibiting foulers to grow on the surface and could hence be a model organism for new kinds of antifouling strategies for technical surfaces like ship hulls and harbour facilities. For a time period of 12 weeks during the summers of 2009 and 2010, drifting seeds of 50 different species were exposed to the North Sea. Additionally, surface properties (e.g. elasticity, hardness, contact angle, microstructure) of the seeds were measured. The results show that 24% of all tested species did not show any foulers at all. With micro- and macroscopic analyses, a large spectrum of microstructures were identified, ranging from hairy and curly over folded to smooth but uneven. Further analyses of the surface characteristics including chemical aspects as well as first drifting seed inspired surfaces samples are in progress. Email address for correspondence: [email protected] 14:55 Monday 4th July 2011

shape as well as in the extent of stem-clasping and indention. Although indentations are usually interpreted as detrimental for mechanical properties, the ramifications show an extraordinary stability under static and dynamic loading. To explain this behaviour, modern engineering techniques and modern imaging methods are applied to gain detailed knowledge of the functional morphology and the material properties on all hierarchical levels of the stem-branch attachment. Concluding from our results, the combined optimization of outer shape and inner fibre arrangement causes the high load-bearing capacity. In addition to basic botanic research, another objective of these investigations is the interdisciplinary knowledge transfer to a technical implementation in biomimetic braided branched fibre-reinforced composites. Email address for correspondence: [email protected] 14:15 Monday 4th July 2011

A12.12 Morphological and anatomical load adaptations of cactus ramifications

Hannes Schwager (Institute for Botany, Technische Universität Dresden, Germany) and Christoph Neinhuis (Institute for Botany, Technische Universität Dresden, Germany) The morphology and anatomy of ramified arborescent columnar cacti differ significantly from other non-succulent tree-like plants. A constriction can be observed at the branch base and even beneath the succulent cortex indentations can be found on the adaxial and abaxial side of the vascular tissue. Although the junction faces the highest bending and torsion stresses it features the smallest diameter, and does not allow large deformations. The implementation of biomechanical models helps understanding the structure-function relationships of the columnar cacti ramifications, especially the mechanical role of their anatomical peculiarities. Therefore the elastic constants of the different tissues were determined in material tests with optical deformation measurements. The latter were used to evaluate representative finite element models of cacti ramifications. Under self-weight conditions the results showed that the load adaptation does not follow the simple rule of stress homogenization and minimization by secondary growth in highly stressed areas. Due to the succulent cortex it seems more advantageous to tune the stress state by geometric changes to already predominant fibre directions. Even if the results indicated the compression stresses on the abaxial side to be partly dissipated by the parenchymatous cortex, the lignified vascular tissue remains the main mechanical support. Under lateral wind drag the tension stress maximum in the vascular tissue occurs in the same region as under dead load and again the cortex partly dissipates the compression stresses on the backside. Technically applied this finding might enhance the use of fibrereinforced composites for structures with limited design space. Email address for correspondence: [email protected] 14:35 Monday 4th July 2011

A12.14 Biologically-inspired reversible adhesives: Where are we now?

Stanislav Gorb (Functional Morphology and Biomechanics Zoological Institute, University of Kiel, Germany) Biological hairy attachment systems demonstrate their excellent adhesion and high reliability of contact. The structural background of various functional effects of such systems is discussed in the present paper. Additionally, it is demonstrated here, how comparative experimental biological approach can aid in development of novel adhesives. Experimental studies show that the effective elastic moduli of fibre arrays and spatula-like terminal elements are low, and this is of fundamental importance for adhesion enhancement on rough substrata and for an increased tolerance to defects at the level of individual contacts. Based on the broad structural and experimental studies of biological attachment devices, the first industrial bioinspired reversible adhesive foil was developed, which adhesive properties were characterised using variety of measurement techniques and compared with the flat surface made of the same polymer. The microstructured foil demonstrates considerably higher pull off force per unit contact area. The foil is less sensitive to contamination by dust particles, and after washing with water, its adhesive properties can be completely recovered. This glue-free, reversible adhesive is applicable in dynamic pick-and-drop processes, climbing robots, and other systems even under vacuum conditions. The foil represents therefore a considerable step towards development of industrial dry adhesives based on the combination of several principles previously found in biological attachment devices. Email address for correspondence: [email protected] 15:35 Monday 4th July 2011

A12.13 Inspired by drifting seeds: towards a new antifouling strategy for technical use

Antje Clasen (Biomimetics-Innovation-Centre (B-I-C), University of Applied Sciences, Germany), Katrin B Mühlenbruch (B-I-C, University of Applied Sciences, Germany), Stefanie Wuttke (B-I-C, University of Applied Sciences, Germany) and Antonia B Kesel (B-I-C, University of Applied Sciences, Germany) Due to the specific characteristics of their habitat, seeds of plants growing at beaches or in marsh areas are exposed to biofouling. These

A12.15 The influence of surface roughness and ambient humidity on the performance of spider adhesion pads

Jonas O Wolff (Zoological Institute, CAU, Kiel, Germany) and Stanislav N Gorb (Zoological Institute CAU, Kiel, Germany) Many spiders are capable of walking on smooth surfaces due to "dry" adhesion. In hunting spiders that do not build webs to catch

Abstracts 2011 prey, each pretarsus bears a hierarchically built hairy adhesive pad consisting of a dense array of flattened setae covered with numerous microtrichia at the substrate facing side. Microtrichia carry spatulate end tips that provide a close contact to the substrate. Previous studies on the hairy attachment devices of beetles, flies, and lizards showed that attachment forces are influenced by surface profile. In this study, traction forces of running spider Philodromus dispar (Araneae, Philodomidae) were estimated on rough epoxy resin surfaces (asperity size 0.3, 1, 3, 9, 12 µm) and on smooth surface as a control. A strong reduction in adhesion was observed for substrates with an asperity size of 0.3 and 1 µm. Comparison of the present data with previous results on different organisms demonstrates that different amount of the force reduction on rough substrata depends on the dimension of terminal contact elements (spatulae). Additionally, the effect of ambient humidity on the attachment ability of P. dispar was revealed for the first time: spider attached stronger at intermediate humidities (50, 70%) than in dry (15%) or humid (99%) air. Water condensation on the substrate strongly reduced adhesion. Email address for correspondence: [email protected] 16:20 Monday 4th July 2011

161 W Jon P Barnes (University of Glasgow, UK), Aranzazu Del Campo (MPI for Polymer Research Mainz, Germany) and Hans-Juergen Butt (MPI for Polymer Research Mainz, Germany) Tree and torrent frogs are able to adhere and move about their wet or even flooded environments without falling. Their toe pad structure, including channels at different length scales, curved contact geometries and a fluid secretion with particular characteristics seems to play a major role for their adhesive and frictional performance. Our aim is to understand and translate nature's design into new adhesion concepts for artificial systems.The attachment pads show a complex surface structure including channels with hexagonal geometries at both microand nano-scales. There are mucus glands producing a fluid secretion, extensive fibres running at right angles to the surface, and an inner capillary network that probably acts as a shock-absorber. Adhesion is by a combination of capillary and hydrodynamic forces.Properties that make frog toe pads smart adhesives include an ability to adhere repeatedly to both hydrophilic and hydrophobic surfaces (tree frogs), the ability to adhere to rough surfaces under flooded conditions (torrent frogs), the ability to self-clean, and effortless detachment by peeling.We have prepared structured surfaces with different geometries, combining micro and nano-patterning techniques including both optical and soft lithography with polymeric materials, to obtain hierarchical structures with a hierarchical contact geometry that resembles the structure of tree frog toe pads. Preliminary experiments show that, as in the natural system, such toe pad mimics perform better in friction experiments than unstructured (smooth) surfaces under wet conditions. Toe pad mimics also generate larger pull-off forces when the intervening fluid is toe pad mucus rather than water. Email address for correspondence: [email protected] 17:00 Monday 4th July 2011

A12.16 Stress analysis and parameter study of permanent attachment pads of Parthenocissus tricuspidata by finite element simulations

Sven Bundschuh (Institute for Applied Materials, Germany), Tina Steinbrecher (Institute for Applied Materials, KIT Plant Biomechanics Group, University of Freiburg, Germany), Ruth Schwaiger (Institute for Applied Materials, KIT, University of Freiburg, Germany), Oliver Kraft (Institute for Applied Materials, KIT, University of Freiburg, Germany), Thomas Speck (Plant Biomechanics Group, University of Freiburg, Germany) and Christoph Eberl (Institute for Applied Materials, KIT, University of Freiburg, Germany) Descriptions of permanent attachment systems of climbing plants can be found in literature since Darwin's essay of climbing plants in 1865. Darwin described different kinds of climbing strategies and divided them into four classes: twining plants, leaf climbers, tendril bearers and hook and root climbers. We are focusing on Parthenocissus tricuspidata (Siebold and Zucc) Planch which belongs to the second class, the leaf climbers. An attachment pad is grown, which uses a combination of two different mechanisms to attach to the surface. One is the release of glue-like liquid that is secreted right after the first contact with the substrate is established. The second is a mechanical connection by a tight form closure with the surface and subsequent shrinkage during the desiccation process. Since the upcoming of bionic and biomimetic science few mechanical tests have been accomplished on the different length scale, from nanoindentation to small scale tensile testing. Our previous studies of the structure and the mechanical behaviour indicate a highly complex interaction between the different structural components of the pad. In order to get more insight into the inside of the pad we created finite element models to simulate the mechanical behaviour and compare them to experimental results, e.g. micro tensile tests. The simulation challenges can be divided into a geometrical part, where optimization has taken place during evolution, and material properties part to understand the inhomogeneous and anisotropic mechanical behaviour of the lignified attachment structure. In this paper we will discuss our simulation results and mechanical tests. Email address for correspondence: [email protected] 16:40 Monday 4th July 2011

A12.18 Biotechnology of mineral composites

Eva Weber (Leibniz-Institute for New Materials, Germany) and Ingrid M Weiss (Leibniz-Institute for New Materials, Germany) Animal and plant biominerals are an attractive source of inspiration to generate materials with extraordinary properties. From a material science point of view, many fundamental processes in biomineralization are challenging to mimic. This refers in particular to the biocomposite nature of biominerals, which consist of organic and inorganic compounds and are assembled into hierarchical structures in a functional way. In order to control processes for technological applications, biomineralization proteins can be extracted from organic matrices from different species. Primary structures of some attractive candidates involved in biomineralization processes have been identified. These proteins can serve as catalytic units to build up bioinspired materials in artificial systems by more or less specific interactions with inorganic precursors. However, bottle-necks towards application are given by the low abundance and extremely difficult extraction procedures of biomineralization proteins from natural resources. Here, we report on our latest achievements of prokaryotic expression and purification of partially insoluble biomineralization proteins. Biotechnologically produced proteins from animals and plants were characterized with respect to calcium carbonate polymorphism and mineralization kinetics. We investigated protein interference with crystal formation in situ using Raman spectroscopy, fluorescence microscopy, LCPolScope birefringence analysis and field-flow-fractionation. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

A12.17 Wet but not slippery: biomimetics of tree and torrent frog adhesion

A12.19 Insects as an idea generator for bio-inspired adhesives: An interaction of tarsal morphology and chemistry

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Society for Experimental Biology

Christian Schmitt (Eberhard-Karls Universität Tübingen), Manuela Gradl (Eberhard-Karls Universität Tübingen), Tim Nicholson (EberhardKarls Universität Tübingen), Klaus Albert (Eberhard-Karls Universität Tübingen), Oliver Betz (Eberhard-Karls Universität Tübingen) Nature has formed a diversity of numerous processes, mechanisms, and materials that can be adopted in aspiring biomimetic research and the development of new technologies. In this regard, our interest is focused on the tarsal attachment system of insects, since in the course of their evolution insects have generated various adhesive devices with remarkable properties like fast, strong, and reversible adherence. The mechanism by which insects attach to different kinds of surfaces is a complex interaction between tarsal morphology and chemistry, which is not yet completely understood. Hence, great effort is put into the investigation of the microstructure and the chemical composition of the tarsal attachment systems and their secretions. To investigate the attachment system, we obtained secretion samples of different insects (Schistocerca gregaria, Gromphadorrhina portentosa, Lampyris noctiluca) by solvent extraction and solid-phase microextraction (SPME) of these insects' smooth adhesive organs. First chromatographic analyses of this fluid were achieved by hyphenation of gas chromatography to mass-spectrometry (GC-MS). Further structure elucidation is provided by microprobe NMR spectroscopy. For characterization of the adhesive pad surface we used a combination of SEM and cryo SEM. The structures of the glands were elucidated by means of TEM studies. Microscopic studies show that the deformable cuticula of the adhesive pads is covered by various grooves and scale-like structures enclosing small openings through which the fluid is secreted. The subcuticular tissue is highly differentiated and show single-cell as well as multicellular glands. The chromatographic approach indicates the presence of a large diversity of hydrocarbons and amino acids. Email address for correspondence: [email protected] de Poster Session - Sunday 3rd July 2011

A12.20 Shooting silk: Techniques to study silk fibres under high speed impact

Elizabeth R Mortimer (Oxford University), Daniel Drodge (Oxford University), David Porter (Oxford University), Fritz Vollrath (Oxford University), Clive Siviour (Oxford University), Christopher Holland (Oxford University) Silks have evolved over the aeons to be intrinsically resistant to impact loading, providing biological roles from predation (spider webs) to protection (silkworm cocoons). As a result they have undergone strong selection to absorb large amounts of energy before breaking, making them extremely tough. Additionally silk is a lightweight and environmentally friendly material, so these properties, as well as how it responds to high speed impact, serve as a source of bioinspiration for engineering science. Using cocoon silk from the domesticated silkworm Bombyx mori, techniques have been developed to measure the relative stiffness (moduli) of single fibres when impacted by a projectile at hundreds of metres per second. State of the art high speed cameras are used in the set-up, capable of taking pictures in less than one millionth of a second. These pictures can detect the propagation of sound waves along a fibre, giving an estimation of the stiffness of the fibre. By altering the tension on the fibre by adding weights, changes in the moduli of the silk fibre can be investigated as the fibre is under load (i.e. a spider sitting in a web). The natural variation in the silk will be discussed as well as how quantitative modelling will aid in both the analyses and application of these techniques. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

Abstracts 2011

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SEB Main Meeting, Glasgow 2011

C1 - Tip growth in plant biology

C1.1 RSL4 regulates growth in tip-growing root hairs

Sourav Datta (University of Oxford, UK), Keke Yi (Zhejiang University, China), Priya Vijayakumar (University of Oxford, UK) and Liam Dolan (University of Oxford, UK) Root hairs are tubular projections arising from root epidermal cells that undergo tip growth. Arabidopsis root hairs provide a good system to study cell growth and morphogenesis in plants because of the ease with which mutants with growth defects can be identified. We discovered a basic helix-loop-helix (bHLH) transcription factor called RSL4 (ROOT HAIR DEFECTIVE 6-LIKE 4) that is sufficient to promote postmitotic cell growth in Arabidopsis thaliana root hair cells. Loss of RSL4 function results in the development of very short root hairs whereas constitutive RSL4 expression induces constitutive growth, resulting in the formation of very long root hairs. Auxin and low phosphate availability modulate hair cell extension by regulating RSL4 transcript and protein levels. RSL4 protein is present in growing cells and our results suggest that its degradation signals the cessation of growth. Currently we are characterizing the mechanism of RSL4 protein turnover. We are also using an inducible form of RSL4 to identify downstream targets of RSL4 involved in root hair growth. We anticipate that these experiments will define the mechanism of RSL4regulated cell growth. Email address for correspondence: [email protected] 10:30 Friday 1st July 2011 Plant cells display a variety of shapes that usually form an adaptation to perform specific functions. Root hairs are elongated cylindrical outgrowths of root epidermal cells and their presence significantly increases the surface of the root, which is beneficial for nutrient and water uptake. In contrast to most other plant cells these tubular outgrowths are created by growth that is confined to their tip only and that is coordinated by a wide range of internal and external factors. The network that controls the tip-focused calcium gradient, vesicle transport and polarization of the actin cytoskeleton is highly complex and not fully understood. Combining micro-array data of different root hair mutants, we identified the Root Hair Specific 4 (RSH4) gene of Arabidopsis. RSH4 contains root hair-specific cis-elements (RHE) in the promoter region and encodes an unknown protein that is predicted to reside in the nucleus. Expression analysis in promotor-GUS and GFP plants revealed that the expression was limited to trichoblasts only and that it starts from the moment the root hair bulge starts to form. The effect of different treatments (e.g. phosphate and iron deficiency) on the expression pattern is in progress. T-DNA insertion mutants for the studied gene exhibit a root hair phenotype when compared to wild type plants. In order to further uncover the role of RSH4 in root hair formation the effect of constitutive and own promotor-driven overexpression is currently under study. Furthermore, protein-GFP fusion constructs will reveal whether the protein is located in the nucleus as predicted. Email address for correspondence: [email protected] 12:10 Friday 1st July 2011

C1.4 C1.2 A network approach to root hair development

Claire Grierson (School of Biological Sciences, University of Bristol, UK) We have used a combination of bioinformatic and traditional laboratory approaches to identify and characterise 151 genes expressed during root hair growth. eQTL analysis identified new candidate root hair transcription factors. Coexpression and protein interaction network analysis predicted roles for over 40 genes with no previously known function or recognisable sequence domains. Our results shed light on many aspects of root hair tip growth and produce numerous avenues for future investigation. Email Address for correspondence: [email protected] 11:30 Friday 1st July 2011

A role for CSLD3 during cell wall synthesis in apical plasma membranes of tip-growing root hair cells

Erik Nielsen (University of Michigan, USA) In plants, cell walls play essential roles controlling cell shape and size. In a common form of cell expansion called diffuse growth, synthesis and orientation of cellulose in new cell walls is integral to control of cell shape. In root hairs, cell walls also control expansion, but by a distinct process called tip-growth. We were interested in the mechanisms controlling cellulose deposition in root hairs. Using immunofluorescence and in vivo growth assays with cellulose synthase inhibitors or cell wall degrading enzymes we show that cellulose-like polysaccharides are present in root hair tips, and its de novo synthesis is required for tip-growth. We examined cellular distributions of fluorescently-tagged CESA cellulose synthases and the CSLD3 cellulose-synthase-like protein. While neither EGFP-CESA3 nor EYFP-CESA6 localized to plasma membranes in growing root hairs, EYFP-CSLD3 localized to a polarized plasma membrane domain in the root hair apex. EYFP-CSLD3 was found in plasma membranes in growing root hairs, but was restricted to cytoplasmic organelles in non-hair forming root epidermal cells. We also showed that YFP-CSLD3 is a surface exposed plasma membrane protein, and, like CESA proteins, can be cross-linked into cell wall fractions upon treatment with cellulose synthase inhibitors. Finally, we replaced the CSLD3 catalytic domain with a CESA6 catalytic domain and this CSLD3::CESA6 fusion protein functionally rescued atcsld3 mutant plants. These results provide evidence that CSLD3 represents a distinct (1->4)-beta-glucan synthase activity in apical plasma membranes during tip-growth in root hair cells. Email address for correspondence: [email protected] 13:30 Friday 1st July 2011

C1.3 Functional Root Hair Specific 4 (RSH4) is required for normal Arabidopsis root hair development

Daria Balcerowicz (University of Antwerp, Belgium), Thanaa Doubbo (University of Antwerp, Belgium), Agnieszka Boron (University of Antwerp, Belgium), Gordon Breen (University of Bristol, School of Biological Sciences, UK), Claire S Grierson (University of Bristol, School of Biological Sciences, UK) and Kris Vissenberg (University of Antwerp, Belgium)

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Society for Experimental Biology Pavlína Brettlová (Institute of Experimental Botany AS CR), Roman Pleskot (Institute of Experimental Botany AS CR), Lukasz Cwiklik (Institute of Organic Chemistry and Biochemistry AS CR), Martin Potocký (Institute of Experimental Botany AS CR), Pavel Jungwirth (Institute of Organic Chemistry and Biochemistry AS CR), Viktor Zárský (Institute of Experimental Botany AS CR) The exocyst is an evolutionary conserved tethering complex involved in exocytosis of eukaryotes. It was described as an effector of small G-proteins and comprises eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. Recently, we have proven the exocyst presence and function also in Arabidopsis and at the tobacco pollen tube tip (Hála et al., Plant Cell, 2008).In this work we focus on Sec3 subunit and its possible role as a membrane-binding protein. We constructed 3D model of N-terminal domain of Arabidopsis protein by homology modeling technique using crystal structure of N-terminal part of yeast Sec3 (rcsb3A58) as a template. N-terminal part of yeast Sec3 has got a PH-domain fold and binds phosphatidylinositol (4,5)-bisphosphates (PIP2). Based on analysis of our molecular docking results of PIP2 head group inositol trisphosphates we found that binding site of yeast and plant domain is overlapping but there are differences in binding amino acid residues. To further investigate interaction of N-terminal domain of Sec3 with membrane containing PIP2 we performed molecular dynamics study. We also prepared Arabidopsis Sec3 N-terminal part as a GST and GFP-fusion protein to get in vitro and in planta insight into the role of this domain. Financial support from grants LC06034, MSM0021620858, Kontakt ME10033 and IAA601110916 is acknowledged. Email address for correspondence: [email protected] 16:20 Friday 1st July 2011

C1.5 Dynamic assembly and organization of plant microtubule bundle arrays

Marylin Vantard (UMR CNRS,CEAINRA, Université Joseph Fourier de Grenoble, France) MT bundling is a crucial step in organizing plant MT arrays and hinges on the ability of this cytoskeleton to self-organize into higher order structures in the absence of a well-defined MT organizing centre. These structures are involved in diverse sets of cellular processes, including cell morphogenesis, establishment of cell polarity and cell division. However, the different parameters that control their spatial organization and the regulation of their growth are poorly documented. This is particularly important as one may anticipate that the dynamic behaviour of MT within bundles affects their self-organization into specific arrays and that their dynamic feature must underlie many of their functions. To understand physical laws and molecular mechanisms that allow the dynamic assembly that control the spatial organization of the cortical MT array, we reconstituted MT bundle arrays in vitro using biomimetic assays. MT zippering proteins used in our experimental set up are members of the evolutionary conserved MAP65 family as they have been emerged as key players in the self-assembly of acentrosomal MT arrays in eukaryotes. In the MT bundle arrays we reconstituted, we could investigate at the molecular level the physical interactions between MTs and/or MT bundles, MT dynamics within bundles, etc. The different data that define some molecular mechanisms by which MAP65s induce MT bundles and coordinate MT growth within bundles and that we obtained at the experimental levels and by mathematical simulations will be discussed. Email address for correspondence: [email protected] 14:30 Friday 1st July 2011

C1.8 The proline rich protein-like AtPRPL1 controls elongation of root hairs and dark-grown hypocotyl cells of Arabidopsis thaliana

Agnieszka Boron (University of Antwerp, Belgium), Jürgen Van Orden (University of Antwerp, Belgium), Grégory Mouille (Laboratoire de Biologie Cellulaire, INRA, France), Herman Höfte (Laboratoire de Biologie Cellulaire, INRA, France), Jean-Pierre Verbelen (University of Antwerp, Belgium) and Kris Vissenberg (University of Antwerp, Belgium) The synthesis and composition of cell walls is dynamically adapted in response to many developmental and environmental signals. In this respect cell wall proteins controlling cell elongation are critical for decent cell development. Transcription analysis identified a gene which was named proline rich protein-like AtPRPL1 based on sequence similarities. Phylogenetic analysis showed most resemblance with AtPRP1 and AtPRP3 proteins from Arabidopsis thaliana which are known to be involved in root hair growth and development. Proline-rich proteins are structural cell wall proteins that play a role in building up the cross connections between cell wall components. In Arabidopsis thaliana four proline-rich cell wall protein genes are distinct. AtPRPL1 is a small gene that contains a N-terminal putative signal sequence that most likely directs the protein to the ER. High promoter::GUS expression was observed in trichoblast cells and in the collet. Chemical or mutational interference with root hair formation inhibited expression. Altered expression levels interfered with proper root hair development and aetiolated hypocotyl development. Colocalization analysis of the AtPRPL1-GFP fusion protein and different RFP-labeled markers confirmed the presence of AtPRPL1GFP in small vesicles moving over the ER. FT-IR analysis on cell walls from overexpression lines showed differences in the methylation grade of pectins. Together, all these data indicate that the AtPRPL1 gene is involved in the cell's elongation process and somewhere links structural proteins with pectin modification. How exactly this is achieved, remains unclear for the moment.

C1.6 Tip growth in root hairs: exocytosis and the cytoskeleton in cell wall deposition

Ying Zhang (Wageningen University, The Netherlands) and Tijs Ketelaar (Wageningen University, The Netherlands) Anne-Mie C Emons (Wageningen University, The Netherlands) and

Root hairs are outgrowths of root epidermal cells and elongate at their tips only. The actin cytoskeleton brings Golgi bodies containing Golgi vesicles to the growing hair tip, and microtubules play a role in ensuring a straight growth direction. Root hairs of terrestrial plant have axial/ helical cellulose microfibrils in the hair tube. In Arabidopsis thaliana we investigated the cell wall texture, i.e. the combined cellulose microfibrils forming the load-bearing structures of the cell wall. These crystalline structures are made by cellulose synthase complexes (CSCs). We show velocity, density, and direction of these complexes as well as their insertion into the plasma membrane, and their relation to the cortical microtubules. CSCs enter the plasma membrane in an exocytotic event. We studied a protein, SEC3A, which, in yeast and mammals, is part of the exocyst, a complex needed for exocytosis to occur. Sec3A mutants are embryo-lethal, and sec3A is expressed constitutively. It localizes to the plasma membrane including that of the cell plate. We report the localization reaction of this protein to treatment with cytoskeleton- and membrane drugs. Email address for correspondence: [email protected] 15:40 Friday 1st July 2011

C1.7 The role of exocyst Sec3 subunit as a membrane binding domain

Abstracts 2011 Email address for correspondence: [email protected] ac.be 16:40 Friday 1st July 2011

165 revealed at least 2 PM H+ ATPase isoforms (LilHA1 and LilHA2) and 4 main 14-3-3 isoforms (Lil1433_0 to Lil1433_3) being expressed. To elucidate more details of the interaction between the PM H+ ATPase and 14-3-3 isoforms during pollen tube germination and growth, the time-dependence of their expression and putative interactions are investigated by real-time RT-PCR and mass spectrometry analysis of interaction complexes. Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

C1.9 Space and time coordination of cellular growth processes in pollen tubes

José A Feijo (University of Lisbon, Portugal) Pollen tubes are favourable models for fundamental understanding of cellular growth and morphogenesis in apical growing cells. Transcriptomics reveals the expression of about 7000 genes, but modelling suggests that the cooperation of all of these into the processes of wall surface and cytoplasmic volume production, is a sufficient condition to explain most characteristics of these cells. Spatial and temporal integration of extended biochemical and biophysical processes is mandatory, and in the past we have demonstrated that ion dynamics may function as regulator of fundamental growth processes. In order to test this hypothesis we are developing a number of genetic, imaging and electrophysiological approaches to define the membrane transporters that could underly the transduction necessary coordination. We have uncovered original data in terms of proton pumping, and will describe new mechanisms for calcium and chloride (anion) transport. We will provide data suggestive that the feed-back mechanisms by which these ions could affect fundamental cell biology mechanisms is centred on the membrane recycling mechanisms. In fact advanced imaging methods and Monte-Carlo simulations suggest that sorting of exocytic vesicles might be achieved by direct electric interaction with ion fluxes. Finally we developed 3-D models of ion fluxes and cytosolic diffusion based on the current knowledge of the system. These models are instrumental to define the minimal needs for channels to explain all the available evidence. Hopefully they will allow us to expand these conclusions to a broader understanding of the fundamental basis that govern cellular growth and morphogenesis by directed exocytosis. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011

C1.11 Distinct signalling phospholipids regulate the initiation and maintenance of plant tip growth

Premysl Pejchar (Institute of Experimental Botany, Czech Republic), Roman Pleskot (Institute of Experimental Botany, Czech Republic), Jan Martinec (Institute of Experimental Botany, Czech Republic), Chris Staiger (Purdue University, USA), Viktor Zárský (Institute of Experimental Botany, Czech Republic) and Martin Potocký (Institute of Experimental Botany, Czech Republic) Plant cell morphogenesis relies upon a dynamic network of processes integrating cell-wall dynamics with signal transduction, changes in ion transports, membrane (phospho)lipid modifications, regulation of the secretory pathway and cytoskeleton dynamics. He we present data showing that different lipid-signalling pathways are necessary for the establishment and propagation of plant tip growth, represented by germinating tobacco pollen and growing pollen tubes. We labelled tobacco pollen with phospholipid fluorescent analogues and assayed the production of signalling lipid messengers. This approach revealed massive activation of phospholipase A2 (PLA2) and phospholipase (PLD) during pollen germination and tube growth, respectively. We found that secretory PLA2 (sPLA2) is responsible for high PLA2 activity and is required for pollen germination. Analysis of tobacco sPLA2s by qRT-PCR, subcellular localisation of sPLA2-YFP fusion proteins, antisense-mediated knockdown and employment of specific inhibitors identified NtsPLA2 as isoform responsible for correct pollen germination. We then studied the importance of several PLD isoforms in pollen tube growth regulation focusing on control of actin dynamics. We identified NtPLD1 as a regulatory partner of actin and demonstrated that antisense-mediated suppression of NtPLD1 compromises pollen tube F-actin dynamics. Moreover, the activity of tobacco PLD is specifically enhanced by F-actin and inhibited by G-actin. The actininteracting domain NtPLD1 was identified and amino-acids crucial for actin-binding were mapped. The positive-feedback loop created by mutual activation of PLD and F-actin provides a mechanism to locally increase membrane-F-actin dynamics. Grant support: GAAV-IAA601110916, GACR-522/09/P299, GACR522/07/1614 and MSMT-LC06034. Email Address for correspondence: [email protected] 11:50 Saturday 2nd July 2011

C1.10 Insights from the pollen plasma membrane proteome: H+ pump activity and tip growth

Gerhard Obermeyer (University of Salzburg, Germany), Veronika Lang (University of Salzburg, Germany), Katharina Schmidt (University of Salzburg, Germany), Verena Kuchler (University of Salzburg, Germany), Waltraud Schulze (MPI Molecular Plant Physiology) and Heidi Pertl-Obermeyer (University of Salzburg, Germany) Upon landing on a stigma, pollen grains start to generate a pollen tube that penetrates the stigma tissue and grows towards the ovule where fertilization takes places. The tip growth mechanism of pollen tubes is a highly regulated network of various cellular processes involving the cytoskeleton, exocytosis, cytosolic gradients of pH and Ca2+, ion transporter activity, signal transduction pathways and water uptake. Especially, the uptake of ions and nutrients across the plasma membrane is energized by an electrochemical gradient of H+ generated by the plasma membrane H+ ATPase. The importance of the PM H+ ATPase activity for germination and growth of pollen tubes is demonstrated by ATPase inhibitors and stimulators as well as by compounds affecting the regulation of PM H+ ATPase by 14-3-3 proteins (fusicoccin and AICAR). In Arabidopsis, transcriptome studies showed that 4 PM H+ ATPase isoforms (total 11) are expressed in pollen with AHA8 being the most abundant and 3 main 14-3-3 isoforms (total 12) are expressed (highest expression for 14-3-3phi/chi). A proteomic study of membrane and membrane-associated proteins

C1.12 An osmotic model of tip growth in pollen

Adrian E Hill (Cambridge University), Yair Y Shachar-Hill (MSU East Lansing USA), Janet P Powell (Cambridge University), Jeremy N Skepper (Cambridge University), Bruria Shachar-Hill (Cambridge University) A prime example of cellular tip growth is the pollen cell but selfcontained models of the process in terms of the basic driving forces, osmotic and turgor pressures, have not yet been made. We present a detailed model involving the regulation of turgor and its effect on the extension of the tip, treated as a viscous liquid film. The basic elements

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Society for Experimental Biology of activated Rac/Rop GTPases at the plasma membrane at the pollen tube apex. In addition to further characterizing the molecular mechanisms responsible for the apical polarization of Rac/Rop activity, we also want to learn more about the Rac/Rop dependent signalling network that coordinates the different cellular processes underlying tip growth. To this end, we have identified and are functionally characterizing a number of putative Rac/Rop effectors. Tobacco pollen tubes and Physcomitrella patens protonema are both excellent experimental systems to study tip growth. Employing these two systems for our work, we are benefitting from the distinct advantages each of them offers, and we hope to achieve a better understanding of how the molecular control of tip growth has evolved. Email address for correspondence: [email protected] 14:30 Saturday 2nd July 2011

of the model are general enough to apply to tip growth in fungal hyphae and root hairs. The model is run as a computer program to simulate the growth and behaviour of Lilium tubes and is supported by experimental and structural studies in our laboratories together with data from the literature. Among novel predictions of the model are : (i) pollen tubes require a restricted area for osmotic water entry near the apex, and that this area - the `osmotic zone' - must be maintained by a dynamic process; (ii) the turgor pressure is largely self-regulated and very constant whilst other variables are changing; (iii) regulation of the thickness of the tip wall, which is also remarkably constant, is an essential pre-requisite for stable growth, and can only be fully achieved by a sensor that detects the inward driving forces on water and controls the exocytosis of wall polymers; and (iv) oscillation in linear growth may serve no biological purpose but arises from the operation of a delay between a limited number of cell variables. Email address for correspondence: [email protected] 12:10 Saturday 2nd July 2011

C1.15 The rhizobial infection thread, a special case of inverted polar growth

Jeremy D Murray (John Innes Centre), Nicola Stacey (John Innes Centre), Andrew Breakspear (John Innes Centre) Nodulation in legumes is comprised of the synchronised yet genetically separable events of epidermal infection and nodule organogenesis. During infection Nod factor-producing rhizobia induce a series of events through the Nod factor signalling pathway. Rhizobia attach near the tip of a growing root hair which then undergoes a brief period of isotropic growth followed by resumption of anisotropic but now asymmetric growth leading to the formation of a curl which entraps a colony of rhizobia in a so-called `infection pocket'. The bacteria enter the root at the site of the infection pocket where polarised exocytosis leads to localized cell wall degradation and the formation of a tubular invagination, called an infection thread, which extends down the root hair by a process of inverted polar growth that is dependent on the proximity and movement of the nucleus through the root hair. Nod factors may activate yet unidentified calcium channels and ROS generating enzymes to drive growth of infection threads. Unlike root hair growth, infection thread growth is inhibited by ethylene. Using transcriptomic and reverse-genetic approaches, we seek to characterize the elements required for the transcriptional reprogramming of the root hair cell required for infection thread formation. Email address for correspondence: [email protected] 15:40 Saturday 2nd July 2011

C1.13 Molecules and networks controlling polar growth in the filamentous fungus Aspergillus niger

Vera Meyer (Berlin University of Technology, Germany) and Arthur F Ram (Leiden University Molecular Microbiology, the Netherlands) Aspergillus niger is of considerable economic importance as cell factory for the production of proteins, bulk chemicals and pharmaceuticals. In terms of protein production, the morphology it adopts in a bioreactor is critical for the productivity of the process. Whereas the preferred morphology would consist of highly branched dispersed mycelia, A. niger either produces long hyphae with relative low branching frequencies or pellets which are also not desirable. To understand in more detail the genetic basis of the morphogenetic program of A. niger we have undertaken different genomic, transcriptomic and bioinformatic approaches and have identified several signalling networks, including TORC2, phospholipid, calcium and cell wall integrity signalling, that likely regulate the morphogenetic program of A. niger (1,2). We have furthermore implemented different functional genomics approaches to query the function and biological role of predicted signalling molecules for growth and morphogenesis of A. niger. For example, we could show that Cdc42/Rho GTPases exert distinct and overlapping functions during the life cycle of A. niger (3). Surprisingly, these functions are only partially conserved among related fungi, suggesting that these molecular switches are variably used. References: 1) Meyer et al. (2009) Reconstruction of signalling networks regulating fungal morphogenesis by transcriptomics. Eukaryot Cell 8 2) Meyer et al. (2010) The Aspergillus niger RmsA protein: A node in a genetic network? Commun Integr Biol 3 3) Kwon et al. (2011) Functional characterization of Rho GTPases in Aspergillus niger uncovers conserved and diverged roles of Rho proteins within filamentous fungi. Mol Microbiol 79 Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

C1.16 Reactive oxygen species and calcium signalling in the legume-rhizobia symbiosis

Sarah Shailes (John Innes Centre), Allan Downie (John Innes Centre), Giles Oldroyd (John Innes Centre) Members of the legume family of plants can form symbioses with nitrogen-fixing bacteria known as rhizobia. An early step required to establish the symbiosis is the production of Nod factors (NF) by the bacteria. NFs induce a Ca2+ influx in the tip of root hair cells. The role of the Ca2+ influx is likely to be with the induction of the structures associated with bacterial infection. A NF-induced reactive oxygen species (ROS) transient has been reported to be temporally and spatially coincident with the Ca2+ influx1. In root hair cells tip-focussed NADPH oxidase-dependent ROS production and a Ca2+ gradient interact to drive polar tip growth. Using the Ca2+ -sensitive Cameleon YC2.1 reporter and the ROS-sensitive dye CM-H2DCFDA we aim to investigate whether the NF-induced Ca2+ influx and ROS transient are involved in a common pathway. The Medicago truncatula NF receptor mutant nfp1-1 is defective for the Ca2+ influx2. We treated nfp1-1 seedlings with NF and found that they

C1.14 Control of tip growth by Rac/Rop signalling

Benedikt Kost (Cell Biology Division, Department of Biology, ECROPS, University of Erlangen-Nuremberg, Germany) Rac/Rop GTPases play a central role in the control of the tip growth of pollen tubes and of apical cells of moss protonema. We have characterized complex functional interactions between different regulatory factors, which are required for the specific accumulation

Abstracts 2011 were also defective for the ROS transient. Pretreatment of wildtype seedlings with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited both the NF-induced ROS transient and the Ca2+ influx. We will continue to characterise the NF-induced ROS transient in M. truncatula using inhibitors and mutant lines. We will also examine the NF-induced Ca2+ and ROS responses in mutant lines defective for components of polar tip growth machinery. References: 1. Cardenas et al. (2008) Plant Journal. 56(5): p. 802-813.2. 2. Ben Amor et al. (2003) Plant Journal. 34(4): p. 495-506. Email address for correspondence: [email protected] 16:00 Saturday 2nd July 2011

167 It was found that D4476 affects primary root growth and caused alterations of general root morphology. Root hair growth and development were extremely sensitive to CK1 inhibitor treatment in a dose-dependent manner. Inhibitions of root hair growth were accompanied by their branching or cessations of new root hair formation. The microtubules in epidermal and cortex cells of the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts of the differentiation zone reoriented from transverse/ oblique to chaotic /longitudinal after D4476 treatment. Our studies provide evidence for the involvement of CK1 in the regulation of primary root development, especially root hair formation and growth, through the regulation of cortical microtubules organization, possibly through the tubulin phosphorylation in plant cell. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.17 The effects of air pollution on pollen of Chenopodium album

Leila Amjad (Department of Biology, Falavarjan Branch Islamic Azad University, Iran) and Mahsa Shafighi (Young Researchers Club, Falavarjan Branch, Islamic Azad University, Iran) Air pollution is the introduction of chemicals, particulate matter, or biological materials that cause harm or discomfort to humans or other living organisms, or cause damage to the natural environment or built environment, into the atmosphere. The aim of this project was study the effect of air pollutions on structure and pollen grains development in Chenopodium album. Anthers of Chenopodium album L. were collected at different stages of development from control (less polluted) and polluted areas (mainly SO2, NO2, CO and APM). Structure and development of pollen grains were studied and compared. The studying pollen structure by light and scanning electron microscopy showed that when pollen grains were exposed to polluted air they became abnormal in form and covered with large amounts of pollutants. Pollen abnormalities were seen as irregularity, shrinkage, thinning and breakage of the exine. Cellular material release was induced also. The data presented suggest that prolonged exposures of plants to air pollution may cause different biological effects at the cellular tissue and organ levels. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.19 Study of two genes involved in Arabidopsis root development Thanaa Doubbo (University of Antwerp Biology Department Plant Growth and Development Antwerp Belgium), Kris Vissenberg (University of Antwerp Biology Department Plant Growth and Development Antwerp Belgium), Claire S Grierson (University of Bristol School of Biological Sciences Bristol UK), Jean-Pierre Verbelen (University of Antwerp Biology Department Plant Growth and Development Antwerp Belgium), Gordon Breen (University of Bristol School of Biological Sciences Bristol UK), Daria Balcerowicz (University of Antwerp Biology Department Plant Growth and Development Antwerp Belgium)

Studies of plant growth often exploit the model plant Arabidopsis thaliana. Along its root zones of division, elongation and differentiation, seen as the presence of root hairs, can be found. Root hairs are tip-growing structures derived from specialized epidermal cells, the trichoblasts, and they aid to take up water and nutrients. Until present, a wide variety of genes have been described to be involved in root hair development. So a complex network of molecular mechanisms responsible for trichoblast cell fate determination, root hair initiation and elongation starts to be formed. Based on a reverse genetics approach, we study a gene whose knock out plants had shorter roots and a second one with a 80% reduction in root hair length. In silico analysis identified the first gene as a glucosylceramidase and the second one as a protein-serine/threonine kinase. The expression patterns of the genes were analysed using promotor:GUS and GFP constructs. Reporter gene expression was found in several cell types in the root, the cotyledons and in the pollen or was strictly confined to trichoblast cells in the genes respectively. Hormone and condition (e.g. iron deficiency) treatments are in progress. Furthermore, transgenic plants with constructs for overexpression of the genes and protein-GFP fusions will be analysed. In addition a complementation of the knockout lines is foreseen. These approaches will give more insight into the role and functions of both genes. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.18 Casein kinase 1 is involved in Arabidopsis root hair formation and growth via regulation of microtubules organization

Yarina Sheremet (Institute of Food Biotechnology and Genomics, NAS, Ukraine), Alla Yemets (Institute of Food Biotechnology and Genomics, NAS, Ukraine) and Yaroslav Blume (Institute of Food Biotechnology and Genomics, NAS, Ukraine) Our recent experiments on Arabidopsis thaliana line expressing chimeric gene gfp-map4 identified that various types of serine/threonine and tyrosine kinases are involved in regulation of microtubules organization in primary root cells (Yemets et al., Cell Biol. Int.,2008,32:630­637; Sheremet et al., Cell Tissue Biol.,2010,4(4): 399­409). Nevertheless the regulation of microtubules by direct phosphorylation of tubulins and its functional role in plant cell have not been widely observed. It was shown recently that A. thaliana casein kinase 1 (CK1) member CKL6 is associated with cortical microtubules in vivo and phosphorylates tubulin in vitro (Ben-Nissan et al.,Plant Phys.,2008,148:1897-1907). To examine a functional role of CK1 through its involvement in modulation of tubulin phosphorylation the effects of specific inhibitor D4476 on the microtubule organization in A. thaliana primary root cells were investigated.

C1.20 Expression of GFP-talin reveals an actin-related role for AtFH12, an outlier Arabidopsis Class II formin

Fatima Cvrckova (Department of Experimental Plant Biology, Charles University, Prague, Czech Republic), Michal Grunt (Department of Experimental Plant Biology, Charles University, Prague, Czech Republic) and Viktor Zarsky (Charles University and Inst. Exp. Botany, ASCR, Prague Czech Republic)

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Society for Experimental Biology apex of Arabidopsis seedlings grown in sterile conditions under 16/8 h photoperiod and 20ºC. Contrary, the root hairs length in the upper part of the root was higher than in light-grown plants. We propose that this difference may be related to the different reactive oxygen species (ROS) production in these zones. Previously, we have observed the absence of the ROS production in the root apex of the dark-grown Arabidopsis seedlings, but slight ROS-dependent nitroblue tetrazolium staining in the root upper region with the high density of the root hairs. We have found that ROS production could be induced by light. The question is to be solved whether the root hairs tip growth could be affected by light or different hormonal balance in the dark grown Arabidopsis influence the length of the root hairs. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

Formins (FH2 proteins) are implicated in multiple aspects of cytoskeletal organization, especially F-actin nucleation. Plants possess two formin clades, relatively well-described Class I formins and so far poorly characterized Class II formins exhibiting usually low expression levels and a complex gene structure. Comparison of Class II formins of two Arabidopsis species, A. thaliana and A. lyrata, indicates dynamic evolution within the Class II formin clade. Lack of stronger mutant phenotype manifestations hampers progress in functional analysis of large and redundant (degenerate) gene families, including plant formins. Indeed, disruption of an outlier A. thaliana Class II formin gene, AtFH12, whose expression is induced by NaCl, produced only negligible phenotypic effects under a variety of conditions, including salt stress, suggesting functional redundancy among Class II formins. Here we show that the same mutation massively aggravated toxic effects of the expression of a fluorescent actin marker, GFP-tagged mouse talin (GFP-mTalin), which is known to cause extensive actin bundling. Abnormal actin structures were observed in atfh12 mutants expressing GFP-mTalin as compared to wild type. This not only demonstrates an actin-associated function for AtFH12, but also documents the feasibility of using the heterologous actin marker to "stress-test" the actin cytoskeleton in phenotyping "weak" actin related mutant alleles. This work was supported by the MSM LC06004, MSM 0021620858 and GACR P305/10/0433 projects. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.23 An osmotic model of tip growth in pollen

Adrian E Hill (Cambridge University, UK), Yair Y Shachar-Hill (MSU, East Lansing, USA), Janet P Powell (Cambridge University, UK), Jeremy N Skepper (Cambridge University, UK) and Bruria Shachar-Hill (Cambridge University, UK) A prime example of cellular tip growth is the pollen cell but selfcontained models of the process in terms of the basic driving forces, osmotic and turgor pressures, have not yet been made. We present a detailed model involving the regulation of turgor and its effect on the extension of the tip, treated as a viscous liquid film. The basic elements of the model are general enough to apply to tip growth in fungal hyphae and root hairs. The model is run as a computer program to simulate the growth and behaviour of Lilium tubes and is supported by experimental and structural studies in our laboratories together with data from the literature. Among novel predictions of the model are: (i) pollen tubes require a restricted area for osmotic water entry near the apex, and that this area ­ the `osmotic zone' ­ must be maintained by a dynamic process; (ii) the turgor pressure is largely self-regulated and very constant whilst other variables are changing; (iii) regulation of the thickness of the tip wall, which is also remarkably constant, is an essential pre-requisite for stable growth, and can only be fully achieved by a sensor that detects the inward driving forces on water and controls the exocytosis of wall polymers; and (iv) oscillation in linear growth may serve no biological purpose but arises from the operation of a delay between a limited number of cell variables. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.21 Infection thread initiation and progression in the model legume Medicago truncatula

Andrew Breakspear (John Innes Centre, UK), Jeremy D Murray (John Innes Centre, UK), Nicola Stacey (John Innes Centre, UK) and Christian Rogers (John Innes Centre, UK) Polarly growing infection threads in the model legume Medicago truncatula allow its nitrogen-fixing symbiont Sinorhizobium meliloti to traverse root hair cells and invade developing nodules. Following the establishment of a rhizobial microcolony at a curled root hair, polar growth is initiated resulting in the emergence of a nascent infection thread. The infection thread continues to extend in a polar manor towards the root cortex releasing the symbiont in the nodule primordium. We have established time-points that correspond to both infection thread initiation and progression and are using microarray analysis to further our understanding of this polar growth process. To enhance detection of transcripts involved in the polar growth of infection threads, we are extracting RNA from pure populations of root hairs harvested from infected plants. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

C1.22 Root hairs tip growth is altered in the dark-grown Arabidopsis root

Irina Strizh (Faculty of Biology, M.V. Lomonosov Moscow State University, Russia) Mechanisms of the root hairs tip growth are typically studied in the completely illuminated plants. We have suggested that mechanisms of the root growth and development in the dark may differ from those established for the light-grown plants. We have observed unusual root development of the Arabidopsis grown for 5-6 days in the dark. The most striking difference was observed in the root hairs length: in the root apex it was lower than the root hairs length in the root

Abstracts 2011

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SEB Main Meeting, Glasgow 2011

C2 - Frontiers in Algal Biology

C2.1 Cellular homeostasis, signalling and ion channels in algae: Probing the origins of excitability in eukaryotes

Colin Brownlee (Marine Biological Association of the UK, Plymouth, UK), Alison Taylor (Marine Biological Association of the UK, UK, and University of North Carolina, USA), Glen Wheeler (Marine Biological Association of the UK and Plymouth Marine Laboratory, UK) and Abdul Chrachri (Marine Biological Association of the UK, Plymouth, UK) The marine eukaryotic algae represent a phyletically very diverse group of organisms represented by the red, green and heterokont algal lineages. A number of representatives have provided excellent models for the study of signalling in relation to early development and responses to environmental cues. For example, studies of brown algal zygotes have revealed the role of fast propagating calcium signals and intracellular gradients in the regulation of polarity. Electrophysiological studies have uncovered the presence of fast "animal-like" action potentials in single-celled diatoms and coccolithophores. Genomic studies have supported these findings in showing the presence of a number of typically animal-like ion channel genes, normally associated with nerve or muscle function, in a range of algal species but which are absent in higher plants. More recently we have characterized a proton-selective plasma membrane channel, previously only found in mammalian cells, that is required for shortterm cytosolic pH regulation in the calcifying coccolithophorid phytoplankton. The characterization of this channel is allowing new insights into the mechanisms of calcification in this biogeochemically important group. Taken together, these studies are beginning to provide tantalising clues about the evolution of membrane excitability more generally in eukaryotes. Email address for correspondence: [email protected] 10:30 Sunday 3rd July 2011

C2.3 The systematics of the brown seaweeds

John H Bothwell (Queen's University Belfast), Gabriel Marais (University of Lyon), Mark Cock (Station Biologique de Roscoff), Susana M Coelho (Station Biologique de Roscoff) The brown macroalgae, or seaweeds, determine the productivity and biodiversity of coastal ecosystems. They are also of great evolutionary interest as they have evolved complex multicellularity independently of many other eukaryotic lineages (e.g. plants, animals, fungi) and survive in the harsh intertidal zone. In this talk, we will consider the extent to which their systematics ­ specifically, questions about their evolutionary histories and adaptations ­ can benefit from the raft of new techniques being developed in other model organisms. Using case studies drawn from the past decade of genomic work in the brown algae ­ particularly patterns of gene family loss and gain and the importance of sex chromosome evolution in brown algal speciation ­ we will discuss the evolutionary and ecological scales over which brown seaweed systematics should be considered. Email address for correspondence: [email protected] 12:00 Sunday 3rd July 2011

C2.4 Genetic manipulation of micro-algae: progress, prospects and applications

Saul Purton (Institute of Structural and Molecular Biology, UCL, UK) There is a growing interest in the industrial cultivation of micro-algae for a wide range of different products. These include: naturally derived compounds for the food, cosmetic, nutraceutical and pharmaceutical industries; lipids and other high-energy hydrocarbons as sources of liquid biofuels; whole biomass for animal feedstock, and novel recombinant proteins and bioactive compounds from genetically modified algae. However, such exploitation requires the development of routine and high-throughput molecular genetic techniques for strain improvement and for the introduction of foreign genes for novel proteins or new metabolic pathways. Currently, techniques for the genetic engineering of algae is limited to just a handful of species, with the freshwater green alga Chlamydomonas reinhardtii and the marine diatom Phaeodactylum tricornutum being the most genetically tractable. Even for these species, the available molecular tools are rather rudimentary making any genetic manipulation programme slow and unpredictable. In my group, we have been focusing on improving the tools and strategies for introducing and expressing foreign genes in both the nuclear and chloroplast genomes of C. reinhardtii. Furthermore, we are attempting to transfer the technology into a number of commercially important species of green algae with a view to combining metabolic engineering approaches with classical mutant screens. In this talk, I will review the challenges of algal transgenics and present our progress to-date with examples of possible applications in the areas of biofuels, nutraceuticals and therapeutic proteins. Email address for correspondence: [email protected] 13:30 Sunday 3rd July 2011

C2.2 Surfaces and signals: dissecting the biology/ materials interface in the marine environment

James Callow (School of Biosciences, University of Birmingham, UK) The search for `environmentally-friendly' solutions to the problem of marine biofouling has stimulated basic research efforts in an attempt to understand which interfacial properties (e.g. roughness, wettability, charge, friction, elasticity) are important in influencing the adhesion of fouling organisms. This has been facilitated by the advent of novel technologies, such as various forms of lithography and selfassembly, which enable the production of test surfaces and coatings with systematic variations in structure and properties at the micro- and nano-scales. The aim of this presentation is to illustrate our increasing understanding of this marine biointerface by reference to recent studies on surface selection and adhesion of spores of the green alga Ulva. Examples of how this understanding may translate to the development of novel antifouling coatings that destabilize surface cue-sensing mechanisms or reduce interfacial adhesion strength will be discussed. Email Address for correspondence: [email protected] 11:30 Sunday 3rd July 2011

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C2.5 Botryococcus braunii - a saviour or red herring as a source of algal biofuels

Gordon W Beakes (Newcastle University, UK), Caldwell Gary (Newcastle University, UK), Hack Ethan (Newcastle University, UK), Rawdhan Alanoud (Newcastle University, UK) and Senousy H Hoda (Newcastle University, UK) Botrycoccus braunii, is a green alga that has been intensively studied because of its ability to synthesize (and secrete) copious amounts of hydrocarbon. Historically, Botrycoccus appears to have been a major contributor to "boghead coals" and some petroleum deposits. Using traditional light microscopy, Botrycoccus can be seen to be composed of densely packed clusters of ovoid to spherical cells, loosely held together by fine strands of extracellular `mucilage'. We have used confocal microscopy (in conjunction with vital dyes) and transmission electron microscopy to obtain a better understanding of both cell and colony structure. The cellular distribution of lipid in Botryoccus will be illustrated and discussed in relation to other lipid producing algal species, such as Nanochloropsis and Dunaniella. Molecular studies have revealed the genus Botrycoccus is an extremely diverse species (if not genus) complex, in which there are several divergent clades, which correspond well with the lipid biochemistry of the isolates. The alkadiene A races from a separate clade from both the lycopodiene synthesizing L races and the botryococcene producing B races, which themselves have diverged from a common ancestor. It is also clear that Botrycoccus is a Trebouxiophyte (not Chlorophyte) alga, closely related to the nanoplanktonic coccoid genus, Chlorocystis. I will conclude with a brief summary of a recent analysis carried out by Dr Gary Caldwellon the practicality of using micro-algae as sources of fuel. Emailaddress for correspondence: [email protected] 14:30 Sunday 3rd July 2011

C2.7 Endotransglycosylases in charophytes: key wallremodelling enzymes?

Lenka Frankova (IMPS, University of Edinburgh, UK) and Stephen C Fry (IMPS, University of Edinburgh, UK) Invasion of the land by the earliest bryophyte-grade plants required inevitable changes in the structure and function of the cell wall. To explore transitions in cell wall biochemistry which took place during plant evolution we focused on the modern charophytic algae, the closest extant relatives of land plants. Unlike higher plants, nothing is known about charophyte wall-modifying enzymes, information that would promote our appreciation of the `primordial' plant cell wall. We tested an in vivo approach for discovering wall-remodelling enzymes that act on endogenous algal polysaccharides (as donor substrates), using a set of diverse exogenous radiolabelled oligosaccharides as acceptor substrates. The results revealed that wall polysaccharides of all charophytes tested become covalently linked to exogenous 3 H-labelled oligosaccharides suggesting that hemicelluloses and pectins in charophytes may not be linked only by non-covalent bonds, as assumed in earlier cell wall models. We also screened a collection of enzyme extracts prepared from various pteridophytes and charophytes and detected several novel homo- and heterotransglycosylation activities. In contrast to pteridophytes, charophytes possess considerable mannan-endotransglycosylase activity, implying that the mannan-based polymers of charophyte walls might be continuously re-modelled according to growth demands or covalently linked to other wall polysaccharides. These findings accompanied by florescent microscopy observations of in-situ transglycosylation will be presented, shedding new light on charophytic algal cell wall biology. Supported by the Leverhulme Foundation. Email address for correspondence: [email protected] 16:20 Sunday 3rd July 2011

C2.6 Algal-bacterial interactions - the evolution of vitamin B12-auxotrophy

Alison G Smith (University of Cambridge, UK), Katherine E Helliwell (University of Cambridge, UK), Elena Kazamia (University of Cambridge, UK) and Glen Wheeler (Plymouth Marine Laboratory, UK) Remarkably for photosynthetic organisms, many algae have a requirement for an exogenous supply of organic micronutrients, in particular vitamins B1 (thiamine), B7 (biotin) and B12 (cobalamin). Indeed over 50% of microalgal species surveyed require vitamin B12, and this auxotrophy is found in all phyla, with no phylogenetic relationship between species that are B12-dependent. This observation suggests that auxotrophy has arisen multiple times throughout the algal kingdom, and that it is likely the result of the loss of a single gene. We have used the data from whole genome sequencing of several algae to gain insight into this auxotrophy, and have obtained evidence that algal species that have no requirement for B12 for growth encode a gene for the B12-independent isoform of methionine synthase, METE, whereas B12-requirers encode only a METH isoform, which needs B12 as a cofactor. Some species encode both isoforms, but the METE gene is repressed by the presence of B12, and two species have been found to have METE pseudogenes, which likely represent the evolution of B12-auxotrophy in action. We have demonstrated that B12-synthesizing bacteria supply the vitamin to algae in return for fixed carbon in cocultures, and the bacteria also repress the METE gene. Our observations provide a model to explain the widespread nature of B12-auxotrophy, and have implications for algal community biology. Email address for correspondence: [email protected] 15:40 Sunday 3rd July 2011

C2.8 Metagenomics of algal culture using illumina sequencing

Karen Moore (College of Life and Environmental Sciences, University of Exeter, UK), Christine Sambles (College of Life and Environmental Sciences, University of Exeter, UK), Thomas M Lux (College of Life and Environmental Sciences, University of Exeter) , UK, George R Littlejohn (College of Life and Environmental Sciences, University of Exeter, UK), Julio Illanes (College of Life and Environmental Sciences, University of Exeter, UK), Rob Lee (College of Life and Environmental Sciences, University of Exeter, UK) and John Love (College of Life and Environmental Sciences, University of Exeter, UK) Understanding the environmental niche of the hydrocarbon-producing alga Botryococcus braunii is important for its successful exploitation as a source of biofuel. B. braunii associated microorganisms may have unpredictable effects on biomass and hydrocarbon yield which are essential for reliable biofuel production. We used a combination of bacterial culture with 16S rDNA analysis, and ultra-high throughput illumina sequencing of meta-DNA to profile the microflora within a well established, stable B. braunii consortium. To identify which bacteria might be more closely associated with B. braunii, we characterized the demographic changes to the microbial flora following physical and chemical perturbation to the algal culture. Metagenomic comparisons between the initial B. braunii culture with one that had been repeatedly filtered and one that had also been treated with ciprofloxacin confirmed that DNA sequences of predicted Botryococcus origin were present in equal quantities in all three treatments but the associated microbial community was dramatically altered between the three cultures. Email address for correspondence: [email protected] 16:40 Sunday 3rd July 2011

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C2.9 Algal and human clocks connect, in the dark night of the cell

Andrew J Millar (Centre for Systems Biology at Edinburgh, UK), Sarah Martin (Centre for Systems Biology at Edinburgh, UK), Martin BarriosLlerena (Centre for Systems Biology at Edinburgh, UK), Thierry LeBihan (Centre for Systems Biology at Edinburgh, UK), Gerben Van Ooijen (Centre for Systems Biology at Edinburgh, UK), Carl Troein (University of Lund, Sweden), Maria-Luisa Guerriero (Centre for Systems Biology at Edinburgh, UK) and Jane E Hillston (Centre for Systems Biology at Edinburgh, UK) The circadian clock system generates 24-hour rhythms in very many biological processes, timed appropriately relative to the day/night cycle. The interlocking feedback loops of clock gene regulation have been a paradigm for systems biology. My lab has combined molecular data, mutants and computational methods to develop mathematical models of the clock, originally in Arabidopsis (www.amillar.org) and with CSBE, in the Prasinophyte alga Ostreococcus tauri. The models have predicted unidentified components of the plant clock and photoperiod sensor (Salazar et al., Cell, 2009; Pokhilko et al., Mol Syst Biol, 2010, and refs therein). O. tauri is ~1 micron diameter, marine micro-alga with a very compact genome of 12Mbp (Derelle et al., PNAS, 2006), which lacks the plant genome's multigene families. Mathematical models of the algal clock (Troein et al., Plant J, 2011; Akman et al., FBTC, 2010), based on molecular data from the Bouget lab (Corellou et al., Plant Cell, 2009), indicated that a single feedback loop was largely responsible for circadian timekeeping. Chemical biology approaches and a novel, peroxiredoxin marker revealed that its timing mechanisms include a non-transcriptional timer that is strikingly conserved (O'Neill et al., Nature, 2011) among eukaryotes and beyond. It operates in a different cellular state from the genetically-accessible plant circadian clock (van Ooijen et al., Curr. Biol., 2011). I will discuss the first proteomic survey of this species. Supported by BBSRC awards E015263, F010583 and ANR/BBSRC BBF0054661. Troein is supported by HFSP. The Centre for Systems Biology at Edinburgh funded by BBSRC and EPSRC award D019621. Email Address for correspondence: [email protected] 10:30 Monday 4th July 2011

C2.11 Ca2+ signalling in eukaryote flagella Chlamydomonas as a model system

Glen L Wheeler (Plymouth Marine Laboratory, UK), Peter Collingridge (Marine Biological Association, UK) and Colin Brownlee (Marine Biological Association, UK) Cilia and flagella are responsible for many essential processes in cell biology, from the propulsion of individual sperm cells to the coordinated beating of cilia in our respiratory tracts. Recent evidence suggests that these organelles also play important roles as cellular sensors and even in regulating the cell cycle. Ca2+-dependent signalling mechanisms are associated with many of these processes, although in many cases the underlying mechanisms remain unclear. The motile green alga, Chlamydomonas, has long been used as a model system to understand flagella structure and function. However, imaging Ca2+ in many algae, including Chlamydomonas, has been problematic. We have developed a system to introduce Ca2+ dyes into Chlamydomonas cells via biolistics which enables us to routinely and robustly image Ca2+ in both the cytosol and in the flagella. Using this technique, we have characterized cytosolic Ca2+ signals associated with the process of flagellar excision. More recently, we have examined the nature of Ca2+ signals within the flagella using Total Internal Reflectance Fluorescence (TIRF) microscopy. This approach allows us to specifically image flagella at high spatial and temporal resolution in the absence of interfering fluorescence from the cell body. We show that Chlamydomonas flagella exhibit the ability to generate Ca2+ elevations independently from each other, suggesting a level of control in flagella signalling processes not previously demonstrated. We are currently using RNAi approaches to characterise the contribution of different classes of flagella-localised ion channels to these Ca2+ signalling processes. Email address for correspondence: [email protected] 12:00 Monday 4th July 2011

C2.12 Identification of a cell wall analogous structure in the haptophyte Emiliania huxleyi

Magdalena A Gutowska (Christian Albrechts University, Germany), Nadja Fischer (Christian Albrechts University, Germany), Kerstin Suffrian (Christian Albrechts University, Germany), Maria Mulisch (Christian Albrechts University, Germany) and Markus Bleich (Christian Albrechts University, Germany) Calcifiying haptophytes mineralise CaCO3 liths nearly equal in diameter to the cell body at a rate of 1 hr-1. The high intracellular calcification rate is supported by one of the largest Ca2+ influxes in eukaryotic cells. An extensive peripheral endoplasmic reticulum (PER) has been hypothesized to mediate Ca2+ transport to the site of coccolithogenesis. A better understanding of the morphology of the PER would help support the hypothesis of store operated calcium entry regulating Ca2+ influx in coccolithophores. Concentric rings at the cell periphery have previously been interpreted as PER in transmission electron micrographs. Our TEM work in E. huxleyi indicates the presence of a PER network, however the outermost concentric layers of the cell appear to make up a cell wall analogous structure. This would be a unique morphology for haptophytes. The structural properties of the outermost layers indicate a rigid form. Following freeze thaw cycles and homogenisation these layers still maintain their shape despite evident cell lysis. A separation between the outermost layers and a membrane that constrains the cytoplasm is evident when E. huxleyi are exposed to hyperosmotic conditions. Two concentric spheres can be imaged in cells stained with the lipophilic styryl dye FM1-43. A cell wall analogous morphology in haptophytes would have consequences for our understanding of calcification related substrate transport and lith exocytosis. Ongoing confocal microscopy work with ER retention peptide antibodies and ER specific vital dyes is characterizing the morphology of the PER in coccolithophores.

C2.10 Applications of mechanistic models of microalgae to the biofuels agenda

Kevin J Flynn (Centre for Sustainable Aquatic Research Swansea University) Developing the potential for microalgal biofuels is complicated by the absence of complete robust data sets, and the myriad of potential experimental / logistic options. A solution is to employ mechanistic models of microalgal growth and physiology. The detail that such models can contain is limited by data availability and the intended application; extant models already employ descriptions of enzyme (de)repressions, for example. While there are various caveats that must be applied to the application of these models, these models now have a long established pedigree in applications to environmental and predator-prey systems. Acceptance of such deployment for exploring the biofuels agenda allows many options to be considered in a timely fashion. These include strain selection, potential for genetic modification, optimising the physical environment, nutrient and light consumption. It also enables a consideration of the environmental risks should such an organism escape, or if the cultivation system became contaminated with a competing alga and/or with a predator. The scope and application of such models will be explored. Email Address for correspondence: [email protected] 11:30 Monday 4th July 2011

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Society for Experimental Biology (Environmental and Materials Engineering, University of Leeds, UK) and Joseph A Gallagher (IBERS, Aberystwyth University, UK) Research to date in biofuel production has been almost exclusively conducted on land-produced biomass, with little work on marine biomass. However, approximately 50% of net primary production of global biomass is produced in aquatic environments providing an under-utilised resource for potential future bioenergy production which importantly also avoids the `food versus fuel' and land use issues. This paper reports on the production of biofuels from thermochemical and biological conversion routes, producing ethanol, methane and bio-oil from the macroalgae Laminaria digitata. This kelp was harvested monthly throughout the year to provide information on seasonal variation in relation to biofuel production. Composition analysis including metal content, proximate and ultimate analysis, sugar and carbohydrate content were also conducted to provide an understanding of the changing composition. Comparisons were then drawn between the composition and the biofuel yields which provided further insights into the optimal harvesting periods for the macroalgae for the respective biofuels. Email address for correspondence: [email protected] 15:40 Monday 4th July 2011

Email for correspondence: [email protected] 12:30 Monday 4th July 2011

C2.13 Towards understanding the thermal sensitivity of Synechocystis PCC6803

Toni Slabas (School of Biological and Biomedical Sciences, University of Durham, UK) Cyanobacteria are a group of organisms which are capable surviving in a wide range of environmental conditions. They are believed to be a progenitor of higher plant chloroplasts and have a highly developed endomembrane system dedicated to harvesting light energy for photosynthesis. The genome of Synechocystis sp PCC6803 has been completely sequenced and contains 3264 ORF's. Targeted gene disruption can be readily achieved, by homologous recombination, a property which makes this organisms particularly attractive to study as a model system. In this paper I will discuss experiments we have conducted, in conjunction with our Japanese collaborators, aimed at elucidating components involved in thermal acclimation of Synechocystis. We have used whole-genome microarray and proteomics to identify components involved in the thermal tolerance of cells. The acclimation of photosystem 2 to higher temperatures has been investigated using a "reconstituted thylakoid system" and iTRAQ technology, coupled to selective inhibitor studies. Results from these investigations will be presented. References: Slabas et al. [2006] Proteomics 6:845-8642. Rowland et al. [2010] PLoS One 5:5[5]:e105113. Rowland et al. [2010] Proteomics 10:1917-19294. Nanjo et al. [2010] Biochim. Biophys Acta 1797:1483-1490 Email address for correspondence: [email protected] 13:30 Monday 4th July 2011

C2.16 The genomics of pathogen recognition, or why brown algae may not have resistance genes

Claire M Gachon (Scottish Association for Marine Science, UK), Antonios Zambounis (University of Thessaly, Greece) and Marek Elias (Charles University, Prague, Czech Republic) As for land plants, disease and parasitism have a huge impact on algae, ranging from spectacular outbreaks in natural populations down to significant losses in multibillion dollar crops such as nori. In order to identify candidates potentially involved in algal defence, we mined the genome of the brown alga Ectocarpus siliculosus for homologues of animal and plant defence genes. Whilst homologues of plant resistance genes are absent from the genome, we identified two families of candidate pathogen receptors (LRR-ROCO and NB-ARCTPR proteins) that apparently evolve new ligand-binding specificities by a highly original controlled and dynamic exon shuffling mechanism. Moreover, hypervariable solvent-exposed amino acid residues are subject to positive selection, an unusual feature reflecting strong evolutionary pressures, such as the ones imposed by a host-pathogen arms race. The genomic organization, structural and evolutionary features of these candidate pathogen receptors are strikingly similar to the pathogen recognition systems described in plants and animals. In conclusion, we hypothesize that brown algae might generate their immune repertoire via controlled somatic recombination. We will discuss these findings in the context of the evolution of adaptive immunity in early eukaryotes. Email address for correspondence: [email protected] 16:20 Monday 4th July 2011

C2.14 Algal biophotovoltaic systems

Christopher J Howe (University of Cambridge) The principle of microbial fuel cells is well established, in which microorganisms metabolizing organic molecules `excrete' electrons into an electrode, causing a current to flow in an external circuit. More recently it has been shown that photosynthesis-based versions of these cells can be constructed. In such `biophotovoltaic' systems (also known as photosynthetic microbial fuel cells) prokaryotic or eukaryotic algae transfer electrons to an electrode in a light-dependent manner. This therefore offers the possibility of direct electricity generation from photosynthetic microorganisms. I shall discuss the behaviour of biophotovoltaic systems in more detail, and assess the feasibility of their providing a source of renewable energy. Email address for correspondence: [email protected] 14:30 Monday 4th July 2011

C2.17 Ionomic analysis in Chlamydomonas reinhardtii during phosphorus deficiency and cadmium stress reveals changes in metal accumulation

Andrew P Dean (University of Manchester, UK), Rachel E Webster (University of Manchester, UK) and Jon K Pittman (University of Manchester, UK) The characteristics of metal accumulation in the microalgae Chlamydomonas reinhardtii are important to elucidate for a full understanding of metal homeostasis in this model photosynthetic microbe. This study has utilized an elemental profiling approach to

C2.15 Macroalgae - a new source of biomass for biofuel production?

Jessica M Adams (IBERS, Aberystwyth University, UK), Andrew B Ross (Environmental and Materials Engineering, University of Leeds, UK), Konstantinos Anastasakis (Environmental and Materials Engineering, University of Leeds, UK), Edward M Hodgson (IBERS, Aberystwyth University, UK), Trisha A Toop (IBERS, Aberystwyth University, UK), Iain S Donnison (IBERS, Aberystwyth University, UK), Jenny M Jones

Abstracts 2011 investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation over seven days in batch culture-grown Chlamydomonas. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sub-lethal concentrations of Cd (up to 15 µM) caused increased accumulation of Co, Cu, Fe and Zn. The Cd-induced metal increase was largely abolished when P availability was high (1 mM compared to 0.01 mM P). In contrast to Cd exposure, P availability substantially affected cellular metal content. P limitation increased the accumulation of various essential trace metals and macronutrients including Co, Fe, K, Na and Zn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on Zn accumulation was the same when either inorganic P (PO43-) or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae when P availability is limiting, and which can be exacerbated by Cd pollution. Further investigations using Chlamydomonas mutants and analysis of gene expression changes have begun to reveal some of the mechanisms responsible for these stress-induced changes in metal uptake. Email address for correspondence: [email protected] 16:40 Monday 4th July 2011

173 involved in the cell cycle regulation are essential, the genetic approach dissecting the molecular mechanisms of the cell cycle has relied on the isolation of conditional mutants. Chlamydomonas reinhardtii is haploid unicellular green alga. For decades, it has been a favourite genetic model within the algae. It divides by a specific mechanism called multiple fission. The cell cycle is characterized by long G1 phase followed by several alternating rounds of S and M phases (S/M phase). Isolation of cdc mutants can provide unique insight into the control and coordination of this so far almost uncharacterized cell cycle. Here, we used ethyl-methane sulphonate (EMS) to mutagenize C. reinhardtii cells and we isolated temperature sensitive mutants able to grow and divide at permissive (24°C) and unable to do so at restrictive (36°C) temperature. We screened approximately 20 000 mutants and isolated eight that arrested within the first cell cycle upon transfer to restrictive temperature. The mutants show block either at G1 (early or late) or during the S/M phase. We have characterized cell cycle progression of some of the mutants as well as changes in the global cell cycle gene expression. This work was supported by the GA CR (grant no. 204/09/0111) and the Institutional Research Concept (no. AV0Z50200510). Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.18 Ganoderma lucidum: A source for novel bioactive lectin

Shivayogeeshwar Neelgund (PG Department of Biochemistry), Sanna Durgappa (Indian Institute of Science, India), G U Vinay (PG Department of Biochemistry) and M Krishnappa (PG Department of Biochemistry) Ganoderma lucidum is popularly known for its high medicinal value against various infectious diseases; specifically this species is abundantly grown in the Western Ghat region of India. We have exploited this fungus for isolation of novel bioactive lectins. The crude extract of Ganoderma lucidum prepared in 0.0 1M Tris-HCl buffer was subjected to DEAE- cellulose ion-exchange chromatography, followed by Affi-gel blue gel affinity chromatography to obtain pure form of lectin. Purified lectin was characterized as glycoprotein of 15 kDa, using SDSPAGE and it was confirmed by MALDI-ToF. It has shown antifungal activity against following pathogens: Fusarium oxysporium, Penicillium chrysogenum, Aspergillus niger, Colletotrichum musae, Botrytis cinerea, Trichophyton rubrum, Trichophyton tonsurans, Trichophyton interdigitale, Epidermophyton floccosum and Microsporum canis. Purified lectin did not show inhibition activity against the human cancer cell lines PC3 and MCF-7. The good haemagglutination activity of purified lectin against human and animal erythrocytes was observed. The excellent lectin activity was observed in the pH range 4 to 9 and temperature up to 600ºC. The haemagglutination activity was stable even in the presence of 10mM EDTA and other divalent metal cations such as CaCl2, MgCl2, ZnCl2, and MnCl2. Email Address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.20 Application of proteomics to assess the impact of elevated levels of CO2 on marine organisms

Penelope J Donohue (University of Glasgow, UK), Heidi Burdett (University of Glasgow, UK), Elena Aloisio (University of Plymouth, UK), Piero Calosi (University of Plymouth, UK), Helen Findlay (Plymouth Marine Laboratory, UK), Steve Widdicombe (Plymouth Marine Laboratory, UK), Maggie Cusack (University of Glasgow, UK) and Nicholas Kamenos (University of Glasgow, UK) All living organisms respond to environmental changes through changes in the expression of multiple genes and proteins. Ongoing environmental changes, in particular decreasing ocean pH (ocean acidification or OA, as a result of increasing seawater pCO2), represent additional environmental stimuli which may induce expression changes in marine organisms. Maerl (red coralline algae or rhodoliths), Lithothamnion glaciale, are a marine biogenic calcite which is likely to be structurally very sensitive to increasing seawater pCO2. Significantly, maerl performs a crucial role in maintaining marine biodiversity, ecosystem provision and impacts on the climate system including cloud nucleation and ozone stability. Therefore any OA induced changes at the molecular level, in particular expression changes related to the biomineralisation, may have an unprecedented impact on marine ecosystems. Here we examined the maerl proteome under control conditions (seawater pH 8.0, T = 12ºC, salinity 35) and analysed quantitative expression changes of distinct proteins known to be involved in biomineralisation, in response to acidified conditions (pH 7.7). This study presents evidence of how OA may affect this calcifying marine organism based on molecular level analysis and highlights the benefits of using this systems based approach to investigate the effects of OA in marine systems. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.19 Isolation of thermosensitive cell division cycle mutants of Chlamydomonas reinhardtii

Katerina Bisova (Institute of Microbiology, Academy of Sciences of the Czech Republic), Monika Hlavova (Institute of Microbiology, Academy of Sciences of the Czech Republic), Maria Cizkova (Institute of Microbiology, Academy of Sciences of the Czech Republic), Milada Vitova (Institute of Microbiology, Academy of Sciences of the Czech Republic) and Vilem Zachleder (Institute of Microbiology, Academy of Sciences of the Czech Republic) Cell division cycle (cdc) mutants played a key role in establishment of our understanding of the cell cycle regulation. Since many of the genes

C2.21 Analysis of the CAX1 and CAX2 calcium transporters from Chlamydomonas reinhardtii as possible regulators of cell signaling and cation homeostasis

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Rachel E Webster (University of Manchester, UK), Peter Bickerton (University of Manchester, UK) and Jon K Pittman (University of Manchester, UK) Changes in cytosolic Ca2+ concentration within a cell are an early response to environmental stresses but the mechanisms responsible for shaping Ca2+ signals are poorly understood. In plant cells, cation/ H+ exchangers (CAX) are one of the key Ca2+ transport pathways that mediate Ca2+ export from the cytosol. They are located at the tonoplast where they perform high-capacity transport of Ca2+ and other cations into the vacuole. Previous studies attempting to understand mechanisms of Ca2+ regulation have proved difficult due to the complexity of higher plant signalling networks and redundancy due to many Ca2+ transporter isoforms. These problems may be overcome by using algae as models to study these processes. CAX genes have been identified and characterized from the unicellular model alga Chlamydomonas reinhardtii. Chlamydomonas CAX1 and CAX2 encoding putative Ca2+/H+ exchangers were identified. Both genes could suppress the Ca2+ hypersensitive phenotype of a yeast mutant demonstrating Ca2+ transport activity. This activity was enhanced following truncation of the N-terminal tail of each transporter, suggesting the existence of an N-terminal auto-regulatory mechanism. Both CAX transporters could also transport other cations including Na+ by CAX1 and Cd2+ by CAX2. Knockdown and over-expression mutants for CAX1 and CAX2 have been generated which allow the physiological roles, including the possible Ca2+ signalling functions of these transporters to be determined. Initial analysis has observed phenotypes in the CAX1 and CAX2 mutant lines including an alteration in sensitivity to Ca2+ and cation stress. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.23 Evidence for the lack of -oxidation in cyanobacteria

George N Taylor (University of Exeter), Rob Lee (University of Exeter), John Love (University of Exeter), Nicholas Smirnoff (University of Exeter), Christoph Edner (University of Exeter) -oxidation is the main fatty acid degradative pathway in living organisms. However searches of the genomes of a range of cyanobacterial species using known E. coli and plant -oxidation protein sequences, reveal that various essential enzymes of this pathway are missing. Genome analysis therefore suggests a lack of -oxidation capacity in cyanobacteria. To investigate the occurrence of -oxidation in cyanobacteria, evidence for the occurrence of acyl CoA dehydrogenase activity, the first enzyme of the -oxidation pathway, was sought along with the occurrence of acyl-CoAs, the intermediates in fatty acid oxidation and ability of oxidation of labeled fatty acids. Using palmitoyl-CoA as a substrate, acyl-CoA dehydrogenase activity could not be detected in cell-free extracts of three cyanobacterial strains but could be detected in E. coli. Acyl-CoAs were detected by LC-MS/MS in E. coli, but were undetectable in three cyanobacterial strains. Breakdown of radiolabelled lauric acid was observed in the pulse-chase experiment in E. coli. However, in cyanobacteria lauric acid was incorporated into complex lipids and very little labeled CO2 was produced. It is concluded that cyanobacteria lack predicted -oxidation genes, do not have the ability to oxidise acyl-CoAs and do not contain acyl-CoAs. The implications of lack of -oxidation is discussed in relation to if or how cyanobacteria are able to degrade fatty acids and whether they can recycle fatty acids via an ACP thioester intermediate. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.22 RDP proteins as putative CDC25 phosphatases in Chlamydomonas reinhardtii

Maria Cizkova (Institute of Microbiology, Czech Republic), Monika Hlavova (Institute of Microbiology, Czech Republic), Milada Vitova (Institute of Microbiology, Czech Republic), Vilem Zachleder (Institute of Microbiology, Czech Republic) and Katerina Bisova (Institute of Microbiology, Czech Republic) Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in most eukaryotic cellular processes including the cell cycle control. Cdc25, highly conserved from yeast to man, is a key phosphatase involved in the regulation of G2/M transition. It dephosphorylates and activates the CDK-cyclin complexes inhibited by Wee1 kinase. With the exception of green alga Ostreococcus tauri, no CDC25 homologues were identified in plant kingdom so far. Here, we used unicellular green alga Chlamydomonas reinhardtii as a model organism to study the enigma of CDC25 phosphatases in plant kingdom. Three predicted proteins, in the Chlamydomonas genome sequence, designated RDP1, RDP2, and RDP3 show homology to the conserved rhodanese domains from the CDC25 family and contain the core catalytic motifs necessary for phosphatase activity. To see whether RDP1/2/3 are functional homologues of Cdc25, we cloned RDP genes into yeast vectors and tested if they can complement temperature sensitive cdc25 mutation in Schizosaccharomyces pombe. To further characterize the proteins we have been analyzing their phosphatase activities in vitro. To identify the role of RDP1/2/3 in the regulation of Chlamydomonas cell cycle, we have prepared recombinant algal strains with up or down-regulated expression of CrRDP1/2/3. We are following the phenotypes during the cell cycle progression. This work was supported by the GA CR (grant nos. P501/10/ P258, 204/09/0111) and the Institutional Research Concept (no. AV0Z50200510). Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C2.24 Metagenomics of slgal vulture using illumina dequencing

Karen Moore (College of Life and Environmental Sciences University of Exeter), Christine Sambles (College of Life and Environmental Sciences University of Exeter), Thomas M Lux (College of Life and Environmental Sciences University of Exeter), George R Littlejohn (College of Life and Environmental Sciences University of Exeter), Julio Illanes (College of Life and Environmental Sciences University of Exeter), Rob Lee (College of Life and Environmental Sciences University of Exeter), John Love (College of Life and Environmental Sciences University of Exeter) Understanding the environmental niche of the hydrocarbon-producing alga Botryococcus braunii is important for its successful exploitation as a source of biofuel. B. braunii associated micro-organisms may have unpredictable effects on biomass and hydrocarbon yield which are essential for reliable biofuel production. We used a combination of bacterial culture with 16S rDNA analysis, and ultra-high throughput Illumina sequencing of meta-DNA to profile the microflora within a well established, stable B. braunii consortium. To identify which bacteria might be more closely associated with B. braunii, we characterized the demographic changes to the microbial flora following physical and chemical perturbation to the algal culture. Metagenomic comparisons between the initial B. braunii culture with one that had been repeatedly filtered and one that had also been treated with ciprofloxacin confirmed that DNA sequences of predicted Botryococcus origin were present in equal quantities in all three treatments but the associated microbial community was dramatically altered between the three cultures. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

Abstracts 2011

175

SEB Main Meeting, Glasgow 2011

C3 - Nuclear envelope

C3.1 Chromatin interactions with the nuclear periphery

Maarten Fornerod (Erasmus Medical Center, The Netherlands) The nuclear envelope represents an important boundary within the eukaryotic cell. The inner face of the nuclear envelope directly contacts the genome at the nuclear lamina and at nuclear pore complexes (NPCs). We have characterized both types of interactions in Drosophila, and found that they have completely different characteristics. Whereas nuclear lamina interacting genes are extremely inactive, NPC interacting genes are much more active. Also it appeared that NPC components not only interact with chromatin at the NPC, but also inside the nucleoplasm where they stimulate gene expression. I will discuss the implications of these interactions for chromatin structure and function. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011

Sponsored by:

C3.2 Plant nuclear envelope dynamics in mitotic division

Katja Graumann (Oxford Brookes University, UK) and David E Evans (Oxford Brookes University, UK) Higher plants undergo an open cell division, in which the nuclear envelope (NE) breaks down in prophase and reforms in late anaphase around the newly formed decondensing sister chromatids. Both the fate of the NE membranes between breakdown and reformation as well as the functional association of NE membrane intrinsic proteins during division remain unexplored. To address these questions, we have used the functional NE intrinsic proteins AtSUN1 and AtSUN2 as well as the non-functional NE marker LBR-GFP and investigated their localisation and dynamics in living, dividing BY-2 cells. Our results provide evidence for the ER retention model and reveal that plant NE reformation is spatially organised. The likely functional significance of the plant SUN domain proteins will also be discussed in relation to other processes, including nuclear architecture and gene regulation. Email address for correspondence: [email protected] 11:10 Saturday 2nd July 2011

Cell polarization is a pivotal process during organismal development and tissue homeostasis, which requires the functional interplay of nuclear, cytoskeletal and centrosomal structures. The underlying molecular mechanisms are however, poorly understood. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies that span the entire mammalian nuclear envelope function in cell polarization, by mediating the anchorage of cytoskeletal structures to the nuclear lamina. Specifically we demonstrate that nesprin-2 forms complexes with the kinesin-1 motor protein apparatus, by associating and recruiting kinesin light chain 1 to the outer nuclear membrane. Moreover, nesprin-2 regulates the kinesin light chain 1 content and its perinuclear localization, which additionally requires the presence of lamins A/C. Disruption of either nesprin-2 or kinesin light chain 1, cause both microtubule and centrosomal detachment from the nucleus, which results in defective cell polarization. Immunofluorescence analysis of nesprin-2 silenced cells reveals profound defects in cytoskeleton architecture, cell substratum adhesion and Golgi-apparatus organization. These findings unravel nuclear envelope-associated proteins as major cell architecture determinants and provide novel insights into how the nucleus regulates important cytoskeleton-associated processes that govern tissue morphogenesis. Email address for correspondence: [email protected] ac.uk 11:50 Saturday 2nd July 2011

C3.3 Molecular mechanisms of nuclear envelope mediated cell polarization

Maria Schneider (School of Biological and Biomedical Sciences, Durham University, UK), Iakowos Karakesisoglou (School of Biological and Biomedical Sciences, Durham University, UK), Angelika A Noegel (Center for Biochemistry, Centre for Molecular Medicine Cologne (CMMC), Cologne University, Germany), Josef Gotzmann (Max F. Perutz Laboratories, Medical University of Vienna, Austria), Andreas Brachner (Max F. Perutz Laboratories, Medical University of Vienna, Austria), Sascha Neumann (Centre for Biochemistry, CMMC, Cologne University, Germany) and Wenshu Lu (School of Biological and Biomedical Sciences, Durham University, UK)

C3.4 A novel function for the Nup133 subunit of the vertebrate Nup107-160 nuclear pore subcomplex at mitotic entry

Stephanie Bolhy (Institut Jacques Monod, France), Valerie Doye (Institut Jacques Monod, France), Richard Vallee (Columbia University, USA), Jan Ellenberg (EMBL Heidelberg, Germany), Xavier Gatti (Institut Jacques Monod, France), Michela Zuccolo (Institut Jacques Monod, France), Tania Nayak (Columbia University, USA), Elisa Dultz (EMBL Heidelberg, Germany) and Imene Bouhlel (Institut Jacques Monod, France)

176

Society for Experimental Biology The identification of plant SUN proteins AtSUN1 and AtSUN2 in combination with the fact that no KASH DNA homologues have been identified suggested that there are some KASH-like proteins in plants, which do not share DNA homology with KASH proteins, but have similar functions to them. The newly identified protein AtNE1 was shown to be localized to the plant nuclear envelope periphery. It is a small nucleoplasmic protein, which freely diffuses through the nuclear pore complex (NPC) and is anchored to the INM by interacting with an unknown INM-intrinsic protein. AtNE1 was predicted to have a nuclear localization signal (NLS), two coiled coil domains and one transmembrane (TM) domain. Domain deletion and truncation fluorescent protein constructs were observed by confocal microscopy. The subcellular localization of mutants implied the putative NLS is not essential for AtNE1 to diffuse through NPC but increases efficiency of targeting; both coiled coil domains participate in the interaction of AtNE1 with its INM interaction partner, and the putative TM domain appeared to be non-functional. The function of AtNE1 in plants will be studied by observing tDNA lines and BY-2 cell stable expression lines. Email address for correspondence: [email protected] 14:50 Saturday 2nd July 2011

In eukaryotes, exchanges between the cytoplasm and the nucleus occur through the nuclear pores complexes (NPCs), large protein assemblies composed of approximately 30 distinct proteins (termed nucleoporins or Nups). Over these last years, studies from our laboratory have been focused on the Nup107-160 complex (also termed the Y-complex) a prominent NPC subcomplex composed of nine subunits in vertebrates. We previously reported that this complex plays a crucial role in post mitotic NPC reassembly in vertebrates. In addition, a fraction of the mammalian Y-complex localizes at kinetochores during mitosis and its efficient depletion from kinetochores impairs mitotic progression in HeLa cells. Our recent data revealed a novel function of the Nup133 subunit of the Y-complex at the G2/M transition. This study was initiated by the observation that CENP-F, a mitosis specific interaction partner of Nup133, localizes at the NE at the G2/M transition prior to its accumulation at kinetochores in mitosis. In addition to CENP-F, the kinetochore constituents NudE, NudEL, dynein and dynactin also accumulate at the NE in G2/prophase cells. We demonstrated that the N-terminal domain of Nup133, while largely dispensable for NPC assembly, is required for efficient anchoring of the dynein-dynactin complex to the NE in prophase and exerts this function through an interaction network via CENP-F and NudE/EL. Finally, our study revealed that this molecular chain is critical for tethering centrosomes to the nuclear surface at the G2/M transition and thereby contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly. Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

C3.7 The role of the Ran system in nuclear envelope formation

Paul Clarke (Biomedical Research Institute, School of Medicine, University of Dundee, UK), Jingyan Fu (College of Life Sciences, Peking University, China), Li Guo (College of Life Sciences, Peking University, China), Ekarat Hitakomate (Biomedical Research Institute, School of Medicine, University of Dundee, UK), Qing Jiang (College of Life Sciences, Peking University, China), Quanlong Lu (College of Life Sciences, Peking University, China), Xuelong Lu (College of Life Sciences, Peking University, China), Khamsah S Mohd (Biomedical Research Institute, School of Medicine, University of Dundee, UK), Helen S Sanderson (Biomedical Research Institute, School of Medicine, University of Dundee, UK) and Chuanmao Zhang (College of Life Sciences, Peking University, China) The small nuclear GTPase Ran determines the direction of macromolecular transport between the nucleus and the cytoplasm in eukaryotic cells. Like other GTPases, Ran acts as a molecular switch that changes interactions with effector proteins depending on its guanine-nucleotide bound state. Ran-GTP is generated at chromatin by the guanine-nucleotide exchange factor RCC1, while GTP hydrolysis on Ran stimulated by RanGAP occurs in the cytoplasm. Ran-GTP controls nucleocytoplasmic transport by interaction with import and export proteins of the karyopherin family in the nucleus, causing them to release or bind their cargos, respectively. During mitosis, when the nucleus is reorganised to allow chromosome segregation into two daughter cells, Ran has sequential functions in mitotic spindle assembly and nuclear envelope formation. The mechanisms of these functions provide insights into the spatial and temporal control of the changes in intracellular organisation during the cell division cycle. I will briefly review the functions of Ran in the coordination of nuclear functions and describe the biochemical mechanisms of its actions. I will discuss how Ran coordinates nuclear envelope formation and the assembly of nuclear pore complexes at the end of mitosis. Finally, I will present our new data on the role of Ran and karyopherins in the spatial organisation of nuclear envelope formation through the recruitment of multiprotein complexes to chromatin. Email address for correspondence: [email protected] 15:40 Saturday 2nd July 2011

C3.5 Function of the TREX-2 complex in the integration of transcription and nuclear processing with mRNA export

Murray Stewart (MRC, Laboratory of Molecular Biology, UK), Divyang Jani (MRC, Laboratory of Molecular Biology, UK) and Andrew M Ellisdon (MRC Laboratory of Molecular Biology, UK) There is an emerging consensus that the nuclear steps of the gene expression pathway are integrated and that the TREX-2 complex is central to this process. In yeast, TREX-2 is based on a Sac3 scaffold to which Sus1, Cdc31, Thp1 and Sem1 bind. TREX-2 binds to nuclear pores and is important in gene gating as well as in coordinating the generation of export-competent mRNPs. The structures of the Sac3:Sus1:Cdc31 and Sac3:Thp1:Sem1 complexes show how these proteins function in this process. Email address for correspondence: [email protected] 14:10 Saturday 2nd July 2011

C3.6 Identification of a plant nuclear envelope protein and its characterization

Ting Lu (Oxford Brookes University, UK), Katja Graumann (Oxford Brookes University, UK) and David E Evans (Oxford Brookes University, UK) In animal and yeast cells, a cross nuclear envelope complex termed LINC (The Linker of Nucleoskeleton and Cytoskeleton) complex is formed by interacting outer nuclear membrane (ONM) intrinsic KASH (Klaricht/ANC-1/Sync homology) proteins and inner nuclear membrane (INM) intrinsic SUN (Sun1/UNC-84) proteins. The LINC complex provides a physical signalling between nucleoskeleton and cytoskeleton and participates in nucleus positioning and nucleus movement in cell division.

Abstracts 2011

177

C3.8 Nuclear envelope structure in frogs, fungi, flies and flowers

Martin W Goldberg (Durham University, UK) The separation of the genome from the translation machinery by the nuclear envelope (NE) was a pivotal event in the evolution of complex organisms, enabling new complexity in the control of gene expression. The nuclear envelope forms a double membrane barrier around the chromosomes. The nuclear pore complexes (NPCs), which facilitate and control transport of soluble proteins and RNAs, traverse both membranes, connecting them, enabling, and possibly controlling, the movement of membrane proteins from the ER, where they are synthesized, to the inner membrane where they function. The NPCs are massive protein complexes that must be able to function in these diverse transport pathways and are also physically linked to the nuclear lamina, which lines the inner nuclear membrane, to the chromatin and to the cytoskeleton. We have used high-resolution scanning electron microscopy to study the structure of the NPC and how it interfaces with these different cellular components. This has been combined with transmission electron microscopy of rapidly frozen samples to determine mechanisms of transport of both soluble and membrane proteins. We have shown how different transport pathways take different routes through the NPC channel. We have also shown the structural relationship between NPCs of different organisms and provide evidence that the nuclear lamina, previously thought to only be present in animal cells, has an equivalent structure in plants. Email address for correspondence: [email protected] 16:20 Saturday 2nd July 2011

178

Society for Experimental Biology

SEB Main Meeting, Glasgow 2011

C4 - Gas sensing and signalling in animal cells

C4.1 CO2/HCO3/pH chemosensing via soluble adenylyl cyclase

Lonny R Levin (Weill Medical College of Cornell University, USA) and Jochen Buck (Weill Medical College of Cornell University, USa) Carbon dioxide (CO2) is produced by living organisms as a by product of metabolism. In physiological systems, CO2 is inextricably linked to bicarbonate (HCO3-) and pH via a ubiquitous family of carbonic anhydrases, and numerous biological processes are dependent upon a mechanism for sensing the level of CO2, HCO3, and/or pH. Our discovery that soluble adenylyl cyclase (sAC) is directly regulated by bicarbonate provided a link between CO2/HCO3/pH chemosensing and signaling via the widely used second messenger cyclic AMP. In my talk, I will describe a number of physiological systems where bicarbonateregulated sAC functions as a CO2/HCO3/pH chemosensor. Email address for correspondence: [email protected] 10:30 Sunday 3rd July 2011 As the partial pressure of dissolved CO2 (pCO2) controls the pH of blood, and many physiological processes are sensitive to pH, efficient regulation of pCO2 in blood is a critical homeostatic function. Chemosensors within the brain measure arterial pCO2 and evoke adaptive changes in breathing to control this parameter. While CO2 can be detected indirectly as pH, the idea that it could also be detected directly has largely been overlooked. We have recently discovered a new molecular transducer for CO2 chemoreception ­ the connexin 26 (Cx26) hemichannel. Cx26 is directly sensitive to both increases and decreases of pCO2 and is able to mediate CO2-dependent ATP release. This bidirectional sensitivity of Cx26 to pCO2 alters release of ATP via the hemichannel in a bidirectional manner thus regulating the excitation of the respiratory network and contributing to the adaptive changes during both hyper- and hypocapnia. Interestingly some connexins closely related to Cx26 also exhibit CO2-sensitivity. The widespread distribution of Cx26 in the sub-pial astrocytes and leptomeninges at the brain surface suggests that it may play roles in other CO2 sensitive reflexes. I shall present evidence for CO2-dependent ATP release at the surface of the tuberomammillary nucleus, likely mediated via Cx26, that could contribute to CO2 -induced arousal via the histaminergic system. Our recent finding that CO2 also opens an inwardly rectifying K+ channel at constant pH, suggests that a wider range of physiologically significant channels may exhibit sensitivity to CO2 than has been appreciated hitherto. Email address for correspondence: [email protected] 12:00 Sunday 3rd July 2011

C4.2 NF-B, CO2 sensing, innate immunity and inflammation

Eoin P Cummins (University College Dublin, Ireland), Kathryn M Oliver (University College Dublin, Ireland), Colin R Lenihan (University College Dublin, Ireland), Susan F Fitzpatrick (University College Dublin, Ireland), Ulrike Bruning (University College Dublin), Carsten C Scholz (University College Dublin, Ireland), Craig Slattery (University College Dublin, Ireland), Martin O Leonard (University College Dublin, Ireland), Paul McLoughlin (University College Dublin, Ireland) and Cormac T Taylor (University College Dublin, Ireland) Molecular oxygen (O2) and Carbon dioxide (CO2) are the primary substrate and product of aerobic metabolism respectively. Levels of these physiologic gases in the cell microenvironment vary dramatically both in health and in diseases such as chronic inflammation, ischemia and cancer where significant changes in metabolism occur. The identification of the hypoxia-inducible factor (HIF) led to the discovery of an ancient and direct link between tissue O2 and gene transcription. Here, we demonstrate that mammalian cells also sense changes in local CO2 levels leading to altered gene expression via the NF-B pathway. IKK, a central regulatory component of NF-B, rapidly and reversibly translocates to the nucleus in response to elevated CO2. This response is independent of HIF, extracellular and intracellular pH or pathways which mediate acute CO2-sensing in nematodes and flies and leads to attenuation of bacterial lipopolysaccharide-induced gene expression. These results suggest the existence of a molecular CO2 sensor in mammalian cells which is linked to the regulation of genes involved in innate immunity and inflammation. Email address for correspondence: [email protected] 11:30 Sunday 3rd July 2011

C4.4 CO2 sensing in fungal pathogens of humans

Fritz A Muhlschlegel (University of Kent, UK) The ubiquitous gas carbon dioxide (CO2) is a signal for all organisms ranging from bacteria to mammals. The fungal pathogen Candida albicans senses and specifically responds to changes in environmental CO2 with two major proteins: adenylylcyclase and carbonicanhydrase (CA). Carbonicanhydrase is crucial as it efficiently fixes CO2 inside the cell as bicarbonate. The latter activates adenylylcyclase but also serves as substrate in the cells fundamental carboxylation reactions. The physiological significance for this is shown by CA's requirement for fungal growth. We found that CA expression itself is tightly controlled by the availability of CO2. Low levels, as found in ambient air, strongly induce CA transcript and protein. By contrast high concentrations (5.5%), where sufficient bicarbonate is spontaneously formed to meet the metabolic requirements, suppress CA expression. We identified a previously uncharacterized bZIP transcription factor, as the first CO2 regulator of CA expression in yeast. We show that this TF acts separately from the CO2 switch adenylyl cyclase, previously characterized by us. We also demonstrate that the impact of CO2 on CA expression; CA's requirement for growth; and CA regulation by orthologues is conserved in the yeasts Candida glabrata and Saccharomy cescerevisiae pointing to the broad significance of this novel pathway in fungi. By using high resolution two-photon excitation confocal microscopy we record fluorescent CA signals in single cells and yeast colonies offering insight into the flux of CO2 and nichespecific metabolic adaptation in fungal populations. Email address for correspondence: [email protected] 12:30 Sunday 3rd July 2011

C4.3 Direct sensing of CO2 in the brain

Nicholas Dale (University of Warwick, UK)

Abstracts 2011

179 Gaseous exchange of O2 and CO2 in the nematode C. elegans, like in many small animals, occurs by diffusion across the body wall. This means the concentrations of these gases in body fluids reflect their concentrations outside the animal. C. elegans has developed sophisticated mechanisms to detect small changes inthe concentrations of these gases in its body fluid. I will describe the molecular signalling pathways that mediate of O2 and CO2 sensing in this animal, the resulting properties of gas sensing neurons and neural circuits, and the way these circuits encode escape behaviours, including aggregation. Email address for correspondence: [email protected] 15:45 Sunday 3rd July 2011

C4.5 Hypercapnia (high CO2 level) effects on Caenorhabditis elegans

Yosef Gruenbaum (Hebrew University of Jerusalem, Israel), Jacob I Sznajder (Northwestern University, USA), Inbar Mizrahi (Hebrew University of Jerusalem, Israel) and Kfir Sharabi (Hebrew University of Jerusalem, Israel) Hypercapnia occurs in a number of lung diseases and it is associated with worse outcomes in patients with chronic obstructive pulmonary disease (COPD). It is largely unknown how hypercapnia is sensed and responds in non-neuronal cells. Here we used C. elegans to study the response to non-anaesthetic CO2 levels and show that levels exceeding 9% CO2 induce aberrant motility that is accompanied by age-dependent deterioration of body muscle organization, slowed development, reduced fertility and increased lifespan. These effects occur independently of the IGF-R, dietary restriction, egg laying or mitochondrial-induced aging pathways. However, changes in mitochondria shape after exposure to CO2 suggest involvement of the mitochondria in the CO2 response. Acute exposure of wild type C. elegans to hypercapnia significantly reduces pumping rate of the pharynx. This reduction is significantly rescued if animals are starved prior to CO2 exposure and is mediated by specific genes. In addition, animals are more sensitive to aldicarb, an inhibitor of acetylcholine esterase, and levamisole, a potent agonist of the nicotinic AchR, which suggests a decrease in post synaptic muscle sensitivity. Together, these results suggest specific physiological and molecular responses to hypercapnia, which will hopefully help to better understand the molecular mechanism involved in sensing and responding to hypercapnia. Email address for correspondence: [email protected] 14:00 Sunday 3rd July 2011

C4.8 The influence of carbon dioxide on cAMP and calcium signalling

Martin J Cann (Durham University, UK), Michael A Gray (Newcastle University, UK) and Zara C Cook (Durham University, UK) The importance of CO2 in biology is paramount. CO2 is integral to all life as the substrate for the CO2-fixing enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase in photosynthetic organisms and the pHdependent CO2/bicarbonate equilibrium is critical for cell and systems physiology. While a number of examples are now described of specific sensing mechanisms for CO2/HCO3- that function in the regulation of appropriate physiological processes (e.g. chemosensory control of breathing, sperm maturation) little is known of the molecular mechanism(s) by which elevated CO2 can have non-specific detrimental effects on cell biology. Knowledge of such detrimental effects is crucial to understanding why pathological conditions associated with chronic elevated CO2 (e.g. chronic hypoventilation in obesity and airways disease) predisposes to cardiovascular disease and increased likelihood of mortality (with associated higher healthcare costs). In direct contrast to the current paradigm where CO2 can activate specific physiological processes through accumulation of cyclic nucleotides we demonstrate that CO2 generically blunts cellular activities regulated by cAMP. This effect is independent of pH, cell type, and mechanism of activating the cAMP pathway and requires Ca2+ release via the IP3 receptor. Here we will describe our knowledge of the signalling route from CO2 to cAMP, through Ca2+, and to a defined cAMP dependent physiological process. Given the ubiquity of cAMP signalling in mammalian cells, this work suggests a key mechanism by which CO2 can have a broad spectrum of deleterious effects on cell physiology independent of pH. Email address for correspondence: [email protected] 16:15 Sunday 3rd July 2011

C4.6 Genes and neural circuits controlling sensitivity to carbon dioxide in the mosquito

Conor J McMeniman (The Rockefeller University, USA) and Leslie B Vosshall (The Rockefeller University, USA) Carbon dioxide (CO2) is a key chemosensory cue that activates the mosquito olfactory system, alerting mosquitoes to the presence of human skin odour and heat. Despite the critical importance of CO2 reception in driving the orientation of mosquitoes towards humans, the molecular and cellular basis of how this gas is sensed by this bloodfeeding insect is poorly understood. Previously, two chemosensory receptors Gr21a and Gr63a, were shown to be essential to detect volatile CO2 in Drosophila. We are applying zinc-finger nuclease (ZFN) technology for targeted mutagenesis and homologous recombination at two orthologous genes, AaegGr1 and AaegGr3, in the yellow fever mosquito Aedes aegypti. Progress on the genetic, behavioural, and electrophysiological characterization of mosquito responses to CO2 will be discussed. Application of functional genetics to this model mosquito species stands to provide powerful insight into the genes and neural circuits used by this disease vector for CO2 sensation, and their significance to mosquito host-seeking behaviour. Email address for correspondence: [email protected] 15:00 Sunday 3rd July 2011

C4.9 Lung Injury and carbon dioxide: good versus evil

Daniel O'Toole (National University of Ireland, Ireland), John Laffey (National University of Ireland, Ireland) There is a wealth of evidence to support elevated CO2 levels as being of overall benefit in acute lung injury (ALI) patients, the allowance of which through specific ventilator strategies is termed "permissive hypercapnia". As other groups have also shown, hypercapnic acidosis (HCA) is broadly inhibitory of inflammation, yet it is often associated with poor clearance of pathogenic bacteria and restricted healing of injured lung tissue. The latter can be broadly addressed by reference to bacteria-promoting pH changes, while the mechanism of the former is only now being elucidated. We have shown recently that lung epithelial cells have inhibited migration under HCA, which would complement in vivo data, and have demonstrated that this occurs via perturbation of NF-kB signalling cascade. More recently, we have investigated the effects of CO2 on activation of specific proteins on the canonical NF-kB activation pathway and

C4.7 Neural circuits and molecular mechanisms for sensing O2 and CO2 in C. elegans

Mario De Bono (MRC Laboratory of Molecular Biology Cambridge)

180

Society for Experimental Biology local acidotic conditions experienced within certain tumours prior to angiogenesis, and how correct maintenance of inorganic carbon equilibria facilitates cancer disease progression. Here we describe a pH-independent reduction in levels of the second messenger cAMP on hypercapnia resulting in downstream physiological effects on pH recovery. The effects of hypercapnia on cAMP signalling are independent of cell type and the method of adenylyl cyclase activation. Further, we have demonstrated a requirement for calcium ion release through the IP3 receptor in the endoplasmic reticulum for the reduction in cellular cAMP. Due to the ubiquitous nature of cAMP and Ca2+ signalling within animal cells, our results point toward a critical mechanism by which CO2 causes deleterious effects on the cell, independent of pH. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

the control of downstream events at the NF-kB binding promoter consensus sequence. Our findings would indicate that CO2 can be inhibitory to many aspects of cytosolic signalling, which we hope to translate into more effective therapeutic interventions mimicking the beneficial effect of HCA in the patient. Email address for correspondence: [email protected] 16:40 Sunday 3rd July 2011

C4.10 Hypercapnia reduces CFTR-dependent bicarbonate transport in human secretory airway epithelial cells

Mark J Turner (Newcastle University, UK), Martin Cann (Durham University, UK) and Mike A Gray (Newcastle University, UK) Hypercapnia arises from a variety of lung diseases and also occurs after treatment of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) during which ventilation is reduced mechanically. CO2 acts as a cellular signalling molecule across a broad range of species but its role in human cells is poorly understood. Our aim was to investigate how hypercapnia impacts upon G-protein sensitive adenylyl cyclase signalling by studying the effect of pCO2 on cAMPactivated CFTR-dependent HCO3- transport in polarised cultures of human Calu-3 airway cells. Rates of HCO3- transport was assessed by real-time measurements of intracellular pH using the pH sensitive dye BCECF-AM. In the presence of 5% CO2/25 mM HCO3- exposure to adenosine (10 µM) or forskolin (5 µM) maximally stimulated HCO3secretion across the luminal membrane. Short-term exposure to 10% CO2 reduced secretion by ~50%, an effect which was blocked by the phosphodiesterase 3 inhibitor cilostazol. These results suggest that hypercapnia reduces CFTR-dependent HCO3- secretion via a CO2-induced inhibition of adenylyl cyclise and decrease in cAMP levels. Hypercapnia would therefore be predicted to reduce the volume of liquid secretion as well as the pH of secreted fluid which could contribute to airways dysfunction. Our work therefore highlights a possible mechanism underlying the cellular effects of hypercapnia in the airways, which may help in understanding the potential harmful effects of CO2 in patients undergoing treatment for ALI/ARDS. This work was supported by a MRC DTG Studentship to MT. Email address for correspondence: [email protected] Poster Session - Sunday 3rd July 2011

C4.11 The influence of hypercapnia on cAMP and calcium interactions

Zara C Cook (School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, UK), Michael A Gray (Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, UK) and Martin J Cann (School of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, UK) Carbon dioxide is essential for all life on Earth, primarily as the key substrate for inorganic carbon fixation and through its pH-dependent equilibrium with bicarbonate ions, critical for acid/base homeostasis and cell physiology. While a number of specific CO2/HCO3- sensing mechanisms are known (e.g. chemosensory control of breathing rate, sperm maturation), little is known about the underlying molecular basis for the classically detrimental effects of CO2 on the cell. Molecular understanding of the signalling events associated with elevated CO2 (hypercapnia) is paramount to defining its physiological impact in a variety of disease states, including airways disorders (e.g. asthma and COPD) and obesity, where hypercapnia occurs as a result of associated hypoventilation. In addition, this understanding may provide further insight into cancer cell survival under the hypercapnic and associated

Abstracts 2011

181

SEB Main Meeting, Glasgow 2011

P1 - Regulation of resource allocation and growth

Sponsored by:

P1.1 Towards mechanistic models of plant organ growth: How to link molecular regulatory mechanisms with spatio-temporal regulation of cell division and expansion to whole organ growth?

Gerrit T Beemster (University of Antwerp), Dirk De Vos (University of Antwerp), Delphine Draelants (University of Antwerp), Abdiravuf Dzhurakhalov (University of Antwerp), Jan Broeckhove (Universiteit Antwerpen), Wim Vanroose (University of Antwerp), Roeland Merks (Netherlands Institute for Systems Biology (NISB) and Centrum Wiskunde Informatica (CWI)) Plant growth results from a complex interplay of molecular regulatory mechanisms, which essentially operate at the level of the individual cells that make up the plant. These cells sense their intrinsic state and local environment and based on this information regulate the cell division and cell expansion processes and the production and transport of signalling molecules that are sensed by neighbouring and also cells located at greater distances. Although this is general knowledge, how these regulatory mechanisms interact mechanistically is difficult to understand intuitively and can only be addressed by building mechanistic simulation models. These models should implement the regulatory relationships as a set of differential equations at the level of the individual cell and then allow us to evaluate the growth patterns of the organ as an emerging property. This presents us with the challenge of 1. collecting relevant molecular and cellular level measurements in planta and 2. constructing a model that represents the growing cells/ organs realistically. In this presentation an example of how to approach these challenges will be outlined and some of the outcomes and limitations of the models will be presented. Email address for correspondence: [email protected] 10:30 Friday 1st July 2011

can be altered, we can quantify the influence of these parameters on leaf form and function, thus characterise the spatial and temporal limits within which a particular parameter can act to influence the system. Our data suggest that leaf form is generated by a process of local growth suppression set against the background of a growing system and indicate that specific cell cycle regulators acting during an early phase of leaf growth can have a long term influence on leaf cellular architecture, with consequences for organ physiology. Email address for correspondence: [email protected] 11:15 Friday 1st July 2011

P1.3 Diel patterns of leaf and root growth - circadian rhythmicity or environmental response?

Achim Walter (ETH Zurich), Tom Ruts (Forschungszentrum Juelich), Shizue Matsubara (Forschungszentrum Juelich), Michael Mielewczik (ETH Zurich) Leaves and roots live in different habitats, but are parts of the same organism. Whereas the shoot is exposed to pronounced environmental variations throughout the diel cycle (24 h), the root grows in a temporally less fluctuating environment. We hypothesize that this difference in environmental constraints has induced different basic control patterns of the diel growth fluctuations in roots and leaves of mono- and dicotyledonous plants, respectively. Differences between diel leaf growth patterns of mono- and dicotyledonous plants are linked to the different organization and placement of growth zones in these species. In monocots, growth zones are organized linearly, are protected from the environment by sheathes of older leaves and are situated close to the ground. In contrast to this, dicot leaf growth zones are exposed more directly to the environment and show characteristic, species-specific diel growth variations. Dicot leaf growth is to a much higher degree controlled by the circadian clock and hence shows a more indirect connection to oscillations of environmental parameters compared to monocot leaves and roots, thereby optimizing resourceuse efficiency of the whole plant. Email address for correspondence: [email protected] 11:45 Friday 1st July 2011

P1.2 Leaf form and function: Linking cellular architecture and organ physiology

Andrew Fleming (University of Sheffield, UK), Carmen Dorca Fornell (University of Sheffield, UK), Asuka Kuwabara (University of Sheffield, UK), Robert Malinowski (University of Sheffield, UK) and Ania Kasprzewska (University of Sheffield, UK) Significant progress has been made in identifying the transcriptional networks and signalling pathways involved in various aspects of leaf development but we still do not understand the downstream mechanism by which leaf form is generated. In addition, we also have limited insights into the outcome on leaf physiology of changes purely of leaf structure, separate from the changes in biochemistry that generally accompany such changes in morphology. We have set out to identify the rules relating cell division and growth to leaf form and function. By creating artificial systems in which individual parameters

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P1.4 Plant turgor pressure coordinates sink-source relationships via MCA1 in Arabidopsis thaliana

Alexandra Wormit (Imperial College London, UK), Salman Butt (Imperial College London, UK), Issariya Chairam (Imperial College London, UK), Joseph McKenna (Imperial College London, UK), Adriano Nunes-Nesi (Universidade Federal de Vicosa, Brazil), Lars Kjaer (Imperial College London, UK), Alisdair Fernie (MPI Molecular Plant Physiology Golm-Potsdam, Germany), Rüdiger Woscholski (Imperial College London, UK), Laura Barter (Imperial College London, UK) and Thorsten Hamann (Imperial College London, UK) Cellulose is the most abundant biopolymer in the world, the main load-bearing element in plant cell walls and represents a major sink for carbon in plant cells. The coordination of the activity of this carbon sink with the carbon fixation occurring during photosynthesis is not understood. Here we demonstrate that cellulose biosynthesis inhibition (CBI) leads to transcriptional shutdown of genes involved in photosynthesis, the Calvin cycle and starch degradation. We show that CBI causes redirection of metabolic flux from the cell wall towards starch biosynthesis and reduction of Rubisco activity. The observed effects can be suppressed in a concentration dependent manner by providing osmotic support using PEG. In parallel, hyper-osmotic stress treatments (using KCl or NaCl) have opposite effects on starch and sucrose levels compared to CBI (a hypoosmotic stress. In mid1 complementing activity (mca1, putative stretch activated Ca2+ channel), Arabidopsis histidine kinase (ahk4, osmosensor) and respiratory burst oxidase homolog DF (rbohDF, NADPH oxidase) seedlings the CBI induced effects on starch metabolism and gene expression are not suppressed when an osmotic support is provided. Our findings reveal a novel regulatory mechanism coordinating cellulose biosynthesis with primary metabolism and photosynthetic activity. We demonstrate that the osmotic state of the cell affects carbohydrate metabolism and identify MCA1, AHK4 and NADPH oxidase-derived ROS production as components of the signalling mechanism translating osmotic signals into metabolic changes. Email address for correspondence: [email protected] 12:15 Friday 1st July 2011

P1.6 How could a presumably cell autonomous signalling metabolite of the trehalose metabolism control plant carbon allocation and thus growth?

Henriette Schluepmann (Utrecht University, Netherlands) Trehalose is the -1,1-linked disaccharide of glucose. Surprisingly, trehalose feeding is toxic to plants with low trehalase activity. Early reports document trehalose induced growth arrest in and reduction of sucrose allocation to extension zones of Cuscuta reflexa. Reduced sucrose allocation is associated with less carbon integration in cell wall materials in the extension zone. Work with Arabidopsis seedlings confirmed observations from C. reflexa; seedlings grown on trehalose are growth arrested, their root-tips fail tosynthesize starch instead the source cotyledons accumulate starch. pgm1 mutants unable to synthesize starch are similarly affected suggesting that altered starch deposition does not cause the growth arrest. All plant genomes examined encode enzymes for trehalose biosynthesis with a striking radiation of genes encoding enzymes metabolizing the trehalose biosynthetic precursor, trehalose-6-phosphate (T6P). T6Pis required for embryo and seedling carbon utilization and growth, with small changes in T6P effecting large changes plant phenotype. T6P accumulates 10-fold in seedlings on trehalose and expression of T6Phydrolase suppresses growth arrest. Data suggest that T6P is required for growth on sucrose, but ubiquitous T6P accumulation in the absence of available carbon stops growth in the sinks. High T6P levels occur naturally during early wheat grain development in the endosperm. T6P further inhibits SnRK1 activity in growing tissues; SnRK1 is a central kinase regulator integrating energy and stress signalling pathways. T6P was therefore coined a signalling metabolite. Due to its charged nature T6P likely acts cell autonomously; this is consistent with tissue-specific and stress responsive expression of enzymes of T6P metabolism. Email address for correspondence: [email protected] 14:35 Friday 1st July 2011

P1.7 Regulation of maize leaf growth in response to carbon availability

Angelika B Czedik-Eysenberg (Max-Planck-Institute for Molecular Plant Physiology, Germany), Maria Piques (Max-Planck-Institute for Molecular Plant Physiology, Germany), Sunil Kumar Pal (Max-PlanckInstitute for Molecular Plant Physiology (former member), Germany), Beatrice Encke (Max-Planck-Institute for Molecular Plant Physiology, Germany), Nicole Krohn (Max-Planck-Institute for Molecular Plant Physiology, Germany), Björn Usadel (Max-Planck-Institute for Molecular Plant Physiology, Germany), Ronan Sulpice (Max-PlanckInstitute for Molecular Plant Physiology, Germany) and Mark Stitt (MaxPlanck-Institute for Molecular Plant Physiology, Germany) Plants can fix carbon only in the light, but need it through the complete diurnal cycle. To avoid periods of starvation, plants need to tightly regulate the amount of carbon stored in the light, and its usage during the night. In this study, we investigated the role of carbon availability in the regulation of leaf elongation in maize. The maize leaf constitutes an ideal model for such study because: (i) leaf elongation rate can be determined with time resolution of minutes; and (ii) dividing, elongating and mature photosynthetic tissues are spatially separated, allowing separate analyses. A positive correlation between the elongation rate and carbohydrate levels in the dividing and elongating regions was observed during the diurnal cycle and a prolonged period of darkness, inducing starvation. Interestingly, the mature region was depleted faster than the growing regions, suggesting mechanisms protecting growing tissues against starvation in order to maintain growth. Transcript analysis confirmed that growing regions went into starvation later than the mature region. Protein synthesis was investigated as a major and tractable component of cellular growth. Polysome loading analysis showed that translation

P1.5 Control of growth during the development of maize inflorescence architecture

David Jackson (Cold Spring Harbor Lab), Stacy DeBlasio (CSHL), Andrea Eveland (CSHL), Namiko Satoh Nagasawa (CSHL), Alexander Goldshmidt (CSHL), John Lunn (MPI - Golm) Plant morphology is central to crop yields, for example in controlling grain yield. One critical aspect of morphology, inflorescence branching, is determined by groups of stem cells called shoot meristems. Variations in branching lead to diversity in inflorescence shapes and sizes, and impact on traits such as seed number or ease of harvest. The RAMOSA (RA) genes of maize control inflorescence branching through regulation of the identity and determinacy of axillary inflorescence meristems. Two of the genes in this pathway, RA1 and RA2, encode transcription factors, whereas RA3 encodes a trehalose-6-phosphate phosphatase. The trehalose pathway has been implicated in stress protection, control of sugar signalling and regulation of photosynthetic rates. However how the RA3 gene might function in control of inflorescence architecture is unknown. I will describe efforts we have taken to elucidate the molecular role of RA3 in control of inflorescence architecture. Analysis of the pathways by which RA3 controls development may give unique and novel insights into mechanisms of branching in maize inflorescences and in other crop plants. Email address for correspondence: [email protected] 13:50 Friday 1st July 2011

Abstracts 2011 was immediately decreased at the start of night in mature leaf tissue, as previously seen in Arabidopsis. In contrast, high translation rates were maintained in the growing tissues during the night and even into the prolonged period of darkness. Taken together, our results suggest a close link between carbon availability and growth in maize leaves, and point to the existence of mechanisms that protect growing regions against carbon starvation. Email address for correspondence: [email protected] 15:05 Friday 1st July 2011

183 increase the water use efficiency of carbon assimilation. The C4 acids are subsequently decarboxylated in the daytime, with stomata closed, releasing CO2 at high concentrations for direct fixation by Rubisco and the C3 cycle. This highly specialized pathway of enzymes requires tight molecular and biochemical control to avoid futile cycling of CO2. Using sequences obtained from Roche 454 sequencing we have made transgenic plants of Kalanchoë fedtschenkoi, an obligate CAM. These plants carry silenced (RNAi) genes to key CAM genes resulting in reduced enzyme activities. These plants allow us to investigate the relative contribution key enzymes in the CAM pathway to plant productivity, and their regulation and expression. Plants with reduced activites of NAD malic enzyme, phosphoenolpyruvate carboxylase kinase and pyruvate Pi dikinase show interesting phenotypic changes in the four classic phases of CAM photosynthesis and their circadian response to constant (light/temperature) conditions. Significant changes in malate and starch metabolism are also apparent. These transgenic plants grow surprisingly well in well-watered conditions. We are now investigating their growth under drought stress to compare the contribution of a compromised CAM pathway to growth and yield, in drought and well-watered conditions. Email address for correspondence: [email protected] 16:10 Friday 1st July 2011

P1.8 How to achieve an optimal growth? It is not necessarily about what you can get, but also when/ how and where to use it

Ronan Sulpice (Max-Planck-Institute for Molecular Plant Physiology, Germany), Pyl Eva-Theresa (Max-Planck-Institute for Molecular Plant Physiology, Germany), Ishihara Hirofumi (Max-Planck-Institute for Molecular Plant Physiology, Germany), Antonio Carla (Max-PlanckInstitute for Molecular Plant Physiology, Germany), Piques Maria (Max-Planck-Institute for Molecular Plant Physiology, Germany), Gibon Yves (Max-Planck-Institute for Molecular Plant Physiology, Germany), Steinfath Matthias (Max-Planck-Institute for Molecular Plant Physiology, Germany), Usadel Bjorn (Max-Planck-Institute for Molecular Plant Physiology, Germany), Fernie R Alisdair (Max-PlanckInstitute for Molecular Plant Physiology, Germany) and Stitt Mark (MaxPlanck-Institute for Molecular Plant Physiology, Germany) Deeper understanding of the genetic, molecular and physiological determinants of plant growth is required to support breeding for higher plant growth rates. Fast growth can be achieved by increasing photosynthetic rates and the assimilation of nutrients, but also by using more efficiently the resources available in relation with environmental constraints. Using the large natural diversity present in the model plant Arabidopsis, we have been profiling a large set of cellular structural components, metabolites and enzyme activities involved in the primary metabolism to uncover key growth determinants. Our results suggest that several strategies are possible. The first concerns the diurnal regulation of C usage, which is reflected by the starch contents at the end of the day being significantly negatively correlated with biomass. The second involves decreasing the costs of maintenance and growth without impairing the capacity to assimilate resources. This is achieved both by decreasing the amount of proteins per unit biomass, a very costly component of the cells, and by allocating qualitatively more proteins in primary metabolism, so in assimilatory processes. But to allocate more proteins in primary metabolism, plants must decrease other(s) protein pool(s). Ribosome levels, which represent around 7% of the protein pool, are negatively correlated with biomass. Because they are key players of translation, a decrease in their levels was expected to result in a decreased growth. We then investigated their spatial distribution between actively growing and mature leaves, as well as the efficiency of their use in a diurnal cycle. Email address for correspondence: [email protected] 15:40 Friday 1st July 2011

P1.10 The role of sucrose synthase and fructokinase in aspen wood formation

Lorenz Gerber (Umea Plant Science Centre, Sweden), Melissa Roach (Umea Plant Science Centre, Sweden), Andras Gorzas (Umea Plant Science Centre, Sweden), Mattias Hedenström (Umea University, Sweden), Björn Sundberg (Umea Plant Science Centre, Sweden) and Totte Niittylä (Umea Plant Science Centre, Sweden) Sucrose is the main transported form of carbon in most plants including the model tree aspen. In order to be integrated into metabolism sucrose must be cleaved by either sucrose synthase (SuSy) or invertase. Cleavage with SuSy produces UDP-glucose and fructose, whereas invertase produces glucose and fructose. SuSy has previously been hypothesized to channel UDP-glucose into cellulose biosynthesis. To investigate the role of SuSy in wood formation of trees we characterized transgenic lines of aspen with almost absent wood SuSy activity. In-depth chemical and mechanical analyses revealed a decrease in all wall polymers and a dramatically altered cell wall ultrastructure pointing to an important role for SuSy in wood cell wall biosynthesis. However, cellulose biosynthesis was not abolished and no strong growth phenotypes were observed. This suggested that just like in Arabidopsis, SuSy is not essential for cellulose biosynthesis. Regardless of which enzyme cleaves sucrose, half of the sucrosederived carbon is fructose. We have identified a fructokinase (FRK), which is important for fructose phosphorylation and carbon flux to cell walls in aspen. Already a moderate RNAi mediated reduction in FRK activity led to accumulation of soluble neutral sugars and decrease in hexose phosphates and UDP-glucose. Analysis of the cell walls in FRKRNAi trees revealed a similar phenotype as observed for the SuSyRNAi trees. We conclude that SuSy and FRK are important for cell wall synthesis in aspen, but SuSy is not essential for cellulose biosynthesis. Email address for correspondence: [email protected] 16:30 Friday 1st July 2011

P1.9 Investigating the contribution of the CAM pathway to growth and yield in the obligate CAM plant Kalanchoë fedtschenkoi

Louisa V Dever (University of Liverpool, UK), James Hartwell (University of Liverpool, UK), Neil Hall (University of Liverpool, UK), Richard Gregory (University of Liverpool, UK), Jana Knerova (University of Liverpool, UK) and Susanna Boxall (University of Liverpool, UK) Crassulacean acid metabolism (CAM) is a highly successful adaption to drought conditions. In CAM plants a unique series of enzymes fix CO2 into C4 acids at night, this nocturnal fixing of CO2 can dramatically

P1.11 Control of leaf expansion: a developmental switch from metabolics to hydraulics

Florent Pantin (LEPSE, INRA, France), Simonneau Thierry (LEPSE, INRA, France) and Bertrand Muller (LEPSE, INRA, France) Leaf expansion is the central process by which plants colonize space, allowing energy capture and carbon acquisition. Water and carbon

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Society for Experimental Biology a gene, gpt2 (At1g61800) that is essential for plants to acclimate to an increase in growth irradiance. Furthermore, we observed that the accession Columbia-0 (Col-0) is unable to respond to increases in light. Therefore, a quantitative trait locus (QTL) mapping analysis was performed in Landsberg erecta (Ler)/Columbia (Col) recombinant inbred line population to identify novel genes responsible for this variation to acclimation. In order to investigate the reverse acclimation to lower growth irradiance in Arabidopsis thaliana, photosynthetic capacity was measured in plants of the accession Wassileskija (WS) and plants lacking expression of gene At1g61800 (WS-gpt2). There was a decrease in the photosynthetic capacity by about 40% in WS plants and about 20% decrease in the WS-gpt2 plants. This shows that under lower or limiting light, photosynthesis was slowed down and that gpt2 gene was not essential for plants to acclimate. Thus, it is concluded that acclimation from high to low light is not a simple reversal of acclimation from low to high light but is mechanistically distinct process. Email address for correspondence: [email protected] 11:15 Saturday 2nd July 2011

emerge as main limiting factors of leaf expansion but the literature remains controversial about their respective contributions. Here, we tested the hypothesis that the importance of hydraulics and metabolics is organized according to both dark/light fluctuations and leaf ontogeny. For this purpose, we established the developmental pattern of individual leaf expansion during days and nights in the model plant Arabidopsis thaliana. Under control conditions, decreases in leaf expansion were observed at night immediately after emergence, when starch reserves were lowest. These nocturnal decreases were strongly exaggerated in a set of starch mutants, consistent with an early carbon limitation. However, low light treatment on wild-type plants had no influence on these early decreases, implying that expansion can be uncoupled from changes in carbon availability. From four days after leaf emergence onwards, decreases of leaf expansion were observed in the daytime. Using mutants impaired in stomatal control of transpiration as well as plants grown under soil water deficit or high air humidity, we gathered evidence that these diurnal decreases were the signature of a hydraulic limitation that gradually set up as the leaf developed. Changes in leaf turgor were consistent with this pattern. It is concluded that during the course of leaf ontogeny, the predominant control of leaf expansion switches from metabolics to hydraulics. We suggest that the leaf is better armed to buffer variations in the former than in the latter. Email address for correspondence: [email protected] 16:45 Friday 1st July 2011

P1.14 Phenotypic buffering in Arabidopsis: a genetical genomics approach

Joost Keurentjes (Wageningen University) Phenotypic diversity within species can be the consequence of heritable genomic variation. The resulting complex trait variation can be effectively analysed in mapping populations identifying quantitative trait loci (QTL) causal for the observed variation. However, many intermediary steps separate the DNA sequence polymorphisms from the eventual phenotype and genetic regulation can act on each of these subsequent steps. Modern technologies (transcriptomics, proteomics, and metabolomics) now enable us to follow the path from genotype to phenotype in great detail on every level. Similar to classical quantitative traits, variation in gene expression, protein and metabolite abundance can be subjected to QTL analysis, an approach known as genetic genomics. We have performed the first system-wide genetic genomics study of molecular variation in a model organism, in which we integrate transcript, protein and metabolite data with publicly available phenotypic data from a population of recombinant inbred lines (RILs) of Arabidopsis thaliana. Although the parental lines of the population differed in at least 500 000 SNPs expression QTLs (eQTLs) were detected for only 5000 genes, indicating that the majority of SNPs are neutral. Moreover, only a handful of these genetic effects were propagated to the phenotype level. These results suggest that much of the genetic variation is buffered along the way from genotype to phenotype. The findings are in agreement with robustness theories which state that organisms are buffered against perturbations to retain optimal performance in stable environments. At the same time robustness allows the accumulation of mutations which might become beneficial in changing environments. Email address for correspondence: [email protected] 11:30 Saturday 2nd July 2011

P1.12 How functional is the `functional equilibrium' of biomass allocation?

Hendrik Poorter (Forschungszentrum Juelich, Germany), Karl Niklas (Cornell University, USA), Peter Reich (University of Minnesota, USA), Jacek Oleksyn (Polish Academy of Sciences, Poland), Pieter Poot (University of Western Australia, Australia) and Liesje Mommer (Wageningen University, the Netherlands) In this paper we discuss a number of aspects related to the allocation of biomass in plants: 1. When studying environmental effects on allocation, there is a strong tradition of using allometry to correct for differences in plant size. But what are the pitfalls when applying this correction? 2. How strong, actually, are ontogenetic trends in allocation? Are there any rules of thumb to be given? 3. Are there inherent differences in allocation between functionally or phylogenetically similar species? 4. What are the dose-response curves that describe the reaction of plants with respect to allocation for different levels of light quantity, red:far-red ratio, UV-B, CO2, ozone, nutrients, drought, waterlogging, submergence, temperature, salinity and soil compaction? Can we order these environmental factors with respect to the strength of the response? 5. Is there an important role for biomass allocation in competition? These questions will be analysed from a whole-plant-growth perspective. More information can be found at www.metaphenomics.org. Email address for correspondence: [email protected] 10:30 Saturday 2nd July 2011

P1.13 Photoacclimation in Arabidopsis thaliana

Furzani Pa'ee (University of Manchester, UK) and Giles N Johnson (University of Manchester,UK) Photoacclimation is a process by which photosynthetic capacity is regulated in response to environmental adjustments in terms of light regime. Photoacclimation is essential in determining the photosynthetic capacity to optimize light use and to avoid potentially damaging effects. Previous work in our laboratory has identified

P1.15 Natural variation of nitrogen remobilisation efficiency in Arabidopsis

Celine Masclaux-Daubresse (IJPB, INRA, France), Fabien Chardon (IJPB, INRA, France) and Anne Guiboileau (IJPB, INRA, France) Plants have a fundamental dependence on inorganic nitrogen and million metric tons of nitrogenous fertilizers are added to the soil worldwide annually. Nitrogen is one of the most expensive nutrients to supply and commercial fertilizers represent the major cost in crop

Abstracts 2011 production. Furthermore there is serious concern regarding nitrogen loss in fields that pollutes soil and water. The ability of a plant to capture nitrogen from soil, to transport, assimilate and recycle nitrogen efficiently is essential to better nitrogen use efficiency. Albeit Arabidopsis is not a crop of economical importance, the determination of the natural variation of traits related to biomass and NUE using "agronomical type index" was performed in order to assess NUE concept in Arabidopsis and in order to identify similarities and differences between accessions or nitrogen nutrition and to identify parent lines showing atypical features. Variation of many traits as biomass, yield, N-remobilization to the seeds, seed C/N, seed dry weight was studied using a core collection of Arabidopsis accessions. Plants were grown under ample nitrate supply or at chronic limiting nitrate supply. Correlations between traits give an idea of the global feature of Arabidopsis under ample or limiting nitrate conditions. Variations allowed us to determine trait heritability and to identify a robust experimental design for further investigations. On the other hand, genetic variability allowed us to identify good parents of RIL populations suitable for further NUE QTL mapping. Email address for correspondence: [email protected] 12:00 Saturday 2nd July 2011

185 East Anglia, Uk), John Snape (John Innes Centre, UK) amd James Simmonds (John Innes Centre, UK) Epicuticular and intracuticular waxes significantly influence plant water relations, light reflectance, and interactions with fungal pathogens and insects. In wheat, the presence of visible epicuticular wax is described as a glaucous phenotype due to the light scattering effect of the wax. This phenotype is controlled antagonistically by a set of wax producing genes and glaucous suppressors. The exact extent of gene control of epicuticular wax composition and quantity, and their effects on productivity traits, is unclear. We have mapped Iw1, a dominant inhibitor of glaucousness derived from Triticum turgidum ssp. dicoccoides (DIC) and present in the UK variety Shamrock. Major QTL for yield and grain filling period were also detected across this region. Six independent trials in high yielding environments found consistent and significant increases associated with the DIC Iw1 allele on plot yield. The DIC Iw1 allele also extended grain filling independent of flowering time. This suggests that there are at least three major phenotypic effects (non-glaucous, delayed senescence, increased yield) which co-segregate in the DIC Iw1 introgression in the UK environment. Using colinearity and a variety of sequence sources we have fine mapped Iw1 to a sub-cM interval. We have also introgressed the DIC Iw1 interval (20-cM) into six UK elite varieties to validate and explore the wax/senescence/yield effects across different germplasm. Understanding the balance and relationship between these effects will be important to determine strategies to modify cuticular waxes and optimize grain production under current and future growing conditions. Email address for correspondence: [email protected] 14:15 Saturday 2nd July 2011

P1.16 The crop systems biology of photosynthesis, resource allocation and growth

Paul C Struik (Wageningen University, the Netherlands) and Xinyou Yin (Wageningen University, the Netherlands) Crop systems biology (CSB) combines molecular, experimental and computational, plant biology with crop physiology and modelling. We use CSB to analyse photosynthesis, resource allocation and growth, linking key processes across levels of biological organization. We quantify C3 or C4 photosynthesis based on thorough understanding of light capture, electron transport, and diffusion of CO2 from ambient air into carboxylation sites. In C4, we also quantify the CO2 concentration mechanism that minimises photorespiration. Genetic variation in leaf anatomy; in organization of thylakoids, photosynthetic cells and tissues; and in efficiencies of electron and gas transport across 3D microstructures is also evaluated. The theoretical upper limit of the conversion efficiency of solar energy in plants is 12.3%; the actual maximum efficiency under low-light conditions is only about half of that maximum value in both C3 and C4 plants. The gap is caused by intrinsic inefficiencies in photosystem II, alternative electron pathways (especially in C4) and photorespiration (mainly in C3). Photosynthetic enzymatic systems are unable to fully utilize available sunlight, further reducing actual efficiency. Dark respiration of the whole-plant adds another 30% efficiency loss. In natural environments of annual crops, a large portion of incoming solar radiation is lost due to incomplete canopy cover. We describe 3D aspects of light penetration in the crop canopy, and of gas exchange between ambient air and carboxylation sites in the individual leaf, including their temporal dynamics. Moreover, we describe effects of 3D profiles of microclimate on leaf composition, localized photosynthesis, assimilate distribution within the plant and subsequent growth. Email address for correspondence: [email protected] 13:30 Saturday 2nd July 2011

P1.18 Arabidopsis shade avoidance strategy is temperature-dependent and involves the receptorlike kinase ERECTA

Dhaval Patel (University of Bristol, UK) and Keara A Franklin (University of Bristol, UK) Vegetative shading resulting in light limitation is a common problem for plant survival. Light reflected from and transmitted through living vegetation is depleted in photosynthetically active red (R) and blue wavelengths and enriched in green and far-red (FR) wavelengths. Plants detect the presence of neighbouring vegetation through monitoring the ratio of R to FR wavelengths (R:FR) in ambient light. In crowded environments with a low R:FR, plants display a suite of characteristic responses including increased stem and petiole elongation, reduced leaf area and thickness, decreased leaf chlorophyll content, leaf hyponasty and accelerated flowering. Collectively, these responses are termed the shade avoidance syndrome and are regulated by phytochromes. Shade avoidance has largely been studied at growth temperatures higher than 20ºC. Using the Landsberg erecta (La-er) accession of Arabidopsis, we studied shade avoidance at a cooler temperature (16ºC) and found a strikingly different response. In cooler conditions, plants responded to low R:FR by dramatically increasing leaf area and thickness, rather than elongating petioles. To explore natural genetic variation in temperature-dependent light foraging strategy, we screened multiple Arabidopsis accessions and found that Cape Verde island (CVi) plants displayed conventional shade avoidance responses, irrespective of growth temperature. Comparative analysis using La-er X Cvi NILs resulted in mapping of the ERECTA gene. I have established that ERECTA is a major regulator of petiole elongation in low R:FR at cool temperatures. Further molecular characterisation of the role of ERECTA in temperature-regulated light foraging strategy is underway. Email address for correspondence: [email protected] 14:45 Saturday 2nd July 2011

P1.17 Fine mapping of the dominant Inhibitor of glaucousness 1 gene (Iw1) and its effects on grain filling and yield in the UK environment

Cristobal Uauy (John Innes Centre, UK), Sarah G Mugford (John Innes Centre, UK), Nikolai Adamski (John Innes Centre, UK), Alan Jones (John Innes Centre, UK), Nikolai Pedentchouk (University of

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P1.19 Ethylene as an early neighbour detection cue

Wouter Kegge (Utrecht University, Netherlands), Mieke De Wit (Utrecht University, Netherlands), LACJ Voesenek (Utrecht University, Netherlands) and Ronald Pierik (Utrecht University, Netherlands) Plants need to accurately sense their environmental conditions in order to perform optimally. During above ground competition with neighbouring plants, plants respond to changes in both light quantity and quality. Phytochromes are photoreceptors that are sensitive to changes in the red to far-red (R:FR) ratio and control shade avoidance responses, including upward leaf movement (hyponasty) and shoot elongation. These shade avoidance responses help plants to optimize light capture in dense vegetations. Standing theory dictates that these responses are induced by reduced R:FR ratio's already prior to the onset of actual shading in dense vegetations. In a time series with dense canopies of Arabidopsis thaliana, plant responses to neighbours and to changes in light quality were measured. We observed the occurrence of hyponasty early on in canopy development. R:FR ratio's at this stage of canopy development were found to be too high to induce hyponasty or petiole elongation. This indicates that signals other than R:FR ratio are involved in the very early neighbour responses of this rosette species. Strikingly, ethylene emissions were also enhanced at the time-points at which hyponasty was first observed. Since ethylene is a volatile compound that can induce hyponasty, the observed increase of ethylene emissions might constitute an early signal for plant neighbour detection that occurs prior to significant changes in R:FR ratio. Email address for correspondence: [email protected] 15:00 Saturday 2nd July 2011

P1.21 Nutrient supply, below ground processes and elevated CO2 change the defence-growth relationship in cyanogenic pasture crops

Roslyn M Gleadow (School of Biological Sciences, Monash University, Australia), Timothy R Cavagnaro (Monash University, Australia), Siobhan Isherwood (Monash University, Australia) and Rebecca M Miller (Monash University, Australia) The balance between plant growth and resource allocation to bioactive products is regulated at the molecular and environmental levels. Over 2000 species produce cyanogenic glycosides, which release hydrogen cyanide (HCN) in response to plant tissue damage (cyanogenesis). Many plants grown at elevated CO2 have lower leaf nitrogen, and allocate less of that nitrogen to protein but relatively more to cyanogenic glycosides. We grew sorghum (C4) and clover (C3) in agricultural soil supplemented with different amounts of phosphate and nitrogen in a glasshouse. In parallel experiments the same species were grown at elevated CO2 under field conditions (FACE). Clover is polymorphic for cyanogenesis and represents an ideal system to strengthen knowledge of defence chemistry, allocation and resource trade-offs under different environmental conditions. All naturally occurring varieties of Sorghum (C4) are cyanogenic. Both species form symbiotic associations that enhance nutrient uptake (Rhizopus and/or mycorrhizae). Plant growth and colonization rates were determined and leaves analysed for cyanogenic glycosides, nitrogen, phosphorus and micronutrients. Ratios of stable isotopes of nitrogen were used to indicate the proportion of nitrogen in clover taken up through fixation and subsequently allocated to defence. We found that the change in relative proportions of carbon, nitrogen and phosphorus affect the allocation of resources to defence. We were, however, unable to detect any cost to the plants in the production of cyanogenic glycosides. Understanding what drives resource allocation may be important in determining successful management of agriculture and animal health into the future. Email address for correspondence: [email protected] 16:15 Saturday 2nd July 2011

P1.20 Manipulating pod development in relation to whole plant resource allocation

Carol Wagstaff (University of Reading, UK), Emma J Bennett (University of Reading, UK) and Jeremy A Roberts (University of Nottingham, UK) The ability to manipulate where resources are ultimately stored within a plant could positively impact both the yield and nutritional value of seeds and fruits. Reproductive structures of plants contain proteins, lipids and other bioactive compounds which are an essential part of human and livestock diets. Many of the components act as a source of resources for plant growth upon germination; hence early plant development and seed nutritional quality are intrinsically linked. The current study investigated how manipulating the number of Arabidopsis reproductive structures per plant via selective stem removal, and the use of ethylene mutants, impacted upon pod physiology and protein concentration. Analyses showed that whilst fewer siliques per plant resulted in fewer seeds per pod these seeds were of an increased weight and size; therefore there remains a potential for yield enhancement in Brassicaceous species. Such alterations to seed physiology also correlated with delayed leaf senescence, a likely consequence of having fewer pods acting as sinks for resource allocation. Manipulation of senescence using Arabidopsis lines carrying mutations in individual ethylene receptors has shown that receptors can act in a specific manner and that they have limited functional redundancy. Translation of this knowledge of crop ideotype into Brassica has already begun, with the use of the BraIRRI and Oregin populations. Our findings demonstrate the highly plastic nature of Arabidopsis resource allocation, a process which if further understood could be used to increase the yield and nutritional quality of commercial Brassica crops. Email address for correspondence: [email protected] 15:45 Saturday 2nd July 2011

P1.22 Ascorbate oxidase expression and activity during development and in response to stress in Arabidopsis thaliana

Choon Kiat Lim (University of Exeter, UK) and Nicholas Smirnoff (University of Exeter, UK) The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis it is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. A number of studies have reported high AO activity in expanding tissues. In our study, we have investigated the AO activity and gene expression in various tissues of Arabidopsis at vegetative and reproductive stages. AO activity was high in very young leaves and decreased during leaf maturation. In the reproductive phase, flowers showed the highest activity. Detailed floral organ analysis showed that petals had highest AO activity. RT-PCR showed a positive correlation between AO3 expression and AO activity in various tissues of reproductive stage, but not in the vegetative stage. Also, a positive correlation was observed between expression of AO3 and an expansin gene, EXPA8 in vegetative and reproductive stages.

Abstracts 2011 Our results support the hypothesis that AO is involved in cell expansion. AO3 is the main contributor to AO activity in leaves and reproductive organs. Drought increased AO activity but not AO gene expression, suggesting post-transcriptional control. The phenotype of a double ao1/ ao3 mutant and its response to drought is currently under investigation. Email address for correspondence: [email protected] 16:50 Saturday 2nd July 2011

187 and the control (parent plant) showed that activities of peroxides and esterase enzymes were more in mutants. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.25 The determination of the optimal cauliflower microshoot development stage suitable for capsulation as artificial seeds and the optimization of conversion using semi solid media and commercial substrates

Hail Rihan (University of Plymouth, UK), Michael Fuller (University of Plymouth, UK), Mohammed Al-Issawi (University of Plymouth, UK), and Stephen Burchett (University of Plymouth, UK) Cauliflower microshoots were produced using the meristmatic layer of cauliflower curd in liquid culture and then encapsulated in a sodium alginate matrix. The development stages of microshoots were investigated in order to determine the optimal stage suitable for encapsulation and 13 to 14 day-old microshoots was found to be the optimal in terms of both conversion rate and viability of artificial seeds. Artificial seeds were cultivated in semi solid media and the effect of several types and concentrations of auxins were investigated in terms of their effects on the conversion ability of artificial seeds. While the use of 2 mg L-1 of IBA gave the best results in terms of artificial seeds viability, no significant effects of auxins were found on conversion rate. The capacity of cauliflower artificial seeds for conversion and growth in commercial substrates (compost, vermiculite, perlite and sand) was investigated and the irrigation solution was found to be the key factor for the significant subsequent growth in these substrates. The use of a full nutrient MS solution supplemented with 2 mg L-1 of Kinetin and 2 mg L-1 of NAA gave the optimal interaction with both perlite and compost, resulting in the optimal conversion rate and viability. The results obtained indicate promising possibilities for the direct use of cauliflower artificial seeds under in vivo conditions. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.23 The effect of varying levels of nitrogen fertilizer on safflower physiology, growth performance and seed yield

Shiren Mohammed (University of Plymouth, UK) The effect of nitrogen fertilizer on physiology and yield component were followed in plant of safflower (Carthamus tinctorius L.) grown in standard grade perlite. Plant watered with a standard hydroponic A, B and after month watered with complete Hoagland's solution in the glasshouse at the university of Plymouth. Supplementary lighting was used to maintain a 12 hour photoperiod. The eight levels of nitrogen fertilizer were used (0, 25, 50, 75, 100, 125, 150, 175 kg N.ha-1) in the form of ammonium nitrate. The increased level of nitrogen to 150 kg N. ha-1 markedly increased the assimilation rate (A) by an average of 51.55%, stomatal conductance by 55.21%, leaf area index (LAI) by 41%, leaf chlorophyll content by 55%, plant material nitrogen concentration by 40% and above ground biomass by 46% compared with control ( no nitrogen treatment ) when measured at anthesis. While the175 kg N. ha-1 significantly increased the stem height by an average of 7.38%, leaf number per plant by 41.5%, branch number per plant by 43%, capitula number per plant by 35.86%, above ground biomass by 42.5% and finally, increased seed yield by 93% at harvest compared with control. The positive correlation among the physiological parameters and between physiological parameters and yield component were recorded. The present study indicates that nitrogen fertilizer can consider as a very important nutrient for perlite grown safflower physiology and seed yield increase. Email Address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.24 Enzymology of callus cultures (mutated and non mutated) of garlic Allium sativum infected with basal rot

Zill-e-huma Bilal (Institute of plant pathology, University of the Punjab, Pakistan), Saira Banarus (Institute of Plant Pathology, University of the Punjab Lahore Pakistan) and Iffat Siddique (Lahore College for Women, University of Lahore, Pakistan) The aim of the research work was the selection of garlic variants resistant to basal rot disease caused by Fusarium oxysporum using induced mutations. For callus induction different explants were used and basal portion of inner leaf segment proved best explant for callus induction in garlic. MS (Murashige and Skoogs, 1962) growth media containing 2,4-D at 3 mg L-1 concentration was the best for callus induction. Callus initiated from basal portion of inner leaf segment on MS medium supplemented with 3 mg L-1 2,4-D incubated under light condition was used for further studies. For mutagenesis, calluses were exposed to ultraviolet radiation at a constant distance from the source for one to four hours. The mutants achieved at exposure of 2 and 2.5 hours were screened for the selection of resistant calluses through pathogenecity test using culture filtrates of F. oxysporum. Biochemical studies of the variants (resistant calluses)

P1.26 Embryogenic callus induction and proliferation in cauliflower (Brassica oleracea var. botrytis)

Magda A Al Samari (Plymouth university), Michael P Fuller (Plymouth university), Anita J Jellings (Plymouth university) An efficient protocol was developed for embryogenic callus production and regeneration in cauliflower from three types of explants: cotyledon, hypocotyl and root. Surface sterilized explants were placed on callus induction medium (CIM) which consisted of MS (Murashige and Skoog, 1962) supplemented with various combinations of exogenous hormone. The application of 2, 4-D at 0.15 mgL-1 and Kinetin at 0.1 mgL-1 proved to be the most effective treatment to induce embryogenic callus and it was the best throughout subsequent sub-culture. Good quality of embryogenic callus was induced from all three types of explants on the same CIM medium. Hypocotyl explants showed maximum callus production and the highest growth as measured by callus diameter. Callus was achieved on the edges of hypocotyl explants and on the edges of callus pieces which were used for subculture and was green and friable. Also root explants showed positive response in terms of embryogenic callus but it was yellow and friable. Cotyledon explants gave the least embryogenic callus induction and therefore it was not used for subsequent culture. The best period for subsequent callus sub-culture was 21 days since after this time, callus tissue turned brown and died.

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Society for Experimental Biology

Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.29 Effect of deficit irrigation on resource allocation in the tomato

Dragana Rancic (Faculty of Agriculture, University of Belgrade, Serbia), Ilinka Pecinar (Faculty of Agriculture, University of Belgrade, Serbia), Sofija Pekic Quarrie (Faculty of Agriculture, University of Belgrade, Serbia), Maja Terzic (Faculty of Agriculture, University of Belgrade, Serbia), Radenko Radosevic (Faculty of Agriculture, University of Belgrade, Serbia) and Radmila Stikic (Faculty of Agriculture, University of Belgrade, Serbia) This research aimed to compare the effectiveness of these two water saving techniques: regulated deficit irrigation-RDI and partial root-zone drying-PRD on resource allocation and fruit anatomy of tomato and the possible role of ABA in these effects using ABA deficient tomato mutants. Three tomato genotypes (wild type Ailsa Craig and ABA deficient mutants: notabilis and flacca) were grown in growth chamber conditions. The plants were exposed to three water regimes: control plants were well-watered, plant exposed to RDI treatment got 50% less water than considered optimal to the entire root-zone, while plants exposed to PRD treatment got the same amount of water as RDI but unevenly distributed to the root system so that part is irrigated while the remainder is allowed to dry the soil. Dry weight of leaves, stem and root as well as number of flowers and fruits, fruit diameter, fruit dry weight and total fruit weight per plant were measured, and water use efficiency and root to shoot ration were calculated. Our results showed that even when the same volumes of water were applied, the effect of PRD on the resource allocation is more expressed then in RDI. Transport of assimilates in fruits is not affected in PRD, but is significantly reduced in RDI. The magnitude of these effects differs between genotypes indicating the possible role of ABA. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.27 Genetic diversity and qualitative variation of Rosa damascena in Syria

Tarek Alsemaan (Damascus University, Syria) This study was conducted at Damascus University in Syria and Suleyman Demirel University in Turkey aiming at investigating genetic diversity and qualitative variation within Syrian accessions of Rosa damascena. Eight robust microsatellite markers were used to analyse the genetic diversity of seven accessions of R. damascena collected across major and minor rose oil production areas in Syria and one accession collected from Isparta province in Turkey. Six different genotypes have been obtained from Rosa damascena accessions collected from Syria. Two accesions, Almarah1 and Bab Alnayrab, were identical to the Turkish gynotype. Gas chromatography/mass spectrometry analysis of rose oil extracted from each accession identified the main components of oil such as: geraniol (28-31%), citronellol (26-30%), nerol (12-14%), germacrene-D (6-8%), nonadecane (4-6%) and linalool (1-3%). Besides, many trace compounds were detected such as: eicosane, eugenol, citral, hexadecane and rose oxide. The essential oil of Almarah1 and Ispata accession had got the highest quality. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.28 Vacuolar Ca2+/H+ transport activity is required for systemic phosphate homeostasis involving shootto-root signalling in Arabidopsis

Tzu-Yin Liu (Agricultural Biotechnology Research Centre, Academia Sinica, Taiwan), Kyaw Aung (Plant Biology Department, Michigan State University, USA), Ching-Ying Tseng (School of Biological Sciences, University of Texas, USA), Tzu-Yun Chang (Agricultural Biotechnology Research Centre, Academia Sinica, Taiwan), Ying-Shin Chen (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan) and Tzyy-Jen Chiou (Agricultural Biotechnology Research Centre, Academia Sinica, Taiwan) Calcium ions (Ca2+) and Ca2+-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca2+/ H+ transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (Pi) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated Pi level in shoots as a result of increased Pi uptake in a miR399/PHO2independent signalling pathway. Microarray analysis of the cax1/cax3 mutant suggests the role of CAX1 and CAX3 as negative regulators to suppress the expression of onefifth of Pi starvation responsive genes in the shoot, including genes encoding the PHT1;4 Pi transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not upregulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/cax3 scion is responsible for high Pi accumulation in grafted plants, and that the pht1;1 rootstock is sufficient to moderately repress such Pi accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root Pi transporter system, partly via post-translational regulation of PHT1;1. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.30 Metabolic changes during axillary bud growth in tomato plants

Chloe Steels (University of Sheffield, UK), Steve Coates (Advance Technologies (Cambridge) Limited, UK), Andrew Fleming (University of Sheffield, UK) and Mike Burrell (University of Sheffield, UK) Axillary bud growth can affect the structure of the plant and can have a major outcome on agricultural practice and crop yield. Understanding the regulation of axillary bud development will improve our ability to manipulate plant form and, thus, increase food production. In comparison to molecular changes that occur during bud development very little is known about changes in metabolism. The aim of this research is to combine methods of spatial analysis of gene expression by in situ hybridization with advanced metabolite profiling techniques (nanospray-MS and MALDI-MS) to gain a comprehensive understanding of how axillary bud development is controlled. Axillary bud growth in tomato plants (Lycopersicon esculentum cv Alisa craig) was initiated by removing all aerial parts of the plant above the first leaf axil. At eight-hour intervals over the subsequent 48 hours of growth, the axillary meristem (100 µm in diameter), was dissected, the metabolites extracted and analysed using mass spectrometry. In parallel, the expression pattern of specific cell cycle genes was visualized by in situ hybridization, documenting the temporal progression of the initiation of axillary bud growth. A substantial increase in the activity of the citric acid cycle (TCA) during initiation of axillary meristem growth was observed. These changes were closely paralleled by the increased expression of the cell cycle S phase marker gene Histone H4. I will present data to show the changes of both gene expression and metabolism that occur during the early stages of axillary bud initiation growth in tomato. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

Abstracts 2011

189 (ACS) is still highly active at the end of ripening. This correlates with high ACS gene expression levels. In contradiction to literature, ACS is not the rate limiting step but ACO is. Besides ethylene biosynthesis, the Yang-cycle was also investigated. We noticed that the expression of Yang-cycle genes is synchronized: all Yang-cycle genes are upregulated during ripening. But it was peculiar to observe that during late ripening and postharvest storage, all Yangcycle genes are upregulated to even higher levels. So, when ACO is limiting ethylene biosynthesis after the climacteric peak, recycling by the Yang-cycle is strongly encouraged. The complexity of these multiscale data implies the aid of modelling. A data driven kinetic model, constructed from the ethylene stoichiometry was developed. This model was able to predict fruit total ethylene production solely with gene expression data as input. Email address for correspondence: [email protected]be Poster Session - Saturday 2nd July 2011

P1.31 Characterization of a SAUR-like gene involved in Arabidopsis root cell elongation

Marios N Markakis (University of Antwerp, Belgium), Bram Van Loock (University of Antwerp, Belgium), Agnieszka Boron (University of Antwerp, Belgium), Ashka Jhaveri (University of Antwerp, Belgium), Susanna Cirera (University of Copenhagen, Denmark), Jean-Pierre Verbelen (University of Antwerp, Belgium) and Kris Vissenberg (University of Antwerp, Belgium) Ethylene has been characterized as one of the major plant hormones involved in various developmental processes such as fruit ripening and flower senescence. Characteristic is the well known triple response of seedlings. Moreover, we have described that ethylene is responsible for the very rapid inhibition of cell elongation in the root of Arabidopsis and it exerts its control in a concentration-dependent manner. Furthermore, ethylene is known to affect cell elongation through changes in auxin transport and metabolism. A micro-array analysis has been performed on control roots and roots treated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) for 3 hours to identify differentially expressed genes. This approach identified 240 genes (176 up-regulated and 64 downregulated). Here we describe a gene with an increased expression level after ACC application that was identified as a SAUR (Small Auxin Up RNAs)-like gene. Expression analysis using promotor::GUS and GFP transgenic plants revealed expression in the stele from the differentiation zone on and in developing lateral root primordia. Application of ACC, IAA or NAA shifted the expression towards the start of the elongation zone and concomitantly expression was extended outwards from the stele to the cortex and epidermal cells. qPCR confirmed this high induction by the different hormonal treatments. Significant phenotypic alterations in root growth were detected in overexpression plants, whereas the absence of expression in knock-out plants did not alter root growth, presumably because of redundancy. Using confocal microscopy the protein-GFP fusion was detected in the cytoplasm and nucleus, as predicted. Email address for correspondence: [email protected] be Poster Session - Saturday 2nd July 2011

P1.33 The shape of epidermal pavement cells in the Arabidopsis thaliana leaf - the role of the cytoskeleton and cell wall

Thanaa Doubbo (University of Antwerp, Belgium), Jan Buytaert (University of Antwerp, Belgium), Jean-Pierre Verbelen (University of Antwerp, Belgium) and Kris Vissenberg (University of Antwerp, Belgium) Among other plants, epidermal pavement cells of the Arabidopsis leaf have a very distinctive design. Their initially rod-shaped cell walls modify into a more complex sinuous-waved architecture. To obtain this shape the anticlinal wall must develop in a specific way showing alterations of outgrowth and restriction. The cytoskeleton and cell wall play a pivotal role during the process of cell expansion and are the topic of this study. The focus lies on the morphogenesis of the fourth true leaf, cotyledons not included. The microtubules and actin are visualized using GFP transgenic lines, the double mutant glabrous1/-Tubulin6 (g1/TUA6)GFP and Fimbrin Actin Binding Domain 2 (FABD2)-GFP respectively. To follow lobe formation over a short time period, live cell imaging is performed to monitor changes in cytoskeletal organization. At crucial time points during cell development, specific inhibitors are applied to disrupt microtubule, actin or cellulose microfibril organisation. These include oryzalin which negatively affects microtubules, latrunculin B for interrupting actin organization and dichlorobenzonitrile which inhibits cellulose synthesis. Alternatively the temperature-sensitive mutant mor1-1 is studied, which shows a disturbed microtubule pattern solely at restrictive temperatures. The effects of these inhibitors and the temperatureinduced mutation on leaf (and cell) development will be monitored. One approach to reveal the level of importance of cellulose microfibrils during cell morphogenesis is using the temperature sensitive mutant rsw1-1, which has reduced cellulose synthesis at restrictive temperatures. A second approach is to visualize the cellulose microfibrils or the movement of cellulose synthase complexes in developing cells. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.32 ACC-oxidase is the rate limiting step of ethylene biosynthesis during fruit climacteric fruit ripening A systems biology approach

Bram Van de Poel (Katholieke Universiteit Leuven, Belgium), Inge Bulens (Katholieke Universiteit Leuven, Belgium), Maarten M Hertog (Katholieke Universiteit Leuven, Belgium), Sandy Vandoninck (Katholieke Universiteit Leuven, Belgium), Yasmine Oppermann (University of Kiel, Germany), Etienne Waelkens (Katholieke Universiteit Leuven, Belgium), Maurice P De Proft (Katholieke Universiteit Leuven, Belgium), Margret Sauter (University of Kiel, Germany), Bart M Nicolaï (Katholieke Universiteit Leuven, Belgium) and Annemie H Geeraerd (Katholieke Universiteit Leuven, Belgium) Ethylene is the plant hormone regulating climacteric fruit ripening of e.g. tomato. It determines eventual fruit quality and post-harvest storage capability. We have executed a systems biology methodology to investigate ethylene biosynthesis at the level of metabolites, proteins and genes. This multidisciplinary approach showed new insights in the regulation of ethylene biochemistry. When tomato fruits have reached the orange-red stage, ethylene biosynthesis drops. This decline is caused by a decrease in ACCoxidase (ACO) activity. From this moment on, ACO gene expression and ACO protein levels are limiting. On the contrary, high levels of ACC (the ethylene precursor) and MACC (the ACC derivate) are observed during these stages. Further investigation showed that ACC-synthase

P1.34 Can polyphenol oxidase (PPO) mitigate photooxidative damage under abiotic stress conditions?

Tinne Boeckx (IBERS - Aberystwyth University), Richard Webster (IBERS - Aberystwyth University), Ana Winters (IBERS - Aberystwyth University), Alison Kingston-Smith (IBERS - Aberystwyth University), Judith Webb (IBERS - Aberystwyth University)

190

Society for Experimental Biology Chie Hattori (John Innes Centre, UK), Carmel O'Neill (John Innes Centre, UK) and Ian Bancroft (John Innes Centre, UK) Fatty acids from oilseed rape are important nutrition for human diet, renewable sources of energy and raw materials for industry. The oil consists of different types of FA which make the oil more valuable as it can be used for many different purposes. Among these variations, the degree of desaturation plays important role to determine the oil characteristics. Also alpha-linolenic acid, one of the polyunsaturated FA, is well known essential nutrient which need to be obtained from food as the human body cannot synthesise. Major genes controlling polyunsaturated FA biosynthesis pathway identified in Arabidopsis thaliana have been applied to the oilseed rape breeding programs, however other genetic regulatory mechanisms are sought after for further manipulation of seed oil composition. Although their effects are quantitative and smaller than those of major genes, they may allow end-users to avoid chemical or physical manipulations which increase both cost and other complications. Arabidopsis thaliana, close relative of Brassicaceae is used as a model plant for the search of loci involved in the novel regulatory mechanisms of desaturation pathway of FA and the result will be applied to the genomics of oilseed crop, Brassica napus. This project is based on quantitative trait locus (QTL) analysis and near isogenic lines are produced for each target QTL, subsequently fine mapping will be performed with the aid of genomic sequence information of parental lines to identify loci regulating desaturation pathway. This project is funded by BBSRC, KWS, SAATEN UNION, and MONSANTO. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

Polyphenol oxidase (PPO) activity results in the oxidative complexing of proteins with quinones. This is often observed during browning reactions of fruit, initiated by wounding which enables mixing of vacuolar phenolic substrates with chloroplastic PPO enzymes. However, observations of enhanced biomass yields of field-grown wild-type red clover compared with a mutant (low PPO activity) and the location of PPO on the luminal side of thylakoids, suggest that PPO could also have a role associated with photosynthesis. As PPOs catalyze the oxidation of monophenols to o-diphenols using molecular oxygen, it is probable that this may contribute to regulating the pool of molecular oxygen in the vicinity of the photosystems, which could otherwise disturb the plastid redox state. It has been proposed that PPOs could be involved in maintaining the balance of reactive oxygen species (ROS) in photosynthetically active cells. We are using wild-type red clover (Trifolium pratense L., cv Milvus) and a mutant lacking expression of the PPO4 gene in leaves to assess the implications of PPO activity on development of symptoms of oxidative damage. The photosynthetic performance of the wild type and mutant plants are compared under controlled growth conditions (16/20ºC night/day temperature, 250 µmol m-2 s-1 light, 16h day) and during a four day period of exposure to 2ºC at 500 µmol m-2 s-1. The relationship between photosynthetic parameters including Amax, PSII, ETR, NPQ, qP, yield and dark adapted Fv/Fm and biochemical measurements of PPO activity and protein oxidation will reveal the potential for PPO4 to mitigate photooxidative damage. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.35 Can the size of a root system be estimated by its electrical capacitance?

Ralf C Dietrich (James Hutton Institute, UK), Anthony G Bengough (James Hutton Institute, UK), Hamlyn G Jones (University of Dundee, UK) and Philip J White (James Hutton Institute, UK) There is much interest in the use of capacitance measurement to estimate the size of intact root systems. The equipment required is cheap and easy to apply in field and laboratory studies. Many studies have reported good correlations between capacitance and root mass, and a linear relationship between these variables is predicted by a model that has been proposed by Dalton (Dalton F. N. 1995, Plant and Soil, 173, 157-165). The aim of this study was to test the model on hydroponically grown barley (Hordeum vulgare L. cv. Optic) by using a range of treatments that included: raising whole roots out of solution; cutting roots at different positions below the solution surface; and varying the distance between plant electrode and solution surface. Although good correlations were found between capacitance and mass for intact whole root systems this was not the case for single roots. Critically excision of submerged root parts had negligible effects on the observed capacitance. Overall, the capacitance correlated better with the sum of root cross-sectional areas at the immersion points than with total mass. Apparently the bulk of the root system submerged did not influence the capacitance. The presence of root material at water surface was sufficient. These findings, which indicate that the previously observed correlations between root mass and capacitance are circumstantial and arise from a general correlation between root system mass and root size at the surface, were incorporated into a new model. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.37 Trehalose 6-phosphate (T6P) signalling is gated dependent on developmental stage

Liam E O'Hara (University College London, UK), Thierry Delatte (Utrecht University, Netherlands), Henriette Schluepmann (Utrecht University, Netherlands), Matthew Paul (Rothamsted Research, UK), and Astrid Wingler (University College London,UK) Trehalose 6-phosphate (T6P), the metabolic precursor of the disaccharide trehalose, is, via its inhibitory effect on Sucrose-NonFermenting-Related-Kinase 1 (SnRK1), a signalling molecule in the regulation of plant growth and development. T6P levels reflect the carbon status of plants and T6P has been found to accumulate in senescing tissues, following a similar rise in sugar contents. The levels of T6P can be increased by expressing the bacterial T6P synthase gene, otsA, or decreased compared with wild-type by expressing the bacterial T6P phosphatase gene, otsB. Elevated T6P, in plants expressing otsA, allows greater utilization of carbon for growth and development when supplemented with sucrose. Plants that express otsB flower and senesce later than wild-type. The supplementation with glucose, of plants grown on a low nitrogen (LN) containing medium, accelerates the onset of senescence. However plants expressing otsB appear insensitive to senescence induction by glucose. Corresponding with developmental insensitivity of SnRK1 to T6P, the effectiveness of glucose supplementation in bringing about accelerated senescence appears to be developmentally timed. This was revealed by transferring plants from LN medium with 111 mM glucose to medium with sorbitol or vice versa, such that the sugar treatment was present at different development stage and duration. Further experiments aim to investigate the effect of T6P levels on environmental stress responses that require sugar accumulation, such as cold-acclimation, or adjustment of starch turnover due to extended night. Establishment of these responses may be impaired in plants with altered T6P content. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.36 "Minors come to the lead": searching for novel genes controlling seed polyunsaturated fatty acid (PUFA) composition in Arabidopsis thaliana

Abstracts 2011

191 detailed phenotypic analysis of these transgenic lines. Highlights from the phenotypic analysis of the transgenic lines will be presented, along with an overview of the discoveries achieved through the RNA-seq analysis of the diurnal control of the CAM leaf transcriptome. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.38 Microtubules distribution and cell shape are affected by AtFH1 formin mutation in Arabidopsis

Amparo Rosero (Charles University), Ondrej Sebesta (Charles University), Fatima Cvrckova (Charles University), Viktor Zarsky (Charles University) Microtubules and microfilaments, each of them with specific roles during the morphogenetic process, affect the shape of plant cells. Formins (FH2 proteins) participate in both microtubule and actin organization, and especially their microtubule-related roles are still not clearly understood. AtFH1, the main housekeeping Arabidopsis formin, belongs to the type-I plant formins; it is localized in the membranes and known to nucleate and bundle actin filaments. To understand the biological role of AtFH1, we observed the effects of AtFH1 mutations on microtubules and cell shape after introducing the Map4-GFP microtubule marker and after treatment with cytoskeletal inhibitors (LatB and Oryzalin). Seedlings carrying homozygous T-DNA insertions in AtFH1 (two alleles, fh1-1 and fh1-2) showed cotyledon epidermal cells with more lobes than wild type. fh1/Map4-GFP plants showed reduction in the lobe length with respect to fh1-1 plants, however the differences compared to wild type were still significant. LatB did not affect the cell shape in fh1-1 and wt, the lobe length and number were the same to the control, while oryzalin caused increase in both number and length of epidermal cell. Microfilaments in fh1 showed altered distribution compared to wt. Those results showed the effect of AtFH1 mutation on microtubule organization affecting cotyledon epidermal cell shape. This work was supported by the GACR P305/10/0433 and MSM 0021620858 projects. Email Address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.40 Genetic behaviour of purple colour in sesame (Sesamum indicum L.)

Bülent Uzun (Akdeniz University, Turkey), Seymus Furat (Akdeniz University, Turkey) and Engin Yol (Akdeniz University, Turkey) Green is a traditional colour in sesame stems, capsules and leaves while the purple colour is monitored rarely. During the vegetative and reproductive phases the colour is usually a shade of green, and then as the plants mature and begin to drop their leaves, the colour will turn to many shades of yellow/green, and rarely purple (Langham, 2007). When purple lines reach late bloom stage until the drying phase, plants indicated purple colour, especially in stems and capsules on the contrary of usual sesame plants. The chemical content of ACS 70 accession with purple plant colour was previously studied and it was suitable for commercial production with high total antioxidant capacity (Erbas et al. 2009). The colour is controlled genetically but inheritance of purple colour in sesame has not previously been reported. In 2008 growing season, purple colour genotype, ACS 70 () was crossed with Muganli-57 () cultivar (green), in Antalya, Turkey. F1 and F2 generations were grown at the same location in 2009 and 2010 growing seasons, respectively. The results in the F1 generation indicated that purple colour was dominant over green colour. In F2 progeny, 65 plants indicated purple form while 22 plants were green. Chi-square test showed a good fit for a monogenic inheritance with the F2 phenotypic ratio of 3:1 indicating that purple colour character was controlled by a single dominant gene. The result presented in this study for the first time would be of importance for the improvement of high antioxidant capacity in sesame. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.39 Detailed RNA-seq analysis of the light/dark regulation of the transcriptome of Kalanchoë fedtschenkoi leaves identifies genes with potential functions in the circadian control of Crassulacean acid metabolism

Jana Knerova (University of Liverpool, UK), Susanna Boxall (University of Liverpool, Uk), Louisa Dever (University of Liverpool, UK), Richard Gregory (University of Liverpool, UK), Neil Hall (University of Liverpool, Uk) and James Hartwell (University of Liverpool, UK) Crassulacean acid metabolism (CAM) enhances water use efficiency up to 10-fold relative to C3 photosynthesis. We use functional genomics approaches to study the establishment and circadian coordination of CAM in the Madagascan endemic, Kalanchoë fedtschenkoi. Our longterm goal is to improve the growth and yield of CAM biofuels crops such as Agaves, and to enhance photosynthetic and water use efficiency in C3 crops. Roche 454 sequencing was used for the de novo generation and assembly of the transcriptome sequence of K. fedtschenkoi. This 454-transcriptome assembly provided the reference sequence for quantitative RNA-seq analysis of the light/dark regulation of the transcriptome of C3 and CAM leaves using Applied Biosystems SOLiD sequencing. The RNA-seq data revealed both known and unknown CAM genes. Downstream analysis focused on genes encoding regulatory proteins with potential roles in the circadian coordination of CAM. RT-PCR analysis of the regulation of the discovered genes over circadian time course experiments identified novel clock-controlled CAM genes. These CAM genes have been manipulated in transgenic lines using both silencing with RNAi, and constitutive over-expression. An Agrobacterium-based transformation system has been developed and optimized achieving up to 50% transformation efficiency. Using this transformation procedure, we have also silenced several known steps within the CAM pathway (PPDK, NAD(P)-ME etc), and have begun

P1.41 Expression and biochemical analysis of AtSDL, a putative sorbitol dehydrogenase in Arabidopsis thaliana

Diego Ampuero (Universidad de Chile, Chile), Francisca Aguayo (Universidad de Chile, Chile), Roberto Parada (Universidad de Chile, Chile), Patricio Mandujano (Universidad de Chile, Chile) and Michael Handford (Universidad de Chile, Chile) Sorbitol is the main product of photosynthesis in Rosaceae species (apples, peaches, plums, among others). This sugar alcohol is produced in source organs (mature leaves) and transported through the phloem to sink organs (roots, fruits, immature leaves), where, by the action of sorbitol dehydrogenase (SDH) it is oxidized to fructose, which can be either stored or metabolized. On the other hand, in almost all other plant families, including Solanaceae and Brassicaceae, sucrose is the principle sugar transported. Nevertheless, sorbitol and/or SDH activity have been detected in some of these species, yet their physiological roles have yet to be understood. As an approach to determine the roles of SDH in non-sorbitol translocating species, we have identified a protein in Arabidopsis thaliana with >75% amino acid identity with previously-characterized plant SDHs, named AtSDL. RT-PCR expression analysis showed that AtSDL is expressed in multiple organs. In addition, analysis of Arabidopsis plants which have been stably-transformed with the AtSDL promoter::GUS fusion construct, revealed systemic expression except

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Society for Experimental Biology protein kinase (SnRK1; AKIN10/ AKIN11) which T6P inhibits. Inhibition of SnRK1 by T6P has previously been found to promote sucrose utilization and plant growth and synthesis of products such as starch and cell walls. The harnessing of this mechanism has promise for food security and provides new ideas for overcoming the recent plateauing of yield improvement for crops such as wheat. We examined the interaction of T6P and SnRK1 in wheat grain. T6P levels changed 178-fold from 1-45 days after anthesis, correlating with sucrose content. T6P was particularly elevated in maternal and filial tissues pre-grain filling ranging 49 to 119 nmol/g/FW, more than 100fold higher than previously reported in Arabidopsis. During grain filling T6P accumulation was almost entirely endospermal (43 nmol/g/FW) with 0.6 to 0.8 nmol/g/FW in embryo and pericarp. In contrast to the large changes in T6P, SnRK1 activity ranged only threefold, but was strongly inhibited by T6P in vitro with evidence of regulation of SnRK1 marker genes by T6P in vivo. The data show a correlation between T6P and sucrose overall that belies a marked effect of tissue type and development stage on T6P content, consistent with tissue-specific regulation of SnRK1 by T6P in wheat grain. We conclude that the inhibition of SnRK1 by T6P is a likely key player in determining grain yield in cereals. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

in roots and petals where only the vasculature was stained. An analysis of the promoter region of AtSDL revealed the over-representation of light-responsive elements, and plants are being studied under different light conditions. Additionally, AtSDL has been localized to the cytosol and atsdl mutants have been obtained. Finally, using in vitro translation and yeast expression systems, recombinant AtSDL possesses the ability to oxidise sorbitol and other sugar alcohols found in Arabidopsis. Funding: Fondecyt 1100129, CONICYT Magister 22100522 (Diego Ampuero) and 22110701 (Francisca Aguayo). Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.42 Expression of the APY1 gene during early stages of germination of pea (Pisum sativum L. var. Alaska)

Motohito Yoneda (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Shunnosuke Abe (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Eugene H Morita (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Trivima Sharma (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan) and Vaidurya P Sahi (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan) Apyrases catalyse the hydrolysis of nucleoside tri- and/or diphosphates, but not mono- phosphates or non-nucleoside phosphates. Using non-radioactive hybridization of mRNA in situ, localization of apyrase mRNA was investigated at the organ, tissue, cellular, and sub cellular level during germination of pea seeds. The spatiotemporal expression of the plant paralogue of APY1 was examined during early germination of pea seeds using non radioactive mRNA in situ hybridization on embryos fixed with 4% paraformaldehyde and sections of 30 m thickness cut on a microtome (Yamoto). Samples were examined after imbibition for 10 hours and at 16, 35, 62 and 84 hours after sowing. APY1 was localized in the shoot apical meristem and the radicle at all the stages studied. It was present in the cortex and epidermis of epicotyls at 35 and 62 hours of sowing, while in 84 hour embryos it was present in the shoot apical meristem, parenchymatous cortex of the root and the lateral root primordia. The mRNA encoding a 49 kDa apyrase was induced autonomously in the embryos of germinating pea seeds in both meristematic and differentiated tissues suggests that the enzyme has a likely probable role in differentiation and development of cells and tissues and may function in cyto-energetics Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.44 Agrobacterium-mediated transformation of barley with CKX gene

Katarína Mrízová (Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic), Hana Pospísilová (Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic), Ludmila Ohnoutková (Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic), Wendy Harwood (Department of Crop Genetics, John Innes Centre, UK), Ivo Frébort (Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic) and Petr Galuszka (Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Republic) Hordeum vulgare is one of the major food sources, hence the production of barley plants with increased yield and enhanced stress tolerance remains the most important goal in breeding. New techniques of molecular cloning accelerate classical breeding techniques and produce crop plants with multiply enhanced traits. Cytokinins are plant hormones derived from adenine. Genetic manipulation of cytokinin level may influence many physiological processes, besides others the stress tolerance, root formation and grain yield. The side chain of isoprenoid type of cytokinins is irreversibly cleaved by cytokinin dehydrogenase (EC 1.5.99.12; CKX), which is a principal factor to control cytokinin levels in planta. For modulation of cytokinin levels in barley roots and grains, several constructs were prepared. Four constructs contained chimeric Arabidopsis thaliana AtCKX1 with altered targeting to either cytoplasma, vacuoles, chloroplasts or apoplast under the control of -glucosidase promoter. Three constructs for root-specific degradation of cytokinins contained ZmCKX1 gene from maize under -glucosidase, PHT and RAF promoters. Positive transgenic plants expressing AtCKX1 or ZmCKX1 were already selected by qPCR and activity measurement and transferred to the second generation. In general, the plants transformed with AtCKX1 and ZmCKX1 genes have two to ten times higher CKX activity and show stronger root system contrary to nontransgenic plants. Furthermore, transgenic plants with AtCKX1, ZmCKX1 or gHvCKX2 transgenes under grain specific B-hordein or LTP2 promoters and HvCKX1 silencing cassette regulated by B-hordein promoter have been prepared and will be analysed in near future. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.43 Wheat grain development is characterized by remarkable trehalose 6-phosphate accumulation pregrain filling: tissue distribution and relationship to SNF1-related protein1 kinase activity

Matthew J Paul (Rothamsted Research, UK), Eleazar MartinezBarajas (Rothamsted Research, UK), Thierry Delatte (Utrecht University, Netherlands), Henriette Schluepmann (Utrecht University, Netherlands), Gerhardus J De Jong (Utrecht University, Netherlands), Govert W Somsen (Utrecht University, Netherlands), Catia Nunes (Rothamsted Research, UK), Lucia F Primavesi (Rothamsted Research) and Rowan Mitchell (Rothamsted Research, UK) Trehalose 6-phosphate (T6P) is a sugar signal that regulates metabolism, growth and development. At least some of the basis of this signalling function is through the central regulatory SNF1-related

Abstracts 2011

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P1.45 The metabolism of sorbitol in non-Rosaceae species

Sofía Zamudio (Universidad de Chile, Chile), Jorge Araya (Universidad de Chile, Chile), Yu-wen Tang (Universidad de Chile, Chile) and Michael Handford (Universidad de Chile, Chile) In members of the Rosaceae family, which includes peaches, pears and apples, sorbitol is the main product of photosynthesis, synthesised from glucose 6-phosphate by the action of sorbitol 6-phosphate dehydrogenase (S6PDH) and sorbitol 6-phosphate phosphatase. Sorbitol is the main form of carbon translocated around these species via the phloem. Once in the carbon sink organs, and in a speciesspecific manner, a proportion of this sugar alcohol is metabolized to fructose via sorbitol dehydrogenase (SDH) or to glucose by sorbitol oxidase. These sugars can then be further metabolized or stored, providing amongst other functions, enhanced sweetness especially in the case of fruits. Even though the main phloem-translocated carbon compound in nonRosaceae species is sucrose, sorbitol and other sugar alcohols are present in these species, particularly under drought, saline and cold stress conditions when they act as compatible solutes. Using a reverse genetics approach, we have identified two genes potentially involved in sorbitol metabolism in non-Rosaceae species. First, in Arabidopsis thaliana, AtS6PDH is an enzyme which is potentially involved in sorbitol synthesis which possesses the main molecular characteristics of S6PDHs identified in other organisms. We have determined that AtS6PDH-GFP is localized in the cytosol and ats6pdh mutants have been obtained. Additionally, enzyme activity assays are currently underway. Second, we have found that VvSDH1 is a potential sorbitol dehydrogenase from grapevine (Vitis vinifera) sharing >75% amino acid identity with known plant SDHs. We demonstrate that it is a cytosolic enzyme capable of oxidizing sorbitol, with increased expression levels under specific stress conditions. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.47 Traits associated with relocation of resources during grain filling in defoliated bread wheat varieties: phenotypic and genetic analyses

Dejan Dodig (School of Biology, Newcastle University, UK; permanent address: Maize Research Institute, Zemun), Jeremy Barnes (School of Biology, Newcastle University, UK), Borislav Kobiljski (Institute of Field and Vegetable Crops, Serbia) and Steve Quarrie (Visiting Professor, School of Biology, Newcastle University, UK) When environmental stress develops during reproductive phases of growth, wheat plants have to rely increasingly on remobilisation of previously stored assimilates to maintain grain filling. In a two-year field trial in 2005 and 2006 we examined nearly 30 traits in a collection of 96 wheats from around the world in relation to their capacity to maintain grain filling in plants that were defoliated by cutting off all leaf blades shortly after anthesis. These wheats were also screened for allele variation at 44 SSR marker loci. Yield/ear of defoliated plants varied from about 66 to 96% yield/ear of control plants, and both grain number/ear and thousand grain weight significantly contributed to this. Yield maintenance in defoliated plants was highly significantly correlated with peduncle length (flag leaf node to base of the ear), and especially with extruded peduncle length (flag leaf ligule to base of the ear). High capacity to maintain grain filling was also associated with high grain filling rate, and awn length. Association mapping with 2006 data identified four SSR marker loci which were highly significantly associated with capacity to maintain yield and grain size in defoliated plants: gwm458 (1DC), gwm484 (2DS), gwm539 (2DL) and gwm186 (5AL). Highly significant associations with stem length (to base of the ear), peduncle length and surface area were also associated with these markers, but not maximum grain growth rate. Peduncle length was a key trait determining grain filling in defoliated plants, and several highly significant marker associations with peduncle length were identified. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.48 Evaluation of intervarietal hybrids to increase yield and productivity of maize in Syria

Ayman Almerei (University of Plymouth, UK), Michael Fuller (University of Plymouth, UK), Ahmad Alshekh Kaddor (Aleppo University, Syria), Ahmad Haj Sulaiman (GCSAR) A Reciprocal Diallel Cross among eight diverse genotypes of maize was carried out to try and produce early intervarietal hybrids with the potential to be adapted to Syrian growing conditions and out-yield existing varieties in terms of grain production. The eight genotypes were planted in the first season at Tel-Hadya research station (Agricultural Scientific Research Centre, Aleppo, Syria) using a Reciprocal Diallel Cross with 20% selection intensity. Twenty-eight selected progeny genotypes were tested in the second season and compared with two local varieties (GHOUTA-1 and GHOUTA-82) in three contrasting environments, which form the main planting regions of maize in Syria (Aleppo, Homs and Rakka), using randomized complete block design. Results showed that 6 genotypes were superior in grain yield to the two local varieties (GH-1 and GH-82). The superior genotypes on average out-yielded GH-82 by 36.44% and GH-1 by 46.59%. Four genotypes showed higher 1000 kernel weights with average increases of 16.6% in comparison to GH-82 and 26.84% for GH-1. For leaf area index (LAI), three genotypes were superior and outperformed the mean by 17.60%. Most of the hybrids were superior in terms of seed moisture at harvest indicating earlier maturity and 18 genotypes were superior in respect of shelling (lower rachis content). Email Address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.46 Regulation of photosynthesis in sorghum under drought stress

Chukwuma Ogbaga (University of Manchester, UK) and Giles Johnson (University of Manchester, UK) Changing climate in combination with growing world populations mean that there is need for plants to be grown on land that is currently considered marginal for agriculture. Sorghum is a C4 plant that serves as an important food crop in Africa and India. It is also known to be highly tolerant of environmental stresses such as drought and light. We are investigating the responses of photosynthesis in sorghum to environmental stress. In particular we are examining how regulation of electron transport operates in this plant. Preliminary measurements suggest that sorghum may possess alternative sinks for electrons, possibly involving a plastid terminal oxidase or protein with similar activity. This is being investigated using a combination of gas exchange, chlorophyll fluorescence and absorption spectroscopy Email address for correspondence: [email protected] manchester.ac.uk Poster Session - Saturday 2nd July 2011

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Society for Experimental Biology We hypothesize that changes in the level of IP3, IP4 or IP5 may be the cause of defective Pi homeostasis in ipk1 mutants, which in turn alters protein activities and/or expression of genes involved in Pi signaling and homeostasis. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.49 Identification and expression analyses of cytosolic glutamine synthetase (GS1) isozymes in barley (Hordeum vlugare L.)

Andrew Goodall (University of St. Andrews, UK), Pankaj Kumar (University of St. Andrews, UK) and Alyson Tobin (University of St. Andrews, UK) Nitrogen is an integral component of many compounds like chlorophyll, nucleic acids and enzymes essential for various plant processes. Nitrogen availability within the growth medium limits a plant's productivity. Plants with higher nitrogen use efficiency (NUE) values will yield greater amounts of grain per unit of available nitrogen and reduce the amount of fertiliser required. Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, and plays a major role in productivity and growth, being responsible for the assimilation of ammonium into glutamine as the first step in primary and secondary ammonium assimilation. We have identified three cytosolic (GS1) GS isoforms and a single plastidic form (GS2) in barley. In order to understand their role we investigated their spatial and temporal expression pattern through quantitative PCR. Furthermore their role in NUE was assessed by analysing their response to different nitrogen regimes during hydroponic growth. Grain nitrogen content of barley is an important indicator of malting quality for brewing and dietary protein content of animal feed. We have identified one isoform (GS1_3) specifically expressed during seed development. Localization studies via in situ hybridisation show that all of the GS1 isoforms are localised to the scutellum in the developing grain. We predict that these enzymes have a role in providing the developing embryo with glutamine from the endosperm. We conclude that these GS1 isoforms all have different roles within the plant as shown by the differences in their expression and response to nitrogen. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.51 The expression of the MKRN gene in meristematic regions of pea (Pisum sativum L. var. Alaska) and rice (Oryza sativa L. var. Nipponbare) seeds and its relation to that in mouse embryo

Vaidurya P Sahi (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Hanumant B Wadekar (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Nagganatha Suganthan Ravi (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan), Thangavelu U Arumugam (Cell Free Science and Technology Research Center, Ehime University, Japan), Eugene H Morita (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan) and Shunnosuke Abe (Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Japan) The MKRN gene family encodes zinc ring finger proteins characterised by a unique array of motifs (C3H, RING and a characteristic cys-his motif) in eukaryotes. To elucidate the function of the MKRN gene in plants we compared the gene expression pattern of MKRN in plant seeds with that of mouse embryo. The spatio-temporal expression of MKRN in seeds of pea and rice was carried out using non radioactive mRNA in situ hybridization (NRISH) with DIG and BIOTIN labelled probes for pea and rice embryos respectively. We obtained images of MKRN1 expression in e10.5 whole mount mouse embryo, hybridized with DIG labelled probes, from the database "emage". MKRN transcripts were expressed in the vascular bundle, root apical meristem (RAM) and shoot apical meristem (SAM) in pea and rice embryos and it was also present in the epiblast of the rice embryo. The spatial annotation of the MKRN1 NRISH of whole mount mouse embryo shows prominent localization of MKRN1 in the brain, and its possible expression in spinal cord and the genital ridge. Localization of MKRN in the anterior and posterior ends of pea and rice embryo suggests to the probable role it may have in sculpting the pea and rice plants. The expression of MKRN is similar in functionally and anatomically analogous regions of plant and animal embryos, including the vascular bundle (spinal cord), the RAM (brain) and SAM (genital ridge) thus paving way for further inter-kingdom comparison studies. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.50 The roles of inositol polyphosphates in regulating phosphate homeostasis in Arabidopsis thaliana

Hui-Fen Kuo (Agricultural Biotechnology Research Center, Taiwan), Tzu-Yun Chang (Agricultural Biotechnology Research Center, Taiwan), Su-Fen Chiang (Agricultural Biotechnology Research Center, Taiwan), June-Wei Chen (Agricultural Biotechnology Research Center, Taiwan), Ying-Shin Chen (Agricultural Biotechnology Research Center, Taiwan), and Tzyy-Jen Chiou (Agricultural Biotechnology Research Center, Taiwan) Phytate (inositol hexakisphosphate; IP6) serves as a major storage form of phosphorous reserve in the seeds as well as a factor involved in multiple biological processes, such as remobilization of intracellular calcium and regulation of gene expression. The final step of IP6 biosynthesis is mediated by inositol pentaphosphate kinase 1 (IPK1). Previous studies showed that disruption of IPK1 in Arabidopsis exhibited a significant reduction of IP6 content along with an increased level of inotistol polyphosphate (IP) precursors, IP3, IP4 and IP5. Interestingly, ipk1 mutants display phenotypes of phosphate (Pi) toxicity under replete Pi conditions. ipk1 mutants accumulated excessive Pi specifically in the shoot while maintained normal organic phosphate content, which was attributed to the increased uptake activity of external Pi in accordance with upregulation of several Pi transporters. Under Pi replete conditions, ipk1 mutants exhibited several characteristics of Pi starvation responses, such as shorter primary roots and predominant lateral root proliferation, as well as longer root hairs. Reduction in IP6 content in ipk1 mutants is unlikely the cause for Pi toxicity, because mips1 and mips2 mutants defective in myo-inositol-1-phosphate synthases showed lower IP6 contents but wild-type level of Pi.

P1.52 Growth and metabolic acclimation of Betula pendula to rising night temperatures

Maarit Mäenpää (University of Eastern Finland, Finland), Vladimir Ossipov (University of Turku, Finland), Sari Kontunen-Soppela (University of Eastern Finland, Finland), Markku Keinänen (University of Eastern Finland, Finland), Matti Rousi (Finnish Forest Research Institute, Finland) and Elina Oksanen (University of Eastern Finland, Finland) The climatic change, with increasing average air temperature without a change in photoperiod, poses plants into a changing combination of light and temperature. The response of a plant to environmental factors is connected to the type and the phase of the circadian system. In northern latitudes, night temperatures are expected to increase more than day temperatures.

Abstracts 2011 We were interested in the growth responses and the metabolic acclimation of northern trees to different night temperatures (including evening and morning hours).We made an experiment in growth chambers, stressing the changes emerging from increased night temperatures in silver birch (B. pendula). Gas exchange, growth responses and over 280 low-molecular weight metabolites of three genotypes were analysed. Warmer night temperatures increased dark-respiration and height growth rate, but the plant total biomass was not significantly affected. Genotypes had both unique and shared metabolic responses to night temperatures. Shared metabolic responses were connected to height growth rate and developmental stage of the plant. Changes in growth and metabolite concentrations were non-linear along the temperature gradient, and the sensitivity to react and the critical temperatures were genotype-specific. Thus, with help of several genotypes, the treatment-induced common responses can be identified. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

195 hypothesized that cell elongation at the abaxial side of petiole is a driving force in this process. In our studies we tested whether ethylene-mediated hyponasty in Arabidopsis occurs through local stimulation of cell expansion and if this involves reorientation of CMTs. Since the epidermal cell layer has an important role in both driving and limiting plant growth, we focused our study on this tissue, both on the abaxial and the adaxial sides. By analysing epidermal imprints, we provide the evidence that ethylene primarily drives cell expansion in a restricted abaxial region located in the proximal side of the petiole which induces hyponastic growth. Moreover, the study of the arrangement of cortical microtubules (CMTs) in the epidermis of both ab- and adaxial petiole shows their reorientation in specific parts of a petiole during ethylene treatment. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.55 Plant morphological traits as indicators of nitrogen (N) uptake capacity of selected cultivars of Brussels sprouts (Brassica oleracea var. gemmifera)

Thi Hong Van Nguyen (Institute of Biological Production Systems, Leibniz Universität Hannover, Germany), Hartmut Stützel (Institute of Biological Production Systems, Leibniz Universität Hannover, Germany) and Andreas Fricke (Institute of Biological Production Systems, Leibniz Universität Hannover, Germany) Brussels sprouts is a crop of high N demand. Its N supply therefore must be efficiently managed to produce high yields together with low N residues in the field. Selecting cultivars which can take up and use N efficiently is one important strategy for cultivating this crop. We aimed to find out the relationship between plant morphological traits and N uptake capacity in the field. As a simple screening method for root morphology, seeds of ten early maturing cultivars were grown on agar medium for eight days. Six cultivars were then selected on the base of different root morphological traits (tap root length, lateral root length and distribution of lateral root along the tap root) and grown in the field with three N fertilizer supplies (unfertilized treatment, organic fertilizer treatment with 200 kg N/ha of grounded pig hairs and inorganic fertilizer treatment with Ca(NO3)(NH4) following the KAS system in Germany). N uptake at 48 days after planting (DAP), i.e. before the onset of sprout, showed positive linear relationship with N uptake at harvest date and final yield. Among the root morphological traits on agar medium, tap root length best explained N uptake capacity; however the coefficient of determination was only 0.44. Leaf mass and stem mass at 48 DAP individually showed good linear relationship to N uptake, with leaf mass being the best indicator of N uptake capacity (R2 = 0.9). Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.53 Arabidopsis thaliana ethylene receptors have distinct roles in plant development

Emma J Bennett (University of Reading, UK), Frances Gawthrop (Tozer Seeds Ltd), Jeremy A Roberts (University of Nottingham, UK) and Carol Wagstaff (University of Reading, UK) Ethylene is a gaseous plant hormone known to regulate numerous aspects of plant development and displays a strong association with fruit ripening, the timing of organ senescence and abscission. Arabidopsis perceives ethylene through a family of five cell surface receptors (EIN4, ETR1, ETR2, ERS1 and ERS2) which, when bound to the hormone via copper cofactors, are prevented from eliciting a downstream signalling response. Mutations within the ethylene receptor genes confer dominant ethylene insensitivity such that hormone binding is no longer capable of inhibiting the receptor signalling cascade. The current study investigated the relationship between the ethylene receptor mutants and Arabidopsis thaliana growth by focusing upon silique development and resource compartmentalisation. The distinct phenotypes of the individual mutants in terms of silique and seed number, grain weight and overall leaf biomass indicated that the ethylene receptors have separate roles in hormone perception and the regulation of whole plant resource allocation. This highlights that the ethylene receptors are not functionally redundant and supports the view that they may be capable of mediating more than one response signal. In light of these findings the ability of the ethylene receptors to influence vegetative growth and seed development could prove useful in advancing yield enhancement strategies. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.54 Cell expansion drives ethylene-induced differential petiole growth in Arabidopsis thaliana

Joanna K Polko (Utrecht University, Netherlands), Ronald Pierik (Utrecht University, Netherlands), Anton J Peeters (Utrecht University, Netherlands) and Martijn Van Zanten (Utrecht University, Netherlands) Hyponasty ­ the upward petiole movement ­ is a very rapid reaction of plants in response to various external stimuli. Hyponastic growth, caused by differential growth within a petiole, brings rosette leaves to a more vertical position. The volatile hormone ethylene is one of the factors inducing hyponastic growth in Arabidopsis thaliana. The leaf repositioning is the result of unequal growth rates between the adaxial and abaxial sides of a petiole. Since the response is very rapid, we

P1.56 Effects of Salinity and elevated carbon dioxide on the leaf properties of cucumber (Cucumis sativus L.)

John K Titriku (Leibniz Universität Hannover, Germany), Katrin Kahlen (Leibniz Universität Hannover, Germany), Dirk Wiechers (Leibniz Universität Hannover, Germany) and Hartmut Stützel (Leibniz Universität Hannover, Germany) Salinity and carbon dioxide play vital roles in plant growth. While high salt levels are detrimental to physiological processes such as photosynthesis, elevated carbon dioxide concentrations e[CO2] enhances plant growth and productivity. This study was conducted to analyse the interactions of elevated CO2 and salinity on the growth and productivity of Cucumis sativus. The

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Society for Experimental Biology other organs has not been studied in detail. In order to understand the role of the circadian clock in regulating growth of Arabidopsis plants, we have investigated diel patterns of leaf blade expansion (growth in area) and petiole elongation (growth in length) in several arrhythmic clock mutants of Arabidopsis (CCA1ox#34 and prr9-prr7-prr5). Furthermore, leaf carbohydrate content and several growth parameters for roots (root length, number of lateral roots, root angle) were also determined during the experiments. Over expression of the CCA1 protein in both CCA1ox#34 and prr9prr7-prr5 plants seems to reduce leaf blade growth at the end of the night, leading to higher growth rates at the end of the day and enhanced starch accumulation over the day. Moreover, both mutants exhibited altered root architecture. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

expectation was that elevated CO2 levels might help alleviate the negative consequences of salt stress on cucumber. Hydroponically grown cucumber plants were exposed to three levels of salt stress and CO2 concentrations. Leaf area, fresh weight, dry weight, specific leaf area (SLA), photosynthesis, stomatal conductance, transpiration and water use efficiency were evaluated. Salt stress caused a reduction in photosynthesis, stomatal conductance and transpiration. This consequently caused a reduction in leaf fresh weight, dry weight and leaf area. CO2 had no effects on the latter parameters. SLA decreased with increasing CO2 and salt. Interesting, there was no interaction between salt and CO2 treatments and elevated CO2 did not improve salt tolerance in cucumber as we had anticipated. Another interesting observation was that salt treatments with the highest stomatal conductance made the most efficient use of water. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.57 The effect of cold shock and recovery of photosynthetic capacity in Miscanthus genotypes

Richard J Webster (Aberystwyth University, UK), Sarah Purdy (Aberystwyth University, UK) and John Clifton-Brown (Aberystwyth University, UK) Miscanthus is a C4 perennial grass which has high biomass potential. However yields can be limited by an inability to photosynthesize efficiently at low temperatures. The Miscanthus breeding program at Aberystwyth University has one of the largest Miscanthus germplasm collections in Europe and contains accessions collected from diverse and extreme habitats. These are being screened for a range of traits including cold tolerance. Miscanthus x giganteus is closely related to sugar cane and uses the same C4 (NADP-ME type) pathway, but in contrast to the vast majority of C4 species, it is cold-tolerant. We present data to describe the effect of a cold shock treatment and subsequent recovery, on photosynthetic capacity conducted on four Miscanthus genotypes. The four genotypes were grown at 30/25°C (day/night) and the light response of photosynthesis measured prior to application of a cold shock. The genotypes were then exposed to 12°C, and light saturated photosynthesis and chlorophyll fluorescence measured during the transition and acclimation period. The light response of photosynthesis was measured over subsequent days and during recovery when the four genotypes were returned to 30/25°C (day/night). Our results indicate variation in cold tolerance and maintenance of photosynthetic capacity between Miscanthus genotypes. Email address for correspondence: [email protected] Poster Session - Saturday 2nd July 2011

P1.59 An assessment of somaclonal variation in micropropagated plants of sugarcane by RAPD markers

Kalpana Sengar (MJP Roheilkhand University, India) Sugarcane is a major source of sugar and it is one of the most important commercial crops of several countries in tropical and subtropical zones. Tissue culture techniques are valuable tools for conducting genetic manipulation studies. Micropropagation of sugarcane is being widely adopted and plant regeneration in sugarcane without callusing is the best method to produce plantlets identical to the parental clones. The genetic stability is a major constraint in in vitro propagation. Plantlets derived from tissue culture are known to exhibit somaclonal variation, which is often heritable. Sugarcane variety CoS 99259 early maturing variety is a new variety released in India through the use of somaclonal variation. Significant differences were found in quantitative and qualitative agronomic characters. Another somacl