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SEEING RED:

Daphnia and Hemoglobin

A MIDDLE SCHOOL CURRICULUM UNIT MODELING ECOLOGICAL INTERACTIONS AND THE SIGNIFIGANCE OF ADAPTATIONS

Anne Deken Ste. Genevieve du Bois School Warson Woods, Missouri 2005 Summer Research Fellowship for Science Teachers Sponsored by Howard Hughes Medical Institute Washington University Science Outreach

SEEING RED: DAPHNIA AND HEMOGLOBIN Teacher Manual INTRODUCTION The freshwater zooplankton Daphnia plays an important role in aquatic food chains and has been used as an ecological model for many years. Daphnia is also used in other scientific disciplines including chemistry, physiology, and genetics. Daphnia is useful in many disciplines because of its physical characteristics, such as a transparent outer covering, sensitivity to toxins, and quick development of adaptations. Through this unit, middle school students are able to study this ecological model organism from a variety of different perspectives. They will examine this organism's phenotypic characteristics, observe Daphnia's response to an environmental stress (low oxygen), and investigate the complex role of an invasive Daphnia species in an aquatic food chain. CURRICULUM OVERVIEW The activities in this unit are separated into the following three sections: Part 1 ­ Observing Daphnia Characteristics and Behavior (teacher manual p. 8-11, student manual p. 1-6) Part 2 ­ Seeing Red: Daphnia and Hemoglobin (teacher manual p. 12-13, student manual p. 7-13) Part 3 ­ An Invasive Species of Daphnia (teacher manual p. 14-15, student manual p. 14-15) Students begin by looking at the physical features of Daphnia and understand how these characteristics allow them to survive in their environment. They will then investigate Daphnia's ecological interactions and begin to understand why they are useful for scientific study. Students will see how Daphnia turn red under low oxygen conditions through an increased production of hemoglobin, and thus serves as an ideal model to study the physiological effects of environmental stress. They will also discuss how an invasive species is able to populate a foreign area. Using the invasive species Daphnia lumholtzi, students will predict the type of adaptation that has allowed it to invade North America. Finally, students will also discuss what impacts an invasive species can have on an ecosystem. Through the study of Daphnia, students will understand how adaptations allow an organism to be successful in a particular environment and the importance of balance between organisms in an aquatic ecosystem.

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FIT TO STANDARDS Based on the National Science Education Standards for middle school life science students, this unit focuses on the content areas of population and ecology along with diversity and adaptations. This unit will meet the following National Science Content Standards: o Populations of organisms can be categorized by the function they serve in an ecosystem. Plants and some microorganisms are producers--they make their own food. All animals, including humans, are consumers, which obtain food by eating other organisms. Decomposers, primarily bacteria and fungi, are consumers that use waste materials and dead organisms for food. Food webs identify the relationships among producers, consumers, and decomposers in an ecosystem. o The number of organisms an ecosystem can support depends on the resources available and abiotic factors, such as quantity of light and water, range of temperatures, and soil composition. Given adequate biotic and abiotic resources and no disease or predators, populations (including humans) increase at rapid rates. Lack of resources and other factors, such as predation and climate, limit the growth of populations in specific niches in the ecosystem. o Biological evolution accounts for the diversity of species developed through gradual processes over many generations. Species acquire many of their unique characteristics through biological adaptation, which involves the selection of naturally occurring variations in populations. Biological adaptations include changes in structures, behaviors, or physiology that enhance survival and reproductive success in a particular environment. It will also meet the following Missouri Assessment Program learning objectives: o Identify populations within a community that are in competition with one another for resources. o Predict the possible effects of changes in the number and types of organisms in an ecosystem on the population of other organisms within that ecosystem. o Relate examples of adaptations within a species to its ability to survive in a specific environment. o Predict how certain adaptations may offer a survival advantage to an organism in a particular environment.

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BACKGROUND INFORMATION ON DAPHNIA Physical description Daphnia, commonly called water fleas, are a freshwater zooplankton found in ponds and lakes all over the world. Daphnia received the name water flea due to their jerky swimming motions. Some types of Daphnia can be seen with the human eye, while others must be identified with a microscope. Depending on the species they can range in size from 0.5mm to 1cm. Their outer covering, or carapace, is transparent, so many internal organs can be seen, especially the beating heart. On the head there is a compound eye and a pair of antennae, which are used for swimming. Females are usually larger than males and have a brood chamber under their outer carapace where eggs are carried. Food chain relationships Daphnia are an extremely important part of aquatic food chains. They eat primary producers such as algae, yeast, and bacteria. Daphnia are the prey of tadpoles, salamanders, newts, aquatic insects, and many types of small fish. Fluctuations in Daphnia populations can cause algae overgrowth or even a drastic drop in fish populations. Through the food chain, larger animals caught or eaten by humans can be affected by large changes in Daphnia populations. Reproduction The Daphnia lifespan is approximately eight weeks. Daphnia reach maturity quickly, within five to ten days, and produce multiple offspring each cycle. Daphnia can reproduce both asexually and sexually. During times of plentiful food, females reproduce asexually to produce clones. When food is scarce, Daphnia reproduce sexually in order to produce genetic variation and increase the chance of species survival. During harsh weather conditions, resting eggs are produced. These eggs have a tough outer coating, which can withstand time, heat, drying, and cold temperatures. Reactions to environmental stress Daphnia have a number of natural responses to environmental stress, and due to these qualities are often used as an indicator of watershed health. The term "watershed" refers to all the land and water within a drainage area. Daphnia can detect the presence of kairomones, chemicals released by predators. In response to these chemicals Daphnia can grow larger head and tail spines or develop through a shorter gestation period. They are extremely sensitive to changes in water toxicity. This can be monitored through changes in heartbeat or Daphnia death. When Daphnia are exposed to hypoxic (low oxygen) conditions, they can increase hemoglobin production. Due to their clear outer carapace, they will appear red when hemoglobin production has increased. Species Three species of Daphnia will be used in this unit. Daphnia magna is very rarely found in the wild and is often sold in pet stores as a food source. They can become as large as 1/5 inch and are often less tolerant of environmental changes. Daphnia pulex is very similar to D. magna, but is usually smaller. They are the most common species found across temperate North America. Daphnia lumholtzi is an invasive species originally from

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Africa and Asia, which have extremely large head and tail spines. D. lumholtzi is currently invading watersheds in the United States.

Daphnia magna

Daphnia pulex

Daphnia lumholtzi

Classroom advantages Daphnia can be found in most freshwater lakes and ponds. They are also inexpensive to purchase and cultures are easy to maintain. One culture can easily produce enough individuals for entire class viewing. Organisms can easily be seen through a dissecting or compound microscope. The resting eggs can be kept for months to years.

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DAPHNIA CULTURING PROTOCOL 1. Ordering Daphnia Carolina Biological Supply, http://www.carolina.com Daphnia magna · Catalog #14-2326 Daphnia magna Culture Kit (live) $31.50- includes culture water, plastic aquarium, dried algae powder, and live Daphnia · Catalog #14-2330 Daphnia magna (live) $6.95- Culture for 30 students Daphnia pulex · Catalog #14-2304 Daphnia pulex Culture Kit (live) $26.95- includes culture water, plastic aquarium, food, and live Daphnia · Catalog #14-2314 Daphnia pulex (live) $6.75- Culture for 30 students · Catalog #14-2302 Daphnia pulex (resting eggs) $5.50 · Catalog #14-2316 Daphnia food $5.30 2. Preparing culture water Regardless of the type of culture water used, allow at least 24 hours for water to settle and temperature to stabilize before introducing live Daphnia. · Carolina Biological Supply kits - culture water is provided. Follow directions included in culture kit. · Pond water - Pond water should contain algae, but should not be too thick for organisms to move and obtain oxygen. · Spring water - Fill container and add food or nutrients. · Preparing your own culture water a) Fill a five-gallon bucket with tap water b) Add 5 drops of dechlorinator c) Add nutrients 12mg NaH2PO4 (or Daphnia food) d) Partially cover with lid e) Let bucket sit for at least 24 hours f) Culture water can be transferred to smaller containers such as two liter soda bottles, butter tubs, glass jars, etc. 3. Introducing Daphnia to culture water It may be helpful to test the water by introducing a few Daphnia into the culture water and allowing the culture to sit over night. This will allow you to test water conditions without sacrificing the entire container of Daphnia. a) When Daphnia arrive allow the shipping jar to float in your culture water. (This will allow the Daphnia to acclimate to the temperature) b) After approximately 15 minutes, release the Daphnia into the culture water. c) Remove the lid from the shipping jar. d) Hold the shipping jar upright and gently submerge it. e) Then "pour" underwater to empty the jar. f) Remove empty jar from the culture water.

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4. Feeding and care It should be noted that Daphnia magna are obligate algal feeders. There must be algae in the culture water for them to survive. The dried algae powder provided in the Carolina magna culture kit appears to work better for the magna than the food pellets provided in the pulex culture kit. Some additional care tips: a) Bucket or container can be placed outside in warm weather. The area chosen should receive some shade. b) If the bucket or container must be placed inside, a window that receives indirect sunlight can be used. Fluorescent lights can also be used on a daily night and day schedule. c) Using Carolina food pellets - Add only one pellet (crushed) per week d) Enough food should be added that culture water becomes cloudy, but clears within a day or two. e) Miracle Grow can be used to increase algae growth (1 teaspoon per gallon of water). Algae should not be too thick, or there will not be enough dissolved oxygen. f) Many other items can be used to cause bacteria growth used for food, such as brewer's yeast (1/8 teaspoon), dog food, Gerber's baby food potatoes, blended spinach, alfalfa powder, rabbit pellets, frozen peas, and carrots. 5. Optimal conditions for Daphnia Make sure these optimal conditions are being met in order for culture to survive. a) pH - 6.5-9.5 b) Temperature - 18-22°C (64-72°F). c) Dissolved oxygen - can tolerate low levels, slow aeration is helpful, but not necessary (bubble stone or tiny bubbles will destroy the Daphnia carapace) d) Nutrients - can tolerate high levels of nitrogen and phosphorus. e) Light - indirect sunlight, normal daylight hours. 6. Special care information If you are having trouble getting your culture started, follow these tips. · Replace culture water often. · Harvest culture often to avoid seeing effects of overpopulation. · Do not over-feed (most common cause of Daphnia death). · Place culture water in indirect sunlight (Temperatures over 85-90°F can cause death). · If you order buckets through Carolina Biological Supply they may carry residue that could be toxic to the Daphnia. The buckets need to be thoroughly cleaned before introducing Daphnia. Soaps can leave a residue, so the buckets need to be carefully rinsed before using. Micro 90 is a cleaning product that will not leave a soap residue. · http://www.carolina.com/how_do_I/classroom_critters.asp Includes care guides and videos to assist with Daphnia care.

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7. Purchase information for other helpful materials Observation gel · Carolina Biological Supply, Catalog # 13-2700, $13.25 Dissolved oxygen kits · http://www.drsfostersmith.com, CD-900561, $8.99 (30 tests) · Pet Market Place (8011 Watson Rd. 63119), $14.69 (30 tests) · Acorn Naturalists, #Test-10279 $24.65 (100 tests) Tap water conditioner · Petsmart (87 Brentwood Promenade Ct. 63144), Aquarium Pharmaceuticals Tap Water Conditioner (dechlorinator with no fish coat), 16 Fl Oz, $4.99 Alternate vendors for Daphnia cultures · http://www.sargentwelch.com WL50718 Daphnia Culture Set, $21.49 WL50725 Daphnia Media, $9.19 · http://www.enasco.com LM00039(A)G Daphnia magna, $6.20 LM00754M Small feeder Daphnia, $11.10

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PART 1 ­ OBSERVING DAPHNIA CHARACTERISTICS AND BEHAVIOR Lesson Overview The background section of this unit will familiarize students with both the physical and behavioral characteristics of Daphnia. The background information will be helpful in future activities, as students explore the ways Daphnia respond to stress and the impact of Daphnia on aquatic food chains. If you have access to live Daphnia samples, begin by allowing the students to observe the organisms without receiving any background information. Daphnia are very active organisms with many interesting characteristics, and viewing them gives students the chance to use observation skills and experiment with microscopes. If you do not have access to live samples, provide the students with background information on the organism and start your lesson with the "Daphnia Anatomy: Inside and Out" worksheet. Procedure 1. Recording Behaviors In pairs, students should begin by observing Daphnia behavior while the Daphnia are still in culturing containers. For groups of students, it will be most effective to give each group a clear container with only a few Daphnia in it. Culture water can be transferred to jelly jars, Tupperware, small shipping containers, etc. Each pair of students should choose one Daphnia to observe. Pairs of students can take turns observing, recording, or completing the questions. Students will record their answers to the following questions (which are also printed in their packet). These questions are meant to stimulate thought, but there are not right or wrong answers. Answers can be discussed in class. 1) List at least three behaviors demonstrated by the Daphnia in your container. For example, does the Daphnia look like it is eating? Is it swimming at the bottom or the top of the container? Is it rolling or flipping? 2) Describe the swimming motion of the Daphnia. Is it similar or different to other organisms that you have seen? How? 3) What body parts does the Daphnia use to swim? 4) Do you think Daphnia live in freshwater or saltwater? Why? 5) What do you think Daphnia eat? Why? 6) What do you think eats the Daphnia? Why? 7) Describe the size of the Daphnia. Are all the Daphnia in the container the same size? 2. Observing Physical Characteristics Students will observe individual Daphnia using a dissecting microscope or compound microscope and answer the questions in their lab manuals. Daphnia should be removed from the culture with a pipette and given a small amount of water to keep them alive. A number of methods can be used to keep the Daphnia relatively still during observation ­ deep well slides, s rubber washer coated with petroleum jelly and sealed to a microscope slide, Carolina Observation Gel, or giving the Daphnia an extremely small drop of water in a Petri dish or watch glass.

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3. Daphnia Anatomy Worksheet The "Daphnia Anatomy: Inside and Out" worksheet in the student manual can be completed by students as homework or as preparation for the lab.

A B C D H G F

E

Directions: On the lines write the letter that matches with the correct Daphnia body part on the drawing. 1. 2. 3. 4. 5. 6. 7. 8. D E A B F H G C Carapace - protective outer layer, allows oxygen to pass through, contains brood chamber for eggs Antennae - two sets of antennae used for swimming and sensing the environment Intestine - ground up food particles are digested Heart - clear circulatory fluid is pushed around the body Eye - compound eye controlled by muscles with nerve connections to the brain Spinules - produces water currents, which helps carry food and oxygen molecules to the mouth and gills Legs - used for collecting food and stabilizing Brood chamber - holds eggs 9

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4. Interesting Facts Worksheet The questions on the "Interesting Facts About Daphnia" worksheet in the student manual can be used for group discussion, independent work, or the questions can be divided among groups of students. Answers to the questions will vary, but students should understand the role of Daphnia in ecology and other sciences. 1) Without Daphnia the algae population will explode and cover the surface of the pond. Over time, the fish population will go down because there will be a food shortage. Eventually, the algae covered surface will cause less light to reach other plants and animals, and the fish will eat all other food sources in the pond. Larger fish will also lose their food sources. 2) In a lab, Daphnia can be placed in 10 buckets or jars in order to control the environment. In five jars, the Daphnia will be placed with a predator, such as fish. In another five jars, the Daphnia will be placed without a predator. After two to three weeks, Daphnia from each of the jars can be sampled and the body shape, especially the size of head and tail spines, can be compared. In a natural setting, samples can be taken from ponds containing fish or without fish and the Daphnia can be compared. 3) Resting eggs have a tough outer coating that allows them to survive for years without water and withstand drastic temperature changes. They are also very small and light and can be transported by flooding, animals, and boats. For example, if a fisherman has used his or her boat in more than one location without rinsing equipment, eggs could be transported from one area to another. 4) Using a dissecting microscope the organs of the Daphnia can be seen right through the outer carapace. The heart can be seen pumping and circulating fluid throughout the body. The number of heartbeats per minute can be counted and recorded. If the environment of the Daphnia is changed or a toxin is introduced, the number of Daphnia heartbeats per minute will also change. 5) On a small scale, the effect of a toxin on a Daphnia can be monitored by observing the change in heartbeats per minute or the death of the Daphnia. On a larger scale, Daphnia from lakes and ponds can be sampled to determine the effect of toxins. When toxins are added the pH level of the water will change and can be unfavorable for Daphnia. If the Daphnia population drops drastically the water may have been polluted. 6) Increased hemoglobin production allows the Daphnia to transport a larger amount of oxygen throughout their bodies. If the water conditions are only temporarily low in oxygen, the Daphnia can increase oxygen circulation within their bodies in order to survive. 7) You can identify Daphnia by their jerky swimming motion. When they are kept in a container, they can be seen moving up and down vertically or rolling

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on the bottom. You can distinguish them from other aquatic organisms by their antennae used for swimming and large dark eye. 8) Daphnia can easily be cultured in a laboratory. They are useful for scientific study because they reproduce asexually, produce multiple offspring, and have a short gestation period. Experiments can be run using many organisms and can easily be conducted over multiple generations. 9) A plankton net or fine mesh can be used to collect small organisms from the pond. Once organisms are collected, you can identify the Daphnia in your sample using a microscope and a taxonomic key. If you wish to move them to another sample, they can be picked up with a small amount of water in a pipette. 10) Daphnia are considered a model organism for ecology. They are small, reproduce quickly and in great numbers, and they can easily be cultured. Due to their sensitivity to changes in the environment, they can be used to study the impact of changes in food chains and water quality. Their organs can easily be observed and heartbeat can be monitored.

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PART 2 ­ SEEING RED: DAPHNIA AND HEMOGLOBIN Background Information Hemoglobin is an important protein found in all vertebrates and some invertebrates. Hemoglobin transports oxygen throughout the organism's respiratory system. In complex animals such as humans, hemoglobin carries oxygen from the lungs to the body's tissues and returns carbon dioxide from the tissues to the lungs. Hemoglobin can be found in the red blood cells of vertebrates and the iron contained in hemoglobin is responsible for the red color of blood. Simple invertebrates, such as Daphnia, do not have lungs or red blood cells. Because Daphnia do not have a complex respiratory system, they generally obtain oxygen for respiration through diffusion. Oxygen molecules along with water are carried in and out through the outer carapace of the Daphnia. Hemoglobin in these organisms is extracellular and helps to carry oxygen to body tissues. Daphnia, unlike vertebrates, do not need to maintain a high level of hemoglobin to survive. However, synthesis of hemoglobin does provide a survival advantage when water conditions are low in oxygen. Interestingly, under stressful environmental conditions Daphnia can increase hemoglobin production. This allows the Daphnia to survive in hypoxic (low oxygen) water conditions. Daphnia can develop high amounts of hemoglobin in a few days and can maintain this status for a couple of weeks. Hemoglobin can be produced in brood pouches, and offspring in hypoxic conditions may also produce more hemoglobin. If water conditions return to normal, Daphnia can lower hemoglobin production within a few days, as well. The development of hemoglobin will allow the Daphnia to carry more oxygen to body systems, but the synthesis of hemoglobin does use energy that would normally be used for other life processes. This energy trade-off causes the amount of energy invested in growth and development to decrease. Due to Daphnia's transparent carapace, hemoglobin can be seen as it develops. Daphnia with high hemoglobin content will appear red in color. Daphnia with more hemoglobin have access to food sources found in low oxygen areas, but their red color may be more obvious to predators. The study of Daphnia hemoglobin can be traced all the way back to the 1700's. In 1758, Dutch naturalist J. Swammerdam noticed that the species of Daphnia that live in ponds are often pink or even red. He described the color as "like that of beef, which has been time steeped in water" (Fox 1948). In the 1940's and 1950's scientists studied pond and laboratory conditions that caused Daphnia to increase hemoglobin production. In the years that followed, Daphnia was used as an ecological bioindicator and for toxicology bioassays. Currently, the physiological impact of hemoglobin development on Daphnia is being studied. The genetic sequence for Daphnia hemoglobin has been constructed and has many genetic and ecological applications. Lesson Overview Daphnia will produce hemoglobin in hypoxic water conditions. In this activity, you will be increasing population in order to lower the oxygen content in the water. As the Daphnia reproduce, the larger population will require more dissolved oxygen. In order to survive, some Daphnia will produce hemoglobin and appear red, while others will die.

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Materials 2 small beakers (250 mL recommended) 1 gallon spring water 75 Daphnia magna Pipette for transferring Daphnia Powdered Daphnia food TetraTest Dissolved Oxygen Kit Procedure 1. Fill both beakers with 100 mL of spring water. 2. Using the pipette transfer 25 Daphnia to one of the beakers. 3. Using the pipette transfer 50 Daphnia to the other beaker. 4. Add a small pinch of food to both of the containers. Sprinkle the pinch of food over the surface of the water. Food should cover the surface in a thin layer. 5. Place both containers in indirect sunlight. 6. Continue to feed Daphnia every other day. (When food is added, the surface of the water will become green and cloudy. Once the water begins to clear, you should add another pinch of food. ) 7. Allow at least a week for hemoglobin to be produced. 8. Measure dissolved oxygen in the water by following the directions for the dissolved oxygen kit. These kits can be purchased at many pet stores in their fish departments. The worksheet in the student manual contains places to record the dissolved oxygen from the two Daphnia populations. 9. Students can make observations daily or the observations can be spread out over a longer time period. A total of five observations should be made and recorded on the Observation Summary Sheet in the student manual. Students can remove individual Daphnia from the beakers in order to make observations under the microscope. As described in the previous activity, a number of methods can be used to keep the Daphnia relatively still during observation. · Deep well slides · A rubber washer coated with petroleum jelly and sealed to a microscope slide · Carolina Observation gel · Giving the Daphnia an extremely small drop of water in a Petri dish or watch glass

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PART 3 ­ AN INVASIVE SPECIES OF DAPHNIA Background Information An invasive species is a plant, animal, or other organism that is introduced to an environment where it is not normally found. It may be introduced accidentally or intentionally, but it is likely to cause environmental harm, economic harm, or harm to human health. Invasive species are primarily introduced and spread by humans. When an organism is taken out of its native environment and placed in another, it often has no predators in its new environment. This allows the new species to thrive and threatens the natural diversity of the area. There is an invasive species of Daphnia called Daphnia lumholtzi. This species originated in Africa and Asia and is currently invading North American waterways. This species is believed to be successful in foreign environments due to its tolerance of high temperatures, and ability to avoid predation due to large head and tail spines. Daphnia lumholtzi can grow to be as large as one inch, and may have a competitive advantage over native Daphnia. The head and tail spines of Daphnia lumholtzi are too large to be eaten by small filter feeding fish. These small fish must then eat the native Daphnia and will eventually run out of food. If the small fish population decreases, the larger fish will also be threatened. This effect will continue throughout the entire food chain, since larger fish are caught and eaten by humans. Fishermen and scientists are concerned that Daphnia lumholtzi will continue to spread across the entire North American continent. The public is asked to help keep this species contained. Suggestions to avoid spreading this species include cleaning all boating and recreational equipment and disposing of bait properly. Procedure 1. Most Wanted Poster Students should have completed background information on Daphnia or will need some basic information about Daphnia characteristics. For this next activity, students will create a "Most Wanted" poster for the invasive species of Daphnia. Students should use the directions in their lab manual for this activity. It may be helpful to discuss animal adaptations with the students, but do not show the picture of Daphnia lumholtzi to them until after they have completed their Wanted Posters. Ask students to think about special characteristics that animals have that allow them to avoid predation. Some examples are camouflage, emitting an odor, or spraying a poison. It also may be helpful to discuss the protective mechanisms of skunks, rattlesnakes, and bees. 2. Wanted Poster Evaluation Checklist 1) Student's picture shows an aquatic environment. 2) Picture includes native Daphnia characteristics (small, transparent, antennae, etc.). 3) Picture includes an adaptation that allows the organism to survive (odor, spikes, speed, etc.). 4) Additional creative elements - "Wanted sign", predators, reward amount, magnifying glass or microscope.

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3. Closing Activity Students will present their pictures and explain why they chose their particular adaptation. The class can discuss which adaptations were common or which one they feel is best suited to this organism. Show the students a picture of Daphnia lumholtzi and discuss the competitive advantage of its large head and tail spines. 4. Extension Questions 1) Why are Daphnia lumholtzi a problem for local fishermen? 2) What advantages does this type of Daphnia have that native Daphnia do not? 3) How can scientists determine whether or not a lake or river has been invaded by Daphnia lumholtzi? 4) How do you think this species managed to invade lakes and ponds that are many miles apart? 5) Because Daphnia lumholtzi is very small and often looks very similar to other species, what tools would a scientist need to identify this species? 6) List three ways that scientists could study the affect of Daphnia lumholtzi on local waterways. 7) What can local residents and fishermen do to protect their waterways from this invading species? 5. Extension Questions Answers Answers will vary and students should have original suggestions for these problems. 1) Small fish cannot filter feed on Daphnia lumholtzi, so they will run out of food sources. This will cause a population decrease for small fish. Larger fish that eat the smaller fish will then run out of food. This will cause their population to decrease. Fishermen will then have a harder time catching larger fish. 2) Daphnia lumholtzi have larger head and tail spines, so they are too large to be filter fed by small fish. 3) Scientists must take water samples of lakes to determine whether or not a lake has been invaded. Using a microscope they identify and count the species found within a lake sample. 4) Resting eggs can be carried by animals, boats, and flooding. 5) Scientists use magnifying glasses, microscopes, taxonomic keys and, if available, DNA sequence similarities. 6) Monitor total Daphnia populations. Compare native Daphnia populations to Daphnia lumholtzi. Monitor changes in fish populations. Monitor changes in amount of algae coverage. 7) Residents and fishermen can clean recreational boating equipment, avoid dumping extra bait into water, and monitor changes in fish populations.

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REFERENCES Atha, Jason T. and Ann M. L. Cavallo. (1999) Aquatic Ecology. The Science Teacher, 2629. BioMEDIA Galleries, http://ebiomedia.com/gall/classics/Daphnia_gen.html BIOWEB, www.bioweb.dk/figurer/magna.gif (June 2005) Cladocera, http://www.cladocera.uoguelph.ca/ (June 2005) Daphnia: An Aquarist's Guide, http://www.caudata.org/Daphnia/ (June 2005) Daphnia Genomics Consortium, http://Daphnia.cgb.indiana.edu/ (June 2005) Environmental Inquiry, http://ei.cornell.edu/ (June 2005) Fox, H. Munro. (1948). The hemoglobin of Daphnia. Proceedings of the Royal Society of London. Series B. Biological Sciences, 135(879), 195-212. Glasgow University Zoology Museum (Biomedia Website) www.biol.paisley.ac.uk/ (June 2005) Goldmann, Torsten, and Bertram Becher, et al. (1999) Epipodite and fat cells at sites of hemoglobin synthesis in the branchiopod crustacean Daphnia Magna, Histochemical Cell Biology, 112, 335-339 Gorr, Thomas A., Joshua D. Cahn, Hideo Yamagata, and Franklin Bunn, (2004). Hypoxiainduced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent. The Journal of Biological Chemistry, (279) 34, 36038-36047. Great Lakes Environmental Research Laboratory, www.glerl.noaa.gov/seagrant/GLWL/zooplankton (June 2005) Havel, John E, Jonathan B Shurin, and John R Jones. (2002). Estimating Dispersal From Patterns of Spread: Spatial and Local Control of Lake Invasions. Ecology, 83(12) 33063318. Institute for Biological Invasions, http://invasions.bio.utk.edu/invaders/images, (June 2005) Johnson, Jennifer, and John E. Havel. (2001). Competition between native and exotic Daphnia: in situ experiments, Journal of Plankton Research, 23(4), 373-387 Pirow, R., C. Baumer, and R.J. Paul. (2001). Benefits of hemoglobin in the cladoceran crustacean Daphnia magna, The Journal of Experimental Biology, 204, 3425-3441

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Torsten, Goldmann, and Bertram Becher, et al. (1999) Epipodite and fat cells at sites of hemoglobin synthesis in the branchiopod crustacean Daphnia Magna, Histochemical Cell Biology, 112, 335-339 University of Michigan Museum of Zoology, Animal Diversity Web, http://animaldiversity.ummz.umich.edu (June 2005)

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ADDITIONAL RESOURCES Toxicology Activities Environmental Inquiry: Daphnia bioassays using salt http://ei.cornell.edu/toxicology/bioassays/Daphnia/ Drugged Out Daphnia http://www.wested.org/werc/earthsystems/biology/Daphnia.html The Charms of Duckweed http://www.mobot.org/jwcross/duckweed/duckweed.htm Aquatic Activities Project Wild Aquatic K-12 Curriculum and Activity Guide, Council for Environmental Education 2001 Science for Ohio: It's a Small World After All! (Pond Microcosms) http://casnov1.cas.muohio.edu/scienceforohio/SfoMain/SiteMap.html "Pond" Cultures of Microscopic Invertebrates http://www.ruf.rice.edu/~bioslabs/studies/invertebrates/pond.html#start Make your own plankton net http://www.biosci.ohiou.edu/faculty/currie/ocean/makeanet.htm Ecosystems: Session 8 Hydra Vs. Daphnia (pdf) http://www.nycenet.edu/DIS/mst/ecosystems/session8.pdf Missouri Stream Team http://www.mostreamteam.org/ Daphnia Information The Zooplankton Project http://www.cnas.smsu.edu/zooplankton/Default.htm Daphnia pictures http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopyuk.org.uk/mag/artjun99/wflea.html Daphnia Information http://www.Daphnia.com/index.html

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SEEING RED:

Daphnia and Hemoglobin

Student Manual

Name(s) ________________________________________________________________ Class Hour _______________ Project Start Date _____________________________

OBSERVING DAPHNIA CHARACTERISTICS AND BEHAVIOR

1. Recording Behavior Observations Directions: Daphnia should be observed while they are still in the container. This will allow you to see their movements in the water. Choose one Daphnia to observe. Carefully record all observations. Each student in the group should take a turn observing, recording, and answering questions. 1) List at least three behaviors demonstrated by the Daphnia in your container. For example, does the Daphnia look like it is eating? Is it swimming at the bottom or the top of the container? Is it rolling or flipping?

2) Describe the swimming motion of the Daphnia. Is it similar or different to other organisms that you have seen? How is it different?

3) What body parts does the Daphnia use to swim? Where are they located?

4) Do you think Daphnia live in freshwater or saltwater? Why?

5) What do you think Daphnia eat? Why?

6) What do you think eats the Daphnia? Why?

7) Describe the size of the Daphnia. Are all the Daphnia in the container the same size?

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2. Recording Physical Characteristics Directions: Choose one Daphnia to observe under the microscope. Carefully use a pipette to transfer the Daphnia and a small amount of water to your slide or Petri dish. Keep your Daphnia wet throughout the activity and return it to the container when you are finished observing. Each member of the group should observe, record, and answer questions. 1) Using a microscope observe one Daphnia. Make a drawing of the physical details that you can see. 2) Choose three specific body parts that you feel you can identify. On your drawing label these parts and predict the function of each.

3) Does the Daphnia have eyes? If so, how many?

4) Do you see a heart? If you do, describe the speed of the heartbeat.

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5) Where do you think the Daphnia digests its food? Draw the shape of this organ.

6) Is your Daphnia carrying any eggs? If it does have eggs, describe where they are located on the body. How many can you see?

7) Compare your Daphnia to other organisms that you have seen or studied. (Resources such as encyclopedias, textbooks, internet searches may be helpful) List two organisms that have one or more characteristic, which is similar to Daphnia, and describe the characteristics that they have in common.

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Daphnia Anatomy: Inside and Out

Directions: On the lines write the letter that matches with the correct Daphnia body part on the drawing. 1. 2. 3. 4. 5. 6. 7. 8. Carapace - protective outer layer, allows oxygen to pass through, contains brood chamber for eggs Antennae - two sets of antennae used for swimming and sensing the environment Intestine - ground up food particles are digested Heart - clear circulatory fluid is pushed around the body Eye - compound eye controlled by muscles with nerve connections to the brain Spinules - produces water currents, which helps carry food and oxygen molecules to the mouth and gills Legs - used for collecting food and stabilizing Brood chamber - holds eggs

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Interesting Facts about Daphnia

· · · · · · · · · · Daphnia are often called water fleas because they swim in a jerky motion. Daphnia are extremely small and range in size from 0.5mm to 1cm. Daphnia can be found in freshwater lakes, ponds, and rivers all over the world. The outer carapace of a Daphnia is transparent so all the internal organs, even the beating heart, can be seen. Daphnia mature in approximately a week and can have thousands of offspring. Daphnia are very important to the food chain. They eat algae and are eaten by insects, water mites, and small fish. In the presence of a predator Daphnia can develop large head and tail spines. Daphnia are very sensitive to changes in their environment. In low oxygen water conditions Daphnia can increase hemoglobin production, which causes them to turn red. During unfavorable weather conditions Daphnia can produce resting eggs, which can withstand time, heat, cold, and drought.

Directions: Use the information above to answer the following questions about the interesting characteristics of Daphnia. 1) Daphnia are an extremely important part of aquatic ecosystems. Explain what would happen to the algae population in a pond if all of the Daphnia died. What would happen to the fish population if there were no Daphnia? What would eventually happen to the food chain in a pond with no Daphnia?

2) In the presence of a predator, Daphnia will have a different pattern of physical development. Describe an experiment where you could test the effect of a predator on the physical development of a Daphnia population.

3) Daphnia can be found all over the world. Describe how their ability to produce resting eggs could be related to transport between lakes, ponds, and rivers.

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4) When studying the physiology of Daphnia, why would the fact that they have a transparent outer carapace be helpful?

5) Daphnia are extremely sensitive to changes in water quality. How could they be used to monitor pollution in waterways?

6) Daphnia can develop hemoglobin in low oxygen water conditions. How can this help them to survive in an unhealthy environment?

7) If you were given a sample of pond water and asked to find Daphnia, what characteristics would you use to identify the organisms?

8) Daphnia can produce thousands of offspring in a short amount of time. Why would this be useful in laboratory study?

9) Describe the method you would use if you were asked to collect and identify Daphnia found in a pond.

10) Daphnia are a commonly studied organism in ecology, physiology, and chemistry. Based on the facts above, list three reasons why you think these organisms would be a good organism for scientific study.

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SEEING RED: DAPHNIA AND HEMOGLOBIN

Daphnia Observation Sheet Date_________________

1. Behavior Observations Directions: You should have 5 copies of this sheet in order to observe the Daphnia on 5 different dates. Be sure to record the date above. While the Daphnia are still in the sealed jars, make observations in pairs. One person should make observations, while the other keeps time and records. Choose one Daphnia to observe for four minutes. Every twenty seconds, make a tally mark to represent the type of behavior you see. Complete the chart for the low oxygen sample and the sample under normal conditions. Low oxygen Normal oxygen Moving vertically on bottom of container Rolling on bottom of container Swimming at edges of container Swimming from bottom of container to the top Remaining still on bottom of container

2. Color Observations Directions: Observe one Daphnia under a microscope or with a magnifying glass. Using the color scale on the next page, decide which color is closest to the actual Daphnia color. Color the Daphnia model to match the color scale and record the number from the color scale below. Low O2 Color Scale Number (1 ­ 6) ______ Normal O2 Color Scale Number (1 ­ 6) ______

3. Dissolved Oxygen Measurement Directions: With help from your instructor, measure the dissolved oxygen levels in each sample then record your results below. Low O2 Dissolved Oxygen _______

Seeing Red: Daphnia and Hemoglobin ­ Student Manual

Normal O2 Dissolved Oxygen _______ 7

Daphnia Color Scale

6

5

4

3

2

1

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SEEING RED: DAPHNIA AND HEMOGLOBIN

Observation Summary Sheet Directions: Complete this sheet after filling out 5 different "Daphnia Observation Sheets." Behavior Observations 1. Determine the total number of times a behavior occurred across the five observations. Enter the totals in the chart below. Find the average number of times a behavior occurred by dividing your totals by five. Enter your averages in the chart below. Low oxygen Normal oxygen Total Average Total Average Moving vertically on bottom of container Rolling on bottom of container Swimming at edges of container Swimming from bottom of container to the top Remaining still on bottom of container

2. Compare the behavior in low oxygen conditions with the behavior in normal conditions by creating a double bar graph (2 variables) for the averages you listed in the chart above. The types of behavior should be listed on the x axis and the average number of times the behavior occurred on the y axis.

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3. In the low oxygen sample, which type of Daphnia behavior occurred most often?

4. In the sample with normal oxygen levels, which type of Daphnia behavior occurred most often?

5. Were there differences in the types of Daphnia behavior in the two samples? If so, how were they different? If not, why were they similar?

6. Did the Daphnia in either container reproduce? How many days did it take for them to reproduce? Were there differences in the numbers of offspring or reproduction rate between the two containers? If there was a difference between the two containers, describe the difference.

7. What was the relationship between the Daphnia behavior and the experimental conditions?

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Color Observations Directions: Fill in the first Daphnia picture with a color that matches the scale color from your observations on day one. Continue the process, coloring the next four Daphnia with the colors from your observation sheets and filling in the scale numbers. Low Oxygen Observations

Scale Number Scale Number Scale Number Scale Number Scale Number

Normal Oxygen Observations

Scale Number Scale Number Scale Number Scale Number Scale Number

Dissolved Oxygen Measurement 1. Did the dissolved oxygen measurements vary between the two experimental conditions? Overall, how did these numbers compare?

2. Was there a change in dissolved oxygen levels from your first measurement to your last? If there was a change, what could have caused this change? If there was not, what caused the conditions to remain constant?

Extension Questions 1. What factor was different in the two experimental conditions?

2. Did the Daphnia in your sample change color over the five observations? If the sample did change color, was it a slow change or did it change drastically from

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one observation to the next? If your sample did not change color, describe your experimental conditions and describe why you think there was no change.

3. Construct a double bar graph to show the color scale numbers for each of the five observations. The x axis should show the five observations and the y should show the color scale values between one and six.

4. Compare the graph you created for Daphnia behavior with your graph for color scale number. Is there a relationship between the variables shown on the two graphs? If there is a relationship, how are they related to each other? If there is not, why is there no correlation between the two graphs?

5. Based on your knowledge of Daphnia physiology, how is the dissolved oxygen level in each sample related to the color of the Daphnia found in that sample?

6. Is there a correlation between color scale number, Daphnia behavior, and dissolved oxygen measurements? If so, what is the relationship? If not, which of the three pieces of data does not correlate with the others?

7. Predict the advantages that a color change provides for the Daphnia.

8. Predict the disadvantages that a color change causes for the Daphnia.

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9. If you collected red Daphnia from a pond, what does this mean about the water found in that pond?

10. How could you use the color of Daphnia found in a pond to determine whether or not the conditions in that pond are healthy?

11. What types of environmental changes could cause a pond to become unhealthy for animal and plant life?

12. Compare your experiment using Daphnia and dissolved oxygen levels to what actually takes place in ponds and lakes. How do you think changes in dissolved oxygen levels affect other organisms in the pond or lake?

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MOST WANTED: DAPHNIA LUMHOLTZI

Background An invasive species is a plant, animal, or other organism that is introduced to an environment where it is not normally found. It may be introduced accidentally or intentionally, but it is likely to cause environmental harm, economic harm, or harm to human health. Invasive species are primarily introduced and spread by humans. When an organism is taken out of its native environment and placed in another, it often has no predators in its new environment. This allows the new species to thrive and threatens the natural diversity of the area. Problem Ecologists are scientists that often study the relationship between plants and animals in natural environments. There is a group of ecologists that is studying the relationship between algae, Daphnia, and fish, and they need your help. There is a species of Daphnia, originally from Asia, which is invading North American waterways. The foreign species is called Daphnia lumholtzi. Daphnia lumholtzi has special characteristics that allow it to avoid being eaten by small fish. Daphnia lumholtzi is causing native Daphnia to be wiped out as it overpopulates and takes over the food supplies in many ponds, lakes, and rivers. Fishermen are outraged because, as Daphnia lumholtzi overpopulates, smaller fish run out of food sources. The death of smaller fish then threatens the food supplies of larger fish. Fishermen desperately want to find this foreign species and to figure out how to save their fish. Assignment Your task is to create a "Wanted Poster" for this mysterious invading species. Keep in mind the things you have heard about native Daphnia species such as they are very small, can hardly be seen with the human eye, and are clear. As you make your drawing, remember that Daphnia lumholtzi has special characteristics that native Daphnia don't have, which helps it to avoid being eaten. Your drawing should look very similar to native Daphnia, except for the special characteristic you will add. As a model, you may want to use other animals that have special body parts or behaviors to avoid predators. Make your drawing very specific because it is very important to scientists and fishermen that we find this creature and identify it. Assignment Evaluation Checklist 1) In the picture the organism is placed in an aquatic environment. 2) Picture includes basic Daphnia characteristics (small, transparent, antennae, etc.). 3) Picture includes an adaptation that allows the organism to survive (odor, spikes, speed, etc.). 4) Additional creative elements - "Wanted" sign, predators, reward amount, magnifying glass or microscope, etc.

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Extension Questions 1) Why are Daphnia lumholtzi a problem for local fishermen?

2) What advantages does this type of Daphnia have that native Daphnia do not?

3) How can scientists determine whether or not a lake or river has been invaded by Daphnia lumholtzi?

4) How do you think this species managed to invade lakes and ponds that are many miles apart?

5) Because Daphnia lumholtzi is very small and often looks very similar to other species, what tools would a scientist need to identify this species?

6) List three ways that scientists could study the affect of Daphnia lumholtzi on local waterways.

7) What can local residents and fishermen do to protect their waterways from this invading species?

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