Teaching Physiology with Daphnia

By Brian Kloepfer
Education Resource Manager

Daphnia, also known as the water flea, is a common microcrustacean inhabiting freshwater ponds and lakes of North America and Europe. Useful for demonstrating many physiological phenomena and easily maintained in culture, Daphnia is an ideal animal for experimentation.

Biology

Species of Daphnia range in size from 0.5 to 5.0 mm in length. The Daphnia’s transparent bivalve carapace encloses the body except for the head and antennae. A large, conspicuous compound eye located in the head is sensitive to change in light quality, quantity, and polarization. The ocellus, a smaller, simpler eye located close to the compound eye, is sensitive to ultraviolet light. The light-influenced movement of Daphnia through a water column is a highly interesting aspect of its activities. The water flea is characterized by its “jerky” method of propulsion, which occurs because the large antennae are used as oars, causing the body to “jump forward” as the antennae are stroked.

Daphnia are filter feeders. The setose thoracic legs act as sieves for filtering algae, bacteria, and small particles of debris from the water. Daphnia transfer food to the mouth and grind it with mandibles before it moves through the gut for digestion. Food retention is from ½ to 3 hr.

As in all arthropods, Daphnia growth occurs immediately following molting. Some water fleas can live at least 100 days under optimum conditions. Preadult instars often molt once a day, while adults molt every 2 or 3 days. Daphnia become reproductively mature in the 3rd to 6th instar (depending on the species), and under favorable conditions produce broods of 4 to 65 young just prior to every molt.

Reproduction is parthenogenetic, giving rise to entirely female populations until environmental stress (such as overcrowding, lack of food, or temperature changes) occurs. Then males are produced and sexual reproduction results in 2 “resting” eggs. These sexual eggs reside in a single darkly pigmented, saddle-enclosed case (ephippium) that is highly resistant to adverse environmental conditions. When conditions again are favorable, these ephippial eggs hatch into females that then reproduce parthenogenetically.

Below are instructions for culturing Daphnia and several experiments using Daphnia. You can observe the heart rate and the effects of various stimuli on heart rate, the feeding process, and hemoglobin synthesis.

Culturing Daphnia

To successfully culture Daphnia you’ll need a suitable container of clean, aerated water kept at an appropriate temperature; food; and a controlled light level.

Materials

• Glass Aquarium
• Glass Plate (large enough to cover the aquarium)
• Filtered Pond Water or Conditioned Tap Water
• Air Compressor (or other means of aerating water)
• Daphnia
• Erlenmeyer Flask (optional)
• Dark Cover (large enough to cover the aquarium, optional)
• Aquarium Heater (optional)

Procedure

1. Fill a clean glass container, such as an aquarium, with filtered pond water or conditioned tap water (water allowed to sit for several days or chemically treated to eliminate chlorine). Add water when the level drops.
2. Cover the container with a glass plate to keep out dust.
3. Gently aerate the container using an air compressor and air line tubing to bubble the air through the water. Note: If the air is supplied by a central compressor, pass the air through an Erlenmeyer flask stuffed with cotton before bubbling it into the aquarium. This will remove any oil or dirt in the air lines. Do not use a standard aquarium filtering system because it removes small particles the Daphnia feed upon.
4. Program the aquarium heater (if one is used) to maintain the water at a temperature of 19 to 23° C. Temperature variation greatly affects the metabolism of Daphnia.
5. Place the aquarium in low-intensity light. If the aquarium is in “average” room lighting, a dark cover placed over the glass plate will sufficiently reduce the light intensity.

Food

Feed the Daphnia 2 or 3 times a week. In general, if a culture remains turbid for longer than 8 hours, it was overfed. Use any of the following foods:

• Motile algae.
• Yeast suspension. Note: Do not foul the tank with too much yeast. Adding about 5 cm3 every 3 days to a 40-L (10-gal) aquarium should be sufficient.
• Bacteria. Induce by adding any of the following:
1. Freshly boiled egg yolk
2. Filtrate of trout chow (ground and passed through fine bolting cloth)
3. Infusion of a boiled lettuce leaf
4. Infusion of “stable tea” (soil and manure)
5. Infusion of soil and soybean meal, soil, and cottonseed meal, or wheat grain

Note: Do not remove the debris that accumulates on the aquarium bottom because it contains microorganisms upon which the Daphnia feed.

Cultures kept and fed in this manner should thrive indefinitely. However, eventually with overcrowding or the accumulation of metabolites, Daphnia begin to produce ephippial eggs. The population then drastically decreases. When this happens, either drain ½ the tank and refill with fresh water or remove the animals with a fine net, clean the aquarium, and begin again. In either case, discard about ½ of the animals.

Transferring and Observing

Transfer cladocerans, such as Daphnia, using a wide-bore pipet to avoid damaging delicate appendages. Introduce them under the water’s surface to prevent air bubbles from catching beneath the carapace, thus trapping the animals on the surface.

Study Daphnia morphology by simply placing a drop of water containing a single daphnid on a slide and withdrawing the water with a fine pipet. Leave barely enough water to cover the animal.

It is simple to restrain Daphnia yet allow free movement of the appendages. Place a small amount of Vaseline® in the bottom of a watch glass and cover with water. Add a Daphnia to the watch glass. Using a pipet, carefully withdraw the water so the animal falls on its side and adheres to the Vaseline®. Cover the entire preparation with water. The Vaseline® holds the Daphnia firmly in place, thus facilitating observation of appendages and internal organs.

Experiments

Prepare a Vaseline® wet mount and observe it under a stereomicroscope (the scanning lens of a compound microscope also can be used). Feed the Daphnia a suspension of Congo-red-stained yeast, prepared as follows:

Materials

• 3 g Compressed Yeast
• 30 mg Congo Red Stain
• 10 mL Dechlorinated Water
• Dissecting Needle
• Heat-Proof Container
• Heat Source
• Daphnia
• pH Measuring Strips (optional)

Procedure

1. Mix together 3 g of compressed yeast, 30 mg of Congo red stain, and 10 mL of water.
2. Boil gently for 10 min. then cool. (To speed the process, extract the mixture before cooling. The amount extracted is so small it cools quickly.)
3. Dip a dissecting needle into the Congo-red-stained yeast and then stir gently above the head of the Daphnia. This should add the proper amount of food. Note: Do not overfeed. Excess yeast will obscure observation of the current flow.
• Observe filter feeding in the same manner. Students may take notes on what occurs and sketch the paths of water currents caused by movement of the appendages.
• Observe movement of the food through the gut using this method.
• Monitor pH changes by the color changes of the Congo red. This stain is bright orange-red above pH 5.0, but is blue at pH 3.0.
• Observe a more graded series of changes in pH by adding daphnids to a dilute aqueous solution of neutral red stain (0.02% or less) for about 15 min. Remove the organisms and observe. The dye in the digestive tube is the color red below pH 6.0, rose at pH 7.0, orange at pH 8.0, and yellow at pH 9.0.

Heartbeat

Prepare a Vaseline® wet mount of Daphnia. Monitor the rapidly beating myogenic heart—located in the dorsal region of the body behind the eye—and measure various effects on biological function. Use a large container such as a culture dish instead of a watch glass.

Materials

• Stopwatch (or a watch with a second hand)
• Dechlorinated Water
• Adrenalin (0.01%)
• Thyroxin
• Heat Source
• Wide-Bore Pipet
• Daphnia
• Slide
• Vaseline®
• Nicotine (optional)
• Caffeine (optional)
• Pesticides (your choice; optional)
• Lamp Bulb (with power source; optional)

Procedure

1. Study the effects of readily observed temperature changes.
• First, determine the “normal” heartbeat of Daphnia in its culture water. Using a stopwatch (or a watch with a second hand), determine the number of heartbeats in 15 sec.
• Withdraw the fluid and add dechlorinated water between 0° and 5° C.
• Allow the water to warm to room temperature.
• Count the heartbeat at every 2° or 3° rise in temperature. Note: Higher temperatures can be reached be adding small amounts of hot dechlorinated water or by heating the preparation. A lamp bulb held close to the container warms the water slowly.
• Make several counts at each temperature and calculate the average number of beats per 15 sec.
• Plot the mean rate of heartbeats versus the temperature.
2. Study the effects of hormones such as adrenalin (use about 0.01%) and thyroxin. Prepare Vaseline® wet mounts and measure the rate of heartbeats. Plot the number of heartbeats versus time.
3. Study the effects of drugs (such as nicotine and caffeine) and pesticides in the same manner.

Hemoglobin Synthesis

When Daphnia are maintained in a well-aerated culture (e.g., a shallow tank), they remain pale in color. If grown in cultures where oxygen concentration is low, they synthesize additional hemoglobin and become distinctly red in color. The major factors influencing the formation of hemoglobin are oxygen availability, temperature, available iron, and food.

For demonstration of hemoglobin synthesis resulting from low oxygen concentration, cover the aquarium water surface with plastic film wrap. Oxygen concentration will slowly drop. Observe daily for the appearance of hemoglobin in the Daphnia. After 2 or 3 weeks, remove the plastic wrap. Note the time required for the gradual reduction of the hemoglobin. Use a digital camera or camera on a phone to document the daily change over the weeks.

Carolina carries several species of Daphnia as well as equipment to support your Daphnia cultures. Visit www.carolina.com for a complete listing of all our products as well as free care and handling guides for a large variety of living organisms commonly used in the classroom.