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Laboratory 8 Agonistic Behavior in Betta splendens

The star of today's laboratory show is Betta splendens, sometimes known as the Siamese Fighting Fish. Although it is important for you to know this species' phylogeny, the focus of today's exercise will be your own experimental manipulation of external stimuli that affect the courtship and/or aggressive physical displays of this interesting animal.

I. Phylogeny and Evolutionary Relationships Fish, like all vertebrates, are deuterostome chordates that share several synapomorphies with other deuterostome taxa. Like protostomes (e.g., Annelida, Mollusca, Arthropoda), deuterostomes are bilaterally symmetrical, triploblastic coelomates. But unlike the protostomes, in deuterostomes:

1. The blastopore becomes the anus (a secondary opening becomes the mouth) 2. The coelom is derived via enterocoely 3. Cleavage is radial and indeterminate 4. The nervous system is primitively dorsal 5. The circulatory system is primitively ventral

The deuterostome phyla with which you are probably most familiar are Echinodermata (e.g., starfish, sea urchins, sea cucumbers) and Chordata. Chordata, includes three subphyla, Urochordata (tunicates), Cephalochordata (lancelets) and Vertebrata (vertebrates). Chordate synapomorphies include:

1. pharyngeal gill slits present during some stage of development 2. dorsal, cartilaginous notochord at some stage of development 3. dorsal, hollow nerve cord 4. having an endostyle (Urochordata, Cephalochordata) or homologous thyroid gland (Vertebrata) at some stage of the life cycle 5. tail posterior to the anus (primitively muscular and used for locomotion) 6. segmentally arranged muscle bundles (sarcomeres)

Synapomorphies that set vertebrates apart from all other Chordates include:

1. skeleton constructed of bone (at least primitively) 2. a bony cranium encasing the brain 3. articulated vertebral column

The vertebrates may be the most recognizable of all animals, including such familiar forms as fish (a vast and diverse assemblage), frogs, lizards, snakes, birds, and mammals. Vertebrates, with nervous systems and brains far more complex than other animals, often exhibit complex behaviors related to foraging, territorial defense, and reproduction. In today's lab, you will examine reproduction-related behaviors of a representative vertebrate, Betta* splendens (Osteichthyes).

*Note that the genus name is pronounced “beh’-tuh” (hear the pronunciation at http://www.thefreedictionary.com/betta), not “bay’-tuh” (compare with the pronunciation of the Greek

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letter β here: http://www.thefreedictionary.com/beta) Bet is Latin for “beet”; the taxonomist describing the fish may have been referring to the fish’s bright color).

Wild Betta splendens are colorful and sexually dimorphic (i.e., males and females look different from one another), with males being more brightly colored than females (Figure BB-1). Because of their attractive colors and interesting behaviors, this species has been subject to many years of artificial selection (particularly the males) for mutant color morphs and very long fins and tails. Despite the domestic variety's showier appearance (Figure BB-2), its courtship and other intraspecific displays have remained relatively unchanged. Both wild and domestic Betta splendens will react strongly and aggressively to the appearance of another fish of the same species.

Figure BB-1. Wild type Betta splendens male (left) and female (right). The male is fanning his fins and tail in the beginnings of an agonistic (aggressive) display.

Figure BB-2. Artificially selected Betta splendens males showing various mutant color morphs and fin morphologies. The fish in the upper lefthand corner and

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lower righthand corner are not displaying. All other fish shown are engaging in agonistic displays of varying intensity. II. Natural History of Betta splendens Betta splendens (Family Belontiidae) commonly known as the "Siamese Fighting Fish" is native to tropical Southeast Asia including the northern Malay Peninsula, central and eastern Thailand, Kampuchea, and southern Vietnam (Witte and Schmidt 1992). They live and breed in rice paddies, shallow ponds, and slow-moving streams, and are known for their ability to survive in rather fetid, oxygen-poor water by gulping air from the surface, when necessary. Adult males reach a total length of about 7cm, and females are slightly smaller. Cultivated Bettas have been artificially selected for many decades so that the "domesticated" males are characterized by colorful pigmentation and long, showy fins. Females are slightly smaller, less brightly colored, and have shorter fins than males. Bettas thrive best in water that is close to neutral in pH (6.8 – 7.4), at a cozy tropical temperature (75-86oF; 24-30oC). Although they can survive in oxygen-depleted water by breathing from the surface, like all fish, they suffer less stress when their water is clean and rich in oxygen that they can breathe by drawing water into the mouth and across the gills by opening of the bony opercula that cover the gills. Bettas are popular as aquarium fish because of their spectacular colors, and because they are relatively easy to keep and breed. They appear to thrive comfortably (long term) in a volume as small as 3 gallons of water (Although Bettas can survive in the very small bowls (16 oz.) in which one often sees them displayed, it's arguable that such a small, unenriched environment is optimal for long term survival and health.), and when living in an appropriate, stress-free environment, will breed. Males build a nest of mucus-coated bubbles on the surface of the water, and as the eggs laid by the female hatch, he will carry each hatchling to the nest in his mouth and spit it into the nest. Hatchlings grow in relative safety in their bubble environment, guarded by the male. Every experienced aquarium hobbyist knows that only one male Betta can occupy an aquarium at a time. The males are extremely aggressive towards males of their own species (though they are total wimps when attacked by a different species, and tend to get their beautiful fins picked to shreds in a "community" aquarium), and will readily fight to complete exhaustion. When fighting, males will nip at each others fins until one of them is too tired to continue, though this usually takes long enough so that the fins of both combatants resemble torn up rags by the time one of the fish concedes defeat by retreating.

III. Agonistic Behavior in Betta splendens Ethology is the study of animal behavior. In the terminology of this branch of biology, a releaser is a specific factor that triggers a fixed action pattern, an innate, stereotyped behavior. For example, the mere sight of an egg outside its nest will trigger innate egg retrieval behavior on the part of a nesting sea gull, whether or not that egg is its own.

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Agonistic (from the Greek agonistes, meaning "champion") behavior in animals is defined as that exhibited during a contest, combat, escape, attack, or appeasement episode between two animals. The term is often used to describe the display behaviors exhibited by male animals when they compete for mating opportunities with females. Male Bettas may fight to claim territory, or to protect their eggs or offspring from rival males. But physical combat is invariably preceded by a display sometimes called "flaring." When stimulated by the sight of a rival male (the releaser), a male Betta will exhibit several types of genetically determined aggressive movements (fixed action patterns). The fish will spread his fins, shudder his body, extend his gill opercula and membranes, and generally appear much larger than his resting size. Bettas do not recognize themselves in a mirror, and will display to their reflections as aggressively as they might to another male.

IV. Betta splendens External Anatomy In order to accurately describe and record data on behavior, you'll need to know the proper names of the body parts your fish use to communicate with their conspecifics (i.e., members of the same species). Refer to Figure BB-1 for an overview of the general external anatomy of Betta splendens (which is pretty much the same as that of any other bony fish, with superficial modifications). Note that male and female Bettas use all their fins in their displays, as well as their opercula and the fleshy, colorful extensions beneath the opercula. Your team will need to decide in advance (1) what anatomical features you will monitor during the fish display behaviors, (2) how you will quantify their use, and (3) what statistical test is most appropriate for analysis of your data set, once your experiment is complete.

Figure BB-3. External anatomy of Betta splendens.

Male fighting fish vary in their aggressive response and you will attain the best results will be from the more aggressive males. The sight of its own reflection in a mirror is enough to stimulate agonistic flaring in a sufficiently aggressive male (they do not recognize themselves, and think they see another male). To assess the relative

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aggressiveness of the animals at your station, and to observe the behaviors associated with agonistic display, slowly and carefully place a small mirror against the flat side of the bowl. Note: 1. Which fins or other structures does the fish use for display? 2. What position does the fish assume with respect to its reflection (head-on? sidelong?) 3. What other movements or behaviors does the fish employ in his display? CAUTION: If the fish is over-stimulated, habituation to the stimulus will occur. This means that the animal has become accustomed to the stimulus, and will not respond as strongly (or respond at all) in subsequent trials. When you are not measuring your fish's display, be sure he cannot see his reflection or other males. Keep the fish as relaxed as possible in between trials, and when you are completely finished with your experiment, be sure to replace the visual barriers on the fish bowls. V. The Effect of Different Releasers on the Agonistic Display: Experimental Design. Each station for a four-member team includes two male Bettas, and one female, each in its own plastic drum fish bowl. DO NOT EVER PLACE TWO FISH IN THE SAME BOWL. SUCH IRRESPONSIBLE BEHAVIOR WILL RESULT IN YOUR BEING DISMISSED FROM THE LAB AND YOUR RECEIVING A ZERO FOR THIS LAB EXERCISE. FURTHER DISCIPLINARY ACTION IS ALSO POSSIBLE. Also available at the front desk are materials such as paper cutout models ("puppents") of male and female Bettas of different colors, different sizes, and different positions. You can use these to pose questions about Betta agonistic behavior, devise null and alternative hypotheses, and then design and execute a rigorous experiment to test your ideas. Examine the puppets available, and use them to ask questions and formulate hypotheses.

Consider some of the following questions, and ask some of your own. 1. What releaser elicits the strongest response in your subject? 2. What fixed action pattern is used in response to particular releasers? 3. What is the possible evolutionary significance of the responses of the fish to various releasers?

Work in teams of four. Take the first 20 minutes of lab to consider Betta splendens natural history (feel free to do additional research before you come to lab!), and design an interesting, relevant experiment with the materials at hand. (If you require something else, please ask the TA. We might have it available.) Once your team has decided on its course of action, complete the four outline items on the following page, and draft a brief description of your experimental methods.

Hypothesis:

Prediction:

Possible Outcome #1:

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Explanation: Possible Outcome #2: Explanation: Possible Outcome #3: Explanation: I your team plans to collect data to be analyzed statistically, remember that you must frame your question via falsifiable null and alternative hypotheses, as appropriate.

Use the space below to describe the methods you will use. Include the type of statistical test you will use to analyze your data. What parameter will you measure? How many replicates? If you have treatment and control groups, what are they? Treatment:

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Control: What statistical test will you employ to analyze your results, and why is it appropriate? When all teams have decided on their experiments and finished the outline of the design, each team should take five minutes to briefly explain to their colleagues/classmates what they intend to do, and why. Critique from the TA and from classmates should be used to fine tune the experiment and make final changes before the experiment is actually performed. General Instructions for a Successful Experiment

1. Some individual fish are particularly sensitive or aggressive, and these may be stimulated even by the sight of brightly colored or patterned clothing. Avoid wearing such clothing to lab this week. Pale-colored clothing is the least likely to interfere with fish behavior.

2. Position your fish where it cannot see neighboring animals until you are ready to begin your experiment. Keep the visual barriers in place unless you are actually making observations of fish behavior.

3. Avoid abrupt movements when near the fish, and speak quietly. Do not tap on the side of the fish bowl.

4. When recording the positions and movements of your Betta's display, note that the male will employ most of its fins, its gill opercula, and the associated branchiostegal membrane. A particularly energetic male may bend his body in tight angles. Note the orientation of the fish to its stimulus and record any changes in the coloration of your fish (for example, watch for color to fade or become brighter, or for color streaks to appear on various areas of the body.

5. Be sure to record the length of time of each behavior you are recording, as well as its subjective aspects (for example, you might rate the strength of the display ("-" for weak, "+" for medium, "++" for strong—or variations on that theme, perhaps using numbers to rank degree of energy shown by the fish.) Do not stimulate the fish for longer than one minute for each trial, as longer trials may result in habituation to the stimulus.

6. Wait least 5 minutes between trials, allowing the fish to calm down completely. Between trials, be sure to block your subject's view of other fish, and avoid fast movements or loud noises. (Yes, they can hear them!)

7. It may be helpful to record the sequence of movements the fish uses in a full display.

8. Replicate each trial at least 3 times. Note any differences between replicates. What might cause such differences as time goes on?

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9. Paper fish models will also elicit a response from males, but because they are stationery, they will not elicit as strong a response. However, the subject will also not habituate as quickly to a static model as to a mirror (why might this be the case?)

10. When using a paper model, move it slowly up to the subject and then wave it slightly to attract the subject's attention. Try to use similar technique and movement in each trial, to avoid introducing human error into your experiment.

11. Obviously, male Bettas will respond strongly to the sight of another member of its own species, whether male or female. You may wish to measure the responses of your fish to same sex or different sex stimulus, and quantify the differences between the displays. The question, hypotheses and predictions are up to you!

12. Determining the relative aggressiveness of different animals can be done by comparing displays recorded by different teams with different animals. You may use Table BB-1 below to record your data any way you wish, or make a different table of your own design. Be sure to note in the table legend (which you will provide) what you are measuring (nonparametric, such as "yes" or "no", or parametric, such as "seconds" or other discrete or continuous measurement) as well as their values. Table BB.1. Fish ID/trial #

Dorsal fin

Caudal fin

Ventral fin

Pelvic fin

Branchio- stegal membrane

Orientation Color change?

VI. Data Analysis and Discussion Your team's presentation on this experiment is due next week. When analyzing your results, consider the following thought questions, and incorporate a discussion of your ideas about them in your discussion, as appropriate.

1. What are the possible functions of agonistic display in male Betta splendens? 2. Why do males bother with display? Why not just launch into battle? 3. What might be the significance of the display between two males if there are females observing the contest? 4. How might certain aspects of the display behavior been adaptive in the wild

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ancestors of these fish? 5. Wild Betta splendens are not as brightly colored, and do not have fins as long and showy as the domestic variety you are working with today. How might artificial selection have affected the behavior of these fish? What do you think you can say about your results, in terms of being able to apply them to wild populations? 6. Natural ecosystems are now being inundated with various pesticides and other chemicals that mimic sex hormones in the body of various vertebrates. What might be the effect of such chemicals on the behavior of wild populations of animals that use agonistic display as part of mating ritual? How might you design an experiment to test your ideas about this? 7. What other environmental factors might affect the agonistic display of Betta splendens? Of what evolutionary or ecological significance are these factors? Feel free to cite outside sources, and go beyond the lab manual for ideas about your experiment and observations. But don't forget to cite all your sources properly and include your references at the end of your presentation, or in the slide in which you are citing that source. VII. Ethical Treatment of Experimental Subjects Be kind to your fish, and we will be kind to you. Any student witnessed abusing animals in the laboratory will be immediately dismissed from the lab, receive a ZERO on the lab and the presentation, and be subject to possible additional disciplinary action. Animals of any species used in experimental studies should always be treated with respect, and given the proper care and maintenance at all times.

Adopt-a-Fish! The fish being used in today’s laboratory will be available for adoption after all the lab experiments have been completed at the end of the week. If you would like to adopt a Betta splendens of your very own, you will need the following:

1. an appropriate vessel (minimum 2.5 gallons) 2. good-quality food (Spectrum brand is the best) 3. dechlorination solution (essential! Tapwater kills fish!) 4. floating live plants (provides cover, comfort, and oxygen) 5. a bit of gravel for the bottom of the bowl 6. a small vessel (e.g., a plastic bowl with a lid) to bring your fish home

Note that females can be highly aggressive, and have been known to kill males if the two are in an aquarium that’s too small. Females will also bully one another if they do not have sufficient space. So unless you have a very large, community aquarium with peaceful, non-aggressive fish, you must have a separate vessel and supplies for each Betta. Betta splendens is beautiful and relatively easy to keep, but this species does require specific care. Please read about care information before you adopt:

http://www.bettatalk.com/betta_care.htm

http://care.betta-fish.com.ar/

http://www.bettacentral.com/care.htm

Please contact Dr. Dana Krempels (dana@miami.edu) for adopt-a-fish information.