1. Methods: For our study, groups of Zebrafish were contained within 10 gallon tanks. They filmed for 3 minutes to establish a baseline behavior, then were exposed to 20 ml of distilled water as a control, then after a 3 minute waiting period were exposed to 20 ml of treatment solution. The fish videos were then analyzed to examine frequency of feeding behaviors and if the fish exhibited the classical alarm response behavior: freezing, schooling and rapid movement. Alarm Substance solutions were extracted from the skin of zebrafish. Saturated solutions from Chondroitin powder, which we obtained from shark or cow cartilage, were prepared. Cell cultures were grown from Creek Chub skin samples. Behavioral Attributes: Our analysis was focused on finding similarities between the videos of fish exposed to Chondroitin and those exposed to alarm cells. we quantified behavior according to three simple criteria: Fish movement (number of gridlines crossed in a 1 minute period after exposure), Surface use (number of fish at the top level of the tank) recorded every 10 seconds, Presence of schooling (presence of 3 or more fish within a single grid) recorded every 10 seconds. Summary and Conclusions 1.Zebrafish displayed stereotypical anti-predatory behavior when exposed to conspecific skin extracts. When exposed to conspecific skin extracts, experimental Zebrafish spent a greater amount of time schooling and darting randomly about the tanks. Use of the upper water column and feeding events also decreased significantly relative to control conditions. These findings indicate that we have an acceptable behavioral assay for Zebrafish. 2.Chondroitin induced changes in Zebrafish behavior, but our results do not support the hypothesis that Chondroitin is the alarm substance. Although Zebrafish exposed to Chondroitin exhibited some behaviors consistent with an alarm response, the most conspicuous behaviors in the Chondroitin trials – increased use of the upper water column and increases in the number of feeding strikes – are suggestive of feeding, and not anti-predatory, behaviors in our test fish. These findings compared to those of Mathuru et. al.indicates that Chondroitin serves a more complex role in the alarm response than originally considered, and further study is needed to rectify these contradicting findings. 3.Cultured Cells from Creek Chub induced behaviors in Zebrafish comparable to those found for Chondroitin. Our goal in using cultured Creek Chub cells was to evaluate the suitability of pure epidermal skin cells for use in future experiments. The results of these experiments demonstrated that these cultured cells contain chemical components responsible for the alarm response, along with other chemical signals. Made evident by the partial alarm response seen in the Zebrafish exposed to them. Works Cited: Mathuru et. al., Chondroitin Fragments Are Odorants that Trigger Fear Behavior in Fish, Current Biology (2012), doi:10.1016/j.cub.2012.061. Speedie, Natasha, and Robert Gerlai. "Alarm Substance Induced Behavioral Responses in Zebrafish (Danio Rerio)." Behavioral Brain Research: 168-77. NIH Public Access. Web. 16 Apr. 2015. Whitlock, Kathleen E. "The Sense of Scents: Olfactory Behaviors in the Zebrafish." Zebrafish 3.2 (2206): 203-13. Print. Acknowledgements: Special Thanks to Dr. Winnifred Bryant for assistance in cell culture growth. Special Thanks to Holly Emebke for assistance in data collection. Results: Our Test Animal, The Zebrafish, Danio rerio The Zebrafish recently became a popular model organism for medical tests, since they have qualities that make them a desirable testing subject. Zebrafish are small, easy to care for and breed readily in captivity, they have a relatively short generation time, and live comfortably alone as well as in both small and large groups. Additionally, they exhibit a number of traits that hold medical potential, such as the fear response (making them a good model of stress) and cellular regeneration. Finally, the Zebrafish has a completely documented genome, and are bred in stocks. For our research they were an ideal candidate due to these combined traits, which made them a good subject for our work. Behavioral Responses of Zebrafish to Selected Chemical Constituents of the Skin By: Philip Schadegg, Cynthia Koenigsberg and Claudia Seravalli. Faculty Mentor: Dr. David Lonzarich Chondroitin Mathuru et al (2012) discovered, through extract fractioning, that alarm substance was a mixture containing Chondroitin, since only fractions that contained chondroitin elicited a fear response in their tests. It was also noted that Chondroitin alone does trigger a fear response in fish. Therefore, the hypothesis was put foreword that Chondroitin is the identity of alarm substance. This study was undertaken to verify that the fear responses in fish exposed to Chondroitin was similar to the fear response to those exposed to alarm substance. In order to potentially validate the claim that Chondroitin was a potential identity for alarm substance. Our evaluation of chondroitin is based on evidence by Mathuru et. al. (2012, thus far not replicated, that it is the alarm response stimulus source. Alarm Substance Karl Von Frish discovered in 1938 that some fishes produce a chemical in the skin that elicited a fear response in other fish. When a related fish is injured, the wound releases chemicals that triggers an olfactory response in non-injured fish, causing them to behave erratically, fleeing the source of the injury, even though they may not have seen the source of danger. This “alarm response” has drawn the attention of the biomedical research community for its potential value in understanding the neural pathways associated with fear and anxiety in humans. The alarm response has also drawn the attention of evolutionary biologist, the evolutionary basis for this behavior has been discussed thoroughly in literature, but the true identity of the chemical agents responsible are still unknown. Fish exhibit the fear response to the skin extract of any fish which exhibits the fear response, but what part or parts of this extract actually achieve the fear response remains unconfirmed. Our study aim was to develop a behavioral assay to characterize the alarm response in Zebrafish and to describe their behavior following exposure to chondroitin, and chemical components of cultured skin cells. In developing a behavioral assay our objective was to evaluate behavioral responses to the different treatments. Fig 1: Effects of Test Substances on Feeding Behavior. Zebrafish showed a sharp increase in feeding behavior during the Chondroitin and Cultured cell trials. By contrast, in the Alarm substance trials, feeding behavior was relatively uncommon. Fig 2: Effects of Test Substances on Fish Distribution. Use of the upper water column by Zebrafish increased in Chondroitin and Cultured cell trials, but decreased in Alarm substance trials. Most Zebrafish moved to (or remained on) the bottom of the tanks following exposure to the Alarm substance. Fig 3: Zebrafish showed a nearly two-fold increase in movement in Alarm substance trials. There also was a small increase in movement in the Chondroitin and Cultured cell trials. Fig 4: Effects of Test Substances on Schooling Behavior. Zebrafish spent more time schooling in Alarm substance trials. There also was a slight, though non-significant increase in schooling in the Chondroitin and Cultured cell trials. Upper Section Middle Section Lower Section Background