3D Tracking of Anti-Predator Behaviour in Guppies by Ani Vanesyan A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Ani Vanesyan (2012) 3D TRACKING OF ANTI-PREDATOR BEHAVIOURS IN GUPPIES Ani Vanesyan Masters of Science Department of Ecology and Evolutionary Biology University of Toronto 2012 Abstract Guppies from high- and low-predation habitats are well known for the differences in their anti- predator behaviours. However, little has been reported on the differences between social versus asocial stimulus responses. In this study, we conducted a detailed analysis of shoaling and other anti-predator behaviours of guppies from two populations, in pairs and as single fish, in three dimensions. Using custom programs in MATLAB, we quantify the behavioral responses before shoaling and during its dissipation. Our 3D reconstruction allowed us to track the inter-fish distance, velocity and orientation of both fish. Our results demonstrate a positive correlation between the relative orientation of the fish and the interfish distance, for pairs from the high- predation population. We also report that the anti-predator behaviour of guppies is comprised of the following sequence: freezing, darting/skittering, and recovery to pre-stimulus swimming behaviour. Upon repeated encounters with the stimulus, a reduced shoaling and anti-predator response was observed. ii Acknowledgements Firstly, I would like to sincerely thank my supervisors Dr. Helen Rodd and Dr. William Ryu, for their guidance and support throughout my academic journey. Helen, thank you for your invaluable mentorship throughout my university career, for introducing me to the world of research, for coaching me every step of the way and for inspiring me to pursue graduate studies in EEB. Will, I owe you my interest and my understanding of the world of biophysics. Thank you for continuously pushing me to extend my knowledge in this field, to acquire new skills and to think critically and independently. I feel very fortunate to have had two coordinators from different backgrounds to learn from and to be guided by, from the point of decision making to the point of thesis completion. In addition, I thank my supervisory committee members Dr. Marla Sokolowski and Dr. Debra McLennan and my examining committee, Ben Gilbert and Shannon McCauley, for their comments and suggestions. I would like to also thank my lab members from both Ryu lab and Rodd lab, for being there on all of my presentations and for always being ready to provide useful suggestions and advice. I thank the Luys Foundation, for their support and their shared vision and faith in Armenian students worldwide. Finally, I would like to thank my family and friends for their continuous support. Thank you for always encouraging me and for knowing that I will get where I am now, before I even did. You always told me that everything will be worth it at the end, and you were right. Vardges Avetisyan, thank you for always showing such an enthusiasm about my work: you have motivated me more than you know. iii Table of Contents Acknowledgements ........................................................................................................................ iii List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ....................................................................................................................... viii 3D Tracking of Anti-Predator Behaviours in Guppies ................................................................... 1 INTRODUCTION .......................................................................................................................... 1 MATERIALS AND METHODS .................................................................................................... 3 Experimental Fish ....................................................................................................................... 3 Analysis of Schooling Behaviour ................................................................................................ 4 Statistical Analyses ..................................................................................................................... 7 RESULTS ....................................................................................................................................... 8 Interfish Distance ........................................................................................................................ 8 Freezing Duration ...................................................................................................................... 10 Velocity Recovery ..................................................................................................................... 11 Variance of Speed ..................................................................................................................... 11 DISCUSSION ............................................................................................................................... 12 Initial Shoaling Response .......................................................................................................... 12 Anti-Predator Behaviours .......................................................................................................... 14 Decline in Responsivenes to the Stimulus ................................................................................ 15 CONCLUSIONS........................................................................................................................... 17 References ..................................................................................................................................... 19 iv Tables ............................................................................................................................................ 24 Figures........................................................................................................................................... 27 Appendices .................................................................................................................................... 33 APPENDIX A ........................................................................................................................... 33 APPENDIX B ........................................................................................................................... 37 v List of Tables Table 1. Separate repeated-measures ANOVAs, by Trial, on the transformed (square root) ratio of the interfish distance after the stimulus to the distance before the stimulus, for all three days. Table 2. Repeated-measures ANOVA on the average time spent frozen (in seconds) after the stimulus, for the first trials of the three days. Table 3. Results of a repeated-measures ANOVA on variance of speed before and after the stimulus. vi List of Figures Figure 1. Repeated-measures ANOVA, by Trial, on the ratios of (Da/Db) (interfish distance after the stimulus to the distance before stimulus), for the first, second and third trials, across the three days, for high- and low-predation population fish. Figure 2. Correlation of interfish distance with orientation of the fish during the first second after the stimulus for high-predation population and low-predation population guppies. Figure 3. Results of a repeated-measures ANOVA on the transformed (sqrt+1) freezing time of fish in the first tests on days 1,2 and 3 for the high- and low-predation population guppies. Figure 4. Repeated-measures ANOVA on the transformed (sqrt+1) freezing time of the fish in the first trials on Days 1, 2 and 3 for guppies in pairs and as singletons. Figure 5. Repeated-measures ANOVA by Population type, on the transformed (ln) velocity recovery time of fish in the first trials on Days 1, 2 and 3 for pairs of fish from high- and low-predation populations. Figure 6. Repeated-measures ANOVA by Population type on the Speed Variance data for Day1 of the high- and low- predation population guppies. Figure 7. Locating and tracking fish within frames, in 3D. Figure 8. 3D plots of the trajectories of a pair of fish, from high predation populations, before and after the stimulus. vii List of Appendices Appendix A. Image acquisition. Image Analysis. Future Directions. Appendix B. MATLAB Algorithms viii 3D Tracking of Anti-Predator Behaviours in Guppies INTRODUCTION Group formation, which occurs in many animal taxa, can enhance the fitness of the members in a number of ways including increased foraging success (e.g., Breder, 1959; Foster et al., 2001; Krause & Ruxton, 2002), enhanced predator evasion and, in some cases, reduced hydro- or aerodynamic costs (e.g., Svedsen et al., 2003; Viscido & Wethey, 2002). Social grouping in fish is termed shoaling, of which schooling is a subset. Schooling is the synchronized swimming of all members in the group, characterized by a uniform separation among individuals, a specific velocity and matching orientation of all the members in the group (Pitcher, 1983). Both the lateral system and vision can be employed during the formation and maintenance of fish schools (Faucher, 2010). Until