Fundamental developments in larval zebrafish (Danio rerio) olfactory mediated behaviour by Angela Lee Shamchuk A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology, Development and Cell Biology Department of Biological Sciences University of Alberta © Angela Lee Shamchuk, 2015 Abstract For many fish, their olfactory system allows for the critical detection of environmental chemical cues indicative of food, predators, kin and mates. Through odourant recognition, fish are able to react appropriately to their environment and elicit behaviours necessary for survival. The central objective of this thesis was to examine odourant-evoked behaviours of larval zebrafish (Danio rerio), who due to their age are not only ecologically vulnerable, but are undergoing rapid olfactory tissue development. Prior to these studies, few larval odourant-evoked behavioural responses had been investigated. Consequently, a portion of this thesis was dedicated to building the foundation for future studies. Two novel apparatuses were constructed: a flow-through system to observe changes in activity and an avoidance-attraction trough to test whether odourants affect area occupancy. Through validation of this equipment, behavioural responses to embryo extract (avoidance) and hypoxanthine-3-N-oxide (alarm) were characterized for the first time in 7 day-old larvae. To date, these are the earliest observed behavioural responses to these odourants in fish. Post-method validation, additional studies were conducted to identify 5, 6 and 7 day old larval responses to nucleobase compounds. While found to be behaviourally active, results indicated that nucleobase compound chemical structure, fish age and exposure naivety influenced occupancy behavioural responses. Overall, the work described in this thesis has expanded knowledge of nucleobase odourants, highlighted the importance of testing multiple behavioural metrics and established the much needed groundwork for future studies on larval olfactory mediated behaviours. ii Preface This thesis is original work by Angela L. Shamchuk and no part of this document has been previously published. Preliminary electrophysiology studies were conducted in collaboration with Brian Blunt. Synthesis and analysis of hypoxanthine-3-N-oxide was done in collaboration with Kirsten Tomlin of Dr. Jeff Stryker’s laboratory. Glass chambers described in Chapter 2 and tanks described in Chapter 3 were custom built by Jason Dibbs. Plexiglas avoidance-attraction troughs were custom built by Jeffrey Johnston. The research herein described was approved by the University of Alberta Animal Care and Use Committee: BioSciences, AUP #052 ‘Olfactory Responses in Fishes’. iii Dedication “Always the beautiful answer who asks a more beautiful question.” ― E.E. Cummings This thesis is dedicated to my friends and family for their unwavering support of my research. I am forever grateful to have had so many coaches in my corner. Thank you especially to my parents David and Jayne for their love and encouragement. Their confidence in me has always been inspiring. To Peter, I want to thank you for standing by me as we made yet another challenging climb. You’ve taught me what it means to persevere. The trail keeps going, but we can stop for a moment to enjoy the view. iv Acknowledgments This research would not have been possible were it not for the generous funding provided by both NSERC and DSM. Thank you to the members of my supervisory committee Drs.William Ted Allison and Declan Ali for lending their time and expertise towards my project. I am extremely grateful to my thesis supervisor Dr. Keith Tierney for his valued guidance and for ultimately affording me this opportunity for personal and academic growth. I must also acknowledge the numerous students that have worked with me on the material described in this document and on many other less fruitful ventures: Brian Blunt, Kirsten Tomlin, Patricia Leighton, Vlatka Vukojevic, Amy Wang, Anastasia Gasheva, Irtiza Oyon, Carlie Lewis and Elielma de Sousa. My sincerest gratitude is also extended to the Aquatics Facility Staff, notably Clarence Gerla, Aleah McCorry and Jesse Edgington for their unparalleled patience and support in helping me to establish our aquatics room. Finally, I greatly appreciate the equipment, favours and knowledge given to me by Jason Dibbs, Jeffrey Johnston, Troy Locke, Ben McDonald and Shelley Scott. This thesis is the culmination of the efforts and generosity of all of the phenomenal individuals listed here. v Table of contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Dedication ..................................................................................................................................... iv Acknowledgments ......................................................................................................................... v Table of contents .......................................................................................................................... vi List of tables................................................................................................................................ viii List of figures ................................................................................................................................ ix List of abbreviations .................................................................................................................. xiii Chapter 1: Introduction ............................................................................................................... 1 Significance of olfaction in fish .................................................................................................. 1 Fish olfactory system .................................................................................................................. 1 Larval zebrafish as the experimental model ................................................................................ 4 Odourant-evoked behavioural endpoints .................................................................................... 5 Thesis objectives ......................................................................................................................... 6 Chapter 2: Developing larval zebrafish methods for testing odourant-evoked behaviour .... 8 Introduction ................................................................................................................................. 8 Methodology background and rationale .................................................................................... 12 Materials and methods .............................................................................................................. 18 Results ....................................................................................................................................... 36 Discussion ................................................................................................................................. 51 Conclusion ................................................................................................................................. 66 Chapter 3: Zebrafish behavioural responses to nucleobase compounds ............................... 68 Introduction ............................................................................................................................... 68 Materials and methods .............................................................................................................. 74 vi Results ....................................................................................................................................... 80 Discussion ............................................................................................................................... 137 Conclusion ............................................................................................................................... 151 Chapter 4: General conclusion ................................................................................................ 153 Bibliography .............................................................................................................................. 156 Appendix A ................................................................................................................................ 168 Flow-through system troubleshooting and operation notes .................................................... 168 Appendix B ................................................................................................................................ 171 Appendix C ................................................................................................................................ 173 vii List of tables  Table 2.1. Odourants of interest for larval zebrafish behavioural investigation.  Table 3.1. Summary of larval nucleobase compound avoidance-attraction testing. viii List of figures  Figure 2.1. Avoidance-attraction apparatus experimental setup.  Figure 2.2. Sample avoidance-attraction dye trial and region of interest (ROI) overlay.  Figure 2.3. Flow-through apparatus schematic.  Figure 2.4. Flow-through apparatus trial procedure.  Figure 2.5. Sample time course images from a dye trial.  Figure 2.6. Dye calibration standard curve.  Figure 2.7. Absorbances of chamber outflow samples during dye trials.  Figure 2.8. Sample image of EthoVision XT arena set up and tracking.  Figure 2.9. Percent occupancy per region for 7 dpf larvae under control EM conditions.  Figure 2.10. Percent occupancy per region for 7 dpf larvae in odourant vs. EM trials.  Figure 2.11. Percent occupancy per region for 7 dpf larvae in food extract (FE) vs. embryo extract (EE) trials.  Figure 2.12. Average distance travelled (mm) during baseline activity pre odourant exposure.  Figure 2.13. Percent change from acclimated distance travelled during odourant exposure. ix  Figure 2.14. Total time spent in border zone during baseline activity pre odourant exposure.  Figure 2.15. Time spent in border zone during odourant exposure.  Figure 2.16. Maximum velocities (mm/s) during baseline activity pre odourant exposure.  Figure 2.17. Maximum velocities (mm/s) pre, during and post odourant exposure.  Figure 3.1. Sample image of adult zebrafish video tracking in EthoVision XT.  Figure 3.2. Adult zebrafish average velocity (cm/s) pre and post odourant exposure.  Figure 3.3. Time spent in bottom ½ of tank pre and post odourant exposure.  Figure 3.4. Spike alignment for time spent in bottom ½ of tank pre and post odourant exposure.  Figure 3.5. Manually scored area occupancy for larvae in control embryo medium (EM) trials. Figure 3.6. Manually scored area occupancy for larval 1x10-5M adenine trials.  Figure 3.7. Centre of mass shift (mm) in 1x10-5M adenine vs. EM larval avoidance- attraction trials.  Figure 3.8. Manually scored area occupancy for larval 1x10-5M adenosine trials  Figure 3.9. Centre of mass shift (mm) in 1x10-5M adenosine vs. EM larval avoidance- attraction trials.  Figure 3.10. Manually scored area occupancy for larval 1x10-5M AMP trials. x