Temperature Modulation of Biological Clocks in a Reef-Building Coral
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University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2018-01-25 Temperature Modulation of Biological Clocks in a Reef-Building Coral Wuitchik, Daniel Michael Wuitchik, D. M. (2018) Temperature modulation of biological clocks in a reef-building coral (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/5465 http://hdl.handle.net/1880/106386 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Temperature Modulation of Biological Clocks in a Reef-Building Coral by Daniel Michael Wuitchik A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA JANUARY 2018 © Daniel Michael Wuitchik 2018 Abstract Reef-building coral of the Great Barrier Reef demonstrate remarkably synchronized and precise timing of reproduction. They achieve this precision through sophisticated biological clock systems entrained by their local environment. This thesis explores how temperature and lunar light modulates the biological clocks of Acropora millepora by measuring transcriptomic changes with RNA-sequencing. Coral colonies were collected and maintained under artificial lunar light at the Heron Island Research Station under cool and warm temperature treatments corresponding to winter and summer averages at this locale. Individuals were sampled over a 24- hour period during a full lunar month to capture long-term daily profiles of gene expression. It was found that numerous biological clock genes were impacted by temperature and lunar light. Furthermore, temperature and lunar phase altered the expression of interesting hormonal pathways involved in reproductive behaviours. These data will help elucidate the mechanisms underlying the precise timing of reproduction in reef-building coral and the effect that different temperatures have on this process. ii Acknowledgements I could not have asked for a better supervisor and mentor in Dr. Peter Vize. You have challenged me to learn an entirely new skill set while providing patient guidance. I would not have been able to do this without your mentorship and the freedom to learn my system and science at my own pace. I am truly grateful for my time in the lab, and I hope we collaborate in the years to come. To my committee members, thank you for helping me to see the bigger picture; your feedback has been pivotal in the design of this project. The staff and facilities at Heron Island Research Center are top notch and without the quick and professional assistance from many people, my fieldwork would not have been possible. In addition, I would like to say thank you to the graduate students who shared my time on Heron as you helped me with fieldwork and the comradery made for some of the best experiences of my life. The department of biology at the University of Calgary has many unsung heroes that have helped me and my colleagues achieve all of our goals; your efforts were certainly noticed and greatly appreciated. There are many students in the Ecology and Evolutionary Biology program who have been instrumental in sharing their ideas, enthusiasm, and contributed greatly to my understanding of varied subjects in ecology. I would like to thank Jessica Hopson, Robert Morgante and Ramon Nagesan for your comradery and late-night coding sessions, which turned a difficult statistic course into an enjoyable experience. Thank you to Kyle Wilson for great intellectual conversations, drinks, and letting me beat you at squash - your philosophy of science and academia is a model that we should all strive for. This project would not have been possible without the contributions and resources provided by the Xenbase group and thoughtful assistance from Kamran Karimi, Vaneet Lotay iii and Praneet Chaturvedi. Thank you for your aid in my bioinformatics and use of computational resources. Lastly, my family has provided unwavering support throughout my career and pursuits of obscure interests. You have granted me the privilege to always remain curious. For this gift, I am truly thankful. Natalie, thank you for reminding me that all we need is a little movement in life. Dad, I appreciate your late-career shift into the marine sciences and have appreciated your depth of knowledge in crayfish husbandry. Mom, you help me to see the colour in life and have demonstrated time and time again that art and science are not mutually exclusive. iv Dedication I have travelled far with my partner in crime and in science, Sara Smith. How cool is it that we can share a deep interest and career pursuing knowledge together? This journey has only been possible because you have pushed me to be better in everything I do. You are one of the few genuine people I have met, and your enthusiasm and fierce intelligence makes you a rock star in science. You are the best partner I could have ever dreamed of. This thesis is dedicated to you. Always. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Dedication ............................................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... viii List of Figures and Illustrations ......................................................................................... ix List of Symbols, Abbreviations and Nomenclature ......................................................... xiii Epigraph ........................................................................................................................... xiv CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Coral primer and reproduction ...................................................................................1 1.2 Biological clocks ........................................................................................................4 1.2.1 Circadian clock ..................................................................................................6 1.2.2 Circalunar clock .................................................................................................8 1.2.3 Circannual clock ..............................................................................................11 1.3 Study species: Acropora millepora ..........................................................................12 1.4 Objectives and Purpose ............................................................................................13 CHAPTER TWO: METHODS ..........................................................................................14 2.1 Site description and collection .................................................................................14 2.2 Experimental Design ................................................................................................15 2.2.1 Aquaria ............................................................................................................15 2.2.2 Moonlight apparatus ........................................................................................16 2.3 Experimental Design and Sampling ........................................................................18 2.4 RNA isolation ..........................................................................................................19 2.4.1 RNA purification .............................................................................................19 2.4.2 DNA contaminant removal ..............................................................................20 2.5 Library Preparation and Sequencing ........................................................................21 2.6 Bioinformatics and Analyses ...................................................................................21 2.6.1 Preprocessing and Quality Control ..................................................................21 2.6.2 Sequence alignment and read counts ...............................................................22 2.6.3 Normalization ..................................................................................................22 2.6.4 Data Distribution .............................................................................................26 2.6.5 Principal Component Analysis and Outlier Removal .....................................28 2.6.6 Differential expression analyses ......................................................................30 2.6.7 Hierarchical Clustering ....................................................................................33 2.6.8 Annotation .......................................................................................................33