INVESTIGATION OF EPISTATIC INTERACTIONS AMONG FORAGING LOCI IN DROSOPHILA MELANOGASTER by Christie Elizabeth DesRoches A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Christie Elizabeth DesRoches 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-38883-9 Our file Notre reference ISBN: 978-0-494-38883-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Christie Elizabeth DesRoches Master of Science Degree Graduate Department of Cell and Systems Biology University of Toronto 2008 Abstract All organisms exhibit a wide array of intricate behavioural phenotypes throughout their lifetime. Understanding the genetic underpinnings of these behaviours will ultimately help to elucidate the cellular and molecular mechanisms that drive these behaviours. In this thesis, I explore genetic contributions to feeding behaviour, using Drosophila melanogaster as a model organism. The foraging gene (for) is a very well known genetic regulator of feeding behaviour in Drosophila melanogaster. While for underlies much of the natural variation in foraging behaviour, I am interested in other genes that may contribute to this complex phenotype. I show that a group of previously isolated mutations influencing larval foraging behaviour also influence food intake in Drosophila. In addition, I demonstrate that many of these functionally related mutations interact with one another in a genetic network to influence foraging behaviour. This thesis employs genetic and behavioural analyses to investigate the pleiotropic nature of complex food-related behaviours. n Acknowledgments First and foremost, I owe a great debt of gratitude to Dr. Maria Sokolowski for giving me the opportunity to study in her lab. Maria, thank you for taking me under your wing as an undergraduate, and for letting me stay on for graduate work when I realized how much I loved what I was doing. I have grown both as a researcher and as a person throughout my time in the lab, and for that I am truly grateful. Thanks also to the two other members of my supervisory committee, Dr. Tim Westwood and Dr. Joel Levine, for their encouragement, support and advice throughout my thesis. I also owe a special thanks to the past and present members of the Sokolowski Lab, and the Levine lab, who have made my tenure so memorable. Thanks for the advice, both about science and about life. The SokoLab is a great environment in which to work and study, and I could not have made it this far without the support of my fellow lab members. Last, but certainly not least, I am forever indebted to my parents and my brother, for things that would fill a larger volume than this. Mom, Dad and Will, thank you for putting up with me in good times and in bad. Your support has meant more to me than you know, and I couldn't have done it without you. in Table of Contents Abstract ii Acknowledgments iii Table of Contents iv List of Tables v List of Figures vi List of Key Terms and Abbreviations vii Introduction 1 Behaviour Genetics 2 Drosophila melanogaster as a Model Organism 6 The foraging Gene 8 Mutagenesis 11 Epistasis 12 Mapping of Mutations 15 Materials and Methods 17 Fly Stocks 17 Aging of Larvae 18 Larval Foraging Related Locomotion Assay 19 Larval Food Intake Assay 20 Generation of Transheterozygotes 21 Statistical Analysis 22 Complementation Analysis 24 Sequencing Analysis 25 Genomic DNA extraction 26 Amplification, Purification and Sequencing 27 Results 28 Larval Foraging Related Locomotion 28 Food Intake 29 Epistasis 30 Complementation Analysis 33 Sequencing Analysis 34 Discussion 45 Larval Foraging Related Locomotion 45 Food Intake 50 Epistasis 52 Mapping of Mutations 56 References 58 iv List of Tables Table 1 | Transheterozygote pathlength scores and estimates of general combining abilities of parental lines 40 Table 2 | Estimates of specific combining abilities of transheterozygote lines 41 Table 3 | List of mutant lines for complementation testing 43 Table 4 | Candidate genes of interest sequenced in homozygous third-instar control and mutant larvae 44 v List of Figures Figure 1 | Larval foraging pathlength of control and mutant larvae 36 Figure 2 | Larval food intake of control and mutant larvae 38 Figure 3 | Diallel crossing scheme 39 Figure 4 | Interaction diagram 42 VI List of Key Terms and Abbreviations for foraging gene QTL Quantitative trait locus r R Rover allele of foraging for for5 Sitter allele offoraging PKG cGMP dependent protein kinase EMS Ethyl methane sulfonate GFP Green fluorescent protein SNK Student-Newman-Keuls GCA General combining ability SCA Specific combining ability Vll 1 Introduction All organisms exhibit a multitude of behavioural phenotypes throughout their lifetime, and these behaviours vary between species, and even among individuals within a species. Some of these behaviours may be used only at certain times, such as courtship, copulation, aggression and oviposition, while others may be employed on a more regular basis, such as learning, memory, olfaction, gustation, mechanosensation, and foraging behaviour. Such behaviours are extraordinarily complex phenotypes integrating a great number and wide array of biological processes. An organism's behavioural response to a stimulus involves the processing of information by the nervous system, the appropriate expression of genes that affect the behaviour, and communication to other cells in the organism to elicit the ultimate behavioural response. Behavioural phenotypes are typically plastic in nature, and can adapt to environmental conditions and to the needs of the organism. There is considerable evidence, from studies of a number of model organisms, to support a significant genetic contribution to variation in many complex behavioural phenotypes (Sokolowski, 2001; Rankin, 2002; Bucan and Abel, 2002). This relationship between genes and behaviour is complicated, and can provide us with powerful insights into how organisms interact with their environments. Due to the intricate nature of behavioural phenotypes, single behaviours are likely polygenic, or under the influence of many different genes (Pflugfelder, 1998; Heisenberg, 1997). At the same time, single genes may show pleiotropic effects, where one gene influences multiple phenotypes (Pflugfelder, 1998). Feeding behaviour in Drosophila melanogaster, while influenced mainly by natural allelic variation in the foraging gene, is thought to be a complex, polygenic behaviour, foraging also 2 exhibits pleiotropy, as it is known to influence a variety of additional phenotypes in Drosophila. This thesis explores the polygenic nature of feeding behaviour, and investigates possible pleiotropic effects of genes involved in this phenotype. The investigation of the genetic basis of feeding behaviour is a well-known example of the study of the relationship between genes and behaviour. Foraging, encompassing food- search activities and food-intake, or consumption, is one such behaviour. Foraging has established genetic influences, as has been observed in animals as disparate as worms (de Bono and Bargmann, 1998), bees (Ben Shahar et al, 2002), ants (Ingram, et al, 2005) and fruit flies (Sokolowski, 1980). To understand the mechanisms driving these differences in foraging behaviour, we must first understand the genes underlying individual differences in food-related behaviours. This thesis explores the genetic underpinnings of foraging and feeding using Drosophila melanogaster as a model organism. Behaviour Genetics The idea that genes influence behaviour effectively originated over a century ago, through the work of Gregor