ABSTRACT GARLAPOW, MEGAN ELIZABETH. Quantitative Genetics of Food Intake in Drosophila melanogaster. (Under the direction of Drs. Trudy F. C. Mackay and David W. Threadgill). Nutrient intake is an essential activity of eukaryotes. Food intake behaviors are typically polygenic in nature and affected by the environment and gene-by-environment interactions. They are exceptionally complex traits and form the mechanistic connection between behavior and physiology. While the genetic architecture of food intake has been studied in a variety of mammals, the model organism Drosophila melanogaster is exceptionally positioned for elucidating our understanding of the genetic basis of variation in food intake as it is highly genetically and experimentally tractable. The aim of my dissertation was to apply quantitative genetics approaches to understand the genetic architecture underlying natural variation in food intake using Drosophila melanogaster as a model system. I accomplished this first by assessing variation in food intake and micro-environmental plasticity of food intake and using genome-wide analysis to identify genetic variants associated with the observed variation, and second by applying mass selection for divergent feeding to determine the effects on phenotypes, genome-wide gene expression, and genetic divergence in selected populations. In both cases, I took advantage of naturally occurring polymorphisms to assess the quantitative genetics of food consumption. Animals perform many decisions affecting the initiation, continuation, and cessation of feeding. These decisions are affected by the available food source, ambient temperature, reproductive status, sensory perception, circadian cycles, and age; and are modulated by signaling pathways acting in various tissues and organs, with the central nervous system serving as the ultimate integrator of internal physiological cues and the environment to determine total food intake. To better understand the genetic architecture underlying variation in food intake and micro-environmental plasticity of food intake, I exploited naturally occurring polymorphisms in the Drosophila melanogaster Genetic Reference Panel (DGRP), a wild-derived, fully inbred, fully sequenced panel of Drosophila melanogaster suitable for genome wide association analysis. I measured food intake under controlled laboratory conditions across the panel and further assessed variation in micro-environmental plasticity of food intake, whereby some lines consume consistent volumes of food and others consume widely varying volumes of food. The predicted genetic variants affecting mean and variation of variance of food intake were overwhelmingly novel, with subsequent functional validation experiments revealing aminopeptidases, neuropeptide signaling, immune system enzymes, metalloproteinase receptors, genes of unknown molecular function, and others as affecting mean and/or variation of variance of food intake. Next, I assessed the response of organismal phenotypes, genome-wide gene expression, and genetic divergence to mass selection for divergent food intake to gain further understanding of this complex trait. Ten generations of twenty percent selection pressure on outbred populations of Drosophila melanogaster derived from crossing 37 maximally divergent DGRP lines enabled analysis of response to selection, realized heritability, body composition, body mass, transcript abundance, and genes under selection. The results indicate that response to selection for low and high feeding is asymmetrical, with only high feeder populations phenotypically separating from the control population. Body mass was not affected by selection. RNA and DNA sequencing analysis revealed several genes functionally validated in the first, DGRP-based project as well as additional novel transcripts and genes affecting food intake. Both projects support the notion that food intake is highly polygenic and sexually dimorphic. Quantitative genetics approaches in D. melanogaster reveal novel genes affecting food intake and elucidate much of the previously unknown genetic architecture of this complex trait. © Copyright 2015 Megan Elizabeth Garlapow All Rights Reserved Quantitative Genetics of Food Intake in Drosophila melanogaster by Megan Elizabeth Garlapow A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Genetics Raleigh, North Carolina 2015 APPROVED BY: _______________________________ _______________________________ Trudy F. C. Mackay David W. Threadgill Committee Co-Chair Committee Co-Chair _______________________________ _______________________________ Eric A. Stone Jack Odle DEDICATION I dedicate this entire project to my rock and my sunshine, my husband and my daughter, Christian and Ada Garlapow. ii BIOGRAPHY My love of biology began as a young child in Colorado, wandering through the protected wetlands in a park near my childhood home in Denver, growing trees and vegetables in the garden, and hiking through many mountain trails, awed by the sheer beauty of life. Science became incredibly interesting to me in middle and high school, and I am forever indebted to Mr. Michael Burnham for instilling in me a passion for genetics in his elective course, “Genetics,” in the spring semester of my senior year at Kent Denver High School. I attended the University of Chicago, where I earned a Bachelor of Arts in Biology with General Honors in June 2007. While an undergraduate student, I worked with Dr. Martha McClintock, assessing the estrus cycles of socially housed and isolated female rats as part of a broader psychobiology project. My introduction to laboratory research was inspirational and resulted in my committed attention to experimental design and sexual dimorphism and my keen interest in behavioral phenotypes. After graduating, I worked in the laboratory of Dr. Jerry Stitzel at the University of Colorado at Boulder, examining the role of circadian cycles and melatonin in nicotine addiction in inbred laboratory mice. I longed for quantitative genetics approaches, so I began my graduate school research in the Program in Genetics at North Carolina State University in August 2009, joining the laboratory of Dr. Trudy Mackay in January 2010. iii ACKNOWLEDGMENTS I would first and foremost like to acknowledge my husband, Mr. Christian Garlapow, for re- routing his entire life to support me and for loving our daughter unconditionally. Ada Garlapow has been a continuous source of inspiration and joy, and my life really only began when I gave birth to her. My parents-in-law, Mr. and Mrs. Richard Garlapow, have encouraged me to continue forward through many obstacles and helped me achieve much more than I ever thought I could. Additionally, my sister, Ms Dilshanie Perera Friedrich, has provided the sort of love and friendship on which my perseverance has often depended, and my Aunt Kris’s faith in me has kept me steadfast. From the guidance of my advisor and my co-advisor, Drs. Trudy Mackay and David Threadgill, I have learned how to think critically to design and execute exciting research projects examining the genetic bases of food intake in Drosophila melanogaster. My committee members, Drs. Eric Stone and Jack Odle, have further lent insight and advice when needed. In the Mackay laboratory, Dr. Richard Lyman deserves special recognition for his individual mentorship. Mackay and Threadgill laboratory members past and present have played integral roles in my dissertation’s success through conversation and inspiration, including Drs. Wen Huang, Susan Harbison, Shanshan Zhou, Megan Carnes, Logan Everett, Kultaran Chohan, Mary Anna Carbone, Akihiko Yamamoto, Tatiana Morozova, Shilpa Swarup, Lauren Dembeck, Rachel Lynch, and Dave Bautz, and John Shorter, Lavanya Turlapati, Gunjan Arya, Fabio Morgante, and Tiffany Garbutt. iv The graduate program director, my course professors, the fly kitchen staff, and the administrative staff all demonstrate that it really takes a village to make a dissertation. Dr. Marian “Betty” Gardner has been an exceptional teaching mentor. Finally, I have been lucky to mentor some of the most capable, enthusiastic undergraduate scientists: Kairsten Fay, Alexander Gearhart, Kara Peterson, and Michael Yarboro are incredible people with bright futures. v TABLE OF CONTENTS LIST OF TABLES.....................................................................................................................x LIST OF FIGURES.................................................................................................................xii CHAPTER ONE: INTRODUCTION........................................................................................1 REGULATION OF FOOD INTAKE............................................................................4 Drosophila melanogaster as a model system to study food intake...............................4 Environment, nutrient content, physiology, and feeding...............................................8 Signaling pathways and food intake............................................................................12 Central nervous system control of feeding..................................................................18 Assays..........................................................................................................................20 CHARACTERISTICS OF QUANTITATIVE TRAITS.............................................25 The D. melanogaster Genetic Reference Panel...........................................................27