MEDIATORS OF FINE-SCALE POPULATION GENETIC STRUCTURE IN THE BLACK BLOW FLY, PHORMIA REGINA (MEIGEN) (DIPTERA: CALLIPHORIDAE) by Charity Grace Owings A Dissertation Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Biological Sciences Indianapolis, Indiana August 2019 2 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Christine Picard, Chair Department of Biology, Indiana University-Purdue University Indianapolis Dr. Susan Walsh Department of Biology, Indiana University-Purdue University Indianapolis Dr. Xianzhong Wang Department of Biology, Indiana University-Purdue University Indianapolis Dr. William Gilhooly, III Department of Earth Sciences, Indiana University-Purdue University Indianapolis Dr. Jeffrey Holland Department of Entomology, Purdue University Approved by: Dr. Theodore Cummins Head of the Graduate Program 3 This dissertation is dedicated to Mims and Granny. Thank you both for being bad-ass grandmothers. 4 ACKNOWLEDGMENTS First and foremost, I would like to thank my parents, Dr. Keith and Traci Owings, brothers, Amos and Elijah Owings, sister-in-law, Taylor Owings, and nephew, Cain Griffin Owings, for their continuous support over the last (seemingly endless) 12 years of school. I could not have done it without all of you and I would not be here today without your selfless love and moral support. Thank you also to Mike Hopkins for being by my side during the most stressful times of my graduate life and for constantly lifting me up and reassuring me when I doubted myself during my studies. Thank you to Anne Andere for being my dearest friend throughout school. Thank you to all of the Picard and Kusmierczyk Labs for your help, guidance, and friendship: Gina Dembinski, Sarah Lewis, Ioanna Koltsidou, Laura Doll, Annie Ullyot, Hillary Veron, Abeer Mohsen, John Whale, Dilraj Panfair, and Lindsay Hammack. Thank you especially to Anais Tuceryan and Mary Huffine for working tirelessly on fly dissections, DNA extractions, and PCRs. I literally could not have finished my PhD without you. Thank you to Christine Picard for giving me a chance as a PhD student when I was down and out. I am so grateful for all of the opportunities you have given me. Thank you to all of the other members of my committee for your guidance and mentorship over the past 5 years: Susan Walsh, Bill Gilhooly, Xianzhong Wang, and Jeff Holland. Thank you to all of my collaborators who have helped mold this project into something that is truly special: Christine Skaggs, Nick Manicke, Rudy Banerjee, and Travis Asher. Thank you to Patricia Clark for being a friend and mentor. Thank you to my friends at the national parks, Rangers Paul Super and Annie Carlson and interns Rudi Boekschoten and Elgin Akin, for helping Dr. Picard and I find the best places to collect flies and for allowing us to experience the beauty of these protected lands. Thank you to Meaghan Pimsler for your undying friendship, moral support, and random arthropod facts that always put a smile on my face. Thank you to all of the friends and mentors that I did not have room to mention here, for there are so many of you who have deeply impacted my life. And finally, thank you to all of the flies that died in order for this research to be possible. You are truly extraordinary creatures and your significance to this planet cannot be understated. Again, thank you ALL. 5 TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... 9 LIST OF FIGURES ...................................................................................................................... 11 ABSTRACT .................................................................................................................................. 15 CHAPTER 1. INTRODUCTION ................................................................................................. 17 1.1 Blow Fly Biology ................................................................................................................ 17 1.2 Carrion Ecology .................................................................................................................. 18 1.3 Identifying Vertebrate Resources ....................................................................................... 19 1.4 Population Genetics ............................................................................................................ 21 1.5 Research Organism ............................................................................................................. 22 1.6 Conclusion .......................................................................................................................... 22 1.7 References ........................................................................................................................... 22 CHAPTER 2. CHEMICAL ASSAY FOR THE DETECTION OF VERTEBRATE FECAL METABOLITES IN ADULT BLOW FLIES (DIPTERA: CALLIPHORIDAE) ........................ 28 2.1 Introduction ......................................................................................................................... 28 2.2 Methods .............................................................................................................................. 31 2.2.1 Feeding Experiment ..................................................................................................... 31 2.2.2 Wild Blow Fly Collections .......................................................................................... 33 2.2.3 Gut Dissections and DNA Extractions ........................................................................ 33 2.2.4 Preliminary Presumptive Fecal Test ............................................................................ 34 2.2.5 Qualitative LC MS/MS Analyses ................................................................................ 34 2.2.6 Statistical Analyses ...................................................................................................... 35 2.3 Results ................................................................................................................................. 35 2.4 Discussion ........................................................................................................................... 39 2.5 Conclusion .......................................................................................................................... 44 2.6 References ........................................................................................................................... 45 CHAPTER 3. FEMALE BLOW FLIES AS VERTEBRATE RESOURCE INDICATORS ....... 51 3.1 Introduction ......................................................................................................................... 51 3.2 Methods .............................................................................................................................. 52 6 3.2.1 Blow Fly Collections ................................................................................................... 52 3.2.2 Vertebrate Diversity Analyses ..................................................................................... 57 3.2.3 Statistical Analyses ...................................................................................................... 57 3.3 Results ................................................................................................................................. 58 3.3.1 Effect of Subsampling .................................................................................................. 58 3.3.2 Wild Sampling ............................................................................................................. 59 3.3.3 Parameters Affecting Vertebrate Resource Detection ................................................. 63 3.4 Discussion ........................................................................................................................... 64 3.5 Conclusion .......................................................................................................................... 68 3.6 References ........................................................................................................................... 69 CHAPTER 4. STABLE ISOTOPE ANALYSIS REVEALS LARVAL RESOURCE OF ADULT PHORMIA REGINA...................................................................................................................... 73 4.1 Introduction ......................................................................................................................... 73 4.2 Materials and Methods ........................................................................................................ 76 4.2.1 General Procedure for Feeding Experiments ............................................................... 76 4.2.2 Experiment 1: Effects of Preservation ......................................................................... 76 4.2.3 Experiment 2: Persistence of Larval Diet Isotopic Signature ...................................... 77 4.2.4 Experiment 3: Effect of Single Adult Feeding Event .................................................. 77 4.2.5 Wild Sample Validation ............................................................................................... 77 4.2.6 Isotope Ratio Mass Spectrometry ...............................................................................