Genomic Platforms and Molecular Physiology of Insect Stress Tolerance DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Justin Peyton MS Graduate Program in Evolution, Ecology and Organismal Biology The Ohio State University 2015 Dissertation Committee: Professor David L. Denlinger Advisor Professor Zakee L. Sabree Professor Amanda A. Simcox Professor Joseph B. Williams Copyright by Justin Tyler Peyton 2015 Abstract As ectotherms with high surface area to volume ratio, insects are particularly susceptible to desiccation and low temperature stress. In this dissertation, I examine the molecular underpinnings of two facets of these stresses: rapid cold hardening and cryoprotective dehydration. Rapid cold hardening (RCH) is an insect’s ability to prepare for cold stress when that stress is preceded by an intermediate temperature for minutes to hours. In order to gain a better understanding of cold shock, recovery from cold shock, and RCH in Sarcophaga bullata I examine the transcriptome with microarray and the metabolome with gas chromatography coupled with mass spectrometry (GCMS) in response to these treatments. I found that RCH has very little effect on the transcriptome, but results in a shift from aerobic metabolism to glycolysis/gluconeogenesis during RCH and preserved metabolic homeostasis during recovery. In cryoprotective dehydration (CD), a moisture gradient is established between external ice and the moisture in the body of an insect. As temperatures decline, the external ice crystals grow, drawing in more moisture which dehydrates the insect causing its melting point to track the ambient temperature. To gain a better understanding of CD and dehydration in Belgica antarctica I explore the transcriptome with RNA sequencing ii and the metabolome with GCMS. I found an up regulation of genes involved in autophagy and down regulation of those involved in apoptosis. I also found coordinated shut down of metabolism during cryoprotective dehydration. Sequencing the genome of an organism is an expensive and time consuming endeavor, but with the advent of next generation sequencing, it is possible for a single lab or a small group of allied labs to undertake the task. Because of its importance as a model for polar biology, low temperature biology, and dehydration tolerance, I present the assembled, annotated, and characterized genome of B. antarctica. Because of its importance as a model for diapause and low temperature biology, I present the assembled, annotated, and characterized genome of S. bullata. iii Dedication To Alexander Edwin who inspires me to be the very best I can be. iv Acknowledgments First, I would like to thank my friends and family who have supported me during this long and difficult process. Without their love and help I never would have achieved my goals. Second, I would like thank my advisor, Dr. David Denlinger, for his great advice and superlative editing. Next, I would like to thank my other committee members, Dr. Zakee Sabree, Dr. Amanda Simcox, and Dr. Joseph Williams, for their valuable comments. Finally, I would like to thank my collaborators and labmates. Together, as a team, is how we push science forward. v Vita 2005............................................................................. B.S. Chemistry, Milligan College 2008.................................................M.S. Mathematics, East Tennessee State University 2008 to present ..........................Graduate Teaching Assistant, The Ohio State University Publications 1. Paquette, C., Joplin, K. H., Seier, E., Peyton, J. T., & Moore, D. (2008) Sex- specific differences in spatial behavior in the flesh fly Sarcophaga crassipalpis. Physiological Entomology, 33, 382-388. 2. Michaud, R. M., Teets, N. M., Peyton, J. T., Blobner, B. M. & Denlinger, D. L. (2011) Heat shock response to hypoxia and its attenuation during recovery in the flesh fly, Sarcophaga crassipalpis. Journal of Insect Physiology, 57, 203-210. 3. Geji, R., Lou, Y., Munther, D. & Peyton, J. (2011) Convergence to ideal free dispersal strategies and coexistence. Bulletin of Mathematical Biology, 74, 257-299 4. Teets, N. M., Peyton, J. T., Ragland, G. J., Colinet, H., Renault, Hahn, D. A., & Denlinger, D. L. (2012) Combined transcriptomic and metabolomic approach uncovers molecular mechanisms of cold tolerance in a temperate flesh fly, Physiological Genomics, 44, 764-777. vi 5. *Teets, N. M., *Peyton, J. T., Colinet, H., Renault, D., Kelley, J. L., Kawarasaki, Y., Lee, R. E. & Denlinger, D. L. (2012) Gene expression changes governing extreme dehydration tolerance in an Antarctic insect. Proceedings of the National Academy of Sciences, 50, 20744–20749. 6. Kelley, J. L., Peyton, J. T., Fiston-Lavier, A., Teets, N. M., Yee M., Johnston, S. J., Bustamante, C. D., Lee, R. E. & Denlinger, D. L. (2014) Compact genome of the Antarctic midge is likely an adaptation to an extreme environment, Nature Communications, 5, 4611. 7. The International Glossina Genome Initiative. (2014). Genome sequence of the tsetse fly (Glossina morsitans): vector of African trypanosomiasis, Science, 344, 380- 366. 8. Kobelkovaa, A., Goto, S. G., Peyton, J. T., Ikeno, T., Lee, R. E. & Denlinger, D. L. (2015) Continuous activity and no cycling of clock genes in the Antarctic midge during the polar summer, Journal of Insect Physiology, 81, 90 – 96. Fields of Study Major Field: Evolution, Ecology and Organizmal Biology vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Table of Contents ............................................................................................................. viii List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xvi Chapter 1: Review of Mechanisms of Insect Cold Tolerance ............................................ 1 1.1 Nature of Cold Stress ................................................................................................ 1 1.2 On Dealing with Ice .................................................................................................. 2 1.3 Preparing for the Cold ............................................................................................... 5 1.4 Rapid Cold Hardening ............................................................................................... 6 1.5 Cryoprotective Dehydration ...................................................................................... 9 1.6 Sarcophaga bullata ................................................................................................. 10 1.7 Belgica antarctica ................................................................................................... 12 1.8 Specific Goals ......................................................................................................... 13 viii CHAPTER 2: Compact Genome of the Antarctic Midge is Likely an Adaptation to an Extreme Environment ....................................................................................................... 14 Citation .......................................................................................................................... 14 Abstract ......................................................................................................................... 14 2.1 Introduction ............................................................................................................. 16 2.2 Results ..................................................................................................................... 18 2.3 Discussion ............................................................................................................... 24 2.4 Methods ................................................................................................................... 26 2.4.1 Biological Sample............................................................................................. 26 2.4.2 DNA Library Preparation and Sequencing ....................................................... 27 2.4.3 PacBio Library Preparation and Sequencing .................................................... 27 2.4.4 De Novo Genome Assembly ............................................................................ 28 2.4.5 Repeat Annotation ............................................................................................ 29 2.4.6 Gene Annotation ............................................................................................... 30 2.4.7 Comparative Analyses ...................................................................................... 31 2.4.8 Functional Enrichment Analysis ...................................................................... 32 2.4.9 Polymorphism Detection .................................................................................