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Cellular and Molecular Mechanisms of Environmental Stress Tolerance in Insects

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Cellular and Molecular Mechanisms of Environmental Stress Tolerance in Insects Cellular and Molecular Mechanisms of Environmental Stress Tolerance in Insects DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nicholas M. Teets Graduate Program in Entomology The Ohio State University 2012 Dissertation Committee: Professor David L. Denlinger, Advisor Professor Thomas G. Wilson Professor P. Larry Phelan Professor Sandor Gyorke Professor Peter Piermarini Copyright by Nicholas M. Teets 2012 Abstract The ability to tolerate environmental stress is a key adaptation for insects across the world. As ectotherms, insects are unable to regulate internal temperature, and their small body size makes them particularly susceptible to extremes in temperature and water availability. Insects rely on numerous physiological adaptations to cope with environmental stress, and recent advances in molecular biology and “omics” technologies have made it possible to study these mechanisms in detail. In this dissertation, I explore molecular mechanisms of stress tolerance in both temperate and polar insects. In a process called rapid cold-hardening (RCH), brief exposure (i.e. minutes to hours) to nonlethal low temperatures significantly enhances tolerance to cold-shock conditions. While the ecological context of RCH is well-established, the underlying mechanisms are poorly understood. Using cDNA microarrays, we measured changes in gene expression accompanying RCH (2 h at 0°C) in the flesh fly, Sarcophaga bullata. To our surprise, no transcripts were differentially expressed during RCH, suggesting RCH occurs without the need for new gene products. Rather, cold-sensing and RCH appear to be primarily governed by second messenger systems, including calcium signaling. In Chapter 3, we show that chilling evoked an increase in intracellular calcium and activated calcium/calmodulin-dependent protein kinase II. Blocking calcium signaling ii pharmacologically prevented RCH, indicating calcium signaling is required during cold- sensing and RCH. In the latter 4 chapters of this dissertation, I investigated physiological and molecular mechanisms of stress tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and the only insect endemic to the continent. In the unpredictable climate of Antarctica, larvae are likely exposed to multiple bouts of both cold and desiccation stress, thus I quantified the survival and energetic consequences of repeated cold and dehydration exposure in B. antarctica. Larvae exposed to five diurnal freeze-thaw cycles experienced significant mortality and energy depletion. However, this was only true if larvae were frozen during repeated cold exposure; supercooled larvae exposed to the same temperatures experienced no significant mortality or energy depletion. Repeated bouts of dehydration and rehydration were also energetically costly, as 5 cycles of dehydration and rehydration caused larvae to consume 67% of their carbohydrate energy reserves. In the final two chapters, I explored transcriptional mechanisms of extreme stress tolerance in B. antarctica. Targeted qPCR experiments revealed significant restructuring of metabolic gene expression during periods of stress. Cold stress caused upregulation of genes involved in glucose mobilization, while dehydration stress increased expression of genes required for glucose, trehalose, and proline synthesis. Finally, using RNA-seq, I measured changes in gene expression accompanying extreme dehydration in larvae of B. antarctica. Expression results identified upregulation of pathways involved in cellular recycling and energy conservation, such as ubiquitin-mediated proteasome and iii autophagy, with concurrent downregulation of numerous genes involved in central metabolism. iv Dedication Dedicated to my wife and best friend, Julie. v Acknowledgments I thank my advisor, Dr. David Denlinger, for his support and guidance during my graduate career. I also thank past and present members of the Denlinger lab for their camaraderie and assistance, particularly Qirui Zhang, Julie Reynolds, Alena Kobelkova, Megan Meuti, and Justin Peyton, and Marie Bontempo, who have been here for all or most of my time at Ohio State. I am indebted to Rob Michaud for teaching me the basics of molecular biology, and I thank several undergraduate researchers, particularly Charles Dean, for their assistance. I thank Richard Lee, my undergraduate advisor, for his continued support and mentoring. I also thank Yuta Kawarasaki, Juanita Constible, and the Palmer Station support staff for two great field seasons in Antarctica. I acknowledge my committee members, Thomas Wilson, Sandor Gyorke, Larry Phelan, and Peter Piermarini for their contributions to this dissertation. I thank various collaborators who have made substantial contributions to the results presented in this dissertation. Collaborators, with the chapters they contributed to in parentheses, include Justin Peyton (2,7), Herve Colinet (2,7), David Renault (2,7), Greg Ragland (2), Dan Hahn (2), Shu-Xia Yi (3), Richard Lee (3-7), Yuta Kawarasaki (4- 7), and Joanna Kelley (7). Additional contributions are listed in the “Acknowledgements” section of each chapter. vi Many thanks to the College of Biological Sciences and the Department of Entomology for financial support. Finally, thanks to family and friends for their support and encouragement these past 5 years. Specifically, I thank my wife Julie for providing welcome distractions at home, my parents, Darcy and Kaye, for encouraging me to pursue my dreams, and my brothers, Tom, Joe, and Jacob for keeping things interesting. vii Vita June 13, 1984 .................................................Born, Lorain, OH 2003................................................................Marion L. Steele High School, Amherst, OH 2003-2004 ......................................................Laboratory Assistant, Department of Zoology, Miami University 2005-2007 .....................................................Undergraduate Research Assistant, Department of Zoology, Miami University 2007 ...............................................................B.S. in Zoology, Miami University 2007-2012 .....................................................Graduate Research Fellow, Department of Entomology, Ohio State University 2009-2011 (intermittent) ...............................Graduate Teaching Assistant, Department of Entomology, Ohio State University Publications 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 of the United States of America, DOI 10.1073/pnas.1218661109 Teets, N.M., Kawarasaki, Y., Lee, R.E., Denlinger, D.L., 2012. Expression of genes involved in energy mobilization and osmoprotectant synthesis during thermal and desiccation stress in the Antarctic midge, Belgica antarctica. Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology, DOI 10.1007/s00360-012-0707-2. viii Teets, N.M., Peyton, J.T., Ragland, G.J., Colinet, H., Renault, D., Hahn, D.A., Denlinger, D.L., 2012. Uncovering molecular mechanisms of cold tolerance in a temperate flesh fly using a combined transcriptomic and metabolomic approach. Physiological Genomics, 44, 764-777. Teets, N.M., Kawarasaki, Y., Lee, R.E., Denlinger, D.L., 2012a. Energetic consequences of repeated and prolonged dehydration in the Antarctic midge, Belgica antarctica. Journal of Insect Physiology, 58, 498-505. Goto, S.G., Philip, B.N., Teets, N.M., Kawarasaki, Y., Lee, R.E., Denlinger, D.L., 2011. Functional characterization of an aquaporin in the Antarctic midge Belgica antarctica. Journal of Insect Physiology, 57, 1106-1114. Teets, N.M., Kawarasaki, Y., Lee, R.E., Denlinger, D.L., 2011. Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica: a comparison between frozen and supercooled larvae. Journal of Experimental Biology, 214, 806-814. Michaud, M.R., 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. Benoit, J.B., Lopez-Martinez, G., Teets, N.M., Phillips, S.A., Denlinger, D.L., 2009. Responses of the bed bug, Cimex lectularius, to temperature extremes and dehydration: levels of tolerance, rapid cold hardening and expression of heat shock proteins. Medical and Veterinary Entomology, 23, 418-425. Teets, N.M., Elnitsky, M.A., Benoit, J.B., Lopez-Martinez, G., Denlinger, D.L., Lee, R.E., 2008. Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 294, R1938-R1946. Fields of Study Major Field: Entomology ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ..................................................................................................................................
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