Western University Scholarship@Western Electronic Thesis and Dissertation Repository 9-12-2018 3:00 PM Mechanisms Underlying Freeze Tolerance in the Spring Field Cricket, Gryllus veletis Jantina Toxopeus The University of Western Ontario Supervisor Sinclair, Brent J. The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Jantina Toxopeus 2018 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biology Commons, Cellular and Molecular Physiology Commons, Comparative and Evolutionary Physiology Commons, and the Entomology Commons Recommended Citation Toxopeus, Jantina, "Mechanisms Underlying Freeze Tolerance in the Spring Field Cricket, Gryllus veletis" (2018). Electronic Thesis and Dissertation Repository. 5708. https://ir.lib.uwo.ca/etd/5708 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Freeze tolerance has evolved repeatedly across insects, facilitating survival in low temperature environments. Internal ice formation poses several challenges, but the mechanisms that mitigate these challenges in freeze-tolerant insects are not well understood. To better understand how insects survive freezing, I describe a novel laboratory model, the spring field cricket Gryllus veletis (Orthoptera: Gryllidae). Following acclimation to six weeks of decreasing temperature and photoperiod (mimicking autumn), G. veletis juveniles becomes moderately freeze-tolerant, surviving freezing at -8 °C for up to one week, and surviving temperatures as low as -12 °C. Acclimation is associated with increased control of the temperature and location of ice formation, accumulation of cryoprotectant molecules (myo-inositol, proline, and trehalose) in hemolymph and fat body tissue, metabolic rate suppression, and differential expression of more than 3,000 genes in fat body tissue. To test cryoprotectant function, I increase their concentration in G. veletis hemolymph (via injection) and freeze isolated fat body tissue with exogenous cryoprotectants. I show that cryoprotectants improve survival of freeze-tolerant G. veletis (proline), their fat body cells (myo-inositol), or both (trehalose) under otherwise lethal conditions, suggesting limited functional overlap of these cryoprotectants. However, no cryoprotectant (alone or in combination) can confer freeze tolerance on freeze-intolerant G. veletis or their cells. During acclimation, G. veletis upregulates genes encoding cryoprotectant transmembrane transporters, antioxidants, and molecular chaperones, which may protect cells during freezing and thawing. In addition, acclimated G. veletis upregulates genes encoding lipid metabolism enzymes, and cytoskeletal proteins and their regulators, which I hypothesize promote membrane and cytoskeletal remodelling. To investigate the function of these genes in freeze tolerance, I develop a method to knock down gene expression in G. veletis using RNA interference. I knock down expression of three genes (encoding a cryoprotectant transporter, an antioxidant, and a cytoskeletal regulator), laying the ground work for others to test whether and how these genes contribute to mechanisms underlying freeze tolerance. By using a combination of descriptive and manipulative experiments in an i appropriate laboratory model, I improve our understanding of the factors that contribute to insect freeze tolerance. Keywords acclimation, cold tolerance, cryoprotectants, insects, mechanisms, metabolome, overwintering, RNAi, thermal biology, transcriptome ii Co-Authorship Statement Chapter 1 was published as a review article in Biological Reviews (see Appendix D for reprint permission). I am the first author, and co-author Brent J. Sinclair (BJS) contributed to conceiving and writing this review article. Chapter 2 is in preparation for publication. I am the first author, and Alexander H McKinnon (AHM), Tomáš Štětina (TS), Kurtis F. Turnbull (KFT), Vladimír Koštál (VK) and BJS are co-authors. AHM developed the acclimation protocol that induces freeze tolerance in Gryllus veletis, and completed much of the initial characterization of this freeze tolerance as part of his MSc thesis (lethal limits, osmolality, ion concentrations). TS and KFT helped design and collect data for the respirometry experiments. VK contributed to design and interpretation of the metabolomics dataset. BJS contributed to experimental conception, design and interpretation. I conceived of and conducted the cell survival assays, metabolomics, and cryoprotectant manipulation experiments, and analyzed and visualized data from all experiments in this chapter. I drafted the manuscript together with BJS and contributions from all authors. Chapter 3 is in preparation for publication. I will be the first author, with Lauren E. Des Marteaux (LED) and BJS as co-authors. LED helped with differential gene expression analysis, interpretation, and figure preparation. I prepared samples for RNA-Seq, and assembled and annotated the transcriptome. BJS and I contributed to experimental conception, design and interpretation. I drafted the manuscript, with contributions from BJS and LED. Chapter 4 is in preparation for publication. I will be the first author, with BJS as co- author. BJS and I contributed to experimental conception, design and interpretation. I conducted and analyzed all experiments in this chapter. I drafted the manuscript, with contributions from BJS. iii Acknowledgments I would like to thank my academic mentors, past and present. I came into this Ph.D. with a solid scientific foundation, thanks to David Garbary and Tom MacRae, my Honours and Master’s advisors (respectively). Brent Sinclair’s mentorship during my Ph.D. has pushed me to develop further as a researcher, for which I am grateful. He got me hooked on freeze tolerance, and I have really enjoyed exploring this important topic during my Ph.D. I would like to thank Vladimír Koštál for hosting me in his lab at the Czech Academy of Sciences for part of my Ph.D. research, and all the guidance he provided. I would also like to thank my Ph.D. advisory committee members, Jim Staples and Louise Milligan, whose advice has been insightful and kind. Much of this thesis was also made possible by the help and support of faculty and staff at Western and other institutions. At Western, thank you to: Hillary Bain, Arzie Chant, Carol Curtis, Sherri Fenton, and Diane Gauley for their efficient administration of the Biology department and graduate program; Chris Guglielmo and Jim Staples for the continued use of their laboratory equipment; and Steve Bartlett, Carrie Hamilton, and Karen Nygard, who keep the Biotron facilities running smoothly. At the Czech Academy of Sciences, thank you to: Irena Vacková and Jarča Korbelová for their technical support, and Martin Moos and the Laboratory of Analytical Biochemistry for assistance processing my metabolomics samples. I have many academic peers to thank, as well as my supportive friends and family outside of academia. I am eternally grateful to former Sinclair lab members, Ruth Jakobs and Zander McKinnon, who showed me the ropes when I was a very new thermal biologist. I’d especially like to thank Laura Ferguson, whose initial observations on overwintering Gryllus veletis sparked the use of this cricket as a freeze tolerance model. Further, I enjoyed collaborating with and learning from my other lab mates: Susan Anthony, John Ciancio, Lauren Des Marteaux, Jackie Lebenzon, Yanira Jiménez Padilla, and Kurtis Turnbull. I would also like to thank Honza Rozsypal, Tomáš Štětina, and rest of the Koštál lab crew for making me feel welcome in their lab. I appreciated the social and iv intellectual support from my peers in the Biology graduate program, particularly those in BGS 2035, the Physiology and Biochemistry stream, and members of the Mustang Bioinformatics Club (with special shout outs to Tian Wu and Maryam Jangjoo). This Ph.D. would have been a lot more challenging without you! One of the highlights of my Ph.D. has been the undergraduate students who have helped with my research – I enjoyed watching you grow as scientists, and am thankful for all the hours you put into research projects with me. I’d especially like to thank Ose Akioyamen, Aisa Kuper-Psenicnik, Claire Newman, Sarah Vo and Meng Zhang, for their hard work on their (and my) experiments. I am grateful to many lab assistants, for their help with cricket maintenance, including Irene Alfred, Haonan (Johnny) Jiang, Michelle Lim, Nicole Martin-Kenny, Aldina Sertovic, and Adam Smith. I am grateful for financial support from a Canada Graduate Scholarship (Natural Sciences and Engineering Research Council of Canada; NSERC), an Ontario Graduate Scholarship, and the University of Western Ontario Department of Biology. I would also like to thank the Company of Biologists and NSERC for travel grants to support my work in other labs. This support provided important financial security, facilitating my professional development. v Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement................................................................................................... iii Acknowledgments.............................................................................................................