Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Sinclair, Brent J.

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 appropriate laboratory model, I improve our understanding of the factors that contribute to insect freeze tolerance.

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