Laboratory acclimation to autumn-like conditions induces freeze tolerance in the spring field cricket Gryllus veletis (Orthoptera: Gryllidae)

Many temperate insects encounter temperatures low enough to freeze their body fluids. Remarkably, some insects are freeze-tolerant, surviving this internal ice formation. However, the mechanisms underlying freeze tolerance are not well-understood, in part due to a lack of tractable model organisms. We describe a novel laboratory model to study insect freeze tolerance, the spring field cricket Gryllus veletis (Orthopera: Gryllidae). Following acclimation to six weeks of decreasing temperature and photoperiod, G. veletis become freeze-tolerant, similar to those exposed to natural autumn conditions in London, Ontario, Canada. Acclimated crickets suppress their metabolic rate by c. 33%, and survive freezing for up to one week at -8°C, and to temperatures as low as -12°C. Freeze-tolerant G. veletis protect fat body cells from freeze injury in vivo, and fat body tissue from freeze-tolerant cricket survives brief freeze treatments when frozen ex vivo. Freeze-tolerant crickets freeze at c. -6°C, which may be initiated by accumulation of ice-nucleating agents in hemolymph or gut tissue. We hypothesize that control of ice formation facilitates freeze tolerance, but initiating ice formation at high subzero temperatures does not confer freeze tolerance on freeze-intolerant nymphs. Acclimation increases hemolymph osmolality from c. 400 to c. 650 mOsm, which may facilitate freeze tolerance by reducing ice content. Hemolymph ion concentrations do not change with acclimation, and we therefore predict that freeze-tolerant G. veletis elevate hemolymph osmolality by accumulating other molecules. Gryllus veletis is easily reared and manipulated in a controlled laboratory environment, and is therefore a suitable candidate for further investigating the mechanisms underlying freeze tolerance