An invitation to measure insect cold tolerance: Methods, approaches, and workflow.

Insect performance is limited by the temperature of the environment, and in temperate, polar, and alpine regions, the majority of insects must face the challenge of exposure to low temperatures. The physiological response to cold exposure shapes the ability of insects to survive and thrive in these environments, and can be measured, without great technical difficulty, for both basic and applied research. For example, understanding insect cold tolerance allows us to predict the establishment and spread of insect pests and biological control agents. Additionally, the discipline provides the tools for drawing physiological comparisons among groups in wider studies that may not be focused primarily on the ability of insects to survive the cold. Thus, the study of insect cold tolerance is of a broad interest, and several reviews have addressed the theories and advances in the field. Here, however, we aim to clarify and provide rationale for common practices used to study cold tolerance, as a guide for newcomers to the field, students, and those wishing to incorporate cold tolerance into a broader study. We cover the 'tried and true' measures of insect cold tolerance, the equipment necessary for these measurement, and summarize the ecological and biological significance of each. Finally, we suggest a framework and workflow for measuring cold tolerance and low temperature performance in insects.

Chill-tolerant Gryllus crickets maintain ion balance at low temperatures.

Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CTmin) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0 °C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CTmin) than G. pennsylvanicus. During cold exposure, hemolymph water and Na(+) migrated to the gut of warm-acclimated G. pennsylvanicus, which increased hemolymph [K(+)] and decreased muscle K(+) equilibrium potentials. By contrast, cold-acclimated G. pennsylvanicus suffered a smaller loss of ion and water homeostasis during cold exposure, and this redistribution did not occur at all in cold-exposed G. veletis. The loss of ion and water balance was similar between RCH and warm-acclimated G. pennsylvanicus, suggesting that different mechanisms underlie decreased CCR and CTmin compared to increased survival at 0 °C. We conclude that increased tolerance of chilling is associated with improved maintenance of ion and water homeostasis in the cold, and that this is consistent for both phenotypic plasticity and evolved cold tolerance.