Cold tolerance is important in defining the distribution of insects. Here, we review the principal physiological mechanisms underlying homeostatic failure during cold exposure in this diverse group of ectotherms. When insects are cooled sufficiently, they suffer an initial loss of neuromuscular function (chill coma) that is caused by decreased membrane potential and reduced excitability of the neuromuscular system. For chill-susceptible insects, chronic or severe chilling causes a disruption of ion and water homeostasis across membranes and epithelia that exacerbate the initial effects of chilling on membrane potential and cellular function, and these perturbations are tightly associated with the development of chill injury and mortality. The adaptation and acclimation responses that allow some insects to tolerate low temperatures are multifactorial and involve several physiological systems and biochemical adjustments. In this review, we outline a physiological model that integrates several of these responses and discuss how they collectively help to preserve cellular, organ, and organismal homeostasis at low temperature.

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Summary 1. Thermal tolerance may limit and therefore predict ectotherm geographic distributions. However, which of the many metrics of thermal tolerance best predict distribution is often unclear, even for drosophilids, which constitute a popular and well-described animal model. 2. Five metrics of cold tolerance were measured for 14 Drosophila species to determine which metrics most strongly correlate with geographic distribution. The species represent tropical to temperate regions but all were reared under similar (common garden) conditions (20°C). The traits measured were: chill coma temperature (CT min ), lethal temperature (LTe 50 ), lethal time at low temperature (LTi 50 ), chill coma recovery time (CCRT) and supercooling point (SCP). 3. Measures of CT min , LTe 50 and LTi 50 proved to be the best predictors to describe the variation in realized latitudinal distributions (R 2 = 0Á699, R 2 = 0Á741 and 0Á550, respectively) and estimated environmental cold exposure (R 2 = 0Á633, R 2 = 0Á641 and 0Á511, respectively).Measures of CCRT also correlated significantly with estimated minimum temperature (R 2 = 0Á373), while the SCP did not. These results remained consistent after phylogenetically independent analysis or when applying nonlinear regression. Moreover, our findings were supported by a similar analysis based on existing data compiled from the Drosophila cold tolerance literature. 4. Trait correlations were strong between LTe 50 , LTi 50 and CT min , respectively (0Á83 > R 2 > 0Á55). However, surprisingly, there was only a weak correlation between the entrance into coma (CT min ) and the recovery from chill coma (CCRT) (R 2 = 0Á256). 5.Considering the findings of the present study, data from previous studies and the logistical constraints of each measure of cold tolerance, we conclude that CT min and LTe 50 are superior measures when estimating the ecologically relevant cold tolerance of drosophilids. Of these two traits, CT min requires less equipment, time and animals and thereby presents a relatively fast, simple and dynamic measure of cold tolerance.

Multiple stressors, both abiotic and biotic, often are experienced simultaneously by organisms in nature. Responses to these stressors may share signaling pathways ("cross-talk") or protective mechanisms ("cross-tolerance"). Temperate and polar insects that must survive the winter experience low temperatures accompanied by additional abiotic stressors, such as low availability of water. Cold and desiccation have many similar effects at a cellular level, and we present evidence that the cellular mechanisms that protect against cold stress also protect against desiccation, and that the responses to cold and dehydration likely evolved as cross-tolerance. By contrast, there are several lines of evidence suggesting that low temperature stress elicits an upregulation of immune responses in insects (and vice versa). Because there is little mechanistic overlap between cold stress and immune stress at the cellular level, we suggest that this is cross-talk. Both cross-talk and cross-tolerance may be adaptive and likely evolved in response to synchronous stressors; however, we suggest that cross-talk and cross-tolerance may lead to different responses to changes in the timing and severity of multiple stress interactions in a changing world. We present a framework describing the potentially different responses of cross-tolerance and cross-talk to a changing environment and describe the nature of these impacts using interaction of cold-desiccation and cold-immunity in overwintering insects as an example.

SUMMARYTo predict the effects of changing climates on insect distribution and abundance, a clear understanding of the mechanisms that underlie critical thermal limits is required. In insects, the loss of muscle function and onset of cold-induced injury has previously been correlated with a loss of muscle resting potential. To determine the cause of this loss of function, we measured the effects of cold exposure on ion and water homeostasis in muscle tissue, hemolymph and the alimentary canal of the fall field cricket, Gryllus pennsylvanicus, during an exposure to 0°C that caused chilling injury and death. Low temperature exposure had little effect on muscle osmotic balance but it dissipated muscle ion equilibrium potentials through interactions between the hemolymph and gut. Hemolymph volume declined by 84% during cold exposure whereas gut water content rose in a comparable manner. This rise in water content was driven by a failure to maintain osmotic equilibrium across the gut wall, which resulted in considerable migration of Na + , Ca 2+ and Mg 2+ into the alimentary canal during cold exposure. This loss of homeostasis is likely to be a primary mechanism driving the cold-induced loss of muscle excitability and progression of chilling injury in chill-susceptible insect species.Supplementary material available online at