Significance: Insects comprise the largest class of animals and include numerous species of direct importance to humans, including pests and pollinators. Cold tolerance is arguably among the most important traits determining the distribution of insects. Hypothermia has been proposed to induce cell injury directly by promoting membrane phase transitions resulting in cell leak. Additionally, hypothermia induces hemolymph hyperkalemia, and it has been proposed that hypothermia induces injury indirectly through the resulting depolarization by inducing Ca²⁺influx promoting apoptosis/necrosis. Here we show that depolarization is a principal mechanism for hypothermic cell injury. Furthermore, we show that intracellular Ca²⁺increases upon depolarization and that this increase must flux through Ca²⁺channels in the membrane to accumulate and induce injury.

Recent studies suggest that projected rises of aquatic CO₂ levels cause acid–base regulatory responses in fishes that lead to altered GABAergic neurotransmission and disrupted behaviour, threatening fitness and population survival. It is thought that changes in Cl⁻ and HCO₃⁻ gradients across neural membranes interfere with the function of GABA-gated anion channels (GABA_A receptors). So far, such alterations have been revealed experimentally by exposing species living in low-CO₂ environments, like many oceanic habitats, to high levels of CO₂ (hypercapnia). To examine the generality of this phenomenon, we set out to study the opposite situation, hypothesizing that fishes living in typically hypercapnic environments also display behavioural alterations if exposed to low CO₂ levels. This would indicate that ion regulation in the fish brain is fine-tuned to the prevailing CO₂ conditions. We quantified pH regulatory variables and behavioural responses of Pangasianodon hypophthalmus, a fish native to the hypercapnic Mekong River, acclimated to high-CO₂ (3.1 kPa) or low-CO₂ (0.04 kPa) water. We found that brain and blood pH was actively regulated and that the low-CO₂ fish displayed significantly higher activity levels, which were reduced after treatment with gabazine, a GABA_A receptor blocker. This indicates an involvement of the GABA_A receptor and altered Cl⁻ and HCO₃⁻ ion gradients. Indeed, Goldman calculations suggest that low levels of environmental CO₂ may cause significant changes in neural ion gradients in P. hypophthalmus. Taken together, the results suggest that brain ion regulation in fishes is fine-tuned to the prevailing ambient CO₂ conditions and is prone to disruption if these conditions change.

Most insects have the ability to alter their cold tolerance in response to temporal temperature fluctuations, and recent studies have shown that insect cold tolerance is closely tied to the ability to maintain transmembrane ion-gradients that are important for the maintenance of cell membrane potential (Vm). Accordingly, several studies have suggested a link between preservation of Vm and cellular survival after cold stress, but none have measured Vm in this context. We tested this hypothesis by acclimating locusts (Locusta migratoria) to high (31°C) and low temperature (11°C) for four days before exposing them to cold stress (0°C) for up to 48 hours and subsequently measuring ion balance, cell survival, muscle Vm, and whole animal performance. Cold stress caused gradual muscle cell death which coincided with a loss of ion balance and depolarisation of muscle Vm. The loss of ion-balance and cell polarisation were, however, dampened markedly in cold-acclimated locusts such that the development of chill injury was reduced. To further examine the association between cellular injury and Vm we exposed in vitro muscle preparations to cold buffers with low, intermediate, or high [K+]. These experiments revealed that cellular injury during cold exposure occurs when Vm becomes severely depolarised. Interestingly we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend Vm whilst exposed to high extracellular [K+]. Together these results demonstrate a mechanism of cold-acclimation in locusts that improves survival after cold stress: Increased cold tolerance is accomplished by preservation of Vm through maintenance of ion homeostasis and decreased K+-sensitivity.

When cooled, insects first lose their ability to perform coordinated movements (CT_min) after which they enter chill coma (chill coma onset, CCO). Both these behaviours are popular measures of cold tolerance that correlate remarkably well with species distribution. To identify and understand the neuromuscular impairment that causes CT_min and CCO we used inter- and intraspecific model systems of Drosophila species that have varying cold tolerance as a consequence of adaptation or cold acclimation. Our results demonstrate that CT_min and CCO correlate strongly with a spreading depolarization (SD) within the central nervous system (CNS). We show that this SD is associated with a rapid increase in extracellular [K⁺] within the CNS causing neuronal depolarization that silences the CNS. The CNS shutdown is likely caused by a mismatch between passive and active ion transport within the CNS and in a different set of experiments we examine inter- and intraspecific differences in sensitivity to SD events during anoxic exposure. These experiments show that cold adapted or acclimated flies are better able to maintain ionoregulatory balance when active transport is compromised within the CNS. Combined, we demonstrate that a key mechanism underlying chill coma entry of Drosophila is CNS shutdown, and the ability to prevent this CNS shutdown is therefore an important component of acute cold tolerance, thermal adaptation and cold acclimation in insects.