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 (V m ). Several studies have therefore suggested a link between preservation of V m and cellular survival after cold stress, but none has measured V m in this context. We tested this hypothesis by acclimating locusts (Locusta migratoria) to high (31°C) and low temperature (11°C) for 4 days before exposing them to cold stress (0°C) for up to 48 h and subsequently measuring ion balance, cell survival, muscle V m , and whole animal performance. Cold stress caused gradual muscle cell death, which coincided with a loss of ion balance and depolarization of muscle V m . The loss of ion balance and cell polarization 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 V m we exposed in vitro muscle preparations to cold buffers with low, intermediate, or. These experiments revealed that cellular injury during cold exposure occurs when V m becomes severely depolarized. Interestingly, we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend V m 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 V m through maintenance of ion homeostasis and decreased K + sensitivity.

Cold tolerance of insects is arguably among the most important traits defining their geographical distribution. Even so, very little is known regarding the causes of cold injury in this species-rich group. In many insects it has been observed that cold injury coincides with a cellular depolarization caused by hypothermia and hyperkalemia that develop during chronic cold exposure. However, prior studies have been unable to determine if cold injury is caused by direct effects of hypothermia, by toxic effects of hyperkalemia, or by the depolarization that is associated with these perturbations. Here we use a fluorescent DNA-staining method to estimate cell viability of muscle and hindgut tissue from Locusta migratoria and show that the cellular injury is independent of the direct effects of hypothermia or toxic effects of hyperkalemia. Instead, we show that chill injury develops due to the associated cellular depolarization. We further hypothesized that the depolarization-induced injury was caused by opening of voltage-sensitive Ca2+ channels, causing a Ca2+ overload that triggers apoptotic/necrotic pathways. In accordance with this hypothesis, we show that hyperkalemic depolarization causes a marked increase in intracellular Ca2+ levels. Furthermore, using pharmacological manipulation of intra- and extracellular Ca2+ concentrations as well as Ca2+ channel conductance, we demonstrate that injury is prevented if transmembrane Ca2+ flux is prevented by removing extracellular Ca2+ or blocking Ca2+ influx. Together these findings demonstrate a causal relationship between cold-induced hyperkalemia, depolarization, and the development of chill injury through Ca2+-mediated necrosis/apoptosis.

When cooled, insects first lose their ability to perform coordinated movements (CT) 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 and CCO we used inter- and intraspecific model systems of species that have varying cold tolerance as a consequence of adaptation or cold acclimation. Our results demonstrate that CT 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 to be 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 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.

Insect chill tolerance is strongly associated with the ability to maintain ion and water homeostasis during cold exposure. Maintenance of K balance is particularly important due to its role in setting the cell membrane potential that is involved in many aspects of cellular function and viability. In most insects, K balance is maintained through secretion at the Malpighian tubules, which balances reabsorption from the hindgut and passive leak arising from the gut lumen. Here, we used the scanning ion-selective electrode technique (SIET) at benign (23°C) and low (6°C) temperatures to examine K flux across the Malpighian tubules and the rectal pads in the hindgut in five species that differ in cold tolerance. We found that chill-tolerant species were better at maintaining K secretion and suppressing reabsorption during cold exposure. In contrast, chill-susceptible species exhibited large reductions in secretion with no change, or a paradoxical increase, in K reabsorption. Using an assay to measure paracellular leak, we found that chill-susceptible species experience a large increase in leak during cold exposure, which could explain the apparent increase in K reabsorption found in these species. Our data therefore strongly support the hypothesis that cold-tolerant species are better at maintaining K homeostasis through an increased ability to maintain K secretion rates and through reduced movement of K towards the hemolymph. These adaptations are manifested both at the Malpighian tubule and at the rectal pads in the hindgut, and ensure that cold-tolerant species experience less perturbation of K homeostasis during cold stress.

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