Abstract
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 Ca(2+) channels, causing a Ca(2+) overload that triggers apoptotic/necrotic pathways. In accordance with this hypothesis, we show that hyperkalemic depolarization causes a marked increase in intracellular Ca(2+) levels. Furthermore, using pharmacological manipulation of intra- and extracellular Ca(2+) concentrations as well as Ca(2+) channel conductance, we demonstrate that injury is prevented if transmembrane Ca(2+) flux is prevented by removing extracellular Ca(2+) or blocking Ca(2+) influx. Together these findings demonstrate a causal relationship between cold-induced hyperkalemia, depolarization, and the development of chill injury through Ca(2+)-mediated necrosis/apoptosis.