Cold exposure causes cell death by depolarization-mediated Ca²⁺overload in a chill-susceptible insect

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 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.

Chilling induces unidirectional solute leak through the locust gut epithelia

“…as dead/alive, or a range of conditions from dead to alive) to indirectly measure the degree of injury sustained (Findsen et al, 2013; Overgaard and MacMillan, 2017). While each of these chill tolerance traits operate via distinct mechanisms, they have all been in some manner associated with a local or systemic loss of ion balance, suggesting that ionoregulatory failure may be a principal cause of chill susceptibility (Armstrong et al, 2012; Bayley et al, 2018; Findsen et al, 2013; Koštál et al, 2006; MacMillan et al, 2015a; Robertson et al, 2017).…”

“…Through the continuous ionoregulatory actions of the alimentary canal, the hemolymph of most insects contains high and low concentrations of Na + and K + , respectively, at optimal or near-optimal thermal conditions (Engel and Moran, 2013; MacMillan et al, 2015b; Maddrell and O’Donnell, 1992). In cold conditions, however, the activity of ionoregulatory enzymes like V-ATPases and Na + /K + -ATPases is suppressed (Bayley et al, 2018; Hosler et al, 2000; Mandel et al, 1980; McMullen and Storey, 2008; Moriyama and Nelson, 1989; Yerushalmi et al, 2018). Over time, this temperature-induced suppression of transcellular ion transport often results in a net leak of hemolymph Na + and water (which follows Na + osmotically) to the gut lumen, effectively concentrating the K + that remains in the hemolymph (MacMillan and Sinclair, 2011b).…”

“…Some intracellular K + simultaneously leaking down its concentration gradient into the extracellular space worsens this problem (Andersen et al, 2017; MacMillan et al, 2014). As K + concentrations rise in the hemocoel (hyperkalemia), the gradient of K + across the cell membrane is lost, and a marked depolarization in membrane potential occurs, eventually resulting in the activation of voltage-gated Ca 2+ channels (Andersen et al, 2017a; Bayley et al, 2018; MacMillan et al, 2015a). The influx of Ca 2+ that ensues is proposed to initiate a crippling cascade which causes a deterioration of cellular integrity, likely through apoptosis (Mattson and Chan, 2003; Nicotera and Orrenius, 1998; Yi and Lee, 2011; Yi et al, 2007).…”

Warm periods in repeated cold stresses protectDrosophilaagainst ionoregulatory collapse, chilling injury, and reproductive deficits

“…When a chill-susceptible insect remains at unfavourable low temperatures for a prolonged period, it can start to accumulate chilling injuries that manifest as motor defects or an inability to recover the ability to move or continue development upon rewarming (Koštál et al, 2006, 2004; MacMillan and Sinclair, 2011). Damage to the muscles, in particular, is thought to be caused by hemolymph hyperkalemia that causes a depolarization of muscle membrane potentials, dysregulation of Ca 2+ balance, and activation of apoptotic signalling cascades (Andersen et al, 2017; Bayley et al, 2018; MacMillan et al, 2015b).…”

An impressive capacity for cold tolerance plasticity protects against ionoregulatory collapse in the disease vector,Aedes aegypti

“…The temperature at which this paralytic state occurs is called the chill coma onset temperature (CCO) (Overgaard & MacMillan, 2017). With time spent at low temperatures, chill susceptible insects lose ion and water balance and suffer from hemolymph hyperkalemia (high [K + ]), which further depolarizes cells and activates voltage-gated calcium channels, driving rampant cellular apoptosis (Bayley et al, 2018; MacMillan, Andersen, Davies, & Overgaard, 2015; MacMillan, Baatrup, & Overgaard, 2015). The severity of this loss of homeostasis increases with longer or lower temperature exposures, and the tissue damage that accrues while an insect is in this state is thought to largely determine its survival and fitness following rewarming (Overgaard & MacMillan, 2017).…”