Hyperkalemia, not apoptosis, accurately predicts chilling injury in individual locusts

Abstract

<jats:title>Abstract</jats:title><jats:p>During prolonged or severe chilling, the majority of insects accrue chilling injuries that are typically quantified by scoring neuromuscular function after rewarming. In the cold, these chill susceptible insects, like the migratory locust (<jats:italic>Locusta migratoria</jats:italic>) suffer a loss of ion and water balance that is hypothesized to initiate cell death. Whether apoptotic or necrotic cell death pathways are responsible for this chilling injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptosis in three locust tissues (muscle, nerves, and midgut) following prolonged chilling and recovery from an injury-inducing cold exposure. Furthermore, we obtain matching measurements of injury, hemolymph [K<jats:sup>+</jats:sup>], and muscle caspase-3 activity in individual locusts to gain further insight into mechanistic nature of chilling injury. We hypothesized that apoptotic cell death in both muscle and nerve tissue drives motor defects following cold exposure in insects, and that there would be a strong association between cold- induced injury, hyperkalemia, and muscle caspase-3 activity. We found a significant increase in muscle caspase-3 activity, but no such increase was observed in either nervous or gut tissue from the same animals, suggesting that chill injury primarily relates to apoptotic muscle cell death. However, the levels of chilling injury measured at the whole animal level prior to tissue sampling were strongly correlated with the degree of hemolymph hyperkalemia, but not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main cell death pathway driving injury in the cold.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>Temperature has profound effects on animal fitness and sets limits to animal distribution. To understand and model insect responses to climate, we need to know how temperature sets limits to their survival. There is strong evidence that a collapse of ion and water balance occurs in insects in the cold, and it is generally held that the resulting cold injury is caused by activation of programmed cell death (apoptosis). Here, we directly test this idea and show for the first time that although the loss of ion balance is a strong predictor of individual survival outcomes, apoptosis is not the primary cause of cold-induced injury.</jats:p></jats:sec>

Thermal acclimation alters Na+/K+-ATPase activity in a tissue-specific manner in Drosophila melanogaster

“…muscle; Overgaard and MacMillan, 2017). This systemic coldand hyperkalemia-induced depolarization is thought to initiate cell death, particularly in the muscle tissue and ultimately leading to organismal chilling injury (Bayley et al, 2018;Carrington et al, 2020;MacMillan et al, 2015b). If an insect is removed from the cold before extensive injury occurs, successful recovery from chill coma is dependent on the rate of hemolymph ion and water balance restoration (MacMillan et al, 2012), which is tightly linked to the capacity of the renal system to restore hemolymph volume, and Na + and K + balance (MacMillan et al, 2014;Overgaard and MacMillan, 2017).…”

Thermal acclimation alters the roles of Na+/K+-ATPase activity in a tissue-specific manner inDrosophila melanogaster

“…muscle; Overgaard and MacMillan, 2017). This systemic cold- and hyperkalemia-induced depolarization is thought to initiate cell death, particularly in the muscle tissue and ultimately leading to organismal chilling injury (Bayley et al, 2018; Carrington et al, 2020; MacMillan et al, 2015b). If an insect is removed from the cold before extensive injury occurs, successful recovery from chill coma is dependent on the rate of hemolymph ion and water balance restoration (MacMillan et al, 2012), which is tightly linked to the capacity of the renal system to restore hemolymph volume, and Na + and K + balance (MacMillan et al, 2014; Overgaard and MacMillan, 2017).…”

Investigating a Link Between Cold Stress and Bacterial Leak from the Gut of Locusta Migratoria

“…Unlike freeze-avoidant and freeze-tolerant insects which incur injuries related to freezing, chill-susceptible species suffer chilling injuries at low temperatures above the freezing point of their body fluids (Overgaard and MacMillan, 2017). These injuries are thought to be driven by cell death as a result of membrane phase transitions and the loss of ion homeostasis (Andersen et al, 2017;Bayley et al, 2018;Carrington et al, 2020;Koštál et al, 2004;MacMillan and Sinclair, 2011b).…”

“…An injury or mortality assay can also be used a period of time after measuring CCRT, usually a day or more post-cold exposure, in order to quantify the extent of chilling injuries. This assay typically employs a point-based scale to assess an insect's movement or behaviour, with more injured insects exhibiting greater degrees of chilling injuries (Brzezinski and MacMillan, 2020;Carrington et al, 2020;El-Saadi et al, 2020;MacMillan et al, 2014). Both CCRT and survival/injury assays are useful tools when used together, as they are commonly used by ecologists to find patterns or differences in thermal tolerance at the population or species level of an insect (Davis et al, 2021;MacMillan et al, 2015a;Ransberry et al, 2015;Slatyer and Schoville, 2016)…”

“…This pathway triggers apoptosis in neighbouring cells and recruits hemocytes to the site of wounding where phagocytosis occurs to clear the area of debris (Marmaras and Lampropoulou, 2009;Shaukat et al, 2015). This may explain the increase in the number of circulating hemocytes following tissue damage or a low temperature exposure in D. melanogaster (Pastor-Pareja et al, 2008;Salehipour-shirazi et al, 2017), as cell death is a common consequence of cold stress in insects, including locusts, and has been measured repeatedly (Andersen et al, 2017;Bayley et al, 2018;MacMillan et al, 2015b;MacMillan et al, 2017;Carrington et al, 2020). Furthermore, immune activation occurs in D. melanogaster upon the detection of actin in the hemolymph.…”