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Abiotic stressors, such as cold exposure, can depolarize insect cells substantially causing cold coma and cell death. During cold exposure, insect skeletal muscle depolarization occurs through a 2-stage process. Firstly, short-term cold exposure reduces the activity of electrogenic ion pumps, which depolarize insect muscle markedly. Secondly, during long-term cold exposure, extracellular ion homeostasis is disrupted causing further depolarization. Consequently, many cold hardy insects improve membrane potential stability during cold exposure through adaptations that secure maintenance of ion homeostasis during cold exposure. Less is known about the adaptations permitting cold hardy insects to maintain membrane potential stability during the initial phase of cold exposure, before ion balance is disrupted. To address this problem it is critical to understand the membrane components (channels and transporters) that determine the membrane potential and to examine this question the present study constructed a mathematical "charge difference" model of the insect muscle membrane potential. This model was parameterized with known literature values for ion permeabilities, ion concentrations and membrane capacitance and the model was then further developed by comparing model predictions against empirical measurements following pharmacological inhibitors of the Na + /K + ATPase, Cl - channels and symporters. Subsequently, we compared simulated and recorded membrane potentials at 0 and 31 °C and at 10-50 mM extracellular [K + ] to examine if the model could describe membrane potentials during the perturbations occurring during cold exposure. Our results confirm the importance of both Na + /K + ATPase activity and ion-selective Na + , K + and Cl - channels, but the model also highlights that additional electroneutral flux of Na + and K + is needed to describe how membrane potentials respond to temperature and [K + ] in insect muscle. While considerable further work is still needed, we argue that this "charge difference" model can be used to generate testable hypotheses of how insects can preserve membrane polarization in the face of stressful cold exposure., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K + concentration ([K + ]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria , has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K + permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na + -K + pump blocker) or 4-aminopyridine (4-AP; voltage-gated K + channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K + permeability, facilitated by opening of 4-AP-sensitive K + channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K + channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

Cold exposure is known to induce stressful imbalances in chill susceptible insects, including loss of hemolymph water, hyperkalemia and cell depolarization. Cold induced depolarization induces uncontrolled Ca 2+ influx and accumulation of injury through necrosis/apoptosis. Conversely cold induced Ca 2+ influx has been shown to induce rapid cold hardening and therefore also play a role to reduce cold injury. Cold acclimation is known to reduce cold injury in insects and due to the involvement of depolarization and Ca 2+ in the pathophysiology of hypothermia, we hypothesized that cold acclimation modulates voltage gated Ca 2+ channels and fiber excitability. Using intracellular electrodes or force transducers, we measured the Ca 2+ currents, fiber excitability and muscle contractility in warm (31 °C) and cold (11 °C) acclimated locusts. Experiments were performed under conditions ranging from mild conditions where the membrane potential is well regulated to stressful conditions, where the membrane potential is very depolarized and the tissue is at risk of accumulating injury. These experiments found that cold acclimation modulates Ca 2+ currents and fiber excitability in a manner that depends on the cold exposure. Thus, under mild conditions, Ca 2+ currents and fiber excitability was increased whilst muscle contractility was unaffected by cold acclimation. Conversely, fiber excitability and muscle contractility was decreased under stressful conditions. Further work is required to fully understand the adaptive effects of these modulations. However, we propose a model which reconciles the dualistic role of the Ca 2+ ion in cold exposure and cold acclimation. Thus, increased Ca 2+ currents at mild temperatures could help to enhance cold sensing capacity whereas reduced fiber excitability under stressful conditions could help to reduce catastrophic Ca 2+ influx during periods of severe cold exposure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)