The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Effects of a high cholesterol diet on chill tolerance are highly context-dependent in Drosophila.

Chill susceptible insects are thought to be injured through different mechanisms depending on the duration and severity of chilling. While chronic chilling causes "indirect" injury through disruption of metabolic and ion homeostasis, acute chilling is suspected to cause "direct" injury, in part through phase transitions of cell membrane lipids. Dietary supplementation of cholesterol can reduce acute chilling injury in Drosophila melanogaster (Shreve et al., 2007), but the generality of this effect and the mechanisms underlying it remain unclear. To better understand how and why cholesterol has this effect, we assessed how a high cholesterol diet and thermal acclimation independently and interactively impact several measures of chill tolerance. Cholesterol supplementation positively affected tolerance to acute chilling in warm-acclimated flies (as reported previously). Conversely, feeding on the high-cholesterol diet negatively affected tolerance to chronic chilling in both cold and warm acclimated flies, as well as tolerance to acute chilling in cold acclimated flies. Cholesterol had no effect on the ability of flies to remain active in the cold or recover movement after a cold stress. Our findings support the idea that dietary cholesterol reduces mechanical injury to membranes caused by direct chilling injury, and that acute and chronic chilling are associated with distinct mechanisms of injury. Feeding on a high-cholesterol diet may interfere with mechanisms involved in cold acclimation, leaving cholesterol augmented flies more susceptible to chilling injury under some conditions.

A neurophysiological limit and its biogeographic correlations: cold-induced spreading depolarization in tropical butterflies.

The physiology of insects is directly influenced by environmental temperature, and thermal tolerance is therefore intrinsically linked to their thermal niche and distribution. Understanding the mechanisms that limit insect thermal tolerance is crucial to predicting biogeography and range shifts. Recent studies on locusts and flies suggest that the critical thermal minimum (CTmin) follows a loss of CNS function via a spreading depolarization. We hypothesized that other insect taxa share this phenomenon. Here, we investigate whether spreading depolarization events occur in butterflies exposed to cold. Supporting our hypothesis, we found that exposure to stressful cold induced spreading depolarization in all 12 species tested. This reinforces the idea that spreading depolarization is a common mechanism underlying the insect CTmin. Furthermore, our results highlight how CNS function is tuned to match the environment of a species. Further research into the physiology underlying spreading depolarization will likely elucidate key mechanisms determining insect thermal tolerance and ecology.

Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance.

The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint exists.

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

Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na+/K+-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. Here, we tested this hypothesis by measuring activity and thermal sensitivity of the Na+/K+-ATPase at the tagma-specific level (head, thorax and abdomen) in warm- (25 °C) and cold-acclimated (15 °C) flies by measuring Na+/K+-ATPase activity at 15, 20, and 25 °C. We relate differences in pump activity to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While thermal sensitivity remained unchanged, cold-acclimated flies had decreased Na+/K+-ATPase activity in the thorax (mainly muscle) and head (mainly composed of brain). We argue that these changes may assist in maintenance of K+ homeostasis and membrane potential across muscle membranes, and discuss how reduced Na+/K+-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.

The Integrative Physiology of Insect Chill Tolerance.

Cold tolerance is important in defining the distribution of insects. Here, we review the principal physiological mechanisms underlying homeostatic failure during cold exposure in this diverse group of ectotherms. When insects are cooled sufficiently, they suffer an initial loss of neuromuscular function (chill coma) that is caused by decreased membrane potential and reduced excitability of the neuromuscular system. For chill-susceptible insects, chronic or severe chilling causes a disruption of ion and water homeostasis across membranes and epithelia that exacerbate the initial effects of chilling on membrane potential and cellular function, and these perturbations are tightly associated with the development of chill injury and mortality. The adaptation and acclimation responses that allow some insects to tolerate low temperatures are multifactorial and involve several physiological systems and biochemical adjustments. In this review, we outline a physiological model that integrates several of these responses and discuss how they collectively help to preserve cellular, organ, and organismal homeostasis at low temperature.

Hyperkalaemia, not apoptosis, accurately predicts insect chilling injury.

There is a growing appreciation that insect distribution and abundance are associated with the limits of thermal tolerance, but the physiology underlying thermal tolerance remains poorly understood. Many insects, like the migratory locust (Locusta migratoria), suffer a loss of ion and water balance leading to hyperkalaemia (high extracellular [K+]) in the cold that indirectly causes cell death. Cells can die in several ways under stress, and how they die is of critical importance to identifying and understanding the nature of thermal adaptation. Whether apoptotic or necrotic cell death pathways are responsible for low-temperature injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptotic cell death 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, extracellular [K+] and muscle caspase-3 activity in individual locusts to gain further insight into the mechanistic nature of chilling injury. 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 muscle cell death. Levels of chilling injury measured at the whole animal level, however, were strongly correlated with the degree of haemolymph hyperkalaemia, and not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main form of cell damage driving low-temperature injury.

Chilling induces unidirectional solute leak through the locust gut epithelia.

Chill-susceptible insects, like the migratory locust, often die when exposed to low temperatures from an accumulation of tissue damage that is unrelated to freezing (chilling injury). Chilling injury is often associated with a loss of ion balance across the gut epithelia. It has recently been suggested that this imbalance is at least partly caused by a cold-induced disruption of epithelial barrier function. Here, we aimed to test this hypothesis in the migratory locust (Locustamigratoria). First, chill tolerance was quantified by exposing locusts to -2°C and recording chill coma recovery time and survival 24 h post-cold exposure. Longer exposure times significantly increased recovery time and caused injury and death. Ion-selective microelectrodes were also used to test for a loss of ion balance in the cold. We found a significant increase of haemolymph K+ and decrease of haemolymph Na+ concentration over time. Next, barrier failure along the gut was tested by monitoring the movement of an epithelial barrier marker (FITC-dextran) across the gut epithelia during exposure to -2°C. We found a significant increase in haemolymph FITC-dextran concentration over time in the cold when assayed in the mucosal to serosal direction. However, when tested in the serosal to mucosal direction, we saw minimal marker movement across the gut epithelia. This suggests that while cold-induced barrier disruption is present, it is apparently unidirectional. It is important to note that these data reveal only the phenomenon itself. The location of this leak as well as the underlying mechanisms remain unclear and require further investigation.

Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia.

Under conditions of stress, many animals suffer from epithelial barrier disruption that can cause molecules to leak down their concentration gradients, potentially causing a loss of organismal homeostasis, further injury or death. Drosophila is a common insect model, used to study barrier disruption related to aging, traumatic injury, or environmental stress. Net leak of a non-toxic dye (Brilliant blue FCF) from the gut lumen to the hemolymph is often used to identify barrier failure under these conditions, but Drosophila are capable of actively transporting structurally-similar compounds. Here, we examined whether cold stress (like other stresses) causes Brilliant blue FCF (BB-FCF) to appear in the hemolymph of flies fed the dye, and if so whether Drosophila are capable of clearing this dye from their body following chilling. Using in situ midgut leak and transport assays as well as Ramsay assays of Malpighian tubule transport, we tested whether these ionoregulatory epithelia can actively transport BB-FCF. In doing so, we found that the Drosophila midgut and Malpighian tubules can mobilize BB-FCF via an active transcellular pathway, suggesting that elevated concentrations of the dye in the hemolymph may occur from increased paracellular permeability, reduced transcellular clearance, or both. SUMMARY STATEMENT: Drosophila are able to actively secrete Brilliant blue FCF, a commonly used marker of barrier dysfunction.

Dissecting cause from consequence: a systematic approach to thermal limits.

Thermal limits mark the boundaries of ectotherm performance, and are increasingly appreciated as strong correlates and possible determinants of animal distribution patterns. The mechanisms setting the thermal limits of ectothermic animals are under active study and rigorous debate as we try to reconcile new observations in the lab and field with the knowledge gained from a long history of research on thermal adaptation. Here, I provide a perspective on our divided understanding of the mechanisms setting thermal limits of ectothermic animals. I focus primarily on the fundamental differences between high and low temperatures, and how animal form and environment can place different constraints on different taxa. Together, complexity and variation in animal form drive complexity in the interactions within and among levels of biological organization, creating a formidable barrier to determining mechanistic cause and effect at thermal limits. Progress in our understanding of thermal limits will require extensive collaboration and systematic approaches that embrace this complexity and allow us to separate the causes of failure from the physiological consequences that can quickly follow. I argue that by building integrative models that explain causal links among multiple organ systems, we can more quickly arrive at a holistic understanding of the varied challenges facing animals at extreme temperatures.

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

The mosquito Aedes aegypti is largely confined to tropical and subtropical regions, but its range has recently been spreading to colder climates. As insect biogeography is tied to environmental temperature, understanding the limits of A. aegypti thermal tolerance and their capacity for phenotypic plasticity is important in predicting the spread of this species. In this study, we report on the chill coma onset (CCO) and recovery time (CCRT), as well as low-temperature survival phenotypes of larvae and adults of A. aegypti that developed or were acclimated to 15°C (cold) or 25°C (warm). Cold acclimation did not affect CCO temperatures of larvae but substantially reduced CCO in adults. Temperature and the duration of exposure both affected CCRT, and cold acclimation strongly mitigated these effects and increased rates of survival following prolonged chilling. Female adults were far less likely to take a blood meal when cold acclimated, and exposing females to blood (without feeding) attenuated some of the beneficial effects of cold acclimation on CCRT. Lastly, larvae suffered from haemolymph hyperkalaemia when chilled, but cold acclimation attenuated the imbalance. Our results demonstrate that A. aegypti larvae and adults have the capacity to acclimate to low temperatures, and do so at least in part by better maintaining ion balance in the cold. This ability for cold acclimation may facilitate the spread of this species to higher latitudes, particularly in an era of climate change.

Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster.

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K+] in the hemolymph (hyperkalemia). Cold acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. Here, we test whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to 7 days at warm (25°C) or cold (10°C) acclimation conditions. The cold-acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K+ balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K+ secretion rates in the cold, as well as reduced rectal K+ reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, we measured the activities of Na+/K+-ATPase and V-type H+-ATPase at the Malpighian tubules, midgut, and hindgut. Na+/K+-ATPase and V-type H+-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold-acclimated flies, and were not predictive of the observed alterations in K+ transport. Our results suggest that modification of Malpighian tubule and gut ion and water transport probably prevents cold-induced hyperkalemia in cold-acclimated flies, and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na+/K+- and V-type H+-ATPases.

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance.

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of CAPA (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. Here, we provide evidence that low (femtomolar) and high (micromolar) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K+ clearance rates and reduce chill tolerance, high doses facilitate K+ clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, we estimated the maximum achievable hormonal titres of CAPA and found further evidence that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster We provide the first evidence of a neuropeptide that can negatively affect cold tolerance in an insect and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model.

Cold acclimation improves chill tolerance in the migratory locust through preservation of ion balance and membrane potential.

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 (Vm). Several studies have therefore suggested a link between preservation of Vm and cellular survival after cold stress, but none has measured Vm 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 Vm, and whole animal performance. Cold stress caused gradual muscle cell death, which coincided with a loss of ion balance and depolarization of muscle Vm 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 Vm we exposed in vitro muscle preparations to cold buffers with low, intermediate, or high [K+]. These experiments revealed that cellular injury during cold exposure occurs when Vm becomes severely depolarized. Interestingly, we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend Vm 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 Vm through maintenance of ion homeostasis and decreased K+ sensitivity.

Cold tolerance of Drosophila species is tightly linked to the epithelial K+ transport capacity of the Malpighian tubules and rectal pads.

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 Drosophila 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 Drosophila 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.

Cold mortality is not caused by oxygen limitation or loss of ion homeostasis in the tropical freshwater shrimp Macrobrachium rosenbergii.

Using the tropical crustacean Macrobrachium rosenbergii we investigate two popular hypotheses proposed to explain loss of function in ectotherms exposed to critically high and low temperatures. Specifically, we examine whether acute cold stress disrupts hemolymph and muscle ion balance or causes a loss of oxygen availability. We found that acute cold stress causes loss of righting response at 13 °C, but that a cold-induced loss of ion-balance only occurs after onset of mortality. In regards to oxygen availability, we found no decrease in hemolymph oxygen content during cold exposure, and no changes in the concentrations of the anaerobic end products l-lactate and succinate in the tail muscle of the shrimp. Therefore, our results support neither of these two popular hypotheses and it remains unknown what physiological perturbations determine the lower limits of thermal tolerance in Macrobrachium rosenbergii.

Heat stress is associated with disruption of ion balance in the migratory locust, Locusta migratoria.

Thermal tolerance is important in determining the spatial and temporal distributions of insects but the mechanisms which determine upper thermal limits remain poorly understood. In terrestrial insects heat tolerance is unlikely to be limited by oxygen supply but in some arthropods, heat stress has been shown to cause haemolymph hyperkalaemia which is known to have detrimental effects on neuromuscular excitability. It is however unresolved if heat-induced hyperkalemia is the cause or the result of cellular heat injury. To address the putative role of heat-induced hyperkalemia we quantified changes in ion and water balance in haemolymph and muscle tissue of the migratory locust during exposure to two static temperatures clustered around the CTmax (48°C and 50°C). We show that heat stress caused a loss of ion balance and severe haemolymph hyperkalaemia which coincided with the onset of heat stupor. Locusts were able to maintain their haemolymph volume throughout exposure, suggesting it is unlikely that osmoregulatory failure is responsible for haemolymph hyperkalaemia. When locusts were allowed to recover from heat stupor, they recovered ion balance quickly but were still unable to function optimally after 24h. The results therefore indicate that both the haemolymph hyperkalaemia and associated depression of muscular function (heat stupor) are secondary results of cellular heat injury and that the cause of heat stupor most be sought elsewhere.

Paralysis and heart failure precede ion balance disruption in heat-stressed European green crabs.

Acute exposure of ectotherms to critically high temperatures causes injury and death, and this mortality has been associated with a number of physiological perturbations including impaired oxygen transport, loss of ion and water homeostasis, and neuronal failure. It is difficult to discern which of these factors, if any, is the proximate cause of heat injury because, for example, loss of ion homeostasis can impair neuromuscular function (including cardiac function), and conversely impaired oxygen transport reduces ATP supply and can thus reduce ion transport capacity. In this study we investigated if heat stress causes a loss of ion homeostasis in marine crabs and examined if such loss is related to heart failure. We held crabs (Carcinus maenas) at temperatures just below their critical thermal maximum and measured extracellular (hemolymph) and intracellular (muscle) ion concentrations over time. Analysis of Arrhenius plots for heart rates during heating ramps revealed a breakpoint temperature below which heart rate increased with temperature, and above which heart rate declined until complete cardiac failure. As hypothesised, heat stress reduced the Nernst equilibrium potentials of both K+ and Na+, likely causing a depolarization of the membrane potential. To examine whether this loss of ion balance was likely to cause disruption of neuromuscular function, we exposed crabs to the same temperatures, but this time measured ion concentrations at the individual-specific times of complete paralysis (from which the crabs never recovered), and at the time of cardiac failure. Loss of ion balance was observed only after both paralysis and complete heart failure had occurred; indicating that the loss of neuromuscular function is not caused by a loss of ion homeostasis. Instead we suggest that the observed loss of ion balance may be linked to tissue damage related to heat death.