Heath A. MacMillan

How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits

Summary 1. Thermal tolerance may limit and therefore predict ectotherm geographic distributions. However, which of the many metrics of thermal tolerance best predict distribution is often unclear, even for drosophilids, which constitute a popular and well-described animal model. 2. Five metrics of cold tolerance were measured for 14 Drosophila species to determine which metrics most strongly correlate with geographic distribution. The species represent tropical to temperate regions but all were reared under similar (common garden) conditions (20 °C). The traits measured were: chill coma temperature (CTmin), lethal temperature (LTe50), lethal time at low temperature (LTi50), chill coma recovery time (CCRT) and supercooling point (SCP). 3. Measures of CTmin, LTe50 and LTi50 proved to be the best predictors to describe the variation in realized latitudinal distributions (R 2 =0 699, R 2 =0 741 and 0550, respectively) and estimated environmental cold exposure (R 2 =0 633, R 2 =0 641 and 0511, respectively). Measures of CCRT also correlated significantly with estimated minimum temperature (R 2 =0 373), while the SCP did not. These results remained consistent after phylogenetically independent analysis or when applying nonlinear regression. Moreover, our findings were supported by a similar analysis based on existing data compiled from the Drosophila cold tolerance literature. 4. Trait correlations were strong between LTe50, LTi50 and CTmin, respectively (083 > R 2 >0 55). However, surprisingly, there was only a weak correlation between the entrance into coma (CTmin) and the recovery from chill coma (CCRT) (R 2 =0 256). 5. Considering the findings of the present study, data from previous studies and the logistical constraints of each measure of cold tolerance, we conclude that CTmin and LTe50 are superior measures when estimating the ecologically relevant cold tolerance of drosophilids. Of these two traits, CTmin requires less equipment, time and animals and thereby presents a relatively fast, simple and dynamic measure of cold tolerance.

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.

The capacity to maintain ion and water homeostasis underlies interspecific variation in Drosophila cold tolerance

Many insects, including Drosophila, succumb to the physiological effects of chilling at temperatures well above those causing freezing. Low temperature causes a loss of extracellular ion and water homeostasis in such insects, and chill injuries accumulate. Using an integrative and comparative approach, we examined the role of ion and water balance in insect chilling susceptibility/ tolerance. The Malpighian tubules (MT), of chill susceptible Drosophila species lost [Na+] and [K+] selectivity at low temperatures, which contributed to a loss of Na+ and water balance and a deleterious increase in extracellular [K+]. By contrast, the tubules of chill tolerant Drosophila species maintained their MT ion selectivity, maintained stable extracellular ion concentrations, and thereby avoided injury. The most tolerant species were able to modulate ion balance while in a cold-induced coma and this ongoing physiological acclimation process allowed some individuals of the tolerant species to recover from chill coma during low temperature exposure. Accordingly, differences in the ability to maintain homeostatic control of water and ion balance at low temperature may explain large parts of the wide intra- and interspecific variation in insect chilling tolerance.

Cold-induced depolarization of insect muscle: Differing roles of extracellular K+ during acute and chronic chilling

Insects enter chill coma, a reversible state of paralysis, at temperatures below their critical thermal minimum (CTmin), and the time required for an insect to recover after a cold exposure is termed chill coma recovery time (CCRT). The CTmin and CCRT are both important metrics of insect cold tolerance that are used interchangeably, although chill coma recovery is not necessarily permitted by a direct reversal of the mechanism causing chill coma onset. Nevertheless, onset and recovery of coma have been attributed to loss of neuromuscular function due to depolarization of muscle fibre membrane potential (Vm). Here we test the hypothesis that muscle depolarization at chill coma onset and repolarization during chill coma recovery are caused by changes in extracellular [K+] and/or other effects of low temperature. Using Locusta migratoria, we measured in vivo muscle resting potentials of the extensor tibialis during cooling, following prolonged exposure to −2°C and during chill coma recovery, and related changes in Vm to transmembrane [K+] balance and temperature. Although Vm was rapidly depolarized by cooling, hemolymph [K+] did not rise until locusts had spent considerable time in the cold. Nonetheless, a rise in hemolymph [K+] during prolonged cold exposure further depressed muscle resting potential and slowed recovery from chill coma upon rewarming. Muscle resting potentials had a bimodal distribution, and with elevation of extracellular [K+] (but not temperature) muscle resting potentials become unimodal. Thus, a disruption of extracellular [K+] does depolarize muscle resting potential and slow CCRT following prolonged cold exposure. However, onset of chill coma at the CTmin relates to an as-yet-unknown effect of temperature on neuromuscular function.

Sodium distribution predicts the chill tolerance of Drosophila melanogaster raised in different thermal conditions

Many insects, including the model holometabolous insect Drosophila melanogaster, display remarkable plasticity in chill tolerance in response to the thermal environment experienced during development or as adults. At low temperatures, many insects lose the ability to regulate Na(+) balance, which is suggested to cause a secondary loss of hemolymph water to the tissues and gut lumen that concentrates the K(+) remaining in the hemolymph. The resultant increase in extracellular [K(+)] inhibits neuromuscular excitability and is proposed to cause cellular apoptosis and injury. The present study investigates whether and how variation in chill tolerance induced through developmental and adult cold acclimation is associated with changes in Na(+), water, and K(+) balance. Developmental and adult cold acclimation improved the chilling tolerance of D. melanogaster in an additive manner. In agreement with the proposed model, these effects were intimately related to differences in Na(+) distribution prior to cold exposure, such that chill-tolerant flies had low hemolymph [Na(+)], while intracellular [Na(+)] was similar among treatment groups. The low hemolymph Na(+) of cold-acclimated flies allowed them to maintain hemolymph volume, prevent hyperkalemia, and avoid injury following chronic cold exposure. These findings extend earlier observations of hemolymph volume disruption during cold exposure to the most ubiquitous model insect (D. melanogaster), highlight shared mechanisms of developmental and adult thermal plasticity and provide strong support for ionoregulatory failure as a central mechanism of insect chill susceptibility.

Concurrent effects of cold and hyperkalaemia cause insect chilling injury.

Chilling injury and death are the ultimate consequence of low temperature exposure for chill susceptible insects, and low temperature tolerance is considered one of the most important factors determining insect distribution patterns. The physiological mechanisms that cause chilling injury are unknown, but chronic cold exposure that causes injury is consistently associated with elevated extracellular [K+], and cold tolerant insects possess a greater capacity to maintain ion balance at low temperatures. Here, we use the muscle tissue of the migratory locust (Locusta migratoria) to examine whether chill injury occurs during cold exposure or following return to benign temperature and we specifically examine if elevated extracellular [K+], low temperature, or a combination thereof causes cell death. We find that in vivo chill injury occurs during the cold exposure (when extracellular [K+] is high) and that there is limited capacity for repair immediately following the cold stress. Further, we demonstrate that that high extracellular [K+] causes cell death in situ, but only when experienced at low temperatures. These findings strongly suggest that that the ability to maintain ion (particularly K+) balance is critical to insect low temperature survival, and highlight novel routes of study in the mechanisms regulating cell death in insects in the cold.

Muscle membrane potential and insect chill coma

ABSTRACT Chill-susceptible insects enter a reversible paralytic state, termed chill coma, at mild low temperatures. Chill coma is caused by neuromuscular impairment, allegedly triggered by cold-induced depolarization of muscle resting membrane potential (Vm). We used five Drosophila species that vary in cold tolerance (chill coma temperature spanning ∼11°C) and repeatedly measured muscle Vm during a downward temperature ramp (20 to −3°C). Cold-tolerant species were able to defend their Vm down to lower temperatures, which is not explained by species-specific differences in initial Vm at 20°C, but by cold-tolerant drosophilids defending Vm across a broad range of temperatures. We found support for a previously suggested ‘critical threshold’ of Vm, related to chill coma, in three of the five species. Interestingly, the cold-tolerant Drosophila species may enter coma as a result of processes unrelated to muscle depolarization as their Vm was not significantly depolarized at their chill coma temperatures. Summary: Cold-tolerant Drosophila species defend their muscle resting membrane potential at low temperatures and may enter chill coma as a result of different physiological mechanisms than those in less cold-tolerant species.

Hemolymph metabolites and osmolality are tightly linked to cold tolerance of Drosophila species: a comparative study

ABSTRACT Drosophila, like most insects, are susceptible to low temperatures, and will succumb to temperatures above the freezing point of their hemolymph. For these insects, cold exposure causes a loss of extracellular ion and water homeostasis, leading to chill injury and eventually death. Chill-tolerant species are characterized by lower hemolymph [Na+] than chill-susceptible species and this lowered hemolymph [Na+] is suggested to improve ion and water homeostasis during cold exposure. It has therefore also been hypothesized that hemolymph Na+ is replaced by other ‘cryoprotective’ osmolytes in cold-tolerant species. Here, we compared the hemolymph metabolite profiles of five drosophilid species with marked differences in chill tolerance. All species were examined under ‘normal’ thermal conditions (i.e. 20°C) and following cold exposure (4 h at 0°C). Under benign conditions, total hemolymph osmolality was similar among all species despite chill-tolerant species having lower hemolymph [Na+]. Using NMR spectroscopy, we found that chill-tolerant species instead have higher levels of sugars and free amino acids in their hemolymph, including classical ‘cryoprotectants’ such as trehalose and proline. In addition, we found that chill-tolerant species maintain a relatively stable hemolymph osmolality and metabolite profile when exposed to cold stress while sensitive species suffer from large increases in osmolality and massive changes in their metabolic profiles during a cold stress. We suggest that the larger contribution of classical cryoprotectants in chill-tolerant Drosophila plays a non-colligative role for cold tolerance that contributes to osmotic and ion homeostasis during cold exposure and, in addition, we discuss how these comparative differences may represent an evolutionary pathway toward more extreme cold tolerance of insects. Summary: Small increases in the abundance of compatible osmolytes improve osmotic balance and cold tolerance in Drosophila.

Temperate Drosophila preserve cardiac function at low temperature.

Most insects are chill susceptible and will enter a coma if exposed to sufficiently low temperature. This chill coma has been associated with a failure of the neuromuscular system. Insect heart rate (HR) is determined by intrinsic regulation (muscle pacemaker) with extrinsic (nervous and humoral) input. By examining the continually active heart of five Drosophila species with markedly different cold tolerance, we investigated whether cardiac performance is related to the whole animal critical thermal minimum (CTmin). Further, to separate the effects of cold on extrinsic and intrinsic regulators of HR, we measured HR under similar conditions in decapitated flies as well as amputated abdomens of Drosophila montana. Cardiac performance was assessed from break points in HR-temperature relationship (Arrhenius break point, ABP) and from the HR cessation temperature. Among the five species, we found strong relationships for both the HR-ABP and HR cessation temperatures to whole animal CTmin, such that temperate Drosophila species maintained cardiac function at considerably lower temperatures than their tropical congeners. Hearts of amputated abdomens, with reduced extrinsic input, had a higher thermal sensitivity and a significantly lower break point temperature, suggesting that central neuronal input is important for stimulating HR at low temperatures.

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.