A rapid return to normal: temporal gene expression patterns following cold exposure in the bumble bee Bombus impatiens.

Bumble bees are common in cooler climates and many species likely experience periodic exposure to very cold temperatures, but little is known about the temporal dynamics of cold response mechanisms following chill exposure, especially how persistent effects of cold exposure may facilitate tolerance of future events. To investigate molecular processes involved in the temporal response by bumble bees to acute cold exposure, we compared mRNA transcript abundance in Bombus impatiens workers exposed to 0°C for 75 min (inducing chill coma) and control bees maintained at a constant ambient temperature (28°C). We sequenced the 3' end of mRNA transcripts (TagSeq) to quantify gene expression in thoracic tissue of bees at several time points (0, 10, 30, 120 and 720 min) following cold exposure. Significant differences from control bees were only detectable within 30 min after the treatment, with most occurring at the 10 min recovery time point. Genes associated with gluconeogenesis and glycolysis were most notably upregulated, while genes related to lipid and purine metabolism were downregulated. The observed patterns of expression indicate a rapid recovery after chill coma, suggesting an acute differential transcriptional response during recovery from chill coma and return to baseline expression levels within an hour, with no long-term gene expression markers of this cold exposure. Our work highlights the functions and pathways important for acute cold recovery, provides an estimated time frame for recovery from cold exposure in bumble bees, and suggests that cold hardening may be less important for these heterothermic insects.

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.

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.

Heat stress survival and thermal tolerance of Australian stingless bees.

Stingless bees (Meliponini) are important pollinators throughout the world's tropical and subtropical regions. Understanding their thermal tolerance is key to predicting their resilience to changing climates and increasingly frequent extreme heat events. We examined critical thermal maxima (CTmax), survival during 1-8 h heat periods, chill coma recovery and thermal preference for Australian meliponine species that occupy different climates across their ranges: Tetragonula carbonaria (tropical to temperate regions), T. hockingsi (tropical and subtropical regions only) and Austroplebeia australis (widely distributed including arid regions). We found interspecific differences in thermal tolerance consistent with differences in the climate variability observed in each species' range. Foragers of A. australis had a faster chill coma recovery (288 s) than foragers of T. hockingsi (1059 s) and T. carbonaria (872 s). Austroplebeia australis also had the highest CTmax of 44.5 °C, while the CTmax of the two Tetragonula species was ∼43.1 °C. After a 1-h heat exposure, T. carbonaria foragers experienced 95% mortality at 42 °C, and 100% at 45 °C. Surprisingly, larvae and pupae of both Tetragonula species were more resistant to heat exposure than foragers. Within an enclosed temperature gradient apparatus (17-38 °C), no clear preference was found for foragers; however, they were most frequently observed at ∼18 °C. Results indicate that in some regions of Australia, meliponines already experience periodic heat events exceeding their thermal maxima. Employing effective management strategies (such as nest site insulation and habitat preservation) may be crucial to colony survival under continued climate change.

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.

Cold adaptation drives population genomic divergence in the ecological specialist, Drosophila montana.

Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here, we analysed patterns of geographic variation in the cold-adapted species Drosophila montana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and fourth chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and fourth chromosomes, which also showed highest interpopulation FST . This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome-wide effects.

Evidence for genetic isolation and local adaptation in the field cricket Gryllus campestris.

Understanding how species can thrive in a range of environments is a central challenge for evolutionary ecology. There is strong evidence for local adaptation along large-scale ecological clines in insects. However, potential adaptation among neighbouring populations differing in their environment has been studied much less. We used RAD sequencing to quantify genetic divergence and clustering of ten populations of the field cricket Gryllus campestris in the Cantabrian Mountains of northern Spain, and an outgroup on the inland plain. Our populations were chosen to represent replicate high and low altitude habitats. We identified genetic clusters that include both high and low altitude populations indicating that the two habitat types do not hold ancestrally distinct lineages. Using common-garden rearing experiments to remove environmental effects, we found evidence for differences between high and low altitude populations in physiological and life-history traits. As predicted by the local adaptation hypothesis, crickets with parents from cooler (high altitude) populations recovered from periods of extreme cooling more rapidly than those with parents from warmer (low altitude) populations. Growth rates also differed between offspring from high and low altitude populations. However, contrary to our prediction that crickets from high altitudes would grow faster, the most striking difference was that at high temperatures, growth was fastest in individuals from low altitudes. Our findings reveal that populations a few tens of kilometres apart have independently evolved adaptations to their environment. This suggests that local adaptation in a range of traits may be commonplace even in mobile invertebrates at scales of a small fraction of species' distributions.

Evolution of cold tolerance and thermal plasticity in life history, behaviour and physiology during a poleward range expansion.

Many species that are moving polewards encounter novel thermal regimes to which they have to adapt. Therefore, rapid evolution of thermal tolerance and of thermal plasticity in fitness-related traits in edge populations can be crucial for the success and speed of range expansions. We tested for adaptation in cold tolerance and in life history, behavioural and physiological traits and their thermal plasticity during a poleward range expansion. We reconstructed the thermal performance curves of life history (survival, growth and development rates), behaviour (food intake) and cold tolerance (chill coma recovery time) in the aquatic larval stage of the damselfly Ischnura elegans that is currently showing a poleward range expansion in northern Europe. We studied larvae from three edge and three core populations using a common-garden experiment. Consistent with the colder annual temperatures, larvae at the expansion front evolved an improved cold tolerance. The edge populations showed no overall (across temperatures) evolution of a faster life history that would improve their range-shifting ability. Moreover, consistent with damselfly edge populations from colder latitudes, edge populations evolved at the highest rearing temperature (28°C) a faster development rate, likely to better exploit the rare periods with higher temperatures. This was associated with a higher food intake and a lower metabolic rate. In conclusion, our results suggest that the edge populations rapidly evolved adaptive changes in trait means and thermal plasticity to the novel thermal conditions at the edge front. Our results highlight the importance of considering besides trait plasticity and the evolution of trait means, also the evolution of trait plasticity to improve forecasts of responses to climate change.

Durante su expansión hacia los polos, las especies encuentran nuevos regímenes de temperatura a los que tienen que adaptarse. Por esto, una rápida evolución de la tolerancia térmica y de la plasticidad térmica de rasgos fisiológicos clave en las poblaciones del borde del área de distribución es crucial para el éxito y la velocidad de las expansiones de rango. En este estudio testamos la adaptación de la tolerancia la frío y la plasticidad térmica de rasgos de historia de vida, comportamiento y fisiológicos durante una expansión de rango hacia el norte. Reconstruimos las curvas de rendimiento térmico de rasgos de historia de vida (supervivencia, tasa de crecimiento y tasa de desarrollo) y comportamiento (ingestión de alimento), así como la tolerancia al frío (tiempo de recuperación del coma por frío) en la larva acuática de la especie de caballito del diablo Ischnura elegans, especie que muestra un rango de expansión actual en el norte de Europa. Estudiamos larvas de tres poblaciones de la región de expansión norte y tres poblaciones de la región central usando experimentos en laboratorio. En concordancia con las temperaturas más bajas, las larvas del área de expansión mostraron mayor tolerancia al frío. Sin embargo, estas larvas no mostraron en general (a lo largo de las temperaturas estudiadas) evolución hacia una historia de vida más rápida, que aumentaría su habilidad para expandirse. Además, en consistencia con la menor temperatura de la región de expansión, las larvas presentaron mayor tasa de desarrollo a la temperatura experimental más alta (28°C), probablemente para explotar mejor los infrecuentes períodos con altas temperaturas en dicha región. Esto estuvo asociado con una mayor ingestión de alimento y una menor tasa metabólica. En conclusión, nuestros resultados sugieren que los valores medios y la plasticidad de los rasgos estudiados de las poblaciones del frente de expansión evolucionaron rápidamente para adaptarse a las nuevas condiciones térmicas en dicha región. Asimismo, nuestros resultados destacan la importancia de considerar, además de la media y plasticidad de los rasgos, la evolución de esta plasticidad, con el fin de mejorar las predicciones de las respuestas de las especies al cambio climático.

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.

Plastic changes in cold and drought tolerance of Drosophila nepalensis correlate with sex-specific differences in body melanization, cuticular lipid mass, proline accumulation, and seasonal abundance.

Autumn-collected flies of Himalayan Drosophila nepalensis differ in body color phenotypes (males more melanized relative to females) and in their behavior (males abundant in the open sites vs. shelters-seeking females). In contrast, winter-collected flies of both sexes are equally melanized and abundant in the open sites. We tested developmental and adult plasticity changes in cold or drought tolerance in D. nepalensis flies reared under winter or autumn simulated conditions. In D. nepalensis flies reared at 21 °C, male flies were more cold tolerant (as shown by shorter chill-coma recovery time and lower cold-shock mortality). Further, male flies also exhibited greater drought tolerance (increased levels of desiccation resistance, cuticular lipid mass, melanization, hydration level, and dehydration tolerance) as compared to females. We observed sex-specific differences in the adult plasticity responses due to rapid cold or drought hardening (RCH or RDH); and for the persistence of cold acclimation effects. RCH or RDH-induced changes in the level of proline accumulations are negatively correlated with a decrease in the chill-coma recovery time. Therefore, cold or drought hardening treatments are likely to influence cold tolerance through proline accumulation. Developmental acclimation and adult hardening responses revealed significant interaction effects between sexes and thermal treatments. Thus, sex-specific differences in morphological traits (body melanization and cuticular lipid mass) and physiological traits (adult plasticity changes in cold tolerance and proline accumulation) correlate with behavioral divergence (habitat usage) across sexes.

Reversing sodium differentials between the hemolymph and hindgut speeds chill coma recovery but reduces survival in the fall field cricket, Gryllus pennsylvanicus.

Chill-susceptible insects enter the reversible state of chill coma at their critical thermal minimum (CTmin). During chill coma, movement of Na+ and water from the hemolymph to the gut lumen disrupt ion and water balance. Recovery from cold exposure requires re-establishment of this balance, and failure to do so results in chilling injury or death. We hypothesized that the passive leak of Na+ and consequently water during cold exposure is driven by the [Na+] differential between the gut and hemolymph. To determine the extent to which this [Na+] differential affects cold tolerance, we used artificial diets to load the guts of fall field crickets (Gryllus pennsylvanicus) with various concentrations of Na+. Manipulating [Na+] differentials had no effect on the CTmin, agreeing with recent studies demonstrating that chill coma onset precedes loss of ion balance in the cold. A high [Na+] diet reversed the direction of the [Na+] differential between the gut and hemolymph. Crickets fed a high [Na+] diet recovered from 12 h of chill coma nearly twice as fast as those fed low [Na+] diets. However, the high [Na+] diet was detrimental to survival after prolonged cold exposure (three days at 0 °C). Therefore, while a reduced [Na+] differential helps crickets recover from short-term cold exposure, an increased gut Na+ load itself appears to carry longer-term costs and promotes irreversible chilling injury.

Low temperature shock and chill-coma consequences for the red flour beetle (Tribolium castaneum) and the rice weevil (Sitophilus oryzae).

Insects face several (environmental) abiotic stressors, including low temperature, which cause the failure of neuromuscular function. Such exposure leads insects toa reversible comatose state termed chill-coma, but the consequences of this state for the organism biology were little explored. Here, the consequences of the chill-coma phase were investigated in two of the main stored product pest species - the red flour beetle Tribolium castaneum (larvae and adults) and the rice weevil Sitophilus oryzae (adults). For this purpose, a series of low-temperature shocks were used to estimate the chill-coma recovery time (CCRT), survival, nutrition and weight gain/growth of T. castaneum (larvae and adults) and S. oryzae, as well as the development of T. castaneum life stages. The relatively long CCRT was characteristic of beetle larvae, at different low-temperature shocks, and CCRT increased with decreasing temperatures and increasing exposure intervals for both pest species. The survival was little affected by the low-temperature shocks applied, but such shocks affected insect feeding and growth. Tribolium castaneum larvae was more sensitive than adults of both insect species. Moreover, the relative consumption and weight gain of S. oryzae adults were lower than those of T. castaneum adults and mainly larvae, while feeding deterrence was not affected by low temperature shocks, unlike food conversion efficiency. Low-temperature shocks, even under short duration at some temperatures, significantly delayed development. The lower the temperature and the higher the exposure period, the more delayed the development. Thus, the physiological costs of chill-coma are translated into life-history consequences, with potential implications for the management of this insect pest species in stored products and even more so on red flour beetles and rice weevils.

Bilateral auditory processing studied by selective cold-deactivation of cricket hearing organs.

We studied bilateral processing in the auditory ON neurons of crickets using reversible cold-deactivation of the hearing organs by means of Peltier elements. Intracellular recordings of the neurons' activity in response to acoustic stimuli were obtained, while either the ipsilateral or the contralateral hearing organ was cold-deactivated. Afferent activity was abolished at a temperature of approximately 10°C. In ON1, contralateral inhibition had no effect on the latency and amplitude of the phasic onset activity, it enhanced the decline of the onset activity and it decreased the subsequent tonic spiking response to acoustic stimuli. As a consequence, the phasic onset activity became more salient and reciprocal inhibition may support the detection of sound pulses. Contralateral inhibition had a significant impact on the tonic ON1 response, in line with its presumed function to enhance the bilateral auditory contrast. In ON2, experiments confirmed a bilateral excitatory input, with the ipsilateral input dominating the response, and no inhibitory coupling between the ON2 neurons.

Loss of ion homeostasis is not the cause of chill coma or impaired dispersal in false codling moth Thaumatotibia leucotreta (Lepidoptera: Tortricidae).

Dispersal is a central requirement of a successful sterile insect release programme, but field-released false codling moth (FCM) typically suffer from poor dispersal ability, especially at low ambient temperatures. Here we test the hypothesis that poor activity and dispersal in FCM is caused by delayed or perturbed recovery of ion and/or water homeostasis after chilling for handling and transport prior to field release. Hemolymph and flight muscle were collected from two treatment groups at three time points that targeted thermal conditions above and below the chill coma induction threshold of ~ 6 °C: 1) control moths kept at 25 °C, 2) moths exposed to 3 °C or 9 °C for 4 h, and 3) moths allowed to recover at 25 °C for 24 h after exposure to either 3 °C or 9 °C. We measured concentrations of Na+, K+ and Mg2+ in the hemolymph and muscle collected at each time point. Exposure to a chill-coma inducing temperature had little effect overall on ion balance in the hemolymph and flight muscle of false codling moth, but hemolymph [Na+] decreased from 10.4 ±â€¯0.4 mM to 6.9 ±â€¯0.7 mM as moths were chilled to 3 °C and then increased to 10.4 ±â€¯0.9 mM after the 24 h recovery period. In the 9 °C cooling treatment, [K+] increased from 8.2 ±â€¯0.5 mM during chilling to 14.1 ±â€¯1.9 mM after the 24 h recovery period. No changes were seen in equilibrium potentials in either of the ions measured. Thus, we did not find evidence that water and ion homeostasis are lost by the moths in chill coma and conclude that reduced dispersal in field-released moths is not direct a consequence of the costs of re-establishment of homeostasis.

Rapid cold hardening and octopamine modulate chill tolerance in Locusta migratoria.

Temperature has profound effects on the neural function and behaviour of insects. When exposed to low temperature, chill-susceptible insects enter chill coma, a reversible state of neuromuscular paralysis. Despite the popularity of studying the effects of low temperature on insects, we know little about the physiological mechanisms controlling the entry to, and recovery from, chill coma. Spreading depolarization (SD) is a phenomenon that causes a neural shutdown in the central nervous system (CNS) and it is associated with a loss of K+ homeostasis in the CNS. Here, we investigated the effects of rapid cold hardening (RCH) on chill tolerance of the migratory locust. With an implanted thermocouple in the thorax, we determined the temperature associated with a loss of responsiveness (i.e. the critical thermal minimum - CTmin) in intact male adult locusts. In parallel experiments, we recorded field potential (FP) in the metathoracic ganglion (MTG) of semi-intact preparations to determine the temperature that would induce neural shutdown. We found that SD in the CNS causes a loss of coordinated movement immediately prior to chill coma and RCH reduces the temperature that evokes neural shutdown. Additionally, we investigated a role for octopamine (OA) in the locust chill tolerance and found that OA reduces the CTmin and mimics the effects of prior stress (anoxia) in locust.

Environmental and genetic control of cold tolerance in the Glanville fritillary butterfly.

Thermal tolerance has a major effect on individual fitness and species distributions and can be determined by genetic variation and phenotypic plasticity. We investigate the effects of developmental and adult thermal conditions on cold tolerance, measured as chill coma recovery (CCR) time, during the early and late adult stage in the Glanville fritillary butterfly. We also investigate the genetic basis of cold tolerance by associating CCR variation with polymorphisms in candidate genes that have a known role in insect physiology. Our results demonstrate that a cooler developmental temperature leads to reduced cold tolerance in the early adult stage, whereas cooler conditions during the adult stage lead to increased cold tolerance. This suggests that adult acclimation, but not developmental plasticity, of adult cold tolerance is adaptive. This could be explained by the ecological conditions the Glanville fritillary experiences in the field, where temperature during early summer, but not spring, is predictive of thermal conditions during the butterfly's flight season. In addition, an amino acid polymorphism (Ala-Glu) in the gene flightin, which has a known function in insect flight and locomotion, was associated with CCR. These amino acids have distinct biochemical properties and may thus affect protein function and/or structure. To our knowledge, our study is the first to link genetic variation in flightin to cold tolerance, or thermal adaptation in general.

Critical thermal limits of bumblebees (Bombus impatiens) are marked by stereotypical behaviors and are unchanged by acclimation, age or feeding status.

Critical thermal limits often determine species distributions for diverse ectotherms and have become a useful tool for understanding past and predicting future range shifts in response to changing climates. Despite recently documented population declines and range shifts of bumblebees (genus Bombus), the few measurements of thermal tolerance available for the group have relied on disparate measurement approaches. We describe a novel stereotypical behavior expressed by bumblebee individuals during entry into chill coma. This behavioral indicator of minimum critical temperature (CTmin) occurred at ambient temperatures of 3-5°C (approximately 7-9°C core temperatures) and was accompanied by a pronounced CO2 pulse, indicative of loss of spiracle function. Maximum critical temperature (CTmax) was indicated by the onset of muscular spasms prior to entering an unresponsive state and occurred at ambient temperatures of approximately 52-55°C (42-44°C core temperatures). Measurements of CTmin and CTmax were largely unaffected by acclimation, age or feeding status, but faster ramping rates significantly increased CTmax and decreased CTmin This high-throughput approach allows rapid measurement of critical thermal limits for large numbers of individuals, facilitating large-scale comparisons among bumblebee populations and species - a key step in determining current and future effects of climate on these critical pollinators.

Central nervous system shutdown underlies acute cold tolerance in tropical and temperate Drosophila species.

When cooled, insects first lose their ability to perform coordinated movements (CTmin) after which they enter chill coma (chill coma onset, CCO). Both these behaviours are popular measures of cold tolerance that correlate remarkably well with species distribution. To identify and understand the neuromuscular impairment that causes CTmin and CCO we used inter- and intraspecific model systems of Drosophila species that have varying cold tolerance as a consequence of adaptation or cold acclimation. Our results demonstrate that CTmin and CCO correlate strongly with a spreading depolarization (SD) within the central nervous system (CNS). We show that this SD is associated with a rapid increase in extracellular [K+] within the CNS causing neuronal depolarization that silences the CNS. The CNS shutdown is likely to be caused by a mismatch between passive and active ion transport within the CNS and in a different set of experiments we examine inter- and intraspecific differences in sensitivity to SD events during anoxic exposure. These experiments show that cold adapted or acclimated flies are better able to maintain ionoregulatory balance when active transport is compromised within the CNS. Combined, we demonstrate that a key mechanism underlying chill coma entry of Drosophila is CNS shutdown, and the ability to prevent this CNS shutdown is therefore an important component of acute cold tolerance, thermal adaptation and cold acclimation in insects.

Microbiota disruption leads to reduced cold tolerance in Drosophila flies.

It is now acknowledged that bacteria from gut microbiota deeply interact with their host by altering many physiological traits. Such interplay is likely to consequently affect stress tolerance. Here, we compared cold and heat tolerance of Drosophila melanogaster flies with undisrupted (control (Co)) versus disrupted gut microbiota (dechorionated eggs (De)). The disrupting treatment strongly reduced bacterial load in flies' guts, though 16S sequencing analysis did not evidence strong diversity changes in the remaining bacterial community. Both chill coma recovery and acute cold survival were repeatedly lower in De than in Co flies under our experimental conditions. However, heat tolerance was not consistently affected by gut disruption. Our results suggest that microbiota-related effects on the host can alter ecologically relevant traits such as thermal tolerance.

Thermal acclimation is not induced by habitat-of-origin, maintenance temperature, or acute exposure to low or high temperatures in a pit-building wormlion (Vermileo sp.).

Wormlions are sit-and-wait insect predators that construct pit-traps to capture arthropod prey. They require loose soil and shelter from direct sun, both common in Mediterranean cities, and explaining their high abundance in urban habitats. We studied different aspects of thermal acclimation in wormlions. We compared chill-coma recovery time (CCRT) and heat-shock recovery time (HSRT) of wormlions from urban, semi-urban and natural habitats, expecting those originating from the urban habitat to be more heat tolerant and less cold tolerant. However, no differences were detected among the three habitats. We then examined whether maintenance temperature affects CCRT and HSRT, and expected beneficial acclimation. However, CCRT was unaffected by maintenance temperature, while temperature affected HSRT in an opposite direction to our prediction: wormlions maintained under the higher temperatures took longer to recover. When testing with two successive thermal shocks, wormlions took longer to recover from both cold and heat shock after applying an initial cold shock. We therefore conclude that cold shock inflicts some damage rather than induces acclimation. Finally, both cold- and heat-shocked wormlions constructed smaller pits than wormlions of a control group. Smaller pits probably translate to a lower likelihood of capturing prey and also limit the size of the prey, indicating a concrete cost of thermal shock. In summary, we found no evidence for thermal acclimation related either to the habitat-of-origin or to maintenance temperatures, but, rather, negative effects of unfavorable temperatures.