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Winter provides many challenges for insects, including direct injury to tissues and energy drain due to low food availability. As a result, the geographic distribution of many species is tightly coupled to their ability to survive winter. In this review, we summarize molecular processes associated with winter survival, with a particular focus on coping with cold injury and energetic challenges. Anticipatory processes such as cold acclimation and diapause cause wholesale transcriptional reorganization that increases cold resistance and promotes cryoprotectant production and energy storage. Molecular responses to low temperature are also dynamic and include signaling events during and after a cold stressor to prevent and repair cold injury. In addition, we highlight mechanisms that are subject to selection as insects evolve to variable winter conditions. Based on current knowledge, despite common threads, molecular mechanisms of winter survival vary considerably across species, and taxonomic biases must be addressed to fully appreciate the mechanistic basis of winter survival across the insect phylogeny. Expected final online publication date for the

Scoring large amounts of thermal tolerance traits live or with recorded video can be time consuming and susceptible to investigator bias, and as with many physiological measurements, there can be trade-offs between accuracy and throughput. Recent studies show that particle tracking is a viable alternative to manually scoring videos, although it may not detect subtle movements, and many of the software options are proprietary and costly. In this study, we present a novel strategy for automated scoring of thermal tolerance videos by inferring motor activity with motion detection using an open-source Python command line application called DIME (Detector of Insect Motion Endpoint). We apply our strategy to both dynamic and static thermal tolerance assays, and our results indicate that DIME can accurately measure thermal acclimation responses, generally agrees with visual estimates of thermal limits, and can significantly increase the throughput over manual methods.Summary statementMotion detection algorithm for reliable, automatic scoring of thermal limits in insects with open-source tool

Microhabitats with distinct biotic and abiotic properties exist within landscapes, and this microhabitat variation can have dramatic impacts on the phenology and physiology of the organisms occupying them. The Antarctic midge Belgica antarctica inhabits diverse microhabitats along the Western Antarctic Peninsula that vary in macrophyte composition, hygric qualities, nutrient input, and thermal patterns. Here, we compare seasonal physiological changes in five populations of B. antarctica living in close proximity but in different microhabitats in the vicinity of Palmer Station, Antarctica. Thermal regimes among our sample locations differed in both mean temperature and thermal stability. Between the warmest and coldest sites, seasonal mean temperatures differed by 2.6˚C and degree day accumulations above freezing differed by a factor of 1.7. Larval metabolic and growth rates varied among the sites, and adult emergence occurred at different times. Distinct microhabitats also corresponded with differences in body composition, as lipid and carbohydrate content of larvae differed across sites. Further, seasonal changes in carbohydrate and protein content were dependent on site, indicating fine-scale variation in the biochemical composition of larvae as they prepare for winter. Together, these results demonstrate that variation in microhabitat properties influences the ontogeny, phenology, physiology, and biochemical makeup of midge populations living in close proximity. These results have implications for predicting responses of Antarctic ecosystems to environmental change.

Plants and animals use circadian and photoperiodic timekeeping mechanisms to respond to daily and seasonal changes in light:dark and appropriately coordinate their development. Although the mechanisms that may connect the circadian and photoperiodic clock are still unclear in many species, researchers have been using Nanda-Hamner protocols for decades to elucidate how seasonal time is measured and determine whether seasonal responses have a circadian basis in a given species. In this brief tutorial we describe how to design and interpret the results of Nanda-Hamner experiments, and provide suggestions on how to use both Nanda-Hamner protocols and modern molecular experiments to better understand the mechanisms of seasonal timekeeping.

Species distributions are dependent on interactions with abiotic and biotic factors in the environment. Abiotic factors like temperature, moisture, and soil nutrients, along with biotic interactions within and between species, can all have strong influences on spatial distributions of plants and animals. Terrestrial Antarctic habitats are relatively simple and thus good systems to study ecological factors that drive species distributions and abundance. However, these environments are also sensitive to perturbation, and thus understanding the ecological drivers of species distribution is critical for predicting responses to environmental change. The Antarctic midge, Belgica antarctica, is the only endemic insect on the continent and has a patchy distribution along the Antarctic Peninsula. While its life history and physiology are well studied, factors that underlie variation in population density within its range are unknown. Previous work on Antarctic microfauna indicates that distribution over broad scales is primarily regulated by soil moisture, nitrogen content, and the presence of suitable plant life, but whether these patterns are true over smaller spatial scales has not been investigated. Here we sampled midges across five islands on the Antarctic Peninsula and tested a series of hypotheses to determine the relative influences of abiotic and biotic factors on midge abundance. While historical literature suggests that Antarctic organisms are limited by the abiotic environment, our best-supported hypothesis indicated that abundance is predicted by a combination of abiotic and biotic conditions. Our results are consistent with a growing body of literature that biotic interactions are more important in Antarctic ecosystems than historically appreciated. Electronic supplementary material The online version of this article (10.1007/s00442-020-04714-9) contains supplementary material, which is available to authorized users.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Survival of the terrestrial midge, Belgica antarctica, on the Antarctic Peninsula is promoted, not only by their adaptations to prolonged exposures to seasonal stresses, but also by their ability to respond to unpredictable changes in their environments. Rapid cold-hardening (RCH) is an extremely swift acclimatory response of insects that occurs within minutes to hours. While the RCH response is most commonly induced by a brief exposure to mildly low temperatures, a similar rapid acclimatory response can also be elicited by exposure to drought. In this study, we characterized this drought-induced RCH in larvae of B. antarctica. Compared to fully hydrated larvae, those desiccated at various relative humidity (R.H.) conditions between 0 and 99% R.H. for 2 h had a significantly greater survival ( ~ 50%) to freezing at − 14 °C. The amount of water loss varied between 4 and 16% depending on R.H. conditions; however, all treatments were equally effective in eliciting the protective response against freezing stress, and its induction was evident within 30 min of desiccation. Lack of substantial changes in body-fluid osmolality or levels of major cryoprotectants suggest that accumulation of these protective solutes is not a primary mechanism of this response. Interestingly, the RCH protection induced by desiccation persisted after larvae were allowed to recover a significant portion of the lost water. Our results indicate that larval midges are highly sensitive to desiccation, capable of swiftly initiating physiological changes in response to a small reduction in their body water content.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. Unfortunately, irradiation in normal atmospheric conditions can be damaging for males, because it generates substantial oxidative stress that, combined with mass-rearing conditions, may reduce their sexual competitiveness and quality. In this study, oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate the role of oxidative stress and cellular antioxidants in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Microhabitats with distinct biotic and abiotic properties exist within landscapes, and this microhabitat variation can have dramatic impacts on the phenology and physiology of the organisms occupying them. The Antarctic midge Belgica antarctica inhabits diverse microhabitats along the Western Antarctic Peninsula that vary in macrophyte composition, hygric qualities, nutrient input, and thermal patterns. Here, we compare seasonal physiological changes in five populations of B. antarctica living in close proximity but in different microhabitats in the vicinity of Palmer Station, Antarctica. Thermal regimes among our sample locations differed in both mean temperature and thermal stability. Between the warmest and coldest sites, seasonal mean temperatures differed by 2.6˚C and degree day accumulations above freezing differed by a factor of 1.7. Larval metabolic and growth rates varied among the sites, and adult emergence occurred at different times. Distinct microhabitats also corresponded with differences in body composition, as lipid and carbohydrate content of larvae differed across sites. Further, seasonal changes in carbohydrate and protein content were dependent on site, indicating fine-scale variation in the biochemical composition of larvae as they prepare for winter. Together, these results demonstrate that variation in microhabitat properties influences the ontogeny, phenology, physiology, and biochemical makeup of midge populations living in close proximity. These results have implications for predicting responses of Antarctic ecosystems to environmental change.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Tolerance of climatic stressors is an important predictor of the current distribution of insect species, their potential to invade new environments, and their responses to rapid climate change. Cold stress causes acute injury to nerves and muscles, and here we tested the hypothesis that low temperature causes sublethal deficits in locomotor behaviors that are dependent on neuromuscular function. To do so, we applied a previously developed assay, the rapid iterative negative geotaxis (RING) assay, to investigate behavioral consequences of cold stress in Drosophila melanogaster. The RING assay allows for rapid assessment of negative geotaxis behavior by quantifying climbing height and willingness to climb after cold stress. We exposed flies to cold stress at 0°C and assessed the extent to which duration of cold stress, recovery time, and cold acclimation influenced climbing performance. There was a clear dose-response relationship between cold exposure and performance deficits, with climbing height and willingness decreasing as cold exposure increased from 2 to 24 hr. Following cold exposure of an intermediate duration (12 hr), climbing height and willingness gradually improved as recovery time increased from 4 to 72 hr but flies never fully recovered. Finally, cold acclimation improved overall climbing height and willingness in both untreated and cold-stressed flies but did not prevent a reduction in climbing performance. Thus, cold stress causes deficits in locomotor and behavior that are dependent on the dose of cold exposure and persist long after the stress subsides. These results likely have implications for the ecological and evolutionary responses of insect populations to thermally variable environments.

Rapid cold hardening (RCH) is a type of phenotypic plasticity that allows ectotherms to quickly enhance cold tolerance in response to brief chilling (lasting minutes to hours). In this Review, we summarize the current state of knowledge of this important phenotype and provide new directions for research. As one of the fastest adaptive responses to temperature known, RCH allows ectotherms to cope with sudden cold snaps and to optimize their performance during diurnal cooling cycles. RCH and similar phenotypes have been observed across a diversity of ectotherms, including crustaceans, terrestrial arthropods, amphibians, reptiles, and fish. In addition to its well-defined role in enhancing survival to extreme cold, RCH also protects against nonlethal cold injury by preserving essential functions following cold stress, such as locomotion, reproduction, and energy balance. The capacity for RCH varies across species and across genotypes of the same species, indicating that RCH can be shaped by selection and is likely favored in thermally variable environments. Mechanistically, RCH is distinct from other rapid stress responses in that it typically does not involve synthesis of new gene products; rather, the existing cellular machinery regulates RCH through post-translational signaling mechanisms. However, the protective mechanisms that enhance cold hardiness are largely unknown. We provide evidence that RCH can be induced by multiple triggers in addition to low temperature, and that rapidly induced tolerance and cross-tolerance to a variety of environmental stressors may be a general feature of stress responses that requires further investigation.

Pepck is a metabolic enzyme that participates in gluconeogenesis through the conversion of oxaloacetate into phosphoenol pyruvate. Numerous transcriptomic studies have identified Pepck as a potential key player during diapause and various stresses responses. Here, we describe expression patterns of both cytosolic and mitochondrial isoforms of Pepck throughout development, during diapause, and in response to starvation and cold shock in the flesh fly, Sarcophaga bullata. We cloned full-length transcripts for both Pepck isoforms and observed that expression of both genes varied throughout development. Diapausing pupae have the highest relative expression of both isoforms, suggesting participation in the anticipatory production of sugars and sugar alcohols that occurs during this overwintering stage. In response to acute stress, the cytosolic isoform was upregulated whereas the mitochondrial variant remained unchanged. Cytosolic Pepck was strongly upregulated after 2 h recovery from cold shock and returned to baseline levels within 8 h. In response to 24 h of starvation, the cytosolic isoform was similarly upregulated and returned to control levels after 24 h of recovery. Acute stress is known to incur a metabolic cost, and Pepck could be a key player in this response. Although it remains unclear why there is such a dramatic divergence in the expression of the two isoforms, the distinction suggests specific roles for the two isoforms that depend on the developmental status of the fly and the stress conditions to which it is exposed.

Predicting how rapid climate change will affect terrestrial biota depends on a thorough understanding of an organism’s biology and evolutionary history. Organisms at their range boundaries are particularly sensitive to climate change. As predominantly terrestrial poikilotherms, insects are often geographically limited by extremes in ambient temperatures. We compared the cold hardiness strategies of two geographically widespread butterflies, the pine white, Neophasia menapia, and the Mexican pine white, N. terlooii (Lepidoptera: Pieridae), at the near-contact zone of their range boundaries. Eggs are laid on pine needles and are exposed to harsh winter conditions. Eggs were collected from wild-caught butterflies, and we determined the supercooling point (SCP) and lower lethal temperature (LLT50) of overwintering eggs. The SCP of Neophasia menapia eggs (−29.0 ± 0.6 °C) was significantly lower than that of N. terlooii eggs (−21.8 ± 0.7 °C). Both species were freeze-intolerant and capable of surviving down to their respective SCPs (LLT50 of N. menapia between −30 and −31 °C, N. terlooii between −20 and −21 °C). Cold exposure time did not affect the survival of N. menapia, but N. terlooii experienced somewhat greater mortality at sub-freezing temperatures during longer exposures. Our results, coupled with an analysis of microclimate data, indicate that colder winters in northern Arizona may contribute to the northern range limit for N. terlooii. Furthermore, careful analysis of historical weather data indicates that mortality from freezing is unlikely in southern Arizona but possible in northern Arizona. Movements of Neophasia range boundaries could be monitored as potential biological responses to climate change.

Temperature variation is one of the primary challenges facing ectotherms, and the ability to tolerate a range of thermal environments is critical for setting current and future species distributions. Low temperature is particularly challenging for ectotherms because winter conditions have strong latitudinal and temporal variation. Lower lethal temperature (LLT) is a common metric of cold tolerance used in studies of local adaptation and plasticity. Comparisons of LLT across groups typically assume parallel S-shaped survival curves, but genetic variation in the shape of survival vs. temperature curves has not been assessed. Here, we measured the ability of 36 lines of the Drosophila Genetic Reference Panel (DGRP) to survive a 1-h cold shock at seven ecologically relevant low temperatures (-1 to -7 °C) to create a high-resolution response curve for each genotype. We observed surprising variation both in the magnitude of survival and in the shapes of the response curves, with the curves clustering into four distinct shapes. To encompass variation in the shapes of these survival curves, we developed a new cold tolerance metric, cumulative cold tolerance (CCT). By comparing our survival data with climatological data, we propose that variation in the shapes of cold-survival curves arose from weak selection pressure to survive intermediate subzero temperatures in this mid-latitude population of flies. Using publicly available genome sequence and transcript expression data for these lines, we identified several candidate genes associated with CCT, and using transgenic RNAi, we confirmed a functional role for many of these genes.

Insects use rapid acclimation to enhance their tolerance of abiotic stresses within minutes to hours. These responses are critical adaptations for insects and other small ectotherms to tolerate drastic changes in temperature, hydration, or other factors that can fluctuate precipitously with ambient conditions or as a result of behavior. Rapid cold-hardening, where insects use brief exposure to modest chilling as a cue to enhance their cold tolerance, is the most thoroughly-studied of these responses and relatively little is known about rapid acclimation that is either triggered by or enhances tolerance of other abiotic stresses. Here, we used larvae of the Antarctic midge,Belgica antarctica, a polar extremophile that routinely experiences numerous stresses in nature, to investigate how 2 h exposure to modest environmental stresses affect stress tolerance in insects. Brief pretreatment by various stresses, including hyperosmotic challenge, hypoosmotic challenge, acidity, basicity, and UV irradiation enhanced stress tolerance inB. antarcticalarvae relative to untreated controls. These results indicate that numerous environmental cues can trigger rapid acclimation in insects and that these responses can enhance tolerance of multiple stresses.

The ability to quickly respond to changes in environmental temperature is critical for organisms living in thermally variable environments. To cope with sudden drops in temperature, insects and other ectotherms are capable of rapid cold hardening (RCH), in which mild chilling significantly enhances cold tolerance within minutes. While the ecological significance of RCH is well established, the mechanisms underlying RCH are still poorly understood. Previous work has demonstrated that RCH is regulated at the cellular level by post-translational signaling mechanisms, and here we tested the hypothesis that cultured cells are capable of RCH. A 2 h cold shock at -8°C significantly reduced the metabolic viability of

Cold tolerance, the ability to cope with low temperature stress, is a critical adaptation in thermally variable environments. An individual's cold tolerance comprises several traits including minimum temperatures for growth and activity, ability to survive severe cold, and ability to resume normal function after cold subsides. Across species, these traits are correlated, suggesting they were shaped by shared evolutionary processes or possibly share physiological mechanisms. However, the extent to which cold tolerance traits and their associated mechanisms covary within populations has not been assessed. We measured five cold tolerance traits-critical thermal minimum, chill coma recovery, short- and long-term cold tolerance, and cold-induced changes in locomotor behavior-along with cold-induced expression of two genes with possible roles in cold tolerance (heat shock protein 70 and frost)-across 12 lines of Drosophila melanogaster derived from a single population. We observed significant genetic variation in all traits, but few were correlated across genotypes, and these correlations were sex-specific. Further, cold-induced gene expression varied by genotype, but there was no evidence supporting our hypothesis that cold-hardy lines would have either higher baseline expression or induction of stress genes. These results suggest cold tolerance traits possess unique mechanisms and have the capacity to evolve independently.

For insects that overwinter as adults, winter food resources may affect subsequent spring reproduction and abundance. We tested if provision of food supplements to overwintering adult Hippodamia convergens (Guerin) increased energy reserves, winter survival, and spring reproduction. During 2015–2016, H. convergens adults were placed in field cages in December; adults in each cage received water, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs, bee pollen, wheast protein, sugar, honey, or no food (control). In 2016–2017, treatments were reduced to sugar, bee pollen, A. pisum with E. kuehniella eggs, and no food (control). Adults were sampled to quantify weight, lipid, carbohydrate, and protein content. In 2015–2016, A. pisum and E. kuehniella eggs increased adult weight and protein content, but adult carbohydrate content was reduced by A. pisum and wheast protein treatments. Adults receiving honey and sugar supplementation had higher lipid and carbohydrate content relative to controls. The number of live individuals at the end of the experiment in March 2016 did not differ among treatments. In 2016–2017, winter prey supplements had the greatest effect on protein content, weight, and number of live adults recovered, whereas sugar supplementation increased lipid and carbohydrate content, and number of live adults recovered. Spring reproduction of surviving pairs was evaluated among treatments in March 2017. Prey supplementation in 2016–2017 increased the number of eggs laid and decreased preoviposition period, and food treatment did not affect fertility. Our results indicate that prey and sugar resources improve the overwintering success and spring reproduction of H. convergens.

In many species, courtship displays are reliable signals of male quality, and current hypotheses suggest that these displays allow females to choose males with high cellular function. Environmental stressors generate excess reactive oxygen species (ROS) that impair cellular function, and thus antioxidant pathways that remove ROS are probably critical for preserving complex sexual behaviours. Here, we test the hypothesis that enhanced antioxidant activity in mitochondria preserves mating performance following oxidative stress. Using a transgenic approach, we directly manipulated mitochondrial antioxidant activity in the Caribbean fruit fly,Anastrepha suspensa,a lek-mating species with elaborate sexual displays and intense sexual selection that is also a model for sterile insect technique programmes. We generated seven transgenic lines that overexpress mitochondrial superoxide dismutase (MnSOD). Radiation is a severe oxidative stressor used to induce sterility for sterile insect programmes. After radiation treatment, two lines with intermediate MnSOD overexpression showed enhanced mating performance relative to wild-type males. These improvements in mating corresponded with reduced oxidative damage to lipids, demonstrating that MnSOD overexpression protects flies from oxidative stress at the cellular level. For lines with improved mating performance, overexpression also preserved locomotor activity, as indicated by a laboratory climbing assay. Our results show a clear link between oxidative stress, antioxidant capacity and male performance. Our work has implications for fundamentally understanding the role of antioxidants in sexual selection, and shows promise for using transgenic approaches to enhance the field performance of insects released for area-wide pest management strategies and improving performance of biological control agents in general.

Cold stress can reduce insect fitness and is an important determinant of species distributions and responses to climate change. Cold tolerance is influenced by genotype and environmental conditions, with factors such as day length and temperature having a particularly strong influence. Recent studies also indicate that diet impacts cold tolerance, but it is unclear whether diet-mediated shifts in cold tolerance are consistent across distinct genotypes. The goal of this study was to determine the extent to which commonly used artificial diets influence cold tolerance in Drosophila melanogaster, and whether these effects are consistent across genetically distinct lines. Specifically, we tested the impact of different fly diets on 1) ability to survive cold stress, 2) critical thermal minimum (CTmin), and 3) the ability to maintain reproduction after cold stress. Experiments were conducted across six isogenic lines from the Drosophila Genetic Reference Panel, and these lines were reared on different fly diets. Cold shock survival, CTmin, and reproductive output pre- and post-cold exposure varied considerably across diet and genotype combinations, suggesting strong genotype by environment interactions shape nutritionally mediated changes in cold tolerance. For example, in some lines cold shock survival remained consistently high or low across diets, while in others cold shock survival ranged from 5% to 75% depending on diet. Ultimately, these results add to a growing literature that cold tolerance is shaped by complex interactions between genotype and environment and inform practical considerations when selecting a laboratory diet for thermal tolerance experiments in Drosophila.

In response to short-day photoperiods in the autumn, the large milkweed bug Oncopeltus fasciatus Dallas enters a reproductive diapause and migrates south to avoid the adverse environmental conditions and food shortages that prevail in the winter. Milkweed bugs are one of only a few temperate insects that undergo long distance migration during diapause, making them a good model for investigating trade-offs associated with migratory diapause. Although enhanced stress tolerance is typical of diapause, it is unclear whether this aspect of diapause would be retained in a species that migrates to more favourable conditions. The present study tests (i) whether diapause enhances thermal tolerance; (ii) whether food shortage, which is required for maximal expression of diapause, influences thermal tolerance during diapause; and (iii) whether the potential changes in stress tolerance are associated with upregulated heat shock protein expression or metabolic adjustments (including cryoprotectant synthesis), or both. Both cold tolerance at −10 °C and heat tolerance at 43 °C are significantly higher in diapausing O. fasciatus, whereas food restriction has no further effect on thermal tolerance. None of the heat shock protein transcripts measured are significantly upregulated in response to diapause, and the experiments also fail to detect any cryoprotectant accumulation during diapause. Thus, although heat shock proteins and cryoprotectants are common mechanisms for enhancing thermal tolerance in many diapausing insects, the results of the present study suggest that alternative mechanisms are responsible in milkweed bugs.

Winter provides many challenges for terrestrial arthropods, including low temperatures and decreased food availability. Most arthropods are dormant in the winter and resume activity when conditions are favorable, but a select few species remain active during winter. Winter activity is thought to provide a head start on spring growth and reproduction, but few studies have explicitly tested this idea or investigated tradeoffs associated with winter activity. Here, we detail biochemical changes in overwintering winter-active wolf spiders, Schizocosa stridulans, to test the hypothesis that winter activity promotes growth and energy balance. We also quantified levels of putative cryoprotectants throughout winter to test the prediction that winter activity is incompatible with biochemical adaptations for coping with extreme cold. Body mass of juveniles increased 3.5-fold across winter, providing empirical evidence that winter activity promotes growth and therefore advancement of spring reproduction. While spiders maintained protein content throughout most of the winter, lipid content decreased steadily, suggesting either a lack of available prey to maintain lipids, or more likely, an allometric shift in body composition as spiders grew larger. Carbohydrate content showed no clear seasonal trend but also tended to be higher at the beginning of the winter. Finally, we tested the hypothesis that winter activity is incompatible with cryoprotectant accumulation. However, we observed accumulation of glycerol, myo-inositol, and several other cryoprotectants, although levels were lower than those typically observed in overwintering arthropods. Together, our results indicate that winter-active wolf spiders grow during the winter, and while cryoprotectant accumulation was observed in the winter, the modest levels relative to other species could make them susceptible to extreme winter events.

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects’ ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, Belgica antarctica is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against nonlethal freezing injury. Larvae of B. antarctica were exposed to either control (2°C), direct freezing (-9°C for 24 h), or RCH (-5°C for 2 h followed by -9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had significantly higher metabolic rates during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts (hsp60 and hsp90), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly fine-tune their performance in thermally variable environments.

Tolerance of climatic stressors is an important predictor of the current distribution of insect species, their potential to invade new environments, and their responses to rapid climate change. Cold stress causes acute injury to nerves and muscles, and here we tested the hypothesis that low temperature causes sublethal deficits in locomotor behaviors that are dependent on neuromuscular function. To do so, we applied a previously developed assay, the rapid iterative negative geotaxis (RING) assay, to investigate behavioral consequences of cold stress in Drosophila melanogaster. The RING assay allows for rapid assessment of negative geotaxis behavior by quantifying climbing height and willingness to climb after cold stress. We exposed flies to cold stress at 0°C and assessed the extent to which duration of cold stress, recovery time, and cold acclimation influenced climbing performance. There was a clear dose-response relationship between cold exposure and performance deficits, with climbing height and willingness decreasing as cold exposure increased from 2 to 24 hr. Following cold exposure of an intermediate duration (12 hr), climbing height and willingness gradually improved as recovery time increased from 4 to 72 hr but flies never fully recovered. Finally, cold acclimation improved overall climbing height and willingness in both untreated and cold-stressed flies but did not prevent a reduction in climbing performance. Thus, cold stress causes deficits in locomotor and behavior that are dependent on the dose of cold exposure and persist long after the stress subsides. These results likely have implications for the ecological and evolutionary responses of insect populations to thermally variable environments.

The terrestrial midge, Belgica antarctica, occupies a diverse range of microhabitats along the Antarctic Peninsula. Although overwintering larvae have the physiological potential to survive by freezing or cryoprotective dehydration, use of the latter strategy may be constrained by inoculative freezing within hibernacula. To investigate the influence of microhabitat type on larval overwintering, we selected four substrate types that differed markedly in their composition and hydric characteristics. Organic content of these substrates ranged from 14 to 89 %. High organic content was associated with higher values for saturation moisture content (up to 2.0 H2O g−1 dry mass) as well as elevated levels of field moisture content. Seasonal values of field moisture content remained near or above the saturation moisture value for each microhabitat type, and when larvae were cooled in substrates rehydrated to field-based levels, they were unable to avoid inoculation by environmental ice, regardless of substrate type. Consequently, our data suggest that wet hibernacula would force most larvae to overwinter in a frozen state. Yet, dehydrated larvae were collected in April during the seasonal transition to winter suggesting that spatial and temporal variations in precipitation and microhabitat conditions may expose larvae to dehydration and promote larval overwintering in a cryoprotectively dehydrated state.

Developing new soybean (Glycine max) varieties with greater amounts of oil and protein enhances soybean as a renewable source of food and fuel. Soybean expressing highly active ACYL-COA: Diacylglycerol acyltransferase (DGAT) from Vernonia galamensis had oil increased by 4% with no protein reduction. To assess other seed composition changes, seeds of two independent transgenic (VgD) soybean lines and controls were obtained from two field locations in multiple years. Many analyses addressed what seed component(s) was/were reduced to compensate for the oil increase, including carbohydrate analysis (structural and nonstructural), NDF (Neutral detergent fiber), main minerals and proximate analyses. In Princeton, oil increased by 4% for the high oil lines in 2016 and 2017, with no significant reduction in protein, this was slightly different in Spindletop with an increase in oil by 1.6–1.7 %, and an increase in protein by 2.6–2.4 % in comparison to the control, as an average for both high oil lines in 2016 and 2017. Soluble carbohydrate was reduced in VgD by 1.2 % in Princeton location for the high oil lines in 2016 and 2017, whereas only VgD1-2 was significantly reduced in Spindletop in comparison to Jack on 2016 with 2%, Oil in the VgD seeds was greater than in control seeds without a decrease in seed protein in both locations, suggesting that the trait was maintained across environments.

Summary 1. Cryoprotective dehydration is a relatively new addition to our understanding of freeze avoidance strategies employed by polar invertebrates. Although the underlying cellular processes associated with this strategy are similar to those of freeze tolerance, little is known about potential trade-offs of overwintering in these physiological states. 2. This study compares the potential of larvae of the terrestrial midge Belgica antarctica (Diptera, Chironomidae) to overwinter in these two states. As the only insect with the capacity to tolerate freezing and to cryoprotectively dehydrate, it is an ideal model to compare the benefits and costs of these strategies. 3. Compared to summer-acclimated larvae, supercooling points of winter-acclimatized larvae were significantly depressed and were lower than observed minima for their microhabitat temperatures. Thus, if larvae avoid inoculative freezing from environmental ice, they could remain unfrozen via cryoprotective dehydration. 4. Both frozen and cryoprotectively dehydrated larvae readily survived a 32-day exposure to simulated overwintering temperatures. 5. Freezing had little effect on larval body water content and haemolymph osmolality. In contrast, cryoprotective dehydration at 5 °C resulted in a progressive loss of body water, ultimately reducing larval water content by 62%. This level of dehydration corresponded to an increase in haemolymph osmolality to c. 2750 mOsm kg 1 , depressing the haemolymph melting point to 49 °C. 6. Freezing and cryoprotective dehydration resulted in distinctly different patterns of glycogen breakdown. Whereas the glycogen content decreased only during the first 14 days in cryoprotectively dehydrated larvae, frozen larvae continued to break down glycogen throughout the 32-day subzero exposure. However, after recovery at 0 °C for 5 days, glycogen levels were similar in these two groups, as were the levels of total lipids. 7. Our results indicate that freezing and cryoprotective dehydration are both effective in promoting winter survival of larvae, with surprisingly few differences in energetic costs. Whether larvae freeze or become cryoprotectively dehydrated ultimately depends on the hydric condition of their microhabitat. The physiological flexibility of B. antarctica to overwinter in these alternative states likely contributed to its range distribution that extends further south than any other free-living insect.

Abiotic stress is one of the primary constraints limiting the range and success of arthropods, and nowhere is this more apparent than Antarctica. Antarctic arthropods have evolved a suite of adaptations to cope with extremes in temperature and water availability. Here, we review the current state of knowledge regarding the environmental physiology of terrestrial arthropods in Antarctica. To survive low temperatures, mites and Collembola are freeze-intolerant and rely on deep supercooling, in some cases supercooling below −30°C. Also, some of these microarthropods are capable of cryoprotective dehydration to extend their supercooling capacity and reduce the risk of freezing. In contrast, the two best-studied Antarctic insects, the midges Belgica antarctica and Eretmoptera murphyi, are freeze-tolerant year-round and rely on both seasonal and rapid cold-hardening to cope with decreases in temperature. A common theme among Antarctic arthropods is extreme tolerance of dehydration; some accomplish this by cuticular mechanisms to minimize water loss across their cuticle, while a majority have highly permeable cuticles but tolerate upwards of 50–70% loss of body water. Molecular studies of Antarctic arthropod stress physiology are still in their infancy, but several recent studies are beginning to shed light on the underlying mechanisms that govern extreme stress tolerance. Some common themes that are emerging include the importance of cuticular and cytoskeletal rearrangements, heat shock proteins, metabolic restructuring and cell recycling pathways as key mediators of cold and water stress in the Antarctic.

Insects have evolved a number of physiological mechanisms for coping with the detrimental effects of low temperature. As autumn progresses, insects use environmental signals such as shortening day lengths and gradually decreasing temperatures to trigger seasonal cold-hardening adaptations. These mechanisms include dramatic changes in biochemistry, cell function and gene expression that permit improved cell function and viability at low temperature. Insects are also capable of enhancing cold tolerance on a much shorter time scale, in a process called rapid cold-hardening (RCH). Rapid cold-hardening allows insects to improve cold tolerance almost instantaneously (i.e. within minutes to hours) to cope with sudden cold snaps and regularly-occurring diurnal drops in temperature. Initially, it was assumed that RCH would share many of the same basic mechanisms as seasonal cold-hardening, albeit on a shorter time scale. Although there is some evidence supporting this, recent work has called into question some of the original hypotheses concerning the mechanisms of RCH. Also, some mechanisms important for seasonal cold-hardening, such as up-regulation of stress proteins, are unlikely to function at the temperatures and time scales at which RCH occurs. In the present review, the current understanding of the physiological mechanisms governing both seasonal cold-hardening and RCH are summarized. A synthesis of the current literature suggests that these two forms of cold-hardening may be more mechanistically distinct than originally anticipated.

Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype.

Larvae of the Antarctic midge, Belgica antarctica, routinely face periods of limited water availability in their natural environments on the Antarctic Peninsula. As a result, B. antarctica is one of the most dehydration-tolerant insects studied, surviving up to 70% loss of its body water. While previous studies have characterized the physiological effects of a single bout of dehydration, in nature larvae are likely to experience multiple bouts of dehydration throughout their lifetime. Thus, we examined the physiological consequences of repeated dehydration and compared results to larvae exposed to a single, prolonged period of dehydration. For the repeated dehydration experiment, larvae were exposed to 1–5 cycles of 24 h dehydration at 75% RH followed by 24 h rehydration. Each bout of dehydration resulted in 30–40% loss of body water, with a concomitant 2- to 3-fold increase in body fluid osmolality. While nearly 100% of larvae survived a single bout of dehydration

Aquaporins (AQPs) are water channel proteins facilitating movement of water across the cell membrane. Recent insect studies clearly demonstrate that AQPs are indispensable for cellular water management under normal conditions as well as under stress conditions including dehydration and cold. In the present study we cloned an AQP cDNA from the Antarctic midge Belgica antarctica (Diptera, Chironomidae) and investigated water transport activity of the AQP protein and transcriptional regulation of the gene in response to dehydration and rehydration. The nucleotide sequence and deduced amino acid sequence of the cDNA showed high similarity to AQPs in other insects and also showed characteristic features of orthodox AQPs. Phylogenetic analysis revealed that Belgica AQP is a homolog of dehydration-inducible AQP of another chironomid, Polypedilum vanderplanki. A swelling assay using a Xenopus oocyte expression system verified that Belgica AQP is capable of transporting water, but not glycerol or urea. The AQP mRNA was detected in various organs under non-stressed conditions, suggesting that this AQP plays a fundamental role in cell physiology. In contrast to our expectation, AQP transcriptional expression was not affected by either dehydration or rehydration.

In this study, pharate adults of the flesh fly Sarcophaga crassipalpis were exposed to two, four, seven, or ten days of severe hypoxia (3% oxygen) to evaluate its impact on emergence and the expression of genes encoding heat shock proteins (Hsps) and heat shock regulatory elements. A four-day exposure to hypoxia significantly reduced survival, but more than seven days was required to reach the LD(50). Eight genes encoding Hsps, at least one from each major family of Hsps (Hsp90, Hsp70, Hsp60, Hsp40, and sHsps) and two genes encoding proteins involved in Hsp regulation (heat shock factor, hsf, and sirtuin) were cloned, and expression levels were assessed during and after hypoxia using qRT-PCR. Most, but not all hsps studied, were significantly up-regulated during hypoxia, and expression levels for most of the hsps reverted to control levels a few hours after return to normoxia. Hsp70 was the most responsive to hypoxia, increasing expression several hundred fold. By contrast, hsp90 and hsp27 showed little response to hypoxia but did respond to recovery. Neither hsf nor sirtuin were elevated by hypoxia, an observation consistent with their assumed post-transcriptional regulatory roles. These data demonstrate a strong Hsp response to hypoxia, suggesting an important role for Hsps in responding to low oxygen environments.

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately -20 degrees C, all were killed by a direct 1 h exposure to -16 degrees C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4 degrees C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1-h exposure to 0 degrees C improved their subsequent tolerance of -14 and -16 degrees C. In response to heat stress, fewer than 20% of the bugs survived a 1-h exposure to 46 degrees C, and nearly all were killed at 48 degrees C. Dehydration, heat acclimation at 30 degrees C for 2 weeks and rapid heat hardening at 37 degrees C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.

This work was funded by grants from the UK Biotechnology and Biological Sciences Research Council (BB/G020620 and BB/L002647/1) (to S.-A.D., J.A.T.D., and S.T.); US Department of Agriculture/Department of Defense Deployed War Fighters Protection Grant Initiative (#6202-22000-029-00D) and US–Israel Binational Agricultural Research and Development Fund (BARD) (IS-4205-09C) (R.J.N.); and the National Science Foundation (IOS-0840772) (D.L.D.). The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance. Publisher PDF

The midge Belgica antarctica is the only insect endemic to Antarctica and has the southernmost range of any insect. In its natural environment, B. antarctica frequently faces desiccating conditions, as environmental water is frozen for up to 9 months annually. The molecular mechanisms by which B. antarctica tolerates extreme dehydration are poorly understood, but recent work from our laboratory reports genome-wide expression changes in response to extreme dehydration (~40% water loss), the first genome-scale transcriptome reported for an Antarctic animal. Among transcripts differentially regulated during dehydration, there is coordinated upregulation of numerous genes involved in autophagy, including genes responsible for autophagosome synthesis and autophagy-associated transcription factors. Also, several genes and pathways that interact with and regulate autophagy, e.g., sestrins and proteasomal genes, are concurrently upregulated. This suggests that autophagy and related processes are key elements regulating stress tolerance in this extreme environment.

The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment., The Antarctic midge, Belgica antarctica, is the only insect endemic to Antarctica. Here, the authors sequence the B. antarctica genome, the smallest insect genome yet reported, and suggest that genes involved in development, metabolism and stimuli response may have had a role in how this insect adapted to survive in such a harsh environment.