Molecular Mechanisms of Winter Survival.

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.

Scoring thermal limits in small insects using open-source, computer-assisted motion detection.

Scoring thermal tolerance traits live or with recorded video can be time consuming and susceptible to observer bias, and as with many physiological measurements, there can be trade-offs between accuracy and throughput. Recent studies show that automated particle tracking is a viable alternative to manually scoring videos, although some 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 throughput over manual methods.

Fine-scale variation in microhabitat conditions influences physiology and metabolism in an Antarctic insect.

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.

Laboratory diet influences cold tolerance in a genotype-dependent manner in Drosophila melanogaster.

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.

Evidence for a rapid cold hardening response in cultured Drosophila S2 cells.

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 Drosophila S2 cells, but pre-treatment with RCH at 4°C for 2 h prevented this decrease in viability. Thus, S2 cells are capable of RCH in a similar manner to whole insects and provide a new system for investigating the cell biology of RCH.

Rapid cold hardening: ecological relevance, physiological mechanisms and new perspectives.

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.

Environmental factors influencing fine-scale distribution of Antarctica's only endemic insect.

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.

Overexpression of an antioxidant enzyme improves male mating performance after stress in a lek-mating fruit fly.

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.

Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect.

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 non-lethal freezing injury. Larvae of B. antarctica were exposed to 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 a significantly higher metabolic rate 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.

Insect capa neuropeptides impact desiccation and cold tolerance.

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.

Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

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.

Calcium signaling mediates cold sensing in insect tissues.

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Gene expression changes governing extreme dehydration tolerance in an Antarctic insect.

Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world's southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions.

Surviving in a frozen desert: environmental stress physiology of terrestrial Antarctic arthropods.

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.

Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata.

In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. As a natural predator of agricultural pests, the lady beetle Coccinella septempunctata has been commercially mass-cultured and widely employed in pest management. In some insects, insulin signaling, in conjunction with the downstream transcription factor Forkhead box O (FoxO), are master regulators of multiple physiological processes involved in diapause, but it is unclear whether insulin signaling and FoxO affect the diapause of C. septempunctata. In this study, we use a combination of approaches to demonstrate that insulin signaling and FoxO mediate the diapause response in C. septempunctata. In diapausing beetles, application of exogenous insulin and knocking down expression of CsFoxo with RNA interference (RNAi) both rescued beetles from developmental arrest. In non-diapausing beetles, knocking down expression of the insulin receptor (CsInR) with RNA interference (RNAi) arrested ovarian development and decreased juvenile hormone (JH) content to levels comparable to the diapause state. Taken together, these results suggest that a shutdown of insulin signaling prompts the activation of the downstream FoxO gene, leading to the diapause phenotype.

The protective effect of rapid cold-hardening develops more quickly in frozen versus supercooled larvae of the Antarctic midge, Belgica antarctica.

During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as -12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at -5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae.

Survival and nutritional requirements for overwintering Drosophila suzukii (Diptera: Drosophilidae) in Kentucky.

The ability to cope with novel climates is a key determinant of an invasive species' success. Drosophila suzukii (Matsumura, 1931) is an invasive fruit pest, and its seasonality varies across its range. Current evidence suggests that D. suzukii occurs year-round in warmer climates but has low overwintering survival in colder climates and relies on refuges or reinvades each spring. Here, we assessed the capacity of D. suzukii ability to overwinter in Kentucky, a temperate mid-latitude state with relatively mild but variable winters. We tracked year-round population changes for 3 yr and observed the highest populations in early winter months. Following an annual population crash in winter, small numbers of flies remained through the late winter and spring. We also conducted outdoor cage studies to determine the extent to which food resources and microhabitat impact survival and postwinter fecundity under natural conditions. Flies with no food had poor survival during the warmest periods of winter, and flies in all treatments had lower survival in the coldest month. Provisioning flies with either artificial diet or wild berries improved survival. As a follow-up, we determined whether D. suzukii could survive and reproduce after long-term exposure to a typical winter temperature on various wild berries. Drosophila suzukii had the highest survival on privet (Ligustrum sp.), but all berry types yielded higher survival than flies without food. Our results suggest that noncrop berries play an important role for overwintering D. suzukii, and as winters warm the availability of wild berries could influence early-season populations.

Combined transcriptomic and metabolomic approach uncovers molecular mechanisms of cold tolerance in a temperate flesh fly.

The ability to respond rapidly to changes in temperature is critical for insects and other ectotherms living in variable environments. In a physiological process termed rapid cold-hardening (RCH), exposure to nonlethal low temperature allows many insects to significantly increase their cold tolerance in a matter of minutes to hours. Additionally, there are rapid changes in gene expression and cell physiology during recovery from cold injury, and we hypothesize that RCH may modulate some of these processes during recovery. In this study, we used a combination of transcriptomics and metabolomics to examine the molecular mechanisms of RCH and cold shock recovery in the flesh fly, Sarcophaga bullata. Surprisingly, out of ∼15,000 expressed sequence tags (ESTs) measured, no transcripts were upregulated during RCH, and likewise RCH had a minimal effect on the transcript signature during recovery from cold shock. However, during recovery from cold shock, we observed differential expression of ∼1,400 ESTs, including a number of heat shock proteins, cytoskeletal components, and genes from several cell signaling pathways. In the metabolome, RCH had a slight yet significant effect on several metabolic pathways, while cold shock resulted in dramatic increases in gluconeogenesis, amino acid synthesis, and cryoprotective polyol synthesis. Several biochemical pathways showed congruence at both the transcript and metabolite levels, indicating that coordinated changes in gene expression and metabolism contribute to recovery from cold shock. Thus, while RCH had very minor effects on gene expression, recovery from cold shock elicits sweeping changes in gene expression and metabolism along numerous cell signaling and biochemical pathways.

Transcriptional Regulation of Reproductive Diapause in the Convergent Lady Beetle, Hippodamia convergens.

Diapause is an alternate development program that synchronizes an insect's life cycle with seasonally abundant resources and ensures survival in unfavorable conditions. The physiological basis of diapause has been well characterized, but the molecular mechanisms regulating it are still being elucidated. Here, we present a de novo transcriptome and quantify transcript expression during diapause in the convergent lady beetle Hippodamia convergens. H. convergens is used as an augmentative biocontrol agent, and adult females undergo reproductive diapause that is regulated by photoperiod. We sampled females at three stages (early, mid, and late diapause) and compared transcript expression to non-diapausing individuals. Based on principle component analysis, the transcriptomes of diapausing beetles were distinct from non-diapausing beetles, and the three diapausing points tended to cluster together. However, there were still classes of transcripts that differed in expression across distinct phases of diapause. In general, transcripts involved in muscle function and flight were upregulated during diapause, likely to support dispersal flights that occur during diapause, while transcripts involved in ovarian development were downregulated. This information could be used to improve biological control by manipulating diapause. Additionally, our data contribute to a growing understanding of the genetic regulation of diapause across diverse insects.