Integrated transcriptional and biochemical profiling suggests mechanisms associated with rapid cold hardening in adult Liriomyza trifolii (Burgess).

The leafminer Liriomyza trifolii causes severe economic damage on ornamental and horticultural crops in China. Rapid cold hardening (RCH) is a phenomenon where cold tolerance in insects can be significantly enhanced after a short-term acclimation to low temperatures. In this study, the regulation of transcription in response to cold hardening was investigated in L. trifolii adults, and fatty acids and cryoprotectant levels were measured. The composition of fatty acids changed after RCH treatment, and glucose and trehalose levels showed significant accumulation after acclimation, thus indicating that changes in fatty acids and cryoprotectants contribute to RCH in L. trifolii. RNA-seq was used to analyze transcriptional regulation after a 4 h hardening period and showed that differentially expressed genes clustered in multiple metabolic pathways, which indicates the importance of transcriptional regulation in RCH. This study expands our knowledge of biochemical and transcriptional changes in L. trifolii during cold hardening and provides a basis for further investigations aimed at understanding thermal adaptation in insects.

Heat Shock Protein Genes Affect the Rapid Cold Hardening Ability of Two Invasive Tephritids.

Bactrocera dorsalis and Bactrocera correcta are two invasive species that can cause major economic damage to orchards and the fruit import and export industries. Their distribution is advancing northward due to climate change, which is threatening greater impacts on fruit production. This study tested the rapid cold-hardening ability of the two species and identified the temperature associated with the highest survival rate. Transcriptome data and survival data from the two Bactrocera species' larvae were obtained after rapid cold-hardening experiments. Based on the sequencing of transcripts, four Hsp genes were found to be affected: Hsp68 and Hsp70, which play more important roles in the rapid cold hardening of B. dorsalis, and Hsp23 and Hsp70, which play more important roles in the rapid cold hardening of B. correcta. This study explored the adaptability of the two species to cold, demonstrated the expression and function of four Hsps in response to rapid cold hardening, and explained the occurrence and expansion of these two species of tephritids, offering information for further studies.

Trehalose-6-Phosphate Synthase Contributes to Rapid Cold Hardening in the Invasive Insect Lissorhoptrus oryzophilus (Coleoptera: Curculionidae) by Regulating Trehalose Metabolism.

Rapid cold hardening (RCH) is known to rapidly enhance the cold tolerance of insects. Trehalose has been demonstrated to be a cryoprotectant in Lissorhoptrus oryzophilus, an important invasive pest of rice in China. Trehalose synthesis mainly occurs through the Trehalose-6-phosphate synthase (TPS)/trehalose-6-phosphate phosphatase (TPP) pathway in insects. In this study, the TPS gene from L. oryzophilus (LoTPS) was cloned and characterized for the first time. Its expression and trehalose content changes elicited by RCH were investigated. Our results revealed that RCH not only increased the survival rate of adults but also upregulated the expression level of LoTPS and increased the trehalose content under low temperature. We hypothesized that upregulated LoTPS promoted trehalose synthesis and accumulation to protect adults from low-temperature damage. To further verify the function of the LoTPS gene, we employed RNA interference (RNAi) technology. Our findings showed that RCH efficiency disappeared and the survival rate did not increase when the adults were fed dsRNA of LoTPS. Additionally, inhibiting LoTPS expression resulted in no significant difference in trehalose content between the RCH and non-RCH treatments. Furthermore, the expression patterns of trehalose transporter (TRET) and trehalase (TRE) were also affected. Collectively, these results indicate the critical role of LoTPS in L. oryzophilus cold resistance after RCH induction. LoTPS can enhance survival ability by regulating trehalose metabolism. These findings contribute to further understanding the role of TPS in insect cold resistance and the invasiveness of L. oryzophilus. Moreover, RNAi of LoTPS opens up possibilities for novel control strategies against L. oryzophilus in the future.

Rapid cold hardening modifies ion regulation to delay anoxia-induced spreading depolarization in the CNS of the locust.

Insects experience different kinds of environmental stresses that can impair neural performance, leading to spreading depolarization (SD) of nerve cells and neural shutdown underlying coma. SD is associated with a sudden loss of ion, notably K+, homeostasis in the central nervous system. The sensitivity of an insect's nervous system to stress (e.g., anoxia) can be modulated by acute pre-treatment. Rapid cold hardening (RCH) is a form of preconditioning, in which a brief exposure to low temperature can enhance the stress tolerance of insects. We used a pharmacological approach to investigate whether RCH affects anoxia-induced SD in the locust, Locusta migratoria, via one or more of the following homeostatic mechanisms: (1) Na+/K+-ATPase (NKA), (2) Na+/K+/2Cl- co-transporter (NKCC), and (3) voltage-gated K+ (Kv) channels. We also assessed abundance and phosphorylation of NKCC using immunoblotting. We found that inhibition of NKA or Kv channels delayed the onset of anoxia-induced SD in both control and RCH preparations. However, NKCC inhibition preferentially abrogated the effect of RCH. Additionally, we observed a higher abundance of NKCC in RCH preps but no statistical difference in its phosphorylation level, indicating the involvement of NKCC expression or degradation as part of the RCH mechanism.

Rapid cold hardening and cold acclimation promote cold tolerance of oriental fruit fly, Bactrocera dorsalis (Hendel) by physiological substances transformation and cryoprotectants accumulation.

Insect response to cold stress is often associated with adaptive strategies and chemical variation. However, low-temperature domestication to promote the cold tolerance potential of Bactrocera dorsalis and transformation of main internal substances are not clear. Here, we use a series of low-temperature exposure experiments, supercooling point (SCP) measurement, physiological substances and cryoprotectants detection to reveal that pre-cooling with milder low temperatures (5 and 10°C) for several hours (rapid cold hardening) and days (cold acclimation) can dramatically improve the survival rate of adults and pupae under an extremely low temperature (-6.5°C). Besides, the effect of rapid cold hardening for adults could be maintained even 4 h later with 25°C exposures, and SCP was significantly declined after cold acclimation. Furthermore, content of water, fat, protein, glycogen, sorbitol, glycerol and trehalose in bodies were measured. Results showed that water content was reduced and increased content of proteins, glycogen, glycerol and trehalose after two cold domestications. Our findings suggest that rapid cold hardening and cold acclimation could enhance cold tolerance of B. dorsalis by increasing proteins, glycerol, trehalose and decreasing water content. Conclusively, identifying a physiological variation will be useful for predicting the occurrence and migration trend of B. dorsalis populations.

Harnessing thermal plasticity to enhance the performance of mass-reared insects: opportunities and challenges.

Insects are mass-reared for release for biocontrol including the sterile insect technique. Insects are usually reared at temperatures that maximize the number of animals produced, are chilled for handling and transport, and released into the field, where temperatures may be considerably different to those experienced previously. Insect thermal biology is phenotypically plastic (i.e. flexible), which means that there may exist opportunities to increase the performance of these programmes by modifying the temperature regimes during rearing, handling, and release. Here we synthesize the literature on thermal plasticity in relation to the opportunities to reduce temperature-related damage and increase the performance of released insects. We summarize how and why temperature affects insect biology, and the types of plasticity shown by insects. We specifically identify aspects of the production chain that might lead to mismatches between the thermal acclimation of the insect and the temperatures it is exposed to, and identify ways to harness physiological plasticity to reduce that potential mismatch. We address some of the practical (especially engineering) challenges to implementing some of the best-supported thermal regimes to maximize performance (e.g. fluctuating thermal regimes), and acknowledge that a focus only on thermal performance may lead to unwanted trade-offs with other traits that contribute to the success of the programme. Together, it appears that thermal physiological plasticity is well-enough understood to allow its implementation in release programmes.

Transcriptome analysis uncovers different avenues for manipulating cold performance in Chrysomya megacephala (Diptera, Calliphoridae).

Temperature strongly impacts the rates of physiological and biochemical processes, which in turn can determine the survival and population size of insects. At low temperatures performance is limited, however, cold tolerance and performance at low temperature can be improved after short- or long-term acclimation in many insect species. To understand mechanisms underlying acclimation, we sequenced and compared the transcriptome of the blowfly Chrysomya megacephala under rapid cold hardening (RCH) and long-term cold acclimation (LCA) conditions. The RCH response was dominated by genes related to immune response, spliceosome, and protein processing in endoplasmic reticulum with up-regulation during recovery from RCH. In contrast, LCA was associated with genes related to carbohydrate metabolism and cytoskeleton branching and stabilizing. Meanwhile, mRNA levels of genes related to glycerophospholipid metabolism, and some heat shock proteins (Hsps) were collectively up-regulated by both RCH and LCA. There were more genes and pathway adjustments associated with LCA than RCH. Overall, the transcriptome data provide basic information of molecular mechanisms underpinning the RCH and LCA response. The partly independent molecular responses to RCH and LCA suggest that several avenues for manipulating cold performance exist and RCH might be more effective as it only triggers fewer genes and affects the general metabolisms less. These observations provide some appropriate methods to improve cold tolerance of C. megacephala, and hold promise for developing an extended use of mass-reared C. megacephala with better cold performance as a pollinator of crops at low temperatures.

Metabolomic and transcriptomic responses of ticks during recovery from cold shock reveal mechanisms of survival.

Ticks are blood-feeding ectoparasites but spend most of their life off-host where they may have to tolerate low winter temperatures. Rapid cold hardening (RCH) is a process commonly used by arthropods, including ticks, to improve survival of acute low temperature exposure. However, little is known about the underlying mechanisms in ticks associated with RCH, cold shock and recovery from these stresses. In the present study, we investigated the extent to which RCH influences gene expression and metabolism during recovery from cold stress in Dermacentor variabilis, the American dog tick, using a combined transcriptomics and metabolomics approach. Following recovery from RCH, 1860 genes were differentially expressed in ticks, whereas only 99 genes responded during recovery to direct cold shock. Recovery from RCH resulted in an upregulation of various pathways associated with ion binding, transport, metabolism and cellular structures seen in the response of other arthropods to cold. The accumulation of various metabolites, including several amino acids and betaine, corresponded to transcriptional shifts in the pathways associated with these molecules, suggesting congruent metabolome and transcriptome changes. Ticks, D. variabilis and Amblyomma maculatum, receiving exogenous betaine and valine demonstrated enhanced cold tolerance, suggesting cryoprotective effects of these metabolites. Overall, many of the responses during recovery from cold shock in ticks were similar to those observed in other arthropods, but several adjustments may be distinct from the responses in other currently examined taxa.

Glycerol biosynthesis plays an essential role in mediating cold tolerance the red imported fire ant, Solenopsis invicta.

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and known as a global problematic invasive species. At low temperatures, several investigations have demonstrated an increase in glycerol as a primary rapid cold hardening (RCH) component and an increase in the supercooling point. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), have been identified as being involved in the glycerol production process. In this study, one GPDH and two GK sequences were extracted from RIFA transcriptome analysis (Si-GPDH, Si-GK1, and Si-GK2). All three genes were expressed in different body parts and different tissues of S. invicta that Si-GK2 showed a higher expression level than the others. According to gene expression levels by qRT-PCR analysis, the highest expression levels of three genes were observed in fat body tissues. After 1 h of exposure to low temperatures (5°C or lower), the mRNA levels of these genes significantly increased, according to expression analyses. RNA interference (RNAi) of Si-GPDH or Si-GK1 and Si-GK2 exhibited a significant downregulation at the mRNA level. The mortality rate of treated RIFA by double-stranded RNA (dsRNA) specific to GPDH and GK2 significantly increased at low temperatures. This study indicates that GPDH and GK2 as glycerol biosynthesis genes in RIFA have a high expression level to synthesize a high level of glycerol as an RCH factor and they play crucial roles in survival during the cold period.

Differential Response of Leafminer Flies Liriomyza trifolii (Burgess) and Liriomyza sativae (Blanchard) to Rapid Cold Hardening.

Rapid cold hardening (RCH) is a rapid and critical adaption of insects to sudden temperature changes but is often overlooked or underestimated as a component of survival. Thus, interspecific comparisons of RCH are needed to predict how phenotypes will adapt to temperature variability. RCH not only enhances cold survival but also protects against non-lethal cold injury by preserving essential functions such as locomotion, reproduction, and energy balance. This study investigated the difference in basal cold tolerance and RCH capacity of L. trifolii and L. sativae. In both species, the cold tolerance of pupae was significantly enhanced after short-term exposure to moderately cold temperatures. The effect of RCH last for 4 h in L. sativae but only 2 h in L. trifolii. Interestingly, L. trifolii adults had a RCH response but L. sativae adults failed to acclimate. Short-term acclimation also lowered the supercooling point significantly in the pupae of both species. Based on these results, we propose a hypothesis that these differences will eventually affect their competition in the context of climate change. This study also provides the basis for future metabolomic and transcriptomic studies that may ultimately uncover the underlying mechanisms of RCH and interspecific competition between L. trifolii and L. sativae.

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.

Is rapid cold-hardening an aerobic process? Characterization of changes in metabolic activity during its induction and effects of anoxia in flesh fly.

Rapid cold-hardening (RCH) is a type of phenotypic plasticity that promotes a swift improvement of cold tolerance in insects. A brief exposure to mild cold dramatically increases insect survival to a subsequent cold exposure that would be lethal otherwise. In adult male flesh fly, Sarcophaga bullata, as little as 15 min at 5 °C significantly improved organismal survival at -7°C from 0 to 66.7 ± 11.1%. In this study, we investigated whether this RCH response is an aerobic process in S. bullata by characterizing changes in metabolic activity during its induction. At the level of whole organism, CO2 production continued at a level above our detection limit, and a relatively greater rate was observed during the early phase before it stabilized after ~1 h of the RCH induction. Similarly, in isolated flight muscle tissues, those maintained at 5 °C for 10 min exhibited significantly greater rates of oxygen consumption, compared to those maintained at 5 °C for 1 h (2.82 ± 0.29 vs. 1.36 ± 0.22 µl O2 mg-1 DM h-1). When these tissues were exposed to LaCl3, a treatment that should inhibit RCH ex vivo, oxygen consumption rates of the muscles were reduced significantly to a level similar to those that had been maintained at 5 °C for 1 h. Interestingly, however, the RCH response was still evident among individuals exposed to chilling under anoxia. Compared to those exposed to anoxia for 30 min only at 25 °C, flies exposed to 5 °C for 2 h under anoxia following the initial exposure exhibited a significantly greater level of cold tolerance at -7.5 °C (41.7 ± 7.1 vs. 91.8 ± 3.9%). Our results suggest that while relatively greater rates of metabolic activity are associated with the early phase of the RCH induction, it can proceed under the anoxic condition, thereby suggesting its independence to aerobic respiration.

Three novel trehalase genes from Harmonia axyridis (Coleoptera: Coccinellidae): cloning and regulation in response to rapid cold and re-warming.

Trehalose is the main blood sugar in insects. To study the function of trehalase during exposure to low temperatures, three other novel cDNAs of trehalase were cloned from Harmonia axyridis by transcriptome sequencing and rapid amplification of cDNA ends. One of the cloned cDNAs encoded a soluble trehalase, the second trehalase cDNA encoded a transmembrane-like domain, and the third cDNA encoded a membrane-bound protein. Therefore, these cDNAs were, respectively, named HaTreh1-5, HaTreh2-like, and HaTreh2. HaTreh1-5, HaTreh2-like, and HaTreh2 cDNAs encoded proteins containing 586, 553, and 633 amino acids with predicted masses of approximately 69.47, 63.46, and 73.66 kDa, and pIs of 9.20, 5.52, and 6.31, respectively. All three novel trehalases contained signal motifs "PGGINKESYYLDSY", "QWDYPNAWPP", and a highly conserved glycine-rich (GGGGEY) region. The expression levels of HaTreh1-5 and HaTreh2 mRNAs were high during adult stages, whereas HaTreh2-like was expressed in low amounts in the fourth larval stage. The results showed that the activity of membrane-bound trehalases decreased from 25 to 10 °C and from 5 to - 5 °C during cooling. The results also revealed a decreasing trend in expression of the three HaTreh mRNAs during the cooling treatment, and an initial decrease followed by an increase during the process of re-warming.

Rapid Cold Hardening Confers a Transient Increase in Low Temperature Survival in Diapausing Chilo suppressalis Larvae.

The striped stem borer, Chilo suppressalis (Walker), overwinters as a diapausing larva. The diapausing larvae were tested for a rapid cold hardening (RCH) response and its role in the insect’s survival of sub-zero temperatures. When laboratory-reared diapausing larvae were transferred directly from the rearing temperature of 25 °C to −14 °C and maintained there for 2 h, 21% survived. Acclimation of diapausing larvae for 4 h at 5 °C before their exposure for 2 h to −14 °C increased survival to approximately 41%, indicating an RCH response. Durability of RCH effects on low temperature survival was less than 1 h. Although transient in the test, the increased survival acquired through rapid cold hardening may play a role in preparing the diapausing larvae for abrupt temperature drops in the field that would otherwise be lethal.

Identification and analysis of up-regulated proteins in Lissorhoptrus oryzophilus adults for rapid cold hardening.

Rapid cold hardening (RCH) is a central physiological adaptation in which brief chilling significantly enhances the cold tolerance ability of insects. However, the mechanism of rapid cold stress response remains unknown for rice water weevils. To elucidate the mechanism, a comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE) between RCH and control treatment samples. A total of 21 protein spots were found to be up-regulated in rice water weevil adults for the RCH condition. As an initial work, 16 of these proteins were identified using MALDI TOF/TOF MS. In addition, the expression patterns of 9 proteins (including 2 HSPs that were not identified in the 2-DE experiment) were confirmed by qPCR analysis. Using the geneMANIA app in cytoscape, we constructed a protein-protein interaction network analysis of these up-regulated proteins. Gene ontology (GO) function annotation shows that a "response to heat process," including 6 HSPs, was related to the identified proteins. These up-regulated, RCH responsive proteins could possibly serve as potential biomarkers to study the molecular mechanisms of the L. oryzophilus response to cold stress.

Thermal plasticity potentially mediates the interaction between host Chilo partellus Swinhoe (Lepidoptera: Crambidae) and endoparasitoid Cotesia flavipes Cameron (Hymenoptera: Braconidae) in rapidly changing environments.

BACKGROUND: Increasing climatic average temperatures and variability elicit various insect physiological responses that affect fitness and survival and may influence subsequent trophic interactions in agroecosystems. In this background, we investigated short- and long-term plastic responses to temperature of the laboratory-reared stemborer Chilo partellus and its larval endoparasitoid Cotesia flavipes. RESULTS: Rapid cold- and heat-hardening effects in C. partellus larvae, pupae and adults and C. flavipes adults were highly significant (P < 0.001). High-temperature acclimation improved critical thermal limits and heat knockdown time in C. partellus larvae and C. flavipes adults, respectively. Low-temperature acclimation enhanced the supercooling point in C. flavipes and the chill coma recovery time in both C. partellus larvae and C. flavipes adults. CONCLUSION: The results of this study suggest that thermal plasticity may enhance the survival of these two species when they are subjected to lethal low and high temperatures. However, C. partellus appeared to be more plastic than C. flavipes. These results have three major implications: (1) C. partellus may inhabit slightly warmer environments than C. flavipes, suggesting a potential mismatch in biogeography; (2) host-parasitoid relationships are complex and are probably trait dependent, and (3) host-parasitoid differential thermal plastic responses may offset biocontrol efficacy. These results may help inform biocontrol decision making under conditions of global change. © 2017 Society of Chemical Industry.

Rapid Cold-Hardening of a Subtropical Species, Maruca vitrata (Lepidoptera: Crambidae), Accompanies Hypertrehalosemia by Upregulating Trehalose-6-Phosphate Synthase.

A subtropical insect, Maruca vitrata (F.) (Lepidoptera: Crambidae), is invasive to temperate zones, in which low temperatures during winter would be a serious challenge for colonization. This study assessed cold tolerance and cold-hardening of M. vitrata to understand its overwintering mechanism. Supercooling capacity was confirmed in all developmental stages exhibiting body freezing points at lower than -10°C, in which supercooling points (SCPs) were significantly different among developmental stages, with eggs having the lowest SCP (at -22.5°C). However, all developmental stages suffered significant mortality after being exposed to low temperatures much higher than SCPs. Furthermore, nonfreezing injury increased with elapsed time at 25°C after cold shock. One of the nonfreezing symptoms was a darkening on thorax, which was explained by uncontrolled prophenoloxidase activation. Pre-exposure to 8°C for 1 h significantly increased the survival of both young and old larvae to a low-temperature treatment (-5°C for 1 h). Rapid cold-hardening (RCH) was accompanied by significant increase in hemolymph trehalose concentration. During RCH, trehalose-6-phosphate synthase was significantly upregulated in its expression level. These results suggest that M. vitrata is a freeze-susceptible species and becomes cold-hardy via hypertrehalosemia.

Desiccation enhances rapid cold-hardening in the flesh fly Sarcophaga bullata: evidence for cross tolerance between rapid physiological responses.

Many insects use rapid cold-hardening (RCH), a physiological response to sub-lethal exposure to stressors, such as chilling and desiccation, to enhance their cold tolerance within minutes. Recently, drought-induced RCH, triggered by brief, mild desiccation, was described in larvae of the freeze-tolerant gall fly (Eurosta solidaginis). However, its prevalence and ecological significance in other insects is not known. Consequently, we used a freeze-intolerant model, the flesh fly, Sarcophaga bullata, to investigate the effects and mechanisms of drought-induced RCH. In addition, we investigated how drought- and cold-induced RCH interact by exposing flies to both desiccation and chilling. Desiccation for 3 h increased larval pupariation after cold shock from 28 to 40 %-the first example of drought-induced RCH in both a freeze-intolerant insect and in a non-overwintering life stage. We also found that desiccation and chilling together enhanced the cold hardiness of larvae and adults more than either did separately, suggesting that drought and cold trigger distinct physiological mechanisms that interact to afford greater cold tolerance. These results suggest that drought-induced RCH is a highly conserved response used by insects with diverse life history strategies. Furthermore, the protective interaction between drought- and cold-induced RCH suggests that, in nature, insects use multiple cues and physiological mechanisms to fine-tune their response to changing ambient conditions.

Cold hardiness and influences of hibernaculum conditions on overwintering survival of American dog tick larvae.

Understanding how ticks survive the multitude of stresses experienced during winter is integral to predicting population dynamics and transmission of tick-borne pathogens. The American dog tick (Dermacentor variabilis), a predominant vector of Rocky Mountain spotted fever, overwinters in any of its post-egg life stages. In this study, we characterized the cold tolerance of larval D. variabilis and examined the effect of various acclimatory conditions on cold hardiness. Cold-shock survival during 2h exposure to various subzero temperatures was assessed and a lower lethal temperature of -20°C and a 50% mortality temperature near -16°C was established. Larvae exposed to -5°C showed high survival (∼70%) after 14 d and near 50% for up to 56d at -5°C. Larvae cycled between supra- and subzero temperatures showed better long-term survival than at constant -5°C. The temperature of crystallization (Tc) was ∼-23°C and no larvae survived freezing after reaching their Tc. Larvae exposed to inoculative freezing survived brief, mild treatments (70% survival of -5°C for 2h) but survival was reduced compared to larvae cooled in the absence of ice. Reduced photophase, rapid cold hardening, dehydration, and long-term thermal acclimation all improved larval cold hardiness to varying degrees. Survival data were compared to measurements of hibernacula conditions, and we conclude that larvae face little threat from cold-induced mortality but inoculative freezing does pose a risk, and the geographic distribution of D. variabilis is only minimally influenced by the ability of larvae to survive low temperature exposure.

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.