Variation in Thermal Sensitivity of Diapause Development among Individuals and over Time Predicts Life History Timing in a Univoltine Insect.

AbstractPhysiological time is important for understanding the development and seasonal timing of ectothermic animals but has largely been applied to developmental processes that occur during spring and summer, such as morphogenesis. There is a substantial knowledge gap in the relationship between temperature and development during winter, a season that is increasingly impacted by climate change. Most temperate insects overwinter in diapause, a developmental process with little obvious morphological change. We used principles from the physiological time literature to measure and model the thermal sensitivity of diapause development rate in the apple maggot fly Rhagoletis pomonella, a univoltine fly whose diapause duration varies substantially within and among populations. We show that diapause duration can be predicted by modeling a relationship between temperature and development rate that is shifted toward lower temperatures compared with typical models of morphogenic, nondiapause development. However, incorporating interindividual variation and ontogenetic variation in the temperature-to-development rate relationship was critical for accurately predicting fly emergence, as diapause development proceeded more quickly at high temperatures later in diapause. We conclude that the conceptual framework may be flexibly applied to other insects and discuss possible mechanisms of diapause timers and implications for phenology with warming winters.

Simulated climate warming causes asymmetric responses in insect life-history timing potentially disrupting a classic ecological speciation system.

Climate change may alter phenology within populations with cascading consequences for community interactions and on-going evolutionary processes. Here, we measured the response to climate warming in two sympatric, recently diverged (~170 years) populations of Rhagoletis pomonella flies specialized on different host fruits (hawthorn and apple) and their parasitoid wasp communities. We tested whether warmer temperatures affect dormancy regulation and its consequences for synchrony across trophic levels and temporal isolation between divergent populations. Under warmer temperatures, both fly populations developed earlier. However, warming significantly increased the proportion of maladaptive pre-winter development in apple, but not hawthorn, flies. Parasitoid phenology was less affected, potentially generating ecological asynchrony. Observed shifts in fly phenology under warming may decrease temporal isolation, potentially limiting on-going divergence. Our findings of complex sensitivity of life-history timing to changing temperatures predict that coming decades may see multifaceted ecological and evolutionary changes in temporal specialist communities.

Evolutionary ecology of the bark beetles Ips typographus and Pityogenes chalcographus.

Ips typographus (L.) and Pityogenes chalcographus (L.) (Coleoptera: Curculionidae) are two common bark beetle species on Norway spruce in Eurasia. Multiple biotic and abiotic factors affect the life cycles of these two beetles, shaping their ecology and evolution. In this article, we provide a comprehensive and comparative summary of selected life-history traits. We highlight similarities and differences in biotic factors, like host range, interspecific competition, host colonization, reproductive behaviour and fungal symbioses. Moreover, we focus on the species' responses to abiotic factors and compare their temperature-dependent development and flight behaviour, cold adaptations and diapause strategies. Differences in biotic and abiotic traits might be the result of recent, species-specific evolutionary histories, particularly during the Pleistocene, with differences in glacial survival and postglacial recolonization. Finally, we discuss future research directions to understand ecological and evolutionary pathways of the two bark beetle species, for both basic research and applied forest management.

Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation.

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence.

Divergent diapause life history timing drives both allochronic speciation and reticulate hybridization in an adaptive radiation of Rhagoletis flies.

Divergent adaptation to new ecological opportunities can be an important factor initiating speciation. However, as niches are filled during adaptive radiations, trait divergence driving reproductive isolation between sister taxa may also result in trait convergence with more distantly related taxa, increasing the potential for reticulated gene flow across the radiation. Here, we demonstrate such a scenario in a recent adaptive radiation of Rhagoletis fruit flies, specialized on different host plants. Throughout this radiation, shifts to novel hosts are associated with changes in diapause life history timing, which act as "magic traits" generating allochronic reproductive isolation and facilitating speciation-with-gene-flow. Evidence from laboratory rearing experiments measuring adult emergence timing and genome-wide DNA-sequencing surveys supported allochronic speciation between summer-fruiting Vaccinium spp.-infesting Rhagoletis mendax and its hypothesized and undescribed sister taxon infesting autumn-fruiting sparkleberries. The sparkleberry fly and R. mendax were shown to be genetically discrete sister taxa, exhibiting no detectable gene flow and allochronically isolated by a 2-month average difference in emergence time corresponding to host availability. At sympatric sites across the southern USA, the later fruiting phenology of sparkleberries overlaps with that of flowering dogwood, the host of another more distantly related and undescribed Rhagoletis taxon. Laboratory emergence data confirmed broadly overlapping life history timing and genomic evidence supported on-going gene flow between sparkleberry and flowering dogwood flies. Thus, divergent phenological adaptation can drive the initiation of reproductive isolation, while also enhancing genetic exchange across broader adaptive radiations, potentially serving as a source of novel genotypic variation and accentuating further diversification.

Genomically correlated trait combinations and antagonistic selection contributing to counterintuitive genetic patterns of adaptive diapause divergence in Rhagoletis flies.

Adaptation to novel environments can result in unanticipated genomic responses to selection. Here, we illustrate how multifarious, correlational selection helps explain a counterintuitive pattern of genetic divergence between the recently derived apple- and ancestral hawthorn-infesting host races of Rhagoletis pomonella (Diptera: Tephritidae). The apple host race terminates diapause and emerges as adults earlier in the season than the hawthorn host race, to coincide with the earlier fruiting phenology of their apple hosts. However, alleles at many loci associated with later emergence paradoxically occur at higher frequencies in sympatric populations of the apple compared to the hawthorn race. We present genomic evidence that historical selection over geographically varying environmental gradients across North America generated genetic correlations between two life history traits, diapause intensity and diapause termination, in the hawthorn host race. Moreover, the loci associated with these life history traits are concentrated in genomic regions in high linkage disequilibrium (LD). These genetic correlations are antagonistic to contemporary selection on local apple host race populations that favours increased initial diapause depth and earlier, not later, diapause termination. Thus, the paradox of apple flies appears due, in part, to pleiotropy or linkage of alleles associated with later adult emergence and increased initial diapause intensity, the latter trait strongly selected for by the earlier phenology of apples. Our results demonstrate how understanding of multivariate trait combinations and the correlative nature of selective forces acting on them can improve predictions concerning adaptive evolution and help explain seemingly counterintuitive patterns of genetic diversity in nature.

Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages.

Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, we understand very little about how this genetic decoupling occurs. We tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved cellular stress response. We used RNAseq to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and used RNA interference (RNAi) to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5°C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, we analyzed the tissue-specificity of differentially expressed transcripts from FlyAtlas 2 data, and concluded that stage-specific differences in transcription were not simply driven by differences in tissue composition. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages.

Costs of averting or prematurely terminating diapause associated with slow decline of metabolic rates at low temperature.

Diapause, a form of insect dormancy, generally facilitates overwintering by increasing cold tolerance and decreasing energy drain at high temperatures via metabolic rate suppression. Averting or terminating diapause prior to winter is generally assumed to be a lethal phenotype. However, low temperature acclimation can also increase cold tolerance and decrease metabolic rates. Here, we tested the hypothesis that non- and post-diapause individuals in a cold-induced quiescence can achieve a diapause-like phenotype, compensating for the potential costs of averting diapause. We tested this in the apple maggot fly Rhagoletis pomonella, which typically overwinters in the soil as a diapause pupa, but can avert diapause (non-diapause) or terminate diapause early ('weak diapause') when reared at warm temperatures. Metabolic rates were initially higher in non- and post-diapause than diapause pupae at high (25 °C) and low (4 °C) temperatures, but quiescent non- and post-diapause pupae achieved diapause-like metabolic rates slowly over time when incubated at 4 °C for several weeks. We found that diapause and quiescent pupae were freeze-avoidant and had similar tolerance of extreme low temperatures (cooling to c. -18 °C) following 8 weeks acclimation at 4 °C. Despite high tolerance of subzero temperatures, quiescent pupae did not survive well when chilled for prolonged periods (8 weeks or more) at 4 °C. We conclude that cold acclimation can only partially compensate for costs associated with aversion or premature termination of diapause, and that energy drain at low (not just high) temperatures likely contributes to chilling mortality in quiescent insects.

A rapidly evolved shift in life-history timing during ecological speciation is driven by the transition between developmental phases.

For insect species in temperate environments, seasonal timing is often governed by the regulation of diapause, a complex developmental programme that allows insects to weather unfavourable conditions and synchronize their life cycles with available resources. Diapause development consists of a series of distinct phases including initiation, maintenance, termination and post-diapause development. The evolution of insect seasonal timing depends in part on how these phases of diapause development and post-diapause development interact to affect variation in phenology. Here, we dissect the physiological basis of a recently evolved phenological shift in Rhagoletis pomonella (Diptera: Tephritidae), a model system for ecological divergence. A recently derived population of R. pomonella shifted from specializing on native hawthorn fruit to earlier fruiting introduced apples, resulting in a 3-4 week shift in adult emergence timing. We tracked metabolic rates of individual flies across post-winter development to test which phases of development may act either independently or in combination to contribute to this recently evolved divergence in timing. Apple and hawthorn flies differed in a number of facets of their post-winter developmental trajectories. However, divergent adaptation in adult emergence phenology in these flies was due almost entirely to the end of the pupal diapause maintenance phase, with post-diapause development having a very small effect. The relatively simple underpinnings of variation in adult emergence phenology suggest that further adaptation to seasonal change in these flies for this trait might be largely due to the timing of diapause termination unhindered by strong covariance among different components of post-diapause development.

Host plant-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi.

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host-related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America.

Stage-specific genotype-by-environment interactions for cold and heat hardiness in Drosophila melanogaster.

Environments often vary across a life cycle, imposing fluctuating natural selection across development. Such fluctuating selection can favor different phenotypes in different life stages, but stage-specific evolutionary responses will depend on genetic variance, covariance, and their interaction across development and across environments. Thus, quantifying how genetic architecture varies with plastic responses to the environment and across development is vital to predict whether stage-specific adaptation will occur in nature. Additionally, the interaction of genetic variation and environmental plasticity (GxE) may be stage-specific, leading to a three-way interaction between genotype, environment, and development or GxDxE. To test for these patterns, we exposed larvae and adults of Drosophila melanogaster isogenic lines derived from a natural population to extreme heat and cold stress after developmental acclimation to cool (18 °C) and warm (25 °C) conditions and measured genetic variance for thermal hardiness. We detected significant GxE that was specific to larvae and adults for cold and heat hardiness (GxDxE), but no significant genetic correlation across development for either trait at either acclimation temperature. However, cross-development phenotypic correlations for acclimation responses suggest that plasticity itself may be developmentally constrained, though rigorously testing this hypothesis requires more experimentation. These results illustrate the potential for stage-specific adaptation within a complex life cycle and demonstrate the importance of measuring traits at appropriate developmental stages and environmental conditions when predicting evolutionary responses to changing climates.

Reproductive isolation and environmental adaptation shape the phylogeography of mountain pine beetle (Dendroctonus ponderosae).

Chromosomal rearrangement can be an important mechanism driving population differentiation and incipient speciation. In the mountain pine beetle (MPB, Dendroctonus ponderosae), deletions on the Y chromosome that are polymorphic among populations are associated with reproductive incompatibility. Here, we used RAD sequencing across the entire MPB range in western North America to reveal the extent of the phylogeographic differences between Y haplotypes compared to autosomal and X-linked loci. Clustering and geneflow analyses revealed three distinct Y haplogroups geographically positioned within and on either side of the Great Basin Desert. Despite close geographic proximity between populations on the boundaries of each Y haplogroup, there was extremely low Y haplogroup mixing among populations, and gene flow on the autosomes was reduced across Y haplogroup boundaries. These results are consistent with a previous study suggesting that independent degradation of a recently evolved neo-Y chromosome in previously isolated populations causes male sterility or inviability among Y haplotype lineages. Phylogeographic results supported historic contraction of MPB into three separate Pleistocene glacial refugia followed by postglacial range expansion and secondary contact. Distinct sets of SNPs were statistically associated with environmental data among the most genetically distinct sets of geographic populations. This finding suggests that the process of adaptation to local climatic conditions is influenced by population genetic structure, with evidence for largely independent evolution in the most genetically isolated Y haplogroup.

A test of genomic modularity among life-history adaptations promoting speciation with gene flow.

Speciation with gene flow may require adaptive divergence of multiple traits to generate strong ecologically based reproductive isolation. Extensive negative pleiotropy or physical linkage of genes in the wrong phase affecting these diverging traits may therefore hinder speciation, while genetic independence or "modularity" among phenotypic traits may reduce constraints and facilitate divergence. Here, we test whether the genetics underlying two components of diapause life history, initial diapause intensity and diapause termination timing, constrain differentiation between sympatric hawthorn and apple-infesting host races of the fly Rhagoletis pomonella through analysis of 10,256 SNPs measured via genotyping-by-sequencing (GBS). Loci genetically associated with diapause termination timing were mainly observed for SNPs mapping to chromosomes 1-3 in the genome, most notably for SNPs displaying higher levels of linkage disequilibrium (LD), likely due to inversions. In contrast, selection on initial diapause intensity affected loci on all five major chromosomes of the genome, specifically those showing low levels of LD. This lack of overlap in genetically associated loci suggests that the two diapause phenotypes are largely modular. On chromosome 2, however, intermediate level LD loci and a subgroup of high LD loci displayed significant negative relationships between initial diapause intensity and diapause termination time. These gene regions on chromosome 2 therefore affected both traits, while most regions were largely independent. Moreover, loci associated with both measured traits also tended to exhibit highly divergent allele frequencies between the host races. Thus, the presence of nonoverlapping genetic modules likely facilitates simultaneous, adaptive divergence for the measured life-history components.

Divergence of the diapause transcriptome in apple maggot flies: winter regulation and post-winter transcriptional repression.

The duration of dormancy regulates seasonal timing in many organisms and may be modulated by day length and temperature. Though photoperiodic modulation has been well studied, temperature modulation of dormancy has received less attention. Here, we leverage genetic variation in diapause in the apple maggot fly, Rhagoletis pomonella, to test whether gene expression during winter or following spring warming regulates diapause duration. We used RNAseq to compare transcript abundance during and after simulated winter between an apple-infesting population and a hawthorn-infesting population where the apple population ends pupal diapause earlier than the hawthorn-infesting population. Marked differences in transcription between the two populations during winter suggests that the 'early' apple population is developmentally advanced compared with the 'late' hawthorn population prior to spring warming, with transcripts participating in growth and developmental processes relatively up-regulated in apple pupae during the winter cold period. Thus, regulatory differences during winter ultimately drive phenological differences that manifest themselves in the following summer. Expression and polymorphism analysis identify candidate genes in the Wnt and insulin signaling pathways that contribute to population differences in seasonality. Both populations remained in diapause and displayed a pattern of up- and then down-regulation (or vice versa) of growth-related transcripts following warming, consistent with transcriptional repression. The ability to repress growth stimulated by permissive temperatures is likely critical to avoid mismatched phenology and excessive metabolic demand. Compared with diapause studies in other insects, our results suggest some overlap in candidate genes/pathways, though the timing and direction of changes in transcription are likely species specific.

Experimental evidence of genome-wide impact of ecological selection during early stages of speciation-with-gene-flow.

Theory predicts that speciation-with-gene-flow is more likely when the consequences of selection for population divergence transitions from mainly direct effects of selection acting on individual genes to a collective property of all selected genes in the genome. Thus, understanding the direct impacts of ecologically based selection, as well as the indirect effects due to correlations among loci, is critical to understanding speciation. Here, we measure the genome-wide impacts of host-associated selection between hawthorn and apple host races of Rhagoletis pomonella (Diptera: Tephritidae), a model for contemporary speciation-with-gene-flow. Allele frequency shifts of 32 455 SNPs induced in a selection experiment based on host phenology were genome wide and highly concordant with genetic divergence between co-occurring apple and hawthorn flies in nature. This striking genome-wide similarity between experimental and natural populations of R. pomonella underscores the importance of ecological selection at early stages of divergence and calls for further integration of studies of eco-evolutionary dynamics and genome divergence.

Differences in performance and transcriptome-wide gene expression associated with Rhagoletis (Diptera: Tephritidae) larvae feeding in alternate host fruit environments.

Host race formation, the establishment of new populations using novel resources, is a major hypothesized mechanism of ecological speciation, especially in plant-feeding insects. The initial stages of host race formation will often involve phenotypic plasticity on the novel resource, with subsequent genetically based adaptations enhancing host-associated fitness differences. Several studies have explored the physiology of the plastic responses of insects to novel host environments. However, the mechanisms underlying evolved differences among host races and species remain poorly understood. Here, we demonstrate a reciprocal larval performance difference between two closely related species of Rhagoletis flies, R. pomonella and R. zephyria, specialized for feeding in apple and snowberry fruit, respectively. Microarray analysis of fly larvae feeding in apples versus snowberries revealed patterns of transcriptome-wide differential gene expression consistent with both plastic and evolved responses to the different fruit resources, most notably for detoxification-related genes such as cytochrome p450s. Transcripts exhibiting evolved expression differences between species tended to also demonstrate plastic responses to fruit environment. The observed pattern suggests that Rhagoletis larvae exhibit extensive plasticity in gene expression in response to novel fruit that may potentiate shifts to new hosts. Subsequent selection, particularly selection to suppress initially costly plastic responses, could account for the evolved expression differences observed between R. pomonella and R. zephyria, creating specialized races and new fly species. Thus, genetically based ecological adaptations generating new biodiversity may often evolve from initial plastic responses in gene expression to the challenges posed by novel environments.

Seasonality of forest insects: why diapause matters.

Insects have major impacts on forest ecosystems, from herbivory and soil-nutrient cycling to killing trees at a large scale. Forest insects from temperate, tropical, and subtropical regions have evolved strategies to respond to seasonality; for example, by entering diapause, to mitigate adversity and to synchronize lifecycles with favorable periods. Here, we show that distinct functional groups of forest insects; that is, canopy dwellers, trunk-associated species, and soil/litter-inhabiting insects, express a variety of diapause strategies, but do not show systematic differences in diapause strategy depending on functional group. Due to the overall similarities in diapause strategies, we can better estimate the impacts of anthropogenic change on forest insect populations and, consequently, on key ecosystems.

Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly.

Diapause is a widespread adaptation to seasonality across invertebrate taxa. It is critical for persistence in seasonal environments, synchronizing life histories with favorable, resource-rich conditions and mitigating exposure to harsh environments. Despite some promising recent progress, however, we still know very little about the molecular modifications underlying diapause. We used transcriptional profiling to identify key groups of genes and pathways differentially regulated during pupal diapause, dynamically regulated across diapause development, and differentially regulated after diapause was pharmacologically terminated in the flesh fly Sarcophaga crassipalpis. We describe major shifts in stress axes, endocrine signaling, and metabolism that accompany diapause, several of which appear to be common features of dormancy in other taxa. To assess whether invertebrates with different diapause strategies have converged toward similar transcriptional profiles, we use archived expression data to compare the pupal diapause of S. crassipalpis with the adult reproductive diapause of Drosophila melanogaster and the larval dauer of Caenorhabditis elegans. Although dormant invertebrates converge on a few similar physiological phenotypes including metabolic depression and stress resistance, we find little transcriptional similarity among dormancies across species, suggesting that there may be many transcriptional strategies for producing physiologically similar dormancy responses.

Cold tolerance and diapause within and across trophic levels: Endoparasitic wasps and their fly host have similar phenotypes.

Low temperatures associated with winter can limit the survival of organisms, especially ectotherms whose body temperature is similar to their environment. However, there is a gap in understanding how overwintering may vary among groups of species that interact closely, such as multiple parasitoid species that attack the same host insect. Here, we investigate cold tolerance and diapause phenotypes in three endoparasitoid wasps of the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae): Utetes canaliculatus, Diachasma alloeum, and Diachasmimorpha mellea (Hymenoptera: Braconidae). Using a combination of respirometry and eclosion tracking, we found that all three wasp species exhibited the same three diapause duration phenotypes as the fly host. Weak (short duration) diapause was rare, with <5 % of all three wasp species prematurely terminating diapause at 21 °C. Most D.mellea (93 %) entered a more intense (longer duration) diapause that did not terminate within 100 d at this warm temperature. The majority of U.canaliculatus (92 %) and D. alloeum (72 %) averted diapause (non-diapause) at 21 °C. There was limited interspecific variation in acute cold tolerance among the three wasp species: wasps and flies had similarly high survival (>87 %) following exposure to extreme low temperatures (-20 °C) as long as their body fluids did not freeze. The three wasp species also displayed little interspecific variation in survival following prolonged exposure to mild chilling of 8 or more weeks at 4 °C. Our study thus documents a remarkable conservation of cold tolerance and diapause phenotypes within and across trophic levels.

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.