ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

Insect diapause: from a rich history to an exciting future.

Diapause, a stage-specific developmental arrest, is widely exploited by insects to bridge unfavorable seasons. Considerable progress has been made in understanding the ecology, physiology and evolutionary implications of insect diapause, yet intriguing questions remain. A more complete understanding of diapause processes on Earth requires a better geographic spread of investigations, including more work in the tropics and at high latitudes. Questions surrounding energy management and trade-offs between diapause and non-diapause remain understudied. We know little about how maternal effects direct the diapause response, and regulators of prolonged diapause are also poorly understood. Numerous factors that were recently linked to diapause are still waiting to be placed in the regulatory network leading from photoreception to engagement of the diapause program. These factors include epigenetic processes and small noncoding RNAs, and emerging data also suggest a role for the microbiome in diapause regulation. Another intriguing feature of diapause is the complexity of the response, resulting in a diverse suite of responses that comprise the diapause syndrome. Select transcription factors likely serve as master switches turning on these diverse responses, but we are far from understanding the full complexity. The richness of species displaying diapause offers a platform for seeking common components of a 'diapause toolbox'. Across latitudes, during invasion events and in a changing climate, diapause offers grand opportunities to probe evolutionary change and speciation. At a practical level, diapause responses can be manipulated for insect control and long-term storage. Diapausing insects also contain a treasure trove of pharmacological compounds and offer promising models for human health.

Exploiting tools for manipulating insect diapause.

Tools that could be used to subvert the insect diapause response offer potential for insect pest management as well as for the experimental manipulation of insects and the facilitation of mass rearing procedures. In some cases, it is desirable to break diapause on demand and in other cases, it may be attractive to exploit diapause for long-term storage of biocontrol agents or valuable experimental lines. This review highlights some of the diapause disruptors reported in the literature, as well as chemical and physical manipulations that can be used to extend diapause or even induce diapause in an insect not programmed for diapause. The insect hormones are quite effective agents for breaking diapause and in some cases for extending the duration of diapause, but a collection of other chemical agents can also act as potent diapause disruptors, e.g. organic solvents, weak acids and bases, carbon dioxide, imidazole compounds, LSD, deuterium oxide, DMSO, ouabain, cholera toxin, cyclic GMP, heavy metals, and hydrogen peroxide. Physical manipulations such as artificial light at night, anoxia, shaking and heat shock are also known diapause disruptors. Some of these documented manipulations prevent diapause, others terminate diapause immediately, others alter the duration of diapause, and a few compounds can induce a diapause-like state in insects that are not programmed for diapause. The diversity of tools noted in the literature offers promise for the development of new tools or manipulations that possibly could be used to disrupt diapause or manage diapause in controlled laboratory experiments and in mass-rearing facilities.

Rapid stress hardening in the Antarctic midge improves male fertility by increasing courtship success and preventing decline of accessory gland proteins following cold exposure.

Rapid hardening is a process that quickly improves an animal's performance following exposure to potentially damaging stress. In this study of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), we examined how rapid hardening in response to dehydration (RDH) or cold (RCH) improves male pre- and post-copulatory function when the insects are subsequently subjected to a damaging cold exposure. Neither RDH nor RCH improved survival in response to lethal cold stress, but male activity and mating success following sublethal cold exposure were enhanced. Egg viability decreased following direct exposure of the mating males to sublethal cold but improved following RCH and RDH. Sublethal cold exposure reduced the expression of four accessory gland proteins, while expression remained high in males exposed to RCH. Though rapid hardening may be cryptic in males, this study shows that it can be revealed by pre- and post-copulatory interactions with females.

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.

Evolutionary transition from blood feeding to obligate nonbiting in a mosquito.

The spread of blood-borne pathogens by mosquitoes relies on their taking a blood meal; if there is no bite, there is no disease transmission. Although many species of mosquitoes never take a blood meal, identifying genes that distinguish blood feeding from obligate nonbiting is hampered by the fact that these different lifestyles occur in separate, genetically incompatible species. There is, however, one unique extant species with populations that share a common genetic background but blood feed in one region and are obligate nonbiters in the rest of their range: Wyeomyia smithii Contemporary blood-feeding and obligate nonbiting populations represent end points of divergence between fully interfertile southern and northern populations. This divergence has undoubtedly resulted in genetic changes that are unrelated to blood feeding, and the challenge is to winnow out the unrelated genetic factors to identify those related specifically to the evolutionary transition from blood feeding to obligate nonbiting. Herein, we determine differential gene expression resulting from directional selection on blood feeding within a polymorphic population to isolate genetic differences between blood feeding and obligate nonbiting. We show that the evolution of nonbiting has resulted in a greatly reduced metabolic investment compared with biting populations, a greater reliance on opportunistic metabolic pathways, and greater reliance on visual rather than olfactory sensory input. W. smithii provides a unique starting point to determine if there are universal nonbiting genes in mosquitoes that could be manipulated as a means to control vector-borne disease.

Expression of aquaporins in response to distinct dehydration stresses that confer stress tolerance in the Antarctic midge Belgica antarctica.

Larvae of the Antarctic midge Belgica antarctica Jacobs (Diptera: Chironomidae) are highly tolerant of diverse environmental stresses, including freezing, severe desiccation, and osmotic extremes. Furthermore, dehydration confers subsequent desiccation and freeze tolerance. While a role for aquaporins-channels for water and other solutes-has been proposed in these dehydration processes, the types of aquaporins involved in dehydration-driven stress tolerance remain unknown. In the present study, we investigated expression of six aquaporins (Drip, Prip, Eglp1, Eglp2, Aqp12L, and Bib) in larvae of B. antarctica subjected to three different dehydration conditions: desiccation, cryoprotective dehydration, and osmotic dehydration. The expression of Drip and Prip was up-regulated under desiccation and cryoprotective dehydration, suggesting a role for these aquaporins in efficient water loss under these dehydration conditions. Conversely, expression of Drip and Prip was down-regulated under osmotic dehydration, suggesting that their expression is suppressed in larvae to combat dehydration. Larval water content was similarly decreased under all three dehydration conditions. Differences in responses of the aquaporins to the three forms of dehydration suggests distinct water management strategies associated with different forms of dehydration stress.

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.

Reactive oxygen species extend insect life span using components of the insulin-signaling pathway.

Reactive oxygen species (ROS) are well-known accelerants of aging, but, paradoxically, we show that physiological levels of ROS extend life span in pupae of the moth Helicoverpa armigera, resulting in the dormant state of diapause. This developmental switch appears to operate through a variant of the conventional insulin-signaling pathway, as evidenced by the facts that Akt, p-Akt, and PRMT1 are elevated by ROS, but not insulin, and that high levels of p-Akt fail to phosphorylate FoxO through PRMT1-mediated methylation. These results suggest a distinct signaling pathway culminating in the elevation of FoxO, which in turn promotes the extension of life span characteristic of diapause.

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.

Distinct microRNA and mRNA responses elicited by ecdysone, diapause hormone and a diapause hormone analog at diapause termination in pupae of the corn earworm, Helicoverpa zea.

Ecdysone, diapause hormone and a diapause hormone analog are all capable of breaking pupal diapause and prompting initiation of adult development in the cotton earworm, Helicoverpa zea. In this study we asked whether these three chemically-distinct diapause terminators elicit the same effect on expression of a collection of microRNAs and transcripts encoding components of the ecdysone signaling pathway. Injection of all three endocrine agents resulted in downregulation of one miRNA, miR-277-3p, a miRNA previously linked to the insulin/FOXO signaling pathway, and all three agents promoted upregulation of spook, a member of the ecdysone biosynthesis pathway, and iswi, an ecdysone-responsive transcript. Other miRNA and mRNA responses varied depending on the agent used to terminate diapause, thus suggesting that different endocrine pathways and mechanisms can lead to the same final developmental response.

Identification of FOXO targets that generate diverse features of the diapause phenotype in the mosquito Culex pipiens.

Insulin and juvenile hormone signaling direct entry of the mosquito Culex pipiens into its overwintering adult diapause, and these two critical signaling pathways appear to do so by converging on the regulation of forkhead transcription factor (FOXO). Diapause is a complex phenotype, and FOXO emerges as a prime candidate for activating many of the diverse physiological pathways that generate the diapause phenotype. Here, we used ChIP sequencing to identify direct targets of FOXO. The nearest gene in a 10-kb region surrounding a predicted binding site was extracted for each binding site, resulting in a dataset containing genes potentially regulated by FOXO. By selecting candidate genes based on their functional relevance to diapause, we identified five gene categories of potential interest, including stress tolerance, metabolic pathways, lifespan extension, cell cycle and growth regulation, and circadian rhythms. Twelve targets were prioritized for further analysis, 10 of which were validated by ChIP-quantitative PCR and quantitative real-time PCR. These 10 genes activated by FOXO are highly up-regulated during diapause and are thus strong candidates for implementation of the diapause syndrome.

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.

A novel highly divergent protein family identified from a viviparous insect by RNA-seq analysis: a potential target for tsetse fly-specific abortifacients.

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4-10). The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.

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.

Mechanisms of animal diapause: recent developments from nematodes, crustaceans, insects, and fish.

Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages.

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.

Functional circadian clock genes are essential for the overwintering diapause of the Northern house mosquito, Culex pipiens.

The short day lengths of late summer are used to program the overwintering adult diapause (dormancy) of the Northern house mosquito, Culex pipiens. Here, we investigated the role of clock genes in initiating this diapause and asked whether the circadian cycling of clock gene expression persists during diapause. We provide evidence that the major circadian clock genes continue to cycle throughout diapause and after diapause has been terminated. RNA interference (RNAi) was used to knock down the core circadian clock genes and to then assess the impact of the various clock genes on the ability of females to enter diapause. RNAi directed against negative circadian regulators (period, timeless and cryptochrome2) caused females that were reared under diapause-inducing, short day conditions to avert diapause. In contrast, knocking down the circadian-associated gene pigment dispersing factor caused females that were reared under diapause-averting, long day conditions to enter a diapause-like state. Our results implicate the circadian clock in the initiation of diapause in C. pipiens.

Mom Matters: Diapause Characteristics of Culex pipiens-Culex quinquefasciatus (Diptera: Culicidae) Hybrid Mosquitoes.

Females of the northern house mosquito, Culex pipiens L., are capable of entering an adult overwintering diapause characterized by arrested ovarian development, enhanced stress tolerance, and elevated lipid stores. In contrast, the southern house mosquito, Culex quinquefasciatus Say, lacks this capacity and is therefore unable to survive the harsh winters found in northern regions of North America. These two species are capable of forming fertile hybrids in the United States, yet the diapause characteristics of these hybrids have not been extensively investigated. We crossed Cx. pipiens from Columbus, OH, with Cx. quinquefasciatus from Vero Beach, FL, and reared F1 hybrids from all mothers separately under diapause-inducing, short-day conditions (a photoperiod of 8:16 [L:D] h) at 18°C. Egg follicle length and lipid content were used to assess the diapause status of hybrids. Diapause incidence of hybrids varied widely for progeny from different mothers of the same species, but hybrids with Cx. pipiens mothers were consistently more prone to enter diapause than hybrids that had Cx. quinquefasciatus mothers. Our results suggest a strong maternal influence on the diapause phenotype and that a high percentage (45-75%) of Cx. pipiens-Cx. quinquefasciatus hybrids are capable of entering diapause. This implies that many hybrids can successfully overwinter, leading to a possible widening of the hybrid zone of these two species in North America.

Cross-talk between the fat body and brain regulates insect developmental arrest.

Developmental arrest, a critical component of the life cycle in animals as diverse as nematodes (dauer state), insects (diapause), and vertebrates (hibernation), results in dramatic depression of the metabolic rate and a profound extension in longevity. Although many details of the hormonal systems controlling developmental arrest are well-known, we know little about the interactions between metabolic events and the hormones controlling the arrested state. Here, we show that diapause is regulated by an interplay between blood-borne metabolites and regulatory centers within the brain. Gene expression in the fat body, the insect equivalent of the liver, is strongly suppressed during diapause, resulting in low levels of tricarboxylic acid (TCA) intermediates circulating within the blood, and at diapause termination, the fat body becomes activated, releasing an abundance of TCA intermediates that act on the brain to stimulate synthesis of regulatory peptides that prompt production of the insect growth hormone ecdysone. This model is supported by our success in breaking diapause by injecting a mixture of TCA intermediates and upstream metabolites. The results underscore the importance of cross-talk between the brain and fat body as a regulator of diapause and suggest that the TCA cycle may be a checkpoint for regulating different forms of animal dormancy.