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"Denlinger, David L"
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Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens
The short day lengths of late summer program the mosquito Culex pipiens to enter a reproductive diapause characterized by an arrest in ovarian development and the sequestration of huge fat reserves. We suggest that insulin signaling and FOXO (forkhead transcription factor), a downstream molecule in the insulin signaling pathway, mediate the diapause response. When we used RNAi to knock down expression of the insulin receptor in nondiapausing mosquitoes (those reared under long day lengths) the primary follicles were arrested in a stage comparable to diapause. The mosquitoes could be rescued from this developmental arrest with an application of juvenile hormone, an endocrine trigger known to terminate diapause in this species. When dsRNA directed against FOXO was injected into mosquitoes programmed for diapause (reared under short day lengths) fat storage was dramatically reduced and the mosquito's lifespan was shortened, results suggesting that a shutdown of insulin signaling prompts activation of the downstream gene FOXO, leading to the diapause phenotype. Thus, the results are consistent with a role for insulin signaling in the short-day response that ultimately leads to a cessation of juvenile hormone production. The similarity of this response to that observed in the diapause of Drosophila melanogaster and in dauer formation of Caenorhabditis elegans suggests a conserved mechanism regulating dormancy in insects and nematodes.
Journal Article
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 themoth 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.
Journal Article
REGULATION OF DIAPAUSE
Environmental and hormonal regulators of diapause have been reasonably well
defined, but our understanding of the molecular regulation of diapause remains
in its infancy. Though many genes are shut down during diapause, others are
specifically expressed at this time. Classes of diapause-upregulated genes can
be distinguished based on their expression patterns: Some are upregulated
throughout diapause, and others are expressed only in early diapause, late
diapause, or intermittently throughout diapause. The termination of diapause is
accompanied by a rapid decline in expression of the diapause-upregulated genes
and, conversely, an elevation in expression of many genes that were
downregulated during diapause. A comparison of insect diapause with other forms
of dormancy in plants and animals suggests that upregulation of a subset of
heat shock protein genes may be one feature common to different types of
dormancies.
Journal Article
Identification of FOXO targets that generate diverse features of the diapause phenotype in the mosquito Culex pipiens
Significance Diapause is an alternative developmental pathway exploited by insects and other invertebrates to survive inimical seasons. Like many insects that enter an adult diapause, the mosquito Culex pipiens responds to the short day lengths of autumn by feeding extensively on sugar, stockpiling huge fat reserves, halting reproduction, suppressing metabolism, boosting defense responses, and migrating to protected sites where it can safely bridge the winter months. In the experiments presented here, we propose that many of the diverse features of the diapause phenotype are the consequence of activating forkhead transcription factor (FOXO), a transcription factor downstream of insulin and juvenile hormone signaling. Our experimental results reveal how these upstream hormonal signaling pathways act through FOXO to generate the complex phenotype known as diapause.
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.
Journal Article
Obligate diapause and its termination shape the life-cycle seasonality of an Antarctic insect
The Antarctic midge,
Belgica antarctica
, is a unique insect endemic to Antarctica. It has a 2-year life cycle, with larvae overwintering in two different instars and adults emerging the following summer. This seasonality is crucial for adaptation to Antarctica’s harsh climates and ephemeral growing seasons; however, the underlying mechanisms remain unclear. We found that, under summer-like conditions, larvae could develop from egg to the fourth-instar larval stage without interruption, but they never pupated. Spontaneous developmental arrest at this stage suggests that they overwinter in obligate diapause, a genetically determined period of dormancy. The winter cold can terminate this diapause, and long-term cold exposure is more effective. Although this species can utilise two alternative cold tolerance strategies with diapause for overwintering, freezing was more successful than cryoprotective dehydration in allowing survival and developmental resumption in our experimental conditions. In contrast, the first three larval instars continued their development under the same conditions as the fourth-instar larvae. Although we do not exclude the possibility of facultative diapause, they likely overwinter in a quiescent state, an immediate developmental arrest in response to adversity, to maximise exploitation of the short Antarctic summer. Diapause and quiescence ensure developmental and reproductive success in this extremophile insect.
Journal Article
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.
Journal Article
Keeping time without a spine: what can the insect clock teach us about seasonal adaptation?
Seasonal change in daylength (photoperiod) is widely used by insects to regulate temporal patterns of development and behaviour, including the timing of diapause (dormancy) and migration. Flexibility of the photoperiodic response is critical for rapid shifts to new hosts, survival in the face of global climate change and to reproductive isolation. At the same time, the daily circadian clock is also essential for development, diapause and multiple behaviours, including correct flight orientation during long-distance migration. Although studied for decades, how these two critical biological timing mechanisms are integrated is poorly understood, in part because the core circadian clock genes are all transcription factors or regulators that are able to exert multiple effects throughout the genome. In this chapter, we discuss clocks in the wild from the perspective of diverse insect groups across eco-geographic contexts from the Antarctic to the tropical regions of Earth. Application of the expanding tool box of molecular techniques will lead us to distinguish universal from unique mechanisms underlying the evolution of circadian and photoperiodic timing, and their interaction across taxonomic and ecological contexts represented by insects.
This article is part of the themed issue ‘Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals’.
Journal Article
Comparative Transcriptomics Reveals Key Gene Expression Differences between Diapausing and Non-Diapausing Adults of Culex pipiens
Diapause is a critical eco-physiological adaptation for winter survival in the West Nile Virus vector, Culex pipiens, but little is known about the molecular mechanisms that distinguish diapause from non-diapause in this important mosquito species. We used Illumina RNA-seq to simultaneously identify and quantify relative transcript levels in diapausing and non-diapausing adult females. Among 65,623,095 read pairs, we identified 41 genes with significantly different transcript abundances between these two groups. Transcriptome divergences between these two phenotypes include genes related to juvenile hormone synthesis, anaerobic metabolism, innate immunity and cold tolerance.
Journal Article
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.
Journal Article