Daniel A. Hahn

Energetics of insect diapause.

Managing metabolic resources is critical for insects during diapause when food sources are limited or unavailable. Insects accumulate reserves prior to diapause, and metabolic depression during diapause promotes reserve conservation. Sufficient reserves must be sequestered to both survive the diapause period and enable postdiapause development that may involve metabolically expensive functions such as metamorphosis or long-distance flight. Nutrient utilization during diapause is a dynamic process, and insects appear capable of sensing their energy reserves and using this information to regulate whether to enter diapause and how long to remain in diapause. Overwintering insects on a tight energy budget are likely to be especially vulnerable to increased temperatures associated with climate change. Molecular mechanisms involved in diapause nutrient regulation remain poorly known, but insulin signaling is likely a major player. We also discuss other possible candidates for diapause-associated nutrient regulation including adipokinetic hormone, neuropeptide F, the cGMP-kinase For, and AMPK.

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.

Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis

BackgroundFlesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription.ResultsWe obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (E<0.0001) representing approximately 9,000 independent transcripts. Comparison of the distribution of S. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs.ConclusionOur data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic studies of S. crassipalpis and other flesh flies.

Developmental trajectories of gene expression reveal candidates for diapause termination: a key life-history transition in the apple maggot fly Rhagoletis pomonella

SUMMARY The timing of dormancy is a rapidly evolving life-history trait playing a crucial role in the synchronization of seasonal life cycles and adaptation to environmental change. But the physiological mechanisms regulating dormancy in animals remain poorly understood. In insects, dormancy (diapause) is a developmentally dynamic state, and the mechanisms that control diapause transitions affect seasonal timing. Here we used microarrays to examine patterns of gene expression during dormancy termination: a crucial life-history transition in the apple maggot fly Rhagoletis pomonella (Walsh). This species is a model system for host race formation and ecological speciation via changes in diapause regulation of seasonality. Our goal was to pinpoint the timing of the transition from diapause to post-diapause development and to identify candidate genes and pathways for regulation of diapause termination. Samples were taken at six metabolically defined developmental landmarks, and time-series analysis suggests that release from metabolic depression coincides with preparation for or resumption of active cell cycling and morphogenesis, defining the ‘end’ of diapause. However, marked changes in expression, including members of pathways such as Wnt and TOR signaling, also occur prior to the metabolic rate increase, electing these pathways as candidates for early regulation of diapause termination. We discuss these results with respect to generalities in insect diapause physiology and to our long-term goal of identifying mechanisms of diapause adaptation in the Rhagoletis system.

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.

Biphasic metabolic rate trajectory of pupal diapause termination and post-diapause development in a tephritid fly

Metabolic depression is a highly conserved feature of insect diapause, and an increase in metabolism is a reliable indicator of diapause termination and the initiation of post-diapause development. The trajectory of metabolic rate following diapause termination can guide the identification of important physiological and developmental landmarks during this developmental transition, yet quantitative descriptions of these trajectories are relatively rare. Here we track changes in metabolic rate from diapause through diapause termination and pharate adult development in Rhagoletis pomonella (Diptera: Tephritidae), a univoltine tephritid fly that diapauses in the pupal stage. Using respirometric monitoring we show that diapause termination and subsequent pharate adult development is characterized by a biphasic increase in metabolic rate. Respiration rate initially increases logistically, reaching a plateau that is followed by a final, exponential increase terminating in adult eclosion. We develop a non-linear model describing this pattern with easily interpretable landmarks, and we map visible landmarks of morphogenesis onto the trajectory. The bulk of visible morphogenesis in pharate adult development occurs relatively late in the trajectory during the final, exponential phase, that matches the U-shaped trajectory of non-diapause individuals. These results mirror a qualitative description of the same trajectories of diapause and non-diapause flesh flies (Sarcophaga). Overall, our results suggest that (1) the course of diapause breakage and post-diapause development may be evolutionarily conserved in higher flies with pupal diapause, and (2) the biphasic trajectory is distinct from a more continuous trajectory observed during direct development.

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.

Amino acids in nectar enhance longevity of female Culex quinquefasciatus mosquitoes.

Culex mosquitoes feed on a wide range of nectars consisting of mostly carbohydrates and amino acids, however, little is known about the utilization and effects of these different carbohydrates and their accompanying amino acids on longevity. Culex quinquefasciatus larvae were reared on low- and high-quantity food diets to produce adults that were nutritionally representative of wild-caught and laboratory-reared mosquitoes. Emerging adults reared on low- or high-quantity food diets as larvae were then provided Lantana camara nectar mimics containing mixtures of carbohydrates and amino acids to evaluate effects of nectar amino acids on longevity. Carbohydrates (with or without amino acids) were a critical component of the adult diet, and in their absence, adult mosquitoes died within 3-5 days. The nectar mimic that contained both carbohydrates and amino acids did not increase adult longevity of males originating from either poorly or well-fed larvae. However, females receiving adult diets containing both carbohydrates and amino acids lived 5% longer than females fed adult diets with only sugar.

Early changes in the pupal transcriptome of the flesh fly Sarcophagha crassipalpis to parasitization by the ectoparasitic wasp, Nasonia vitripennis

We investigated changes in the pupal transcriptome of the flesh fly Sarcophaga crassipalpis, 3 and 25 h after parasitization by the ectoparasitoid wasp, Nasonia vitripennis. These time points are prior to hatching of the wasp eggs, thus the results document host responses to venom injection, rather than feeding by the wasp larvae. Only a single gene appeared to be differentially expressed 3 h after parasitization. However, by 25 h, 128 genes were differentially expressed and expression patterns of a subsample of these genes were verified using RT-qPCR. Among the responsive genes were clusters of genes that altered the flys metabolism, development, induced immune responses, elicited detoxification responses, and promoted programmed cell death. Envenomation thus clearly alters the metabolic landscape and developmental fate of the fly host prior to subsequent penetration of the pupal cuticle by the wasp larva. Overall, this study provides new insights into the specific action of ectoparasitoid venoms.

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’.