Gregory J. Ragland

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.

Plasticity of Size and Growth in Fluctuating Thermal Environments: Comparing Reaction Norms and Performance Curves

Abstract Ectothermic animals exhibit two distinct kinds of plasticity in response to temperature: Thermal performance curves (TPCs), in which an individuals performance (e.g., growth rate) varies in response to current temperature; and developmental reaction norms (DRNs), in which the trait value (e.g., adult body size or development time) of a genotype varies in response to developmental temperatures experienced over some time period during development. Here we explore patterns of genetic variation and selection on TPCs and DRNs for insects in fluctuating thermal environments. First, we describe two statistical methods for partitioning total genetic variation into variation for overall size or performance and variation in plasticity, and apply these methods to available datasets on DRNs and TPCs for insect growth and size. Our results indicate that for the datasets we considered, genetic variation in plasticity represents a larger proportion of the total genetic variation in TPCs compared to DRNs, for the available datasets. Simulations suggest that estimates of the genetic variation in plasticity are strongly affected by the number and range of temperatures considered, and by the degree of nonlinearity in the TPC or DRN. Second, we review a recent analysis of field selection studies which indicates that directional selection favoring increased overall size is common in many systems—that bigger is frequently fitter. Third, we use a recent theoretical model to examine how selection on thermal performance curves relates to environmental temperatures during selection. The model predicts that if selection acts primarily on adult size or development time, then selection on thermal performance curves for larval growth or development rates is directly related to the frequency distribution of temperatures experienced during larval development. Using data on caterpillar temperatures in the field, we show that the strength of directional selection on growth rate is predicted to be greater at the modal (most frequent) temperatures, not at the mean temperature or at temperatures at which growth rate is maximized. Our results illustrate some of the differences in genetic architecture and patterns of selection between thermal performance curves and developmental reaction norms.

QUANTITATIVE GENETICS OF CONTINUOUS REACTION NORMS: THERMAL SENSITIVITY OF CATERPILLAR GROWTH RATES

Abstract A continuous reaction norm or performance curve represents a phenotypic trait of an individual or genotype in which the trait value may vary with some continuous environmental variable. We explore patterns of genetic variation in thermal performance curves of short‐term caterpillar growth rate in a population of Pieris rapae. We compare multivariate methods, which treat performance at each test temperature as a distinct trait, with function‐valued methods that treat a performance curve as a continuous function. Mean growth rate increased with increasing temperatures from 8 to 35°C, was highest at 35°C, and declined at 40°C. There was substantial and significant variation among full‐sib families in their thermal performance curves. Estimates of broad‐sense genetic variances and covariances showed that genetic variance in growth rate increased more than 30‐fold from low (8–11°C) to high (35–40°C) temperatures, even after differences in mean growth rate across temperatures were removed. Growth rate at 35 and 40°C was negatively correlated genetically, suggesting a genetic trade‐off in growth rate at these temperatures; this trade‐off may represent either a generalist‐specialist trade‐off and/or variation in the optimal temperature for growth. The estimated genetic variance‐covariance function (G function), the function‐valued analog of the variance‐covariance matrix (G matrix), was quite bumpy compared with the estimated G matrix; and results of principal component analyses of the G function were difficult to interpret. The use of orthogonal polynomials as the basis functions in current function‐valued estimation methods may generate artifacts when the true G function has prominent local features, such as strong negative covariances at nearby temperatures (e.g. at 35 and 40°C); this may be a particular issue for thermal performance curves and other highly nonlinear reaction norms.

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.

EVOLUTION IN A CONSTANT ENVIRONMENT: THERMAL FLUCTUATIONS AND THERMAL SENSITIVITY OF LABORATORY AND FIELD POPULATIONS OF MANDUCA SEXTA

Adaptation to temporal variation in environmental conditions is widespread. Whether evolution in a constant environment alters adaptation to temporal variation is relatively unexplored. We examine how constant and diurnally fluctuating temperature conditions affect life-history traits in two populations of the tobacco hornworm, Manduca sexta: a field population that routinely experiences fluctuating temperatures; and a laboratory population (derived from this field population in the 1960s) maintained at a constant temperature for more than 250 generations. Our experiments demonstrate that diurnal fluctuations significantly alter body size and development time in both populations, and confirm that these populations differ in their responses to a mean temperature. However, we found no evidence for population divergence in responses to diurnal temperature fluctuations. We suggest that mean and extreme temperatures may act as more potent selective forces on thermal reaction norms than temperature variation per se.

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.

Rapid population divergence in thermal reaction norms for an invading species: breaking the temperature–size rule

The temperature–size rule is a common pattern of phenotypic plasticity in which higher temperature during development results in a smaller adult body size (i.e. a thermal reaction norm with negative slope). Examples and exceptions to the rule are known in multiple groups of organisms, but rapid population differentiation in the temperature–size rule has not been explored. Here we examine the genetic and parental contributions to population differentiation in thermal reaction norms for size, development time and survival in the Cabbage White Butterfly Pieris rapae, for two geographical populations that have likely diverged within the past 150 years. We used split‐sibship experiments with two temperature treatments (warm and cool) for P. rapae from Chapel Hill, NC, and from Seattle, WA. Mixed‐effect model analyses demonstrate significant genetic differences between NC and WA populations for adult size and for thermal reaction norms for size. Mean adult mass was 12–24% greater in NC than in WA populations for both temperature treatments; mean size was unaffected or decreased with temperature (the temperature–size rule) for the WA population, but size increased with temperature for the NC population. Our study shows that the temperature–size rule and related thermal reaction norms can evolve rapidly within species in natural field conditions. Rapid evolutionary divergence argues against the existence of a simple, general mechanistic constraint as the underlying cause of the temperature–size rule.

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

Abstract 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.

EVOLUTION OF THERMOTOLERANCE IN SEASONAL ENVIRONMENTS: THE EFFECTS OF ANNUAL TEMPERATURE VARIATION AND LIFE‐HISTORY TIMING IN WYEOMYIA SMITHII

Abstract In organisms with complex life cycles, the adaptive value of thermotolerance depends on life-history timing and seasonal temperature profiles. We illustrate this concept by examining variation in annual thermal environments and thermal acclimation among four geographic populations of the pitcher plant mosquito. Only diapausing larvae experience winter, whereas both postdiapause and nondiapause adults occur only during the growing season. Thus, adults experience transient cold stress primarily during the spring. We show that adult cold tolerance (chill coma recovery) is enhanced in spring-like conditions via thermal acclimation but is unaffected by diapause state. Moreover, adult mosquitoes from northern populations were more cold tolerant than those from southern populations largely because acclimation responses were steeper in the north. In contrast to cold tolerance, there was no significant acclimation of heat tolerance (heat knockdown), and no significant differences in heat tolerance between northern and southern populations. Field temperature data show that because of evolved differences in diapause timing, adult exposure to cold stress is remarkably consistent across geography. This suggests that geographic variation in cold tolerance may not be the result of direct selection on adults. Our results illustrate the importance of the interplay between phenological and thermal adaptation for understanding variation along climatic gradients.