Gene expression variation underlying tissue-specific responses to copper stress in Drosophila melanogaster.

Copper is one of a handful of biologically necessary heavy metals that is also a common environmental pollutant. Under normal conditions, copper ions are required for many key physiological processes. However, in excess, copper results in cell and tissue damage ranging in severity from temporary injury to permanent neurological damage. Because of its biological relevance, and because many conserved copper-responsive genes respond to nonessential heavy metal pollutants, copper resistance in Drosophila melanogaster is a useful model system with which to investigate the genetic control of the heavy metal stress response. Because heavy metal toxicity has the potential to differently impact specific tissues, we genetically characterized the control of the gene expression response to copper stress in a tissue-specific manner in this study. We assessed the copper stress response in head and gut tissue of 96 inbred strains from the Drosophila Synthetic Population Resource using a combination of differential expression analysis and expression quantitative trait locus mapping. Differential expression analysis revealed clear patterns of tissue-specific expression. Tissue and treatment specific responses to copper stress were also detected using expression quantitative trait locus mapping. Expression quantitative trait locus associated with MtnA, Mdr49, Mdr50, and Sod3 exhibited both genotype-by-tissue and genotype-by-treatment effects on gene expression under copper stress, illuminating tissue- and treatment-specific patterns of gene expression control. Together, our data build a nuanced description of the roles and interactions between allelic and expression variation in copper-responsive genes, provide valuable insight into the genomic architecture of susceptibility to metal toxicity, and highlight candidate genes for future functional characterization.

Gene expression variation underlying tissue-specific responses to copper stress in Drosophila melanogaster.

Copper is one of a handful of biologically necessary heavy metals that is also a common environmental pollutant. Under normal conditions, copper ions are required for many key physiological processes. However, in excess, copper quickly results in cell and tissue damage that can range in severity from temporary injury to permanent neurological damage. Because of its biological relevance, and because many conserved copper-responsive genes also respond to other non-essential heavy metal pollutants, copper resistance in Drosophila melanogaster is a useful model system with which to investigate the genetic control of the response to heavy metal stress. Because heavy metal toxicity has the potential to differently impact specific tissues, we genetically characterized the control of the gene expression response to copper stress in a tissue-specific manner in this study. We assessed the copper stress response in head and gut tissue of 96 inbred strains from the Drosophila Synthetic Population Resource (DSPR) using a combination of differential expression analysis and expression quantitative trait locus (eQTL) mapping. Differential expression analysis revealed clear patterns of tissue-specific expression, primarily driven by a more pronounced gene expression response in gut tissue. eQTL mapping of gene expression under control and copper conditions as well as for the change in gene expression following copper exposure (copper response eQTL) revealed hundreds of genes with tissue-specific local cis-eQTL and many distant trans-eQTL. eQTL associated with MtnA, Mdr49, Mdr50, and Sod3 exhibited genotype by environment effects on gene expression under copper stress, illuminating several tissue- and treatment-specific patterns of gene expression control. Together, our data build a nuanced description of the roles and interactions between allelic and expression variation in copper-responsive genes, provide valuable insight into the genomic architecture of susceptibility to metal toxicity, and highlight many candidate genes for future functional characterization.

The genetic basis of adaptation to copper pollution in Drosophila melanogaster.

Introduction: Heavy metal pollutants can have long lasting negative impacts on ecosystem health and can shape the evolution of species. The persistent and ubiquitous nature of heavy metal pollution provides an opportunity to characterize the genetic mechanisms that contribute to metal resistance in natural populations. Methods: We examined variation in resistance to copper, a common heavy metal contaminant, using wild collections of the model organism Drosophila melanogaster. Flies were collected from multiple sites that varied in copper contamination risk. We characterized phenotypic variation in copper resistance within and among populations using bulked segregant analysis to identify regions of the genome that contribute to copper resistance. Results and Discussion: Copper resistance varied among wild populations with a clear correspondence between resistance level and historical exposure to copper. We identified 288 SNPs distributed across the genome associated with copper resistance. Many SNPs had population-specific effects, but some had consistent effects on copper resistance in all populations. Significant SNPs map to several novel candidate genes involved in refolding disrupted proteins, energy production, and mitochondrial function. We also identified one SNP with consistent effects on copper resistance in all populations near CG11825, a gene involved in copper homeostasis and copper resistance. We compared the genetic signatures of copper resistance in the wild-derived populations to genetic control of copper resistance in the Drosophila Synthetic Population Resource (DSPR) and the Drosophila Genetic Reference Panel (DGRP), two copper-naïve laboratory populations. In addition to CG11825, which was identified as a candidate gene in the wild-derived populations and previously in the DSPR, there was modest overlap of copper-associated SNPs between the wild-derived populations and laboratory populations. Thirty-one SNPs associated with copper resistance in wild-derived populations fell within regions of the genome that were associated with copper resistance in the DSPR in a prior study. Collectively, our results demonstrate that the genetic control of copper resistance is highly polygenic, and that several loci can be clearly linked to genes involved in heavy metal toxicity response. The mixture of parallel and population-specific SNPs points to a complex interplay between genetic background and the selection regime that modifies the effects of genetic variation on copper resistance.

Pre-adult exposure to three heavy metals leads to changes in the head transcriptome of adult flies.

We examined the effect of developmental exposure to three heavy metals - cadmium, copper, and lead - on gene expression in adult head tissue in the model organism Drosophila melanogaster . All metals affected development time and/or gene expression level. While variation in the response to each metal was apparent, two differentially-expressed genes were upregulated in response to all three metal treatments, and 11 genes were downregulated in two of the three treatments. Our work reveals that developmental metal exposure has the potential to have long-lasting, metal-specific effects on gene expression in adults, even after the metal stress has been removed.

Characterizing the genetic basis of copper toxicity in Drosophila reveals a complex pattern of allelic, regulatory, and behavioral variation.

A range of heavy metals are required for normal cell function and homeostasis. However, the anthropogenic release of metal compounds into soil and water sources presents a pervasive health threat. Copper is one of many heavy metals that negatively impacts diverse organisms at a global scale. Using a combination of quantitative trait locus (QTL) mapping and RNA sequencing in the Drosophila Synthetic Population Resource, we demonstrate that resistance to the toxic effects of ingested copper in D. melanogaster is genetically complex and influenced by allelic and expression variation at multiple loci. QTL mapping identified several QTL that account for a substantial fraction of heritability. Additionally, we find that copper resistance is impacted by variation in behavioral avoidance of copper and may be subject to life-stage specific regulation. Gene expression analysis further demonstrated that resistant and sensitive strains are characterized by unique expression patterns. Several of the candidate genes identified via QTL mapping and RNAseq have known copper-specific functions (e.g., Ccs, Sod3, CG11825), and others are involved in the regulation of other heavy metals (e.g., Catsup, whd). We validated several of these candidate genes with RNAi suggesting they contribute to variation in adult copper resistance. Our study illuminates the interconnected roles that allelic and expression variation, organism life stage, and behavior play in copper resistance, allowing a deeper understanding of the diverse mechanisms through which metal pollution can negatively impact organisms.

Phenology of Drosophila species across a temperate growing season and implications for behavior.

Drosophila community composition is complex in temperate regions with different abundance of flies and species across the growing season. Monitoring Drosophila populations provides insights into the phenology of both native and invasive species. Over a single growing season, we collected Drosophila at regular intervals and determined the number of individuals of the nine species we found in Kansas, USA. Species varied in their presence and abundance through the growing season with peak diversity occurring after the highest seasonal temperatures. We developed models for the abundance of the most common species, Drosophila melanogaster, D. simulans, D. algonquin, and the recent invasive species, D. suzukii. These models revealed that temperature played the largest role in abundance of each species across the season. For the two most commonly studied species, D. melanogaster and D. simulans, the best models indicate shifted thermal optima compared to laboratory studies, implying that fluctuating temperature may play a greater role in the physiology and ecology of these insects than indicated by laboratory studies, and should be considered in global climate change studies.

Dissection of Complex, Fitness-Related Traits in Multiple Drosophila Mapping Populations Offers Insight into the Genetic Control of Stress Resistance.

We leverage two complementary Drosophila melanogaster mapping panels to genetically dissect starvation resistance-an important fitness trait. Using >1600 genotypes from the multiparental Drosophila Synthetic Population Resource (DSPR), we map numerous starvation stress QTL that collectively explain a substantial fraction of trait heritability. Mapped QTL effects allowed us to estimate DSPR founder phenotypes, predictions that were correlated with the actual phenotypes of these lines. We observe a modest phenotypic correlation between starvation resistance and triglyceride level, traits that have been linked in previous studies. However, overlap among QTL identified for each trait is low. Since we also show that DSPR strains with extreme starvation phenotypes differ in desiccation resistance and activity level, our data imply multiple physiological mechanisms contribute to starvation variability. We additionally exploited the Drosophila Genetic Reference Panel (DGRP) to identify sequence variants associated with starvation resistance. Consistent with prior work these sites rarely fall within QTL intervals mapped in the DSPR. We were offered a unique opportunity to directly compare association mapping results across laboratories since two other groups previously measured starvation resistance in the DGRP. We found strong phenotypic correlations among studies, but extremely low overlap in the sets of genomewide significant sites. Despite this, our analyses revealed that the most highly associated variants from each study typically showed the same additive effect sign in independent studies, in contrast to otherwise equivalent sets of random variants. This consistency provides evidence for reproducible trait-associated sites in a widely used mapping panel, and highlights the polygenic nature of starvation resistance.

Costs of cold acclimation on survival and reproductive behavior in Drosophila melanogaster.

Fitness is determined by the ability of an organism to both survive and reproduce; however, the mechanisms that lead to increased survival may not have the same effect on reproductive success. We used nineteen natural Drosophila melanogaster genotypes from the Drosophila Genetic Reference Panel to determine if adaptive plasticity following short-term acclimation through rapid cold-hardening (RCH) affects mating behavior and mating success. We confirmed that exposure to the acclimation temperature is beneficial to survival following cold stress; however, we found that this same acclimation temperature exposure led to less efficient male courtship and a significant decrease in the likelihood of mating. Cold tolerance and the capacity to respond plastically to cold stress were not correlated with mating behavior following acclimation, suggesting that the genetic control of the physiological effects of the cold temperature exposure likely differ between survival and behavioral responses. We also tested whether the exposure of males to the acclimation temperature influenced courtship song. This exposure again significantly increased courtship duration; however, courtship song was unchanged. These results illustrate costs of short-term acclimation on survival and reproductive components of fitness and demonstrate the pronounced effect that short-term thermal environment shifts can have on reproductive success.

Antagonistic pleiotropy and mutation accumulation contribute to age-related decline in stress response.

As organisms age, the effectiveness of natural selection weakens, leading to age-related decline in fitness-related traits. The evolution of age-related changes associated with senescence is likely influenced by mutation accumulation (MA) and antagonistic pleiotropy (AP). MA predicts that age-related decline in fitness components is driven by age-specific sets of alleles, nonnegative genetic correlations within trait across age, and an increase in the coefficient of genetic variance. AP predicts that age-related decline in a trait is driven by alleles with positive effects on fitness in young individuals and negative effects in old individuals, and is expected to lead to negative genetic correlations within traits across age. We build on these predictions using an association mapping approach to investigate the change in additive effects of SNPs across age and among traits for multiple stress-response fitness-related traits, including cold stress with and without acclimation and starvation resistance. We found support for both MA and AP theories of aging in the age-related decline in stress tolerance. Our study demonstrates that the evolution of age-related decline in stress tolerance is driven by a combination of alleles that have age-specific additive effects, consistent with MA, as well as nonindependent and antagonistic genetic architectures characteristic of AP.

Seasonal variation in basal and plastic cold tolerance: Adaptation is influenced by both long- and short-term phenotypic plasticity.

Understanding how thermal selection affects phenotypic distributions across different time scales will allow us to predict the effect of climate change on the fitness of ectotherms. We tested how seasonal temperature variation affects basal levels of cold tolerance and two types of phenotypic plasticity in Drosophila melanogaster. Developmental acclimation occurs as developmental stages of an organism are exposed to seasonal changes in temperature and its effect is irreversible, while reversible short-term acclimation occurs daily in response to diurnal changes in temperature. We collected wild flies from a temperate population across seasons and measured two cold tolerance metrics (chill-coma recovery and cold stress survival) and their responses to developmental and short-term acclimation. Chill-coma recovery responded to seasonal shifts in temperature, and phenotypic plasticity following both short-term and developmental acclimation improved cold tolerance. This improvement indicated that both types of plasticity are adaptive, and that plasticity can compensate for genetic variation in basal cold tolerance during warmer parts of the season when flies tend to be less cold tolerant. We also observed a significantly stronger trade-off between basal cold tolerance and short-term acclimation during warmer months. For the longer-term developmental acclimation, a trade-off persisted regardless of season. A relationship between the two types of plasticity may provide additional insight into why some measures of thermal tolerance are more sensitive to seasonal variation than others.