Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Biological Effects of Single-Nucleotide Polymorphisms in the Drosophila melanogaster Malic Enzyme Locus.

A pair of amino acid polymorphisms within the Drosophila melanogaster Malic enzyme (Men) locus presents an interesting case of genetic variation that appears to be under selection. The two alleles at each site are biochemically distinct, but their biological effects are unknown. One polymorphic site is near the active site and the other is buried within the protein. Strikingly, in twelve different populations, the first polymorphism is always found at approximately a 50:50 allelic frequency, whereas the second polymorphism is always found at approximately 90:10. The consistency of the frequencies between populations suggests that the polymorphisms are under selection and it is possible that balancing selection is at play. We used 16 lines of flies to create the nine genotypes needed to quantify both effects of the polymorphic sites and possible genetic background effects, which we found to be widespread. The alleles at each site differ, but in different biochemical characteristics. The first site significantly influences MEN Km and Vmax, whereas the second site affects the Km and the Vmax/Km ratio (relative activity). Interestingly, the rarest allele is the most biochemically distinct. We also assayed three more distal phenotypes, triglyceride concentration, carbohydrate concentration, and longevity. In all cases, the phenotypes of the heterozygous genotypes are intermediate between those of the respective homozygotes suggesting that if balancing selection is maintaining the observed allele frequencies it is not through non-linear combinations of the biochemical phenotypes.

Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice.

Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter analogous to nitric oxide and carbon monoxide. Cystathionine gamma-lyase (CSE)-derived H2S is implicated in the regulation of insulin resistance and glucose metabolism, but the involvement of CSE/H2S system in energy homeostasis and fat mass has not been extensively explored. In this study, a potential functional role of the CSE/H2S system in in vitro adipocyte differentiation and in vivo adipogenesis and the underlying mechanism was investigated. CSE expression and H2S production were increased during adipocyte differentiation, and that the pattern of CSE mRNA expression was similar to that of CCAAT/enhancer-binding protein (C/EBP) ß and δ, two key regulators for adipogenesis. C/EBPß and γ bind to the CCAAT box in CSE promoter and stimulate CSE gene transcription. H2S induced PPARγ transactivation activity by S-sulfhydrating all the cysteine residues in the DNA binding domain and stimulated adipogenesis. High fat diet-induced fat mass was lost in CSE deficient mice, and exogenously applied H2S promoted fat mass accumulation in fruit flies. In conclusion, CSE/H2S system is essential for adipogenesis and fat mass accumulation through enhancement of PPARγ function in adipocytes. This study suggests that the CSE/H2S system is involved in the pathogenesis of obesity in mice.

Seasonal variation in an acid mine drainage microbial community.

Environmental oxidation and microbial metabolism drive production of acid mine drainage (AMD). Understanding changes in the microbial community, due to geochemical and seasonal characteristics, is fundamental to AMD monitoring and remediation. Using direct sequencing of the 16S and 18S rRNA genes to identify bacterial, archaeal, and eukaryotic members of the microbial community at an AMD site in Northern Ontario, Canada, we found a dynamic community varying significantly across winter and summer sampling times. Community composition was correlated with physical and chemical properties, including water temperature, pH, conductivity, winter ice thickness, and metal concentrations. Within Bacteria, Acidithiobacillus was the dominant genus during winter (11%-57% of sequences) but Acidiphilium was dominant during summer (47%-87%). Within Eukarya, Chrysophyceae (1.5%-94%) and Microbotrymycetes (8%-92%) dominated the winter community, and LKM11 (4%-62%) and Chrysophyceae (25%-87%) the summer. There was less diversity and variability within the Archaea, with similar summer and winter communities mainly comprising Thermoplasmata (33%-64%) and Thermoprotei (5%-20%) classes but also including a large portion of unclassified reads (∼40%). Overall, the active AMD community varied significantly between winter and summer, with changing community profiles closely correlated to specific differences in AMD geochemical and physical properties, including pH, water temperature, ice thickness, and sulfate and metal concentrations.

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc.

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.

Regulation at Drosophila's Malic Enzyme highlights the complexity of transvection and its sensitivity to genetic background.

Transvection, a type of trans-regulation of gene expression in which regulatory elements on one chromosome influence elements on a paired homologous chromosome, is itself a complex biological phenotype subject to modification by genetic background effects. However, relatively few studies have explored how transvection is affected by distal genetic variation, perhaps because it is strongly influenced by local regulatory elements and chromosomal architecture. With the emergence of the "hub" model of transvection and a series of studies showing variation in transvection effects, it is becoming clear that genetic background plays an important role in how transvection influences gene transcription. We explored the effects of genetic background on transvection by performing two independent genome wide association studies (GWASs) using the Drosophila genetic reference panel (DGRP) and a suite of Malic enzyme (Men) excision alleles. We found substantial variation in the amount of transvection in the 149 DGRP lines used, with broad-sense heritability of 0.89 and 0.84, depending on the excision allele used. The specific genetic variation identified was dependent on the excision allele used, highlighting the complex genetic interactions influencing transvection. We focussed primarily on genes identified as significant using a relaxed P-value cutoff in both GWASs. The most strongly associated genetic variant mapped to an intergenic single nucleotide polymorphism (SNP), located upstream of Tiggrin (Tig), a gene that codes for an extracellular matrix protein. Variants in other genes, such transcription factors (CG7368 and Sima), RNA binding proteins (CG10418, Rbp6, and Rig), enzymes (AdamTS-A, CG9743, and Pgant8), proteins influencing cell cycle progression (Dally and Eip63E) and signaling proteins (Atg-1, Axo, Egfr, and Path) also associated with transvection in Men. Although not intuitively obvious how many of these genes may influence transvection, some have been previously identified as promoting or antagonizing somatic homolog pairing. These results identify several candidate genes to further explore in the understanding of transvection in Men and in other genes regulated by transvection. Overall, these findings highlight the complexity of the interactions involved in gene regulation, even in phenotypes, such as transvection, that were traditionally considered to be primarily influenced by local genetic variation.

Molecular evolution of teleost neural isozymes.

Isozymes, homologous enzymes coded by separate loci within a genome, present interesting systems for examining molecular and functional divergence through natural selection. Isozyme pairs for a number of metabolic enzymes, including Triosephosphate isomerase (Tpi), Malate dehydrogenase (Mdh), Phosphoglucose isomerase (Pgi), and Guanylate kinase (Guk), appear to all result from a single, large duplication event early in teleost evolution. These small gene families include two forms, a generally expressed form with no apparent charge and a neurally expressed form with a pronounced negative charge although the canalization of expression of the second form varies across families. Using ancestral sequence reconstructions and standard comparisons of rates of nonsynonymous and synonymous change, combined with the examination of the specific amino acid changes observed and predicted we examined the evolution of the Tpi and Guk families using all available vertebrate sequences and all four families using a smaller, common, dataset. We find that post-duplication, the neural Tpi and Guk isozymes evolved through similar periods of positive selection as evidenced by elevated rates of nonsynonymous change and accumulation of negative amino acids. Over the same evolutionary period our analysis suggests that Mdh and Pgi isozymes appear to have evolved under a less divergent pattern of selection. These distinct results likely reflect functional differences between the isozymes, possibly a result of differences in expression patterns.

A combination of structural and cis-regulatory factors drives biochemical differences in Drosophila melanogaster malic enzyme.

The evolutionary significance of molecular variation is still contentious, with much current interest focusing on the relative contribution of structural changes in proteins versus regulatory variation in gene expression. We present a population genetic and biochemical study of molecular variation at the malic enzyme locus (Men) in Drosophila melanogaster. Two amino acid polymorphisms appear to affect substrate-binding kinetics, while only one appears to affect thermal stability. Interestingly, we find that enzyme activity differences previously assigned to one of the polymorphisms may, instead, be a function of linked regulatory differences. These results suggest that both regulatory and structural changes contribute to differences in protein function. Our examination of the Men coding sequences reveals no evidence for selection acting on the polymorphisms, but earlier work on this enzyme indicates that the biochemical variation observed has physiological repercussions and therefore could potentially be under natural selection.

No water, no mating: Connecting dots from behaviour to pathways.

Insects hold considerable ecological and agricultural importance making it vital to understand the factors impacting their reproductive output. Environmental stressors are examples of such factors which have a substantial and significant influence on insect reproductive fitness. Insects are also ectothermic and small in size which makes them even more susceptible to environmental stresses. The present study assesses the consequence of desiccation on the mating latency and copulations duration in tropical Drosophila melanogaster. We tested flies for these reproductive behavioral parameters at varying body water levels and with whole metabolome analysis in order to gain a further understanding of the physiological response to desiccation. Our results showed that the duration of desiccation is positively correlated with mating latency and mating failure, while having no influence on the copulation duration. The metabolomic analysis revealed three biological pathways highly affected by desiccation: starch and sucrose metabolism, galactose metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. These results are consistent with carbohydrate metabolism providing an energy source in desiccated flies and also suggests that the phenylalanine biosynthesis pathway plays a role in the reproductive fitness of the flies. Desiccation is a common issue with smaller insects, like Drosophila and other tropical insects, and our findings indicate that this lack of ambient water can immediately and drastically affect the insect reproductive behaviour, which becomes more crucial because of unpredictable and dynamic weather conditions.

The Effects of Essential and Non-Essential Metal Toxicity in the Drosophila melanogaster Insect Model: A Review.

The biological effects of environmental metal contamination are important issues in an industrialized, resource-dependent world. Different metals have different roles in biology and can be classified as essential if they are required by a living organism (e.g., as cofactors), or as non-essential metals if they are not. While essential metal ions have been well studied in many eukaryotic species, less is known about the effects of non-essential metals, even though essential and non-essential metals are often chemically similar and can bind to the same biological ligands. Insects are often exposed to a variety of contaminated environments and associated essential and non-essential metal toxicity, but many questions regarding their response to toxicity remain unanswered. Drosophila melanogaster is an excellent insect model species in which to study the effects of toxic metal due to the extensive experimental and genetic resources available for this species. Here, we review the current understanding of the impact of a suite of essential and non-essential metals (Cu, Fe, Zn, Hg, Pb, Cd, and Ni) on the D. melanogaster metal response system, highlighting the knowledge gaps between essential and non-essential metals in D. melanogaster. This review emphasizes the need to use multiple metals, multiple genetic backgrounds, and both sexes in future studies to help guide future research towards better understanding the effects of metal contamination in general.

Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

Quantifying interactions within the NADP(H) enzyme network in Drosophila melanogaster.

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster.

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.

Genetic Diversity in Insect Metal Tolerance.

Insects encounter a variety of metals in their environment, many of which are required at some concentration for normal organismal homeostasis, but essentially all of which are toxic at higher concentrations. Insects have evolved a variety of genetic, and likely epigenetic, mechanisms to deal with metal stress. A recurring theme in all these systems is complexity and diversity; even simple, single gene, cases are complex. Of the known gene families, the metallothioneins are perhaps the best understood and provide good examples of how diverse metal response is. Interestingly, there is considerable diversity across taxa in these metal-responsive systems, including duplications to form small gene families and complex expression of single loci. Strikingly, different species have evolved different mechanisms to cope with the same, or similar, stress suggesting both independent derivation of, and plasticity in, the pathways involved. It is likely that some metal-response systems evolved early in evolutionary time and have been conserved, while others have diverged, and still others evolved more recently and convergently. In addition to conventional genetics, insects likely respond to environmental metal through a variety of epigenetic systems, but direct tests are lacking. Ultimately, it is likely that classical genetic and epigenetic factors interact in regulating insect metal responses. In light of this diversity across species, future studies including a broad-based examination of gene expression in non-model species in complex environments will likely uncover additional genes and genetic and epigenetic mechanisms.

Sex and Genetic Background Influence Superoxide Dismutase (cSOD)-Related Phenotypic Variation in Drosophila melanogaster.

Mutations often have drastically different effects in different genetic backgrounds; understanding a gene's biological function then requires an understanding of its interaction with genetic diversity. The antioxidant enzyme cytosolic copper/zinc superoxide dismutase (cSOD) catalyzes the dismutation of the superoxide radical, a molecule that can induce oxidative stress if its concentration exceeds cellular control. Accordingly, Drosophila melanogaster lacking functional cSOD exhibit a suite of phenotypes including decreased longevity, hypersensitivity to oxidative stress, impaired locomotion, and reduced NADP(H) enzyme activity in males. To date, cSOD-null phenotypes have primarily been characterized using males carrying one allele, cSodn108red, in a single genetic background. We used ANOVA, and the effect size partial eta squared, to partition the amount of variation attributable to cSOD activity, sex, and genetic background across a series of life history, locomotor, and biochemical phenotypes associated with the cSOD-null condition. Overall, the results demonstrate that the cSOD-null syndrome is largely consistent across sex and genetic background, but also significantly influenced by both. The sex-specific effects are particularly striking and our results support the idea that phenotypes cannot be considered to be fully defined if they are examined in limited genetic contexts.

Metabolomic analysis of oxidative stress: Superoxide dismutase mutation and paraquat induced stress in Drosophila melanogaster.

Oxidative stress results in substantial biochemical and physiological perturbations in essentially all organisms. To determine the broad metabolic effects of oxidative stress, we have quantified the response in Drosophila melanogaster to both genetically and environmentally derived oxidative stress. Flies were challenged with loss of Superoxide dismutase activity or chronic or acute exposure to the oxidizing chemical paraquat. Metabolic changes were then quantified using a recently developed chemical isotope labeling (CIL) liquid chromatography - mass spectrometry (LC-MS) platform that targets the carboxylic acid and amine/phenol submetabolomes with high metabolic coverage. We discovered wide spread changes in both submetabolomes in response to all three types of stresses including: changes to the urea cycle, tryptophan metabolism, porphyrin metabolism, as well as a series of metabolic pathways involved in glutathione synthesis. Strikingly, while there are commonalities across the conditions, all three resulted in different metabolomic responses, with the greatest difference between the genetic and environmental responses. Genetic oxidative stress resulted in substantially more widespread effects, both in terms of the percent of the metabolome altered, and the magnitude of changes in individual metabolites. Chronic and acute environmental stress resulted in more similar responses although both were distinct from genetic stress. Overall, these results indicate that the metabolomic response to oxidative stress is complex, reaching across multiple metabolic pathways, with some shared features but with more features unique to different, specific stressors.

Eugenia uniflora leaf essential oil promotes mitochondrial dysfunction in Drosophila melanogaster through the inhibition of oxidative phosphorylation.

Eugenia uniflora L. (Myrtaceae family) has demonstrated several properties of human interest, including insecticide potential, due to its pro-oxidant properties. These properties likely result from the effects on its mitochondria, but the mechanism of this action is unclear. The aim of this work was to evaluate the mitochondrial bioenergetics function in Drosophila melanogaster exposed to E. uniflora leaf essential oil. For this, we used a high-resolution respirometry (HRR) protocol. We found that E. uniflora promoted a collapse of the mitochondrial transmembrane potential (ΔΨm). In addition the essential oil was able to promote the disruption of respiration coupled to oxidative phosphorylation (OXPHOS) and inhibit the respiratory electron transfer system (ETS) established with an uncoupler. In addition, exposure led to decreases of respiratory control ratio (RCR), bioenergetics capacity and OXPHOS coupling efficiency, and induced changes in the substrate control ratio. Altogether, our results suggested that E. uniflora impairs the mitochondrial function/viability and promotes the uncoupling of OXPHOS, which appears to play an important role in the cellular bioenergetics failure induced by essential oil in D. melanogaster.

Natural and synthetic alleles provide complementary insights into the nature of selection acting on the Men polymorphism of Drosophila melanogaster.

Two malic enzyme alleles, Men(113A) and Men(113G), occur at approximately equal frequency in North American populations of Drosophila melanogaster, while only Men(113A) occurs in African populations. We investigated the population genetics, biochemical characteristics, and selective potential of these alleles. Comparable levels of nucleotide polymorphism in both alleles suggest that the Men(113G) allele is not recently derived, but we find no evidence in the DNA sequence data for selection maintaining the polymorphism. Interestingly, the alleles differ in both V(max) and K(m) for the substrate malate. Triglyceride concentration and isocitrate dehydrogenase (IDH) and glucose-6-phosphate dehydrogenase (G6PD) activities are negatively correlated with the in vivo activities of the Men alleles. We examined the causality of the observed correlations using P-element excision-derived knockout alleles of the Men gene and found significant changes in the maximum activities of both IDH and G6PD, but not in triglyceride concentration, suggesting compensatory interactions between MEN, IDH, and G6PD. Additionally, we found significantly higher than expected levels of MEN activity in knockout heterozygotes, which we attribute to transvection effects. The distinct differences in biochemistry and physiology between the naturally occurring alleles and between the engineered alleles suggest the potential for selection on the Men locus.

The structure and population genetics of the breakpoints associated with the cosmopolitan chromosomal inversion In(3R)Payne in Drosophila melanogaster.

We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.