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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cold acclimation wholly reorganizes the Drosophila melanogaster transcriptome and metabolome.

Cold tolerance is a key determinant of insect distribution and abundance, and thermal acclimation can strongly influence organismal stress tolerance phenotypes, particularly in small ectotherms like Drosophila. However, there is limited understanding of the molecular and biochemical mechanisms that confer such impressive plasticity. Here, we use high-throughput mRNA sequencing (RNA-seq) and liquid chromatography - mass spectrometry (LC-MS) to compare the transcriptomes and metabolomes of D. melanogaster acclimated as adults to warm (rearing) (21.5 °C) or cold conditions (6 °C). Cold acclimation improved cold tolerance and led to extensive biological reorganization: almost one third of the transcriptome and nearly half of the metabolome were differentially regulated. There was overlap in the metabolic pathways identified via transcriptomics and metabolomics, with proline and glutathione metabolism being the most strongly-supported metabolic pathways associated with increased cold tolerance. We discuss several new targets in the study of insect cold tolerance (e.g. dopamine signaling and Na(+)-driven transport), but many previously identified candidate genes and pathways (e.g. heat shock proteins, Ca(2+) signaling, and ROS detoxification) were also identified in the present study, and our results are thus consistent with and extend the current understanding of the mechanisms of insect chilling tolerance.

Transvection-based gene regulation in Drosophila is a complex and plastic trait.

Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is potentially widespread in the Drosophila melanogaster genome and varies across cell types and within tissues in D. melanogaster, characteristics of a complex trait. Here, we demonstrate that the trans-interactions at the Malic enzyme (Men) locus are, in fact, transvection as classically defined and are plastic with respect to both genetic background and environment. Using chromosomal inversions, we show that trans-interactions at the Men locus are eliminated by changes in chromosomal architecture that presumably disrupt somatic pairing. We further show that the magnitude of transvection at the Men locus is modified by both genetic background and environment (temperature), demonstrating that transvection is a plastic phenotype. Our results suggest that transvection effects in D. melanogaster are shaped by a dynamic interplay between environment and genetic background. Interestingly, we find that cis-based regulation of the Men gene is more robust to genetic background and environment than trans-based. Finally, we begin to uncover the nonlocal factors that may contribute to variation in transvection overall, implicating Abd-B in the regulation of Men in cis and in trans in an allele-specific and tissue-specific manner, driven by differences in expression of the two genes across genetic backgrounds and environmental conditions.

A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites.

We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC-MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species.

Population differentiation of the African cyprinid Barbus neumayeri across dissolved oxygen regimes.

Population level response to hypoxia has become an issue of global significance because of increased frequency and intensity of hypoxic events worldwide, and the potential for global warming to exacerbate hypoxic stress. In this study, we sequenced two nuclear intronic regions and a single mitochondrial region across seven populations of the African cyprinid, Barbus neumayeri from two river drainages in Uganda: the Rwembaita Swamp-Njuguta River System and the Dura River. We then examined two indices of population structure, G ST and Jost's D, to detect links between oxygen availability and genetic variation and to determine if population genetic structure was associated with (i) dissolved oxygen regime (hypoxia or normoxia), (ii) geographical distance, or (iii) a combination of dissolved oxygen regime and geographical distance. Our results indicate that over a large scale (between drainages), geographical distance significantly affects the genetic structure of populations. However, within a single drainage, dissolved oxygen regime plays a key role in determining the genetic structure of populations. Within the Rwembaita-Njuguta system, gene flow was high between locations of similar oxygen regimes, but low between areas characterized by divergent oxygen regimes. Interestingly, G ST analyses appear to yield less realistic measures of population structure than Jost's D, suggesting that caution must be taken when interpreting and comparing the results from different studies. These results support the idea that aquatic dissolved oxygen can act as a selective force limiting gene flow among populations of aquatic species and therefore should be considered when implementing conservation plans and assessing environmental impact of human activities.

Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques.

We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment.

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.

Interactions of NADP-reducing enzymes across varying environmental conditions: a model of biological complexity.

Interactions across biological networks are often quantified under a single set of conditions; however, cellular behaviors are dynamic and interactions can be expected to change in response to molecular context and environment. To determine the consistency of network interactions, we examined the enzyme network responsible for the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH across three different conditions: oxidative stress, starvation, and desiccation. Synthetic, activity-variant alleles were used in Drosophila melanogaster for glucose-6-phosphate dehydrogenase (G6pd), cytosolic isocitrate dehydrogenase (Idh), and cytosolic malic enzyme (Men) along with seven different genetic backgrounds to lend biological relevance to the data. The responses of the NADP-reducing enzymes and two downstream phenotypes (lipid and glycogen concentration) were compared between the control and stress conditions. In general, responses in NADP-reducing enzymes were greater under conditions of oxidative stress, likely due to an increased demand for NADPH. Interactions between the enzymes were altered by environmental stress in directions and magnitudes that are consistent with differential contributions of the different enzymes to the NADPH pool: the contributions of G6PD and IDH seem to be accentuated by oxidative stress, and MEN by starvation. Overall, we find that biological network interactions are strongly influenced by environmental conditions, underscoring the importance of examining networks as dynamic entities.