Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster

Background

Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.

Methods and Findings

We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79% and 56–66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.

Conclusions

We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

Selective maintenance of Drosophila tandemly arranged duplicated genes during evolution

Background

The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression.

Results

We have catalogued ordered duplicated genes in Drosophila melanogaster, and found that one in five of all genes is organized as tandem arrays. Furthermore, among arrays that have been spatially conserved over longer periods than would be expected on the basis of random shuffling, a disproportionate number contain genes encoding developmental regulators. Using in situ gene expression data for more than half of the Drosophila genome, we find that genes in these conserved clusters are co-expressed to a much higher extent than other duplicated genes.

Conclusions

These results reveal the existence of functional constraints in insects that retain copies of genes encoding developmental and regulatory proteins as neighbors, allowing their co-expression. This co-expression may be the result of shared cis-regulatory elements or a shared need for a specific chromatin structure. Our results highlight the association between genome architecture and the gene regulatory networks involved in the construction of the body plan.     

Odorant receptor (Or) genes: polymorphism and divergence in the D. melanogaster and D. pseudoobscura lineages

Background

In insects, like in most invertebrates, olfaction is the principal sensory modality, which provides animals with essential information for survival and reproduction. Odorant receptors are involved in this response, mediating interactions between an individual and its environment, as well as between individuals of the same or different species. The adaptive importance of odorant receptors renders them good candidates for having their variation shaped by natural selection.

Methodology/Principal Findings

We analyzed nucleotide variation in a subset of eight Or genes located on the 3L chromosomal arm of Drosophila melanogaster in a derived population of this species and also in a population of Drosophila pseudoobscura. Some heterogeneity in the silent polymorphism to divergence ratio was detected in the D. melanogaster/D. simulans comparison, with a single gene (Or67b) contributing ∼37% to the test statistic. However, no other signals of a very recent selective event were detected at this gene. In contrast, at the speciation timescale, the MK test uncovered the footprint of positive selection driving the evolution of two of the encoded proteins in both D. melanogaster —OR65c and OR67a —and D. pseudoobscura —OR65b1 and OR67c.

Conclusions

The powerful polymorphism/divergence approach provided evidence for adaptive evolution at a rather high proportion of the Or genes studied after relatively recent speciation events. It did not provide, however, clear evidence for very recent selective events in either D. melanogaster or D. pseudoobscura.     

Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

Background

The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development.

Methodology/Principal Findings

We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria.

Conclusions/Significance

These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.     

A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.     

The Transcriptional Response of Drosophila melanogaster to Infection with the Sigma Virus (Rhabdoviridae)

Background

Bacterial and fungal infections induce a potent immune response in Drosophila melanogaster, but it is unclear whether viral infections induce an antiviral immune response. Using microarrays, we examined the changes in gene expression in Drosophila that occur in response to infection with the sigma virus, a negative-stranded RNA virus (Rhabdoviridae) that occurs in wild populations of D. melanogaster.

Principal Findings

We detected many changes in gene expression in infected flies, but found no evidence for the activation of the Toll, IMD or Jak-STAT pathways, which control immune responses against bacteria and fungi. We identified a number of functional categories of genes, including serine proteases, ribosomal proteins and chorion proteins that were overrepresented among the differentially expressed genes. We also found that the sigma virus alters the expression of many more genes in males than in females.

Conclusions

These data suggest that either Drosophila do not mount an immune response against the sigma virus, or that the immune response is not controlled by known immune pathways. If the latter is true, the genes that we identified as differentially expressed after infection are promising candidates for controlling the host''s response to the sigma virus.     

Lack of Phenotypic and Evolutionary Cross-Resistance against Parasitoids and Pathogens in Drosophila melanogaster

Background

When organisms are attacked by multiple natural enemies, the evolution of a resistance mechanism to one natural enemy will be influenced by the degree of cross-resistance to another natural enemy. Cross-resistance can be positive, when a resistance mechanism against one natural enemy also offers resistance to another; or negative, in the form of a trade-off, when an increase in resistance against one natural enemy results in a decrease in resistance against another. Using Drosophila melanogaster, an important model system for the evolution of invertebrate immunity, we test for the existence of cross-resistance against parasites and pathogens, at both a phenotypic and evolutionary level.

Methods

We used a field strain of D. melanogaster to test whether surviving parasitism by the parasitoid Asobara tabida has an effect on the resistance against Beauveria bassiana, an entomopathogenic fungus; and whether infection with the microsporidian Tubulinosema kingi has an effect on the resistance against A. tabida. We used lines selected for increased resistance to A. tabida to test whether increased parasitoid resistance has an effect on resistance against B. bassiana and T. kingi. We used lines selected for increased tolerance against B. bassiana to test whether increased fungal resistance has an effect on resistance against A. tabida.

Results/Conclusions

We found no positive cross-resistance or trade-offs in the resistance to parasites and pathogens. This is an important finding, given the use of D. melanogaster as a model system for the evolution of invertebrate immunity. The lack of any cross-resistance to parasites and pathogens, at both the phenotypic and the evolutionary level, suggests that evolution of resistance against one class of natural enemies is largely independent of evolution of resistance against the other.     

Female Drosophila melanogaster gene expression and mate choice: the X chromosome harbours candidate genes underlying sexual isolation

Background

The evolution of female choice mechanisms favouring males of their own kind is considered a crucial step during the early stages of speciation. However, although the genomics of mate choice may influence both the likelihood and speed of speciation, the identity and location of genes underlying assortative mating remain largely unknown.

Methods and Findings

We used mate choice experiments and gene expression analysis of female Drosophila melanogaster to examine three key components influencing speciation. We show that the 1,498 genes in Zimbabwean female D. melanogaster whose expression levels differ when mating with more (Zimbabwean) versus less (Cosmopolitan strain) preferred males include many with high expression in the central nervous system and ovaries, are disproportionately X-linked and form a number of clusters with low recombination distance. Significant involvement of the brain and ovaries is consistent with the action of a combination of pre- and postcopulatory female choice mechanisms, while sex linkage and clustering of genes lead to high potential evolutionary rate and sheltering against the homogenizing effects of gene exchange between populations.

Conclusion

Taken together our results imply favourable genomic conditions for the evolution of reproductive isolation through mate choice in Zimbabwean D. melanogaster and suggest that mate choice may, in general, act as an even more important engine of speciation than previously realized.     

Functional significance of allelic variation at methuselah, an aging gene in Drosophila

Background

Longevity and age-specific patterns of mortality are complex traits that vary within and among taxa. Multiple candidate genes for aging have been identified in model systems by extended longevity mutant phenotypes, including the G-protein coupled receptor methuselah (mth) in D. melanogaster. These genes offer important insights into the mechanisms of lifespan determination and have been major targets of interest in the biology of aging. However, it is largely unknown whether these genes contribute to genetic variance for lifespan in natural populations, and consequently contribute to lifespan evolution.

Methodology/Principle Findings

For a gene to contribute to genetic variance for a particular trait, it must meet two criteria: natural allelic variation and functional differences among variants. Previous work showed that mth varies significantly among wild populations; here we assess the functional significance of wild-derived mth alleles on lifespan, fecundity and stress resistance using a quantitative complementation scheme. Our results demonstrate that mth alleles segregating in nature have a functional effect on all three traits.

Conclusions/Significance

These results suggest that allelic variation at mth contributes to observed differences in lifespan and correlated phenotypes in natural populations, and that evaluation of genetic diversity at candidate genes for aging can be a fruitful approach to identifying loci contributing to lifespan evolution.     

The Chironomus tentans genome sequence and the organization of the Balbiani ring genes

Background

The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes.

Results

The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150–250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations.

Conclusions

We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-819) contains supplementary material, which is available to authorized users.     

RNAi Experiments in D. melanogaster: Solutions to the Overlooked Problem of Off-Targets Shared by Independent dsRNAs

Background

RNAi technology is widely used to downregulate specific gene products. Investigating the phenotype induced by downregulation of gene products provides essential information about the function of the specific gene of interest. When RNAi is applied in Drosophila melanogaster or Caenorhabditis elegans, often large dsRNAs are used. One of the drawbacks of RNAi technology is that unwanted gene products with sequence similarity to the gene of interest can be down regulated too. To verify the outcome of an RNAi experiment and to avoid these unwanted off-target effects, an additional non-overlapping dsRNA can be used to down-regulate the same gene. However it has never been tested whether this approach is sufficient to reduce the risk of off-targets.

Methodology

We created a novel tool to analyse the occurance of off-target effects in Drosophila and we analyzed 99 randomly chosen genes.

Principal Findings

Here we show that nearly all genes contain non-overlapping internal sequences that do show overlap in a common off-target gene.

Conclusion

Based on our in silico findings, off-target effects should not be ignored and our presented on-line tool enables the identification of two RNA interference constructs, free of overlapping off-targets, from any gene of interest.     

A signature inferred from Drosophila mitotic genes predicts survival of breast cancer patients

Introduction

The classification of breast cancer patients into risk groups provides a powerful tool for the identification of patients who will benefit from aggressive systemic therapy. The analysis of microarray data has generated several gene expression signatures that improve diagnosis and allow risk assessment. There is also evidence that cell proliferation-related genes have a high predictive power within these signatures.

Methods

We thus constructed a gene expression signature (the DM signature) using the human orthologues of 108 Drosophila melanogaster genes required for either the maintenance of chromosome integrity (36 genes) or mitotic division (72 genes).

Results

The DM signature has minimal overlap with the extant signatures and is highly predictive of survival in 5 large breast cancer datasets. In addition, we show that the DM signature outperforms many widely used breast cancer signatures in predictive power, and performs comparably to other proliferation-based signatures. For most genes of the DM signature, an increased expression is negatively correlated with patient survival. The genes that provide the highest contribution to the predictive power of the DM signature are those involved in cytokinesis.

Conclusion

This finding highlights cytokinesis as an important marker in breast cancer prognosis and as a possible target for antimitotic therapies.     

Mito-nuclear interactions as drivers of gene movement on and off the X-chromosome

Background

Mito-nuclear gene interactions regulate energy conversion, and are fundamental to eukaryotes. Generally, mito-nuclear coadaptation would be most efficient if the interacting nuclear genes were X-linked, because this maximizes the probability of favorable mito-nuclear allelic combinations co-transmitting across generations. Thus, under a coadaptation (CA) hypothesis, nuclear genes essential for mitochondrial function might be under selection to relocate to the X-chromosome. However, maternal inheritance predisposes the mitochondrial DNA (mtDNA) to accumulate variation that, while male-harming, is benign to females. Numerous nuclear genes were recently reported in Drosophila melanogaster, which exhibit male-specific patterns of differential expression when placed alongside different mtDNA haplotypes, suggesting that nuclear genes are sensitive to an underlying male-specific mitochondrial mutation load. These genes are thus candidates for involvement in mito-nuclear interactions driven by sexual conflict (SC), and selection might have moved them off the X-chromosome to facilitate an optimal evolutionary counter-response, through males, to the presence of male-harming mtDNA mutations. Furthermore, the presence of male-harming mtDNA mutations could exert selection for modifiers on the Y-chromosome, thus placing these mito-sensitive nuclear genes at the center of an evolutionary tug-of-war between mitochondrion and Y-chromosome.We test these hypotheses by examining the chromosomal distributions of three distinct sets of mitochondrial-interacting nuclear genes in D. melanogaster; the first is a list of genes with mitochondrial annotations by Gene Ontologies, the second is a list comprising the core evolutionary-conserved mitochondrial proteome, and the third is a list of genes involved in male-specific responses to maternally-inherited mitochondrial variation and which might be putative targets of Y-chromosomal regulation.

Results

Genes with mitochondrial annotations and genes representing the mitochondrial proteome do not exhibit statistically-significant biases in chromosomal representation. However, genes exhibiting sex-specific sensitivity to mtDNA are under-represented on the X-chromosome, over-represented among genes known to be sensitive to Y-chromosomal variation, and among genes previously associated with male fitness, but under-represented among genes associated with direct sexual antagonism.

Conclusions

Our results are consistent with the SC hypothesis, suggesting that mitochondrial mutational pressure selects for gene movement off-the-X, hence enabling mito-nuclear coadaptation to proceed along trajectories that result in optimized fitness in both sexes.