Drosophila Simulans Research Articles

Complete de novo assembly of Wolbachia endosymbiont of Drosophila willistoni using long-read genome sequencing

Wolbachia is an obligate intracellular α-proteobacterium, which commonly infects arthropods and filarial nematodes. Different strains of Wolbachia are capable of a wide range of regulatory manipulations in their diverse hosts, including the modulation of host cellular differentiation to influence host reproduction. The genetic basis for the majority of these phenotypes is unknown. The wWil strain from the neotropical fruit fly, Drosophila willistoni, exhibits a remarkably high affinity for host germline-derived cells relative to the somatic cells. This trait could be leveraged for understanding how Wolbachia influences the host germline and for controlling host populations in the field. To further the use of this strain in biological and biomedical research, we sequenced the genome of the wWil strain isolated from host cell culture cells. Here, we present the first high quality Nanopore assembly of wWil, the Wolbachia endosymbiont of D. willistoni. Our assembly resulted in a circular genome of 1.27 Mb with a BUSCO completeness score of 99.7%. Consistent with other insect-associated Wolbachia strains, comparative genomic analysis revealed that wWil has a highly mosaic genome relative to the closely related wMel and wAu strains from Drosophila melanogaster and Drosophila simulans, respectively.

Mixed Wolbachia infections resolve rapidly during in vitro evolution.

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.

Phylogenetic Probe of <i>Wolbachia</i> in <i>Bemisia tabaci</i>

The isolated DNA of Wolbachia present in whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleurodidae) collected from six different agroclimatic zones ofAssam, India were amplified through polymerase chain reaction (PCR) using Wolbachia specific primer. A band in the range of 518-528 bp proved the presence of Wolbachia in the samples, which was further confirmed through BLAST analysis using the NCBI database. A phylogenetic analysis of the DNA sequences showed two subgroups of Wolbachia in the collected sample of B. tabaci. TheW2 cluster formed with four samples of Wolbachia showed 91% similarity with Wolbachia from Drosophila simulans.

Molecular Insights into Female Hybrid Sterility in Interspecific Crosses between Drosophila melanogaster and Drosophila simulans.

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDCmel complex in hybrid ovaries was not assembled. However, the activity of the RDCsim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasasim allele and a significant zygotic delay in vasamel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.

Analysis of meiotic recombination in Drosophila simulans shows no evidence of an interchromosomal effect.

Chromosome inversions are of unique importance in the evolution of genomes and species because when heterozygous with a standard arrangement chromosome, they suppress meiotic crossovers within the inversion. In Drosophila species, heterozygous inversions also cause the interchromosomal effect, whereby the presence of a heterozygous inversion induces a dramatic increase in crossover frequencies in the remainder of the genome within a single meiosis. To date, the interchromosomal effect has been studied exclusively in species that also have high frequencies of inversions in wild populations. We took advantage of a recently developed approach for generating inversions in Drosophila simulans, a species that does not have inversions in wild populations, to ask if there is an interchromosomal effect. We used the existing chromosome 3R balancer and generated a new chromosome 2L balancer to assay for the interchromosomal effect genetically and cytologically. We found no evidence of an interchromosomal effect in D. simulans. To gain insights into the underlying mechanistic reasons, we qualitatively analyzed the relationship between meiotic double-stranded break (DSB) formation and synaptonemal complex (SC) assembly. We found that the SC is assembled prior to DSB formation as in D. melanogaster; however, we show that the SC is assembled prior to localization of the oocyte determination factor Orb, whereas in D. melanogaster, SC formation does not begin until the Orb is localized. Together, our data show no evidence that heterozygous inversions in D. simulans induce an interchromosomal effect and that there are differences in the developmental programming of the early stages of meiosis.

Genomes of historical specimens reveal multiple invasions of LTR retrotransposons in Drosophila melanogaster during the 19th century

Transposable element invasions have a profound impact on the evolution of genomes and phenotypes. It is thus an important open question how often such TE invasions occur. To address this question, we utilize the genomes of historical specimens, sampled about 200 y ago. We found that the LTR retrotransposons Blood, Opus, and 412 spread in Drosophila melanogaster in the 19th century. These invasions constitute second waves, as degraded fragments were found for all three TEs. The composition of Opus and 412, but not of Blood, shows a pronounced geographic heterogeneity, likely due to founder effects during the invasions. Finally, we identified species from the Drosophila simulans complex as the likely origin of the TEs. We show that in total, seven TE families invaded D. melanogaster during the last 200y, thereby increasing the genome size by up to 1.2Mbp. We suggest that this high rate of TE invasions was likely triggered by human activity. Based on the analysis of strains and specimens sampled at different times, we provide a detailed timeline of TE invasions, making D. melanogaster the first organism where the invasion history of TEs during the last two centuries could be inferred.

Evolution of Phenotypic Variance Provides Insights into the Genetic Basis of Adaptation.

Most traits are polygenic, and the contributing loci can be identified by genome-wide association studies. The genetic basis of adaptation (adaptive architecture) is, however, difficult to characterize. Here, we propose to study the adaptive architecture of traits by monitoring the evolution of their phenotypic variance during adaptation to a new environment in well-defined laboratory conditions. Extensive computer simulations show that the evolution of phenotypic variance in a replicated experimental evolution setting can distinguish between oligogenic and polygenic adaptive architectures. We compared gene expression variance in male Drosophila simulans before and after 100 generations of adaptation to a novel hot environment. The variance change in gene expression was indistinguishable for genes with and without a significant change in mean expression after 100 generations of evolution. We suggest that the majority of adaptive gene expression evolution can be explained by a polygenic architecture. We propose that tracking the evolution of phenotypic variance across generations can provide an approach to characterize the adaptive architecture.

Wolbachia infection negatively impacts Drosophila simulans heat tolerance in a strain- and trait-specific manner.

The susceptibility of insects to rising temperatures has largely been measured by their ability to survive thermal extremes. However, the capacity for maternally inherited endosymbionts to influence insect heat tolerance has been overlooked. Further, while some studies have addressed the impact of heat on traits like fertility, which can decline at temperatures below lethal thermal limits, none have considered the impact of endosymbionts. Here, we assess the impact of three Wolbachia strains (wRi, wAu and wNo) on the survival and fertility of Drosophila simulans exposed to heat stress during development or as adults. The effect of Wolbachia infection on heat tolerance was generally small and trait/strain specific. Only the wNo infection significantly reduced the survival of adult males after a heat shock. When exposed to fluctuating heat stress during development, the wRi and wAu strains reduced egg-to-adult survival but only the wNo infection reduced male fertility. Wolbachia densities of all three strains decreased under developmental heat stress, but reductions occurred at temperatures above those that reduced host fertility. These findings emphasize the necessity to account for endosymbionts and their effect on both survival and fertility when investigating insect responses to heat stress.

Evolution of compound eye morphology underlies differences in vision between closely related Drosophila species

BackgroundInsects have evolved complex visual systems and display an astonishing range of adaptations for diverse ecological niches. Species of Drosophila melanogaster subgroup exhibit extensive intra- and interspecific differences in compound eye size. These differences provide an excellent opportunity to better understand variation in insect eye structure and the impact on vision. Here we further explored the difference in eye size between D. mauritiana and its sibling species D. simulans.ResultsWe confirmed that D. mauritiana have rapidly evolved larger eyes as a result of more and wider ommatidia than D. simulans since they recently diverged approximately 240,000 years ago. The functional impact of eye size, and specifically ommatidia size, is often only estimated based on the rigid surface morphology of the compound eye. Therefore, we used 3D synchrotron radiation tomography to measure optical parameters in 3D, predict optical capacity, and compare the modelled vision to in vivo optomotor responses. Our optical models predicted higher contrast sensitivity for D. mauritiana, which we verified by presenting sinusoidal gratings to tethered flies in a flight arena. Similarly, we confirmed the higher spatial acuity predicted for Drosophila simulans with smaller ommatidia and found evidence for higher temporal resolution.ConclusionsOur study demonstrates that even subtle differences in ommatidia size between closely related Drosophila species can impact the vision of these insects. Therefore, further comparative studies of intra- and interspecific variation in eye morphology and the consequences for vision among other Drosophila species, other dipterans and other insects are needed to better understand compound eye structure–function and how the diversification of eye size, shape, and function has helped insects to adapt to the vast range of ecological niches.

The condition-dependence of male genital size and shape.

The male genitals of internal fertilisers evolve rapidly and divergently, and sexual selection is generally responsible for this. Many sexually selected traits are condition-dependent-with their expression dependent upon the resources available to be allocated to them-as revealed by genetic or environmental manipulations of condition. However, it is not clear whether male genitals are also condition-dependent. Here we manipulate condition in two ways (via inbreeding and diet) to test the condition-dependence of the genital arch of Drosophila simulans. We found that genital size but not genital shape suffered from inbreeding depression, whereas genital size and shape were affected by dietary manipulation of condition. The differential effects of these treatments likely reflect underlying genetic architecture that has been shaped by past selection: inbreeding depression is only expected when traits have a history of directional selection, while diet impacts traits regardless of historical selection. Nonetheless, our results suggest genitals can be condition-dependent like other sexually selected traits.

Developmental noise and phenotypic plasticity are correlated in Drosophila simulans.

Non-genetic variation is the phenotypic variation induced by the differential expression of a genotype in response to varying environmental cues and is broadly categorized into two types: phenotypic plasticity and developmental noise. These aspects of variation have been suggested to play an important role in adaptive evolution. However, the mechanisms by which these two types of non-genetic variations influence the evolutionary process are currently poorly understood. Using a machine-learning-based phenotyping tool, we independently quantified phenotypic plasticity and developmental noise in the wing morphological traits of the fruit fly Drosophila simulans. Utilizing a rearing experiment, we demonstrated plastic responses in both wing size and shape as well as non-zero heritability of both phenotypic plasticity and developmental noise, which suggests that adaptive phenotypic plasticity can evolve via genetic accommodation in the wing morphology of D. simulans. We found a positive correlation between phenotypic plasticity and developmental noise, while the correlation between the plastic response to three kinds of environmental factors that were examined (nutrient condition, temperature, and light-dark cycle) was poor. These results suggest that phenotypic plasticity and developmental noise contribute to evolvability in a similar manner, however, the mechanisms that underlie the correspondence between these two types of variation remain to be elucidated.

A new hybrid incompatibility locus between Drosophila melanogaster and Drosophila sechellia.

Despite the fundamental importance of hybrid incompatibilities to the process of speciation, there are few cases where the evolution and genetic architecture of hybrid incompatibilities are understood. One of the longest studied hybrid incompatibilities causes F1 hybrid male inviability in crosses between Drosophila melanogaster females and males from the Drosophila simulans clade of species-Drosophila simulans, Drosophila mauritiana, and Drosophila sechellia. Here, we discover dramatic differences in the manifestation of this lethal hybrid incompatibility among the D. simulans clade of species. In particular, F1 hybrid males between D. melanogaster and D. sechellia are resistant to hybrid rescue through RNAi knockdown of an essential hybrid incompatibility gene. To understand the genetic basis of this inter-species difference in hybrid rescue, we developed a triple-hybrid mapping method. Our results show that 2 discrete large effect loci and many dispersed small effect changes across the genome underlie D. sechellia aversion to hybrid rescue. The large effect loci encompass a known incompatibility gene Lethal hybrid rescue (Lhr) and previously unknown factor, Sechellia aversion to hybrid rescue (Satyr). These results show that the genetic architecture of F1 hybrid male inviability is overlapping but not identical in the 3 inter-species crosses. Our results raise questions about whether new hybrid incompatibility genes can integrate into an existing hybrid incompatibility thus increasing in complexity over time, or if the continued evolution of genes can gradually strengthen an existing hybrid incompatibility.

Non-additive effects between genotypes: Implications for competitive fitness assays.

Competitive fitness assays are widely used in evolutionary biology and typically rely on a reference strain to compare different focal genotypes. This approach implicitly relies on the absence of interaction between the competing genotypes. In other words, the performance of the reference strain must not depend on the competitor. This report scrutinized this assumption by competing diverged Drosophila simulans populations against a common reference strain. We detected strong evidence for interaction between the competing genotypes: (1) Frequency-dependent selection was common with opposite effects in genetically diverged populations. (2) Temporal heterogeneity of fitness estimates, which can be partially attributed to a competitor-specific delay in the eclosion of the reference strain. We propose that this inconsistent behavior of the reference strain can be considered a specific case of a genotype × environment interaction. Focal populations could modify the environment of the reference strain, either indirectly by altering the microbiome composition and food availability or directly by genotype-specific cannibalism. Our results provide new insights into the interaction of diverged genotypes and have important implications for the interpretation of competitive fitness assays.

High levels of intra-strain structural variation in Drosophila simulans X pericentric heterochromatin.

Large genome structural variations can impact genome regulation and integrity. Repeat-rich regions like pericentric heterochromatin are vulnerable to structural rearrangements although we know little about how often these rearrangements occur over evolutionary time. Repetitive genome regions are particularly difficult to study with genomic approaches, as they are missing from most genome assemblies. However, cytogenetic approaches offer a direct way to detect large rearrangements involving pericentric heterochromatin. Here, we use a cytogenetic approach to reveal large structural rearrangements associated with the X pericentromeric region of Drosophila simulans. These rearrangements involve large blocks of satellite DNA-the 500-bp and Rsp-like satellites-which colocalize in the X pericentromeric heterochromatin. We find that this region is polymorphic not only among different strains, but between isolates of the same strain from different labs, and even within individual isolates. On the one hand, our observations raise questions regarding the potential impact of such variation at the phenotypic level and our ability to control for such genetic variability. On the other hand, this highlights the very rapid turnover of the pericentric heterochromatin most likely associated with genomic instability of the X pericentromere. It represents a unique opportunity to study the dynamics of pericentric heterochromatin, the evolution of associated satellites on a very short time scale, and to better understand how structural variation arises.

Effects of larval crowding on the transcriptome of Drosophila simulans.

Larval crowding is one common ecological stressor for many insect species. In Drosophila, high larval density alters multiple widely-studied phenotypes including life-history traits, morphology and behavior. Nevertheless, we still miss a holistic view of the full range of phenotypic changes and the underlying molecular mechanisms. In this study, we analyzed the adult transcriptomes of high and low larval density fly cohorts, and highlighted the molecular basis of the plastic traits. Increased cellular energy metabolism and locomotion, along with reduced reproductive investment, are key responses to high larval density. Moreover, we compared the expression changes among cohorts with different developmental delays caused by larval crowding. The majority of genes induced by larval crowding showed the strongest expression alterations in cohorts with intermediate delay. Furthermore, linear expression changes were observed in genes related to nutrition and detoxification. Comparing different high-density cohorts could provide insights into the varied responses to distinct larval crowding-induced stresses such as space competition, food degradation and waste accumulation.

Wolbachia Induces Structural Defects Harmful to Drosophila simulans Riverside Spermiogenesis.

The relationship between cytoplasmic incompatibility and the obligate intracellular alphaproteobacteria Wolbachia has for a long time been reported. Although the molecular mechanisms responsible for this reproductive alteration are beginning to be understood, the effects of Wolbachia on germ cell structure and dynamics have not yet been fully investigated. We report here that the presence of Wolbachia in infected cysts of elongating spermatids is associated with major structural defects that become more evident in mature sperm. We find mitochondrial defects, an improper axoneme structure, reduced sperm numbers, and individualization failures. The large heterogeneous variety of the ultrastructural defects found in elongating spermatids and mature sperm provide the first cytological evidence for the reduced fertility associated with Wolbachia infection in Drosophila simulans males. The observed abnormalities could be the result of the mechanical stress induced by the high bacteria numbers during the process of spermatid elongation, rather than the result of the released factors affecting the proper morphogenesis of the germ cells. Moreover, high Wolbachia densities in male germ cells may not be appropriate for causing cytoplasmic incompatibility as the bacteria are harmful for spermatid differentiation, leading to abnormal sperm that is unlikely to be functional.

A quantitative, genome-wide analysis in Drosophila reveals transposable elements' influence on gene expression is species-specific.

Transposable elements (TEs) are parasite DNA sequences that are able to move and multiply along the chromosomes of all genomes. They can be controlled by the host through the targeting of silencing epigenetic marks, which may affect the chromatin structure of neighboring sequences, including genes. In this study, we used transcriptomic and epigenomic high-throughput data produced from ovarian samples of several Drosophila melanogaster and Drosophila simulans wild-type strains, in order to finely quantify the influence of TE insertions on gene RNA levels and histone marks (H3K9me3 and H3K4me3). Our results reveal a stronger epigenetic effect of TEs on ortholog genes in D. simulans compared to D. melanogaster. At the same time, we uncover a larger contribution of TEs to gene H3K9me3 variance within genomes in D. melanogaster, which is evidenced by a stronger correlation of TE numbers around genes with the levels of this chromatin mark in D. melanogaster. Overall, this work contributes to the understanding of species-specific influence of TEs within genomes. It provides a new light on the considerable natural variability provided by TEs, which may be associated with contrasted adaptive and evolutionary potentials.

Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species.

Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

The genomic distribution of transposable elements is driven by spatially variable purifying selection.

It is widely accepted that the genomic distribution of transposable elements (TEs) mainly reflects the outcome of purifying selection and insertion bias (1). Nevertheless, the relative importance of these two evolutionary forces could not be tested thoroughly. Here, we introduce an experimental system, which allows separating purifying selection from TE insertion bias. We used experimental evolution to study the TE insertion patterns in Drosophila simulans founder populations harboring 1040 insertions of an active P-element. After 10 generations at a large population size, we detected strong selection against P-element insertions. The exception were P-element insertions in genomic regions for which a strong insertion bias has been proposed (2-4). Because recurrent P-element insertions cannot explain this pattern, we conclude that purifying selection, with variable strength along the chromosomes, is the major determinant of the genomic distribution of P-elements. Genomic regions with relaxed purifying selection against P-element insertions exhibit normal levels of purifying selection against base substitutions. This suggests that different types of purifying selection operate on base substitutions and P-element insertions. Our results highlight the power of experimental evolution to understand basic evolutionary processes, which are difficult to infer from patterns of natural variation alone.

The evolutionary history of Drosophila simulans Y chromosomes reveals molecular signatures of resistance to sex ratio meiotic drive.

The recent evolutionary history of the Y chromosome in Drosophila simulans, a worldwide species of Afrotropical origin, is closely linked to that of X-linked meiotic drivers (Paris system). The spread of the Paris drivers in natural populations has elicited the selection of drive resistant Y chromosomes. To infer the evolutionary history of the Y chromosome in relation to the Paris drive, we sequenced 21 iso-Y lines, each carrying a Y chromosome from a different location. Among them, 13 lines carry a Y chromosome that is able to counteract the effect of the drivers. Despite their very different geographical origins, all sensitive Y's are highly similar, suggesting that they share a recent common ancestor. The resistant Y chromosomes are more divergent and segregate in four distinct clusters. The phylogeny of the Y chromosome confirms that the resistant lineage predates the emergence of Paris drive. The ancestry of the resistant lineage is further supported by the examination of Y-linked sequences in the sister species of D. simulans, D. sechellia, and D. mauritiana. We also characterized the variation in repeat content among Y chromosomes and identified multiple simple satellites associated with resistance. Altogether, the molecular polymorphism allows us to infer the demographic and evolutionary history of the Y chromosome and provides new insights on the genetic basis of resistance.