Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors.

Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

Experimental sexual selection reveals rapid evolutionary divergence in sex-specific transcriptomes and their interactions following mating.

Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Chromatin accessibility plays a key role in selective targeting of Hox proteins.

BACKGROUND: Hox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking. RESULTS: Using transient transfection in Kc167 cells, we systematically analyze the binding of all eight Drosophila Hox proteins. We find that Hox proteins show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions. CONCLUSIONS: These studies indicate that chromatin accessibility plays a key role in Hox selectivity. We propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.

Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders.

Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.

Gene expression clines reveal local adaptation and associated trade-offs at a continental scale.

Local adaptation, where fitness in one environment comes at a cost in another, should lead to spatial variation in trade-offs between life history traits and may be critical for population persistence. Recent studies have sought genomic signals of local adaptation, but often have been limited to laboratory populations representing two environmentally different locations of a species' distribution. We measured gene expression, as a proxy for fitness, in males of Drosophila subobscura, occupying a 20° latitudinal and 11 °C thermal range. Uniquely, we sampled six populations and studied both common garden and semi-natural responses to identify signals of local adaptation. We found contrasting patterns of investment: transcripts with expression positively correlated to latitude were enriched for metabolic processes, expressed across all tissues whereas negatively correlated transcripts were enriched for reproductive processes, expressed primarily in testes. When using only the end populations, to compare our results to previous studies, we found that locally adaptive patterns were obscured. While phenotypic trade-offs between metabolic and reproductive functions across widespread species are well-known, our results identify underlying genetic and tissue responses at a continental scale that may be responsible for this. This may contribute to understanding population persistence under environmental change.

Genetic, functional and evolutionary characterization of scox, the Drosophila melanogaster ortholog of the human SCO1 gene.

SCO proteins are copper-donor chaperones involved in the assembly of mitochondrial cytochrome c oxidase (COX). Mutations in the two human SCO-encoding genes, SCO1 and SCO2, produce tissue-specific COX deficiencies associated with distinct clinical phenotypes. Here, we report the identification and characterization of scox, the single Drosophila melanogaster SCO-encoding gene. Null mutations of the scox gene are associated with larval lethality, while mutations in its 5'UTR are associated with motor dysfunction and female sterile phenotypes. All mutant phenotypes may be rescued by a transgene encompassing wild-type scox. The analysis of the phenotypes associated with the D. melanogaster scox mutations shows that unimpaired COX assembly and activity is required for biological processes that specifically depend on an adequate energy supply. Finally, we identified the SCO1 orthologs in 39 eukaryotic species informative for a tentative reconstruction of the evolutionary history of the SCO function. Comparison of the exon/intron structure and other key features suggest that eukaryotic SCO genes descend from an intron-rich ancestral gene already present in the last common ancestor of lineages that diverged as early as metazoans and flowering plants.

The nuclear OXPHOS genes in insecta: a common evolutionary origin, a common cis-regulatory motif, a common destiny for gene duplicates.

BACKGROUND: When orthologous sequences from species distributed throughout an optimal range of divergence times are available, comparative genomics is a powerful tool to address problems such as the identification of the forces that shape gene structure during evolution, although the functional constraints involved may vary in different genes and lineages. RESULTS: We identified and annotated in the MitoComp2 dataset the orthologs of 68 nuclear genes controlling oxidative phosphorylation in 11 Drosophilidae species and in five non-Drosophilidae insects, and compared them with each other and with their counterparts in three vertebrates (Fugu rubripes, Danio rerio and Homo sapiens) and in the cnidarian Nematostella vectensis, taking into account conservation of gene structure and regulatory motifs, and preservation of gene paralogs in the genome. Comparative analysis indicates that the ancestral insect OXPHOS genes were intron rich and that extensive intron loss and lineage-specific intron gain occurred during evolution. Comparison with vertebrates and cnidarians also shows that many OXPHOS gene introns predate the cnidarian/Bilateria evolutionary split. The nuclear respiratory gene element (NRG) has played a key role in the evolution of the insect OXPHOS genes; it is constantly conserved in the OXPHOS orthologs of all the insect species examined, while their duplicates either completely lack the element or possess only relics of the motif. CONCLUSION: Our observations reinforce the notion that the common ancestor of most animal phyla had intron-rich gene, and suggest that changes in the pattern of expression of the gene facilitate the fixation of duplications in the genome and the development of novel genetic functions.

The MitoDrome database annotates and compares the OXPHOS nuclear genes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles gambiae.

The oxidative phosphorylation (OXPHOS) is the primary energy-producing process of all aerobic organisms and the only cellular function under the dual control of both the mitochondrial and the nuclear genomes. Functional characterization and evolutionary study of the OXPHOS system is of great importance for the understanding of many as yet unclear aspects of nucleus-mitochondrion genomic co-evolution and co-regulation gene networks. The MitoDrome database is a web-based database which provides genomic annotations about nuclear genes of Drosophila melanogaster encoding for mitochondrial proteins. Recently, MitoDrome has included a new section annotating genomic information about OXPHOS genes in Drosophila pseudoobscura and Anopheles gambiae and their comparative analysis with their Drosophila melanogaster and human counterparts. The introduction of this new comparative annotation section into MitoDrome is expected to be a useful resource for both functional and structural genomics related to the OXPHOS system.