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Abstract Background Testis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown of ocnus (ocn), a testis-specific gene, resulted in much smaller testis with no germ cells in Drosophila melanogaster. However, the molecular consequences of ocn knockdown in fly testes are unknown. Results In this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways. Conclusions Given the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.

Abstract Background Cytoplasmic incompatibility (CI) is the most common phenotype induced by endosymbiont Wolbachia and results in embryonic lethality when Wolbachia-modified sperm fertilize eggs without Wolbachia. However, eggs carrying the same strain of Wolbachia can rescue this embryonic death, thus producing viable Wolbachia-infected offspring. Hence Wolbachia can be transmitted mainly by hosts’ eggs. One of the models explaining CI is “titration-restitution”, which hypothesized that Wolbachia titrated-out some factors from the sperm and the Wolbachia in the egg would restitute the factors after fertilization. However, how infected eggs rescue CI and how hosts’ eggs ensure the proliferation and transmission of Wolbachia are not well understood. Results By RNA-seq analyses, we first compared the transcription profiles of Drosophila melanogaster adult ovaries with and without the wMel Wolbachia and identified 149 differentially expressed genes (DEGs), of which 116 genes were upregulated and 33 were downregulated by Wolbachia infection. To confirm the results obtained from RNA-seq and to screen genes potentially associated with reproduction, 15 DEGs were selected for quantitative RT-PCR (qRT-PCR). Thirteen genes showed the same changing trend as RNA-seq analyses. To test whether these genes are associated with CI, we also detected their expression levels in testes. Nine of them exhibited different changing trends in testes from those in ovaries. To investigate how these DEGs were regulated, sRNA sequencing was performed and identified seven microRNAs (miRNAs) that were all upregulated in fly ovaries by Wolbachia infection. Matching of miRNA and mRNA data showed that these seven miRNAs regulated 15 DEGs. Wolbachia-responsive genes in fly ovaries were involved in biological processes including metabolism, transportation, oxidation-reduction, immunity, and development. Conclusions Comparisons of mRNA and miRNA data from fly ovaries revealed 149 mRNAs and seven miRNAs that exhibit significant changes in expression due to Wolbachia infection. Notably, most of the DEGs showed variation in opposite directions in ovaries versus testes in the presence of Wolbachia, which generally supports the “titration-restitution” model for CI. Furthermore, genes related to metabolism were upregulated, which may benefit maximum proliferation and transmission of Wolbachia. This provides new insights into the molecular mechanisms of Wolbachia-induced CI and Wolbachia dependence on host ovaries.