Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster

The most common phenotype induced by the endosymbiont Wolbachia in in-sects is cytoplasmic incompatibility,where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females.This suggests that some modi-fications are induced in host sperms during spermatogenesis by Wolbachia.To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster,we applied isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic strategy combined with titanium dioxide(TiO2)enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male re-productive systems in D.melanogaster.We identified 182 phosphopeptides,defining 140 phosphoproteins,that have at least a 1.2 fold change in abundance with a P-value of<0.05.Most of the differentially abundant phosphoproteins(DAPPs)were associated with micro-tubule cytoskeleton organization and spermatid differentiation.The DAPPs included pro-teins already known to be associated with spermatogenesis,as well as many not previously studied during this process.Six genes coding for DAPPs were knocked down,respectively,in Wolbachia-free fly testes.Among them,Slmap knockdown caused the most severe dam-age in spermatogenesis,with no mature sperm observed in seminal vesicles.Immunoflu-orescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted.These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction.By identifying phospho-modulated proteins we also provide a signifi-cant candidate set for future studies on their roles in spermatogenesis.