| Wolbachia is a kind of intracellular symbiotic bacteria which was first discovered in1924by Hertige and Wolbach. It widely exists in arthropods and filamentous nematodes. At present, the insect species infected with Wolbachia accounts for about66%in the worldwide. Wolbachia mainly exists in the host’s reproductive system, which can manipulate host reproductive through a variety of ways that can lead to some reproductive abnormalities, such as sperm-egg cytoplasmic incompatibility (CI)、 parthenogenesis(PI)、feminization and male-killing. While, CI is the most common phenotype of these alterations. It occurs when Wolbachia-infected males mate with females that uninfected or infected with different strains of Wolbachia resulting in very low even zero hatching rate. But females that infected mating with uninfected or same specie Wolbachia-infected males can produce the normal development of embryos. So far, although there are some researches about Wolbachia, the exact molecular mechanism of CI remains unclearly.Our research group has previously used the testis of the third instar larvae of Wolbachia-infected (Dmel wMel) and uninfected Drosophila melanogaster (Dmel T) as the materials to analyse gene expression difference during early spermatiogenesis between them by microarray. And we have identified296genes with differential expression at least1.5times. This study selected two genes, CG8189(ATPsyn-b) and CG17934-RA(Mst84Db),from the296genes which may be associated with sperm sterility, and we carried out further validation and studied their functions in male fertility in Drosophila.By quantitative RT-PCR (qRT-PCR), we found first the expression levels of gene ATPsyn-b were significantly decreased in Wolbachia-infected Drosophila third instar larvae testis, which is consistent with the results from the previous microarray analysis. Next, by qRT-PCR we detected ATPsyn-b expression patterns in various developmental stages in normal Dmel T flies and found that the expression levels were very low in embryonic, larval and pupal stages. However, the transcripts significantly increased in adult stages (P<0.01). Moreover, the gene showed different expression levels between males and females. The mRNA levels in males were significantly higher than that in females (P<0.01). This result suggests that ATPsyn-b may be provided with the male reproductive function of Drosophila. To know more ahout the function of the gene ATPsyn-b, we microinjected dsRNA of the gene synthesized in vitro to5-6day-old Dmel T males, the injection of RNase-free water as a control. We found that the gene expression in experimental group had a certainly declining at each time point (24h,48h, 72h,96h) after injected and the embryo hatchability rate of the experimental group also reduced especially significantly at72hours (P<0.01), descriptied ATPsyn-b knock down severely reducing the fertility of male flies. Previous studies have shown that ATPsyn-b was involved in oxidative phosphorylation, and thus speculated that this genes was associated with ATP synthesis to supply energy for the organisms accompanied by proton transport. Drosophila spermatogenesis is an energy-intensive process.The gene may be associated with the generation of sperm. The gene expression reduced may affect spermatogenesis, thereby the male reproduction is reduced. Currently, we have bought CG8189RNAi transgenic fly lines and have obtained the gene knock down flies by using UAS-Gal4system. The studies on the effect of the gene knockdown on male sterility and the mechanism are further under investigation by using the new CG8189knockdown flies.Our research group has demonstrated that the expression of CG17934-RA (Mst84Db) in Wolbachia-infected Drosophila testis exibited large down-regulation by qRT-PCR and in situ hybridization experiments, which is consistent with the result from microarray analysis. Preliminary research showed that4-6d male flies after injected with dsRNA, Mst84Db gene expression level was significantly decreased. The embryonic hatching rate after72hours injectied in the experimental group was significantly decreased than in the control group, when the male flies which the Mst84Db gene was knocked down mated with the Dmel T virgin fruit flies, indicating that knock down the gene Mst84Db seriously reduce the fertility of male flies. In this study, we used DAP1(4’,6-diamidino-2-phenylindole) to stain the early embryos of experimental group at72hours after injection and to find the reason leading to the embryo death. We found that the embryonic nuclear division was not synchronized, and chromatin bridges were frequently observed. These is similar to the cytological phenotypes appeared in CI embryos. Already cytology studies have shown that CI embryos have abnormal phenotype. Male and female prokaryotic development time inconsistency cause nuclear membrane rupture time of the male pronucleus is later than the female prokaryotic, which makes the male parent segregation abnormal and the chromatin bridge, ultimately the formation of chromosome aneuploidy group causing an embryo to be normal development. Previous studies have shown that Mst84Db specific expresses in the male reproductive system and related to sperm movement. These data suggest that Wolbachia infection might inhibit the Mst84Db gene expression during spermatogenesis, then cause the abnormal structure and function of sperms, eventually leading to embryonic lethality after mating.In this study, using the method of RNA interference (RNAi) to research ATPsyn-b and Mst84Db gene function in the reproductive process of male flies is not only to provide new ideas to study molecular mechanism of Wolbachia-induced CI, but also to explore the interaction between symbiotic bacteria and their hosts. Additionally, this study also provides a broad prospect for the future application of this new type of environment-friendly bio-media for bio-control, and can provide newclues to explore the mechanism of the early animal spermatogenesis and human male infertility. |
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