The Mechanism Of Wolbachia Influencing Male Drosophila Fecundity

Wolbachia is the intracellular symbiotic bacterial that infects a wide range of arthropods and nematodes, it is transmitted to offspring through the cytoplasm of eggs. Recently, studies have shown that there is up to70%insect species that infected with Wolbachia in the world wide. Wolbachia is mainly present in the reproductive tissues of arthropods, it can induce a series of reproductive alterations including sperm-egg cytoplasmic incompatibility(CI)、feminizatio、parthenogenesis and male-killing. And CI is the most common phenotype of these alterations, it occurs when Wolbachia-infected males mate with females that uninfected Wolbachia or infected with different strains of Wolbachia resulting in no or very low numbers of viable offspring. Cytological study found that the development of male pronuclei lagged behind the female pronuclei in the CI embryos, and the asynchronous development of male and female pronuclei eventually led to the death of the embryo.The widespread of Wolbachia and a variety of reproductive manipulation of Wolbachia on host make it a most potential insect vector and agriculture pest control target. Therefore, the study of Wolbachia, especially the mechanisms of CI, and the study of effects of Wolbachia infection on the insect vector have been become one of research focuses at home and abroad. About the molecular mechanisms of CI, although there are some researches, the exact mechanism remains unclear.Our research group study the molecular mechanism associated with CI from the influence of Wolbachia infection on Drosophila sperm fertility perspective.Our research group used the testis of the third instar larvae of Wolbachia-infected and uninfected Drosophila melanogaster as the study object previously, and utilized microarray technology to analyse gene expression difference between them in early period of spermatogenesis. Differential expression was observed in296genes which were up-regulation or down-regulation at least1.5times.The study firstly chose seven genes (CG8627-RA. CG9081-RA、CG5320-RC、CG12052-RP、CG17934-RA、 CG17268-RA and CG7929-RA) which have large differences in expression and maybe associated with reproduction from these differential expression genes to carry out vivo validation though Quantitive RT-PCR.The results suggested that the expression of CG8627-RA、CG9081-RA and CG5320-RC were up-regulation, while the expression of CG12052-RP、CG17934-RA、CG17268-RA and CG7929-RA were down-regulation because of Wolbachia infection according with the results of microarray. The expression level of CG8627-RA and CG12052-RP in Wolbachia-infected and uninfected Drosophila testis is extremely significant difference(P<0.01), while the expression level of CG9081-RA、CG17934-RA and CG17268-RA is significant difference(P<0.05), two additional genes CG5320-RC and CG7929-RA have no significant difference(P>0.05).Due to the expression of CG3767(JHI-26) in Wolbachia-infected Drosophila testis showed large up-regulation in microarray analyses and qRT-PCR tests, and recently juvenile hormone-induced protein26encoded by CG3767(JHI-26) is identified as a sperm protein. In addition, CG17934(Mst84Db) is expressed in the male germ line specially and it is associated with sperm motility. So the study selected these two genes to take in situ hybridization experiments. The results showed that the signals of JHI-26and Mst84Db were detected almost the whole larval testes in both Drosophila with and without Wolbachia.However, the level of JHI-26expression in Wolbachia-infected Drosophila was higher than that of Wolbachia-uninfected Drosophila, while the expression of Mst84Db was opposite, that’s to say, the hybridization signal was weaker in Wolbachia-infected Drosophila than that of Wolbachia-uninfected Drosophila. This suggested that they were indeed express in larval testes, and Wolbachia infection led to the up-regulation expression of JHI-26and the down-regulation.expression of Mst84Db in early period of Drosophila testis. Previous researches found that Wolbachia-infected males which had faster developmental speed in larvae period could induce higher CI level which suggested that it had low embryonic hatching rates when Wolbachia-infected males mated with Wolbachia-uninfected females, while Wolbachia-infected males which had longer larvae developmental period induced lower CI level. Due to JHI-26is one protein which could be induced by JH specifically.Therefore, Wolbachia may induce the expression of JhI-26by mimicking the function of JH. As the high expression of JhI-26may inhibit the development of the larval testes, male flies which develop fastest in larvae period may produce incompletely matured sperm. Finally, immature sperm may then lead to the delayed development of the male pronucleus in fertilized embryo, and the asynchronous development of female and male pronuclei eventually leads to the death of the embryo.Due to CG12052(lola) involved in many biology processes, including axon growth and guidance、immune response、nurse cell apoptosis、morphogenesis of the brain and gonad formation. Evidence exists that the development of nurse cell apoptosis is blocked in lola-mutant flies, in addition, in lola-mutant flies chromatin is not completely condense during programmed cell death in Drosophila ovaries. The study group previously found that the expression of lola-rp was significantly down-regulation in Wolbachia-infected larval testis, therefore, lola-rp may induce abnormal chromatin condensation during spermatogenesis. And lola-rp is one of transcripts of lola, so the study firstly studied the time-course expression of lola-rp which was significantly down-regulation. The tissue specificity results showed that lola-rp was highly expressed in brain and ovaries in Drosophila melanogaster. In addition, in the process of embryo development, lola-rp could be detected from3h embryos and at a high level throughout the embryonic period.These suggests that lola-rp maybe the mother-endogenous gene in Drosophila melanogaster, and it may play an important role in early embryonic development.In order to further study the function of Mst84Db, we used RNAi to interfere the expression of Mst84Db gene in Wolbachia-uninfected male flies. We firstly synthesized the dsRNA of Mst84Db in vitro, then injected it into the males through microinjection. The RNAi results suggested that the expression level of Mst84Db was down-regulation after injecting dsRNA into the4-6days old males, and there was interference effect in24h after injecting dsRNA and gene knock down effect was best in72h after injection. The reproductive capacity of males which the Mst84Db gene was knocked down was decreased significantly and the embryonic hatchability was decreased significantly when the male flies mated with Wolbachia-uninfected virgin flies compared to the control group, which was similar to the CI phenomenon. In addition, the study of the cuticle of the unhatched embryos showed that there was no somites in the cuticle which suggested that the unhatched embryos stopped develop in the early embryonic development stages. Therefore, Wolbachia infection maybe inhibit the transcription of Mst84Db in the spermatogenesis process, then makes sperm normal structure and function abnormal. At last, it results in embryonic death when mated with Wolbachia-uninfected females. For this reason, we speculate the down-regulation of Mst84Db expression that resulted by Wolbachia infection may be one of the molecular mechanisms of CI.The study not only provides a new idea for the molecular mechanisms of Wolbachia-induced CI, but also lays a foundation for the study of the regulatory mechanism of the animal germ cells and the interaction between symbiotic bacteria and their hosts. In addition, the study also has an important application value of basic researches in gender control and the biological control of agricultural pests and insect vector.