Wolbachia-Induced Paternal Defect In Drosophila Is Likely By Interaction With The Juvenile Hormone Pathway

Wolbachia are endosymbionts that infect many insect species. They can manipulate the host’s reproduction such as cytoplasmic incompatibility (CI), parthenogenesis, feminization, male-killing and feminization, to increase their own populations. CI is the most common reproductive modification induced by Wolbachia, which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females or females carrying a different Wolbachia strain. However, mating from both males and females carrying the same Wolbachia strain results in viable progeny with Wolbachia. Despite the molecular mechanisms of CI are currently not clear, several evidences suggest that the modification of sperm by Wolbachia inhibits the paternal chromosomes into anaphase, therefore arrested the development of embryos. Previous microarray analysis in Drosophila melanogaster have identified296genes which have more than1.5fold changes (P-value<5%) in the third instar larval testes when infected by Wolbachia, include167up-regulated genes and129down-regulated genes.In this study, four genes significantly differentially expressed including kenny, Adh, CG12262-RA and CG4988-RA were selected, and verified the difference of expression level between Wolbachia infected and uninfected3rd instar larvae testes in Drosophila by qRT-PCR. Furthermore, we have verified the expression differences of kenny-RA and CG4988-RA transcripts in the larval testes by in situ hybridization, and found that the hybridization signal of kenny probes were significantly enhanced, concentrated in some certain areas of the testes. However, CG4988-RA signal presented weaker and dispersed in the Wolbachia infected testes.To further explore whether these differentially expressed genes have involved in CI, kenny, CG12262-RA, JhI-26and Dbi were selected to research their function in development and reproduction of Drosophila, which have been confirmed up-regulation in infected larvae testes. We first analyzed the spatiotemporal expression patterns of these genes in D. melanogaster by qRT-PCR. These genes showed higher level of expression in male gonads than female gonads, that suggesting their roles on the reproductive of male flies.Furthermore, we have constructed UAS-kenny-RA, UAS-CG12262-RA, UAS-Dbi-RA, UAS-JhI-26-RA and UAS-CG10433-RA transgenic Drosophila strains performed by microinjection, and localised these inserted genes on chromosome through the hybridization method. Then these genes were overexpressed in testes or in whole body by using UAS-GAL4system. We have selected UAS-kenny-RA, UAS-CG12262-RA, UAS-Dbi-RA, UAS-JhI-26-RA and UAS-CG10433-RA overexpression males to cross with Wolbachia uninfected females, and find that1-day-old overexpression male flies can induce different proportions of unhatched embryos among offspring. Therefore, we suggest that the up-regulated expression of all these five genes in males may be related to the mechanism of CI.JhI-26(Juvenile hormone-inducible protein26) was considered a juvenile hormone-induced protein. JhI-26gene can be rapidly and specifically induced by Juvenile hormone (JH). Recently, JhI-26has been identified as a sperm protein in Drosophila. Microarray and in situ hybridization results previously show that JhI-26exhibited significantly up-regulation in the3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia, and qRT-PCR results also showed the expression of JhI-26-RA raised more than12times. These implied that Wolbachia may effect the juvenile hormone (JH) signaling pathway in Drosophila.Considering that Jhamt gene encodes JH acid methyltransferase, a key regulatory enzyme of JH biosynthesis, and that methoprene-tolerant (Met) has been regarded as the best JH receptor candidate, we compared the expression of Jhamt and Met between Wolbachia-’mfected and uninfected fly testes, and found that the expressions of both Jhamt and Met in D. melanogaster were significantly increased in the presence of Wolbachia, suggesting an interaction of Wolbachia with the JH signaling pathway. Then we examined the fertility of JhI-26-RA overexpression males, embryo hatching results show that the hatch rate of the embryos from1-day-old JhI-26overexpression male flies were only32.29■1.25%(nanos-GAL4/UAS-JhI-26) and46.97■3.30%(act-GAL4/UAS-JhI-26) respectively, significantly lower than in control.However, Wolbachia-’mfected females could rescue the egg hatch, which was similar to the phenotype in CI crosses. These results indicated that Wolbachia may effect juvenile hormone pathway in Drosophila host through the induction of JhI-26gene expression, and further affect male fertility in Drosophila.Studies have shown that JhI-26-RA abundantly expressed in adult male accessory glands, implied the interaction between JhI-26and male accessory gland proteins (Acps). In our previous microarry study, we have demonstrated that one Acp gene, CGI0433is significantly upregulated in larval testes induced by Wolbachia infection, and then we analyzed the relationship between JhI-26and CG10433. We found that overexpression of JhI-26caused up-regulation of CGI0433encoding the male accessory gland protein (Acp), but not vice versa. These results suggested that JhI-26might act at the upstream of CGI0433. To know the defects during embryogenesis, we examined early development in fertilized eggs from the crosses between JhI-26said CGI0433overexpressing1-day-old males and Dmel T females. Both JhI-26and CG10433overexpressing males resulted in nuclear division deficient phenotypes in the early embryos, including narrower metaphase spindles, centrosomes dissociated from mitotic spindles, and the spindles exhibited strange morphology with a regular couple of tubulin-containing particles in the middle of the divided chromosomes at telophase.Some studies have shown that Acps have been implicated in triggering postmating response in females, including decrease of propensity to remating. To examine whether JhI-26or CG10433overexpressing males can effect females’receptivity to subsequent mating, Wolbachia-infected males, JhI-26over overexpressing males and CG10433overexpressing males were mated with normal females, and kept females alone for3days. Then the females were individually assigned3naive males, and were counted the proportion of remate within8hours. Data show that80%females that previously mated with Dmel T males (control group) were receptive toward courting males within8hours. However, none of the females mated to Dmel wMel males showed receptive to male courtship. Surprisingly, the females that mated to JhI-26overexpressing males showed the same rejection behavior, only45%of the females previously mated with CG10433overexpressing males displayed sexual receptivity. These results indicated that Wolbachia-infection might affect postmating response in females through juvenile hormone pathway.In conclusion, we found a novel pathway about Wolbachid’s interaction with their Drosophila host. Wolbachia in D. melanogaster testes may enhance the expression of Jhamt, JHAMT converts JH acid into JH. By interacting with its candidate receptor Met, JH induces the expression of JhI-26rapidly, which increases the expression level of CG10433. Males with higher CG10433level thus produce modified sperm, which results in paternal reproductive defects. Taken together, our results provide a working hypothesis whereby Wolbachia induce paternal defects in Drosophila probably by interaction with the JH pathway via JH response genes JhI-26and CG10433, it’s helpful to further explore the mechanism of interaction between Wolbachia and host.