The Study On The Molecular Mechanism Of Wolbachia-induced Cytoplasmic Incompatibility Of Insect

Wolbachia are a widespread negative bacterium of maternally inherited of arthropod, cytoplasmic transferring by host egg. Recent study have proved that Wolbachia are distributed in about 65% insect including Orthoptera, Hymenoptera, Heteroptera, Coleoptera, Diptera and Homoptera, except for ubiquitous in filar nematode, isopod, Araneae and Ticks. Though detecting Wolbachia infection of six distrincts of Drosophila melanogaster line in China, we found that populations from Wuhan in Hubei Province, Tianjin and Liuku in Yunnan Province are infected by Wolbachia, while there was no infected phenomenon in other three areas (Jianfeng mountain in Hainan Province, Jigong hill in Henan Province, Xiachayu in Tibet). The wsp gene sequences were obtained through cloning and submitted to GenBank, the accession numbers assigned are FJ403330, FJ4O3331, FJ403332. Though phylogenetic analyzing these sequences, the genetic relationship of Wolbachia strain infecting Drosophila in these three areas was very similar to each other, all belonging to Mel group in A supergroup of Wolbachia.The reason why Wolbachia has become one of investigative hotspot in domestic and abroad is that it can regulate host reproductive mode through multiple mechanism, such as feminization, parthenogenesis, male-killing and Cytoplasmic Incompatibility (CI) etc, by which way Wolbachia can be extensive disseminated. Among them, CI is the most frequent phenotype, occurs when Wolbachia infected males mate with uninfected females resulting in no or very low numbers of viable offspring. The molecular mechanisms underlying CI are currently unknown. Some study on Cytology showed that male pronucleus can not be formed normally in CI embryo due to constructing chromatin bridge in the karyokinesis process. One point mutation of Histone regulation (Hira) gene could also induce male pronucleus developmental disabilities in Drosophila. In order to study the relationship between CI and Hira gene expression level, firstly we use qRT-PCR to detect Hira gene expression level in Drosophila melanogaster infected Wolbachia with different CI strength. The results showed that in Drosophila males with strong CI strength (progeny had low egg hatch rate), the Hira expression level was significantly lower than that in males with low CI or the uninfected. Because CI strength fall-off by the increasing male age, we compared the Hira expression level between 1 day and 5 day old Drosophila males. The results demonstrated that as the increasing male age, the Hira expression level step-up gradually. In addition, it was proved that there was antagonism relationship between patrogenesis speed and CI strength, through detecting Hira expression level of infected males with different larval developmental time. The offspring embryonal hatch rate increase as the elongation of developmental time. That is, the elder brother with the most development speed had the strongest CI strength, but their Hira expression level was the lowest. All these results indicated that the down-regulation of Hira in Drosophila males caused by Wolbachia infection may be one important reason of inducing CI.In purpose of proving this phenomenon is not just happened in single species-Drosophila melanogaster, we also inspected the Hira gene expression in another host Aedes aegypti (major intermediary of Dengue Virus transmission), the analogical results were obtained. To further verify the conclusion, RNA interference technology was adopt to knock down the Hira gene in uninfected mosquito males, which mimic the infected males, then the mating between Hira-silenced males and uninfected females is performed to turn out a mimic of CI Phenotype. Besides, Wolbachia in females can'rescue'some of the damage resulting from silencing of the Hira gene in males. In vitro cytology study suggest that the expression of a micro RNA (aae-miR-12) which has obviously complementary sequences were difference between Wolbachia infected and uninfected Aedes aegypti cells. The aae-miR-12 signal was significantly stronger in Aag2.wMelPop-CLA cells compared to uninfected cells, which was opposite to the Hira expression. The result suggested that aae-miR-12 may be associated with Wolbachia infection and regulate the expression of the Hira gene. To further test this hypothesis, we adopt synthetic mimics and inhibitors of aae-miR-12 transfection experiment to validate that aae-miR-12 can repress Hira expression. Hira gene expression in Aag2 cells transfected with the aae-miR-12 mimic was significantly lower compared to mock-transfected cells or control-mimic transfected cells. Significantly higher Hira expression was found in Aag2.wMelPop-CLA cells when transfected with the aae-miR-12 inhibitor as compared to mock-transfected cells or control-inhibitor transfected cells. Additional experiments were performed in Aedes cell line using GFP as a reporter gene and constructing PIZ/GFP-Hira vector, proving that aae-miR-12 may suppress Hira expression through complementary pairing. Taken together these results suggest that Hira gene may be negatively regulated by one micro RNA aae-miR-12 in Aedes aegypti, in addition, Wolbachia infection is likely to inhibit Hira gene expression, through up-regulating aae-miR-12, finally induce CI Phenotype of host. These results will supply a firenew insight for the study on the relationship between Wolbachia and their host.Furthermore, in order to fully explore the molecular mechanism of CI caused by Wolbachia infection, we adopt Genechip to identify that a total of 296 genes expression had at least a 1.5 fold change in Wolbachia infected D. melanogaster larval testes, when comparing to the uninfected. Differential expression of genes related to metabolism, immunity, reproduction and other functions were observed. Among them,167 genes were up-regulated and 129 genes were down-regulated. Interestingly, most of the genes putatively involved in immunity were up-regulated in the presence of Wolbachia. In contrast, most of the genes putatively associated with reproduction (especially spermatogenesis) were down-regulated in the presence of Wolbachia. According to these results we infer that, Wolbachia infection may activate host immune path, however, take some adverse effect on spermatogenesis. Specific gene function is being studied.