Effects Of Wolbachia Infection On Hira Expression In Drosophila

Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes, they are gram-negative bacteria and transmitted through the cytoplasm of eggs. Wolbachia can affect the reproductive modes in their insect hosts including cytoplasmic incompatibility (CI), feminization and male-killing. CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females or females infected with different strains of Wolbachia resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown.Hir/Hira (histone regulation) gene is initially identified as the negative regulator of histone gene expression in yeast. A mutation of the Hira gene in Drosophilia results in sterility in females due to abnormal decondensation of sperm nucleus after fertilization, the formation of male pronuclei is arrested in the late chromatin decondensing stage. Therefore the paternal chromatin can not participate in the embryonic development.Considering that Hira is involved in male pronuclei formation and that the phenotype of CI induced by Wolbachia infection also exhibits the abnormal development of male pronuclei, we would like to investigate whether the CI phenotype is correlated with Hira gene expression in the hosts. We initially tested the CI strength in 1-day-old D. simulans males. The results of crossing experiments show that CI is only induced in matings between 1-day-old Dsim wRi males and uninfected Dmel T females (hatch rate of 8.78±1.03). In contrast, no CI is induced by Dsim wAu 1-day-old males (hatch rate of 95.55±1.26). In strong CI level strain (wRi)-infected male flies, the transcriptional level of Hira is lower than that in uninfected males (Dsim wRi/Dsim T:0.42±0.11) (P<0.05), the non CI inducing wAu strain did not result in dramatically decreased Hira expression in males (Dsim wAu/Dsim T:0.86±0.14).It was reported that CI levels declined rapidly with increasing of male age in Drosophila strains infected by Wolbachia. In order to further investigate the correlation of CI intensity with Hira expression in Wolbachia-infected male flies, we compared Hira expression levels between 1-day-old and 5-day-old males of Dsim wRi. We found that 1-day-old Dsim wRi males result in significantly lower hatch rates (7.67±2.18) in comparison to that of the 5-day-old Dsim wRi males (50.89±5.56%) (P<0.01). The level of Hira gene expression increases with male age. Hira expression in 1-day-old males was significantly lower than that in 5-day-old males (P<0.01).To further examine the involvement of Hira expression in the CI phenotype, the crossing experiments with two Hira-mutated D. melanogaster lines (Hirassm and HiraHR1) were assessed similar to that in CI assays. We found that both Hira-mutated males mated to Wolbachia-uninfected Dmel T females resulted in significantly lower egg hatch rates (72.98±5.10%,74.34±4.03%, respectively) relative to Dmel T males (92.44±0.77%) (P<0.05). In the early embryos derived from the crosses of Hira-mutant males with Dmel T females, the asynchronous cleavage and chromatin bridges were observed which is similar to the CI phenotype in D. melanogaster. This suggests that the mutation of Hira in males may mimic the CI phenotype induced by Wolbachia. Since Hira is on theⅩchromosome, we also examined the offspring sex ratio to see whether Hira mutation impacts sperm quantity. We found that mating with both Hira-mutant males resulted in significantly less female progeny in the next generation. In the progeny, the ratio of females to males derived from the crosses with Hirassm and HiraHR1 were 0.68 (±0.08) and 0.60 (±0.11), respectively. These results suggest that many female progeny mortality was associated with Hira mutations on oneⅩchromosome. However, the crosses between Hira-mutated males and wMel-infected females resulted in no significant differences of hatch rate comparing with uninfected crosses. The presence of the wMel strain of Wolbachia in females restored the sex ratio as shown by a sex ratio of～1 when Hira-mutated males were crossed with Dmel wMel females.Taken together, these results indicate that Wolbachia infection may cause Hira expression dramatically decreased in males, thus affect the quality of sperm and result in abnormal embryonic development of offspring. Since HIRA is one of the histone chaperones, it is possible that the low level of Hira in Wolbachia-infected males results in a structural malformation of sperm nucleus, which causes the defects in the early embryonic development. These results may help determine the underlying mechanism of CI and provide further insight in to the important role HIRA plays in the interaction of Wolbachia and its insect host.