Molecular Detection Of Endosymbionts Wolbachia And Its Effection On Ovarian Differentiation In Silkworm

Wolbachia are maternally inherited intracellular bacterial symbionts that often induce various reproductive alterations in the host, such as cytoplasmic incompatibility, feminization, parthenogenesis and male killing. In the past few years, there has been lots of research on the regulation mechanism of Wolbachia bacteria, Population biology and evolution, but mainly focus on fruit flies, mosquitoes and parasitic wasps. In the Lepidoptera, the research on Wolbachia has been reported and mostly concentrated in the butterfly and moth. The reproductive regulations in Lepidoptera are various. Previous studies reported that there were no symbiotic Wolbachia bacteria in silkworm. In this study, we study on the molecular evidence of Wolbachia in silkworm though cloning and southern blotting based on silkworm genome database and Wolbachia genome sequence. Additionally, we explore the functions of Wolbachia on silkworm reproduction by antibiotics treatment. The results are as follows:1. The bioinformatic analysis and cloning of Wolbachia-specific gene in domesticated and wild silkwormWolbachia specific genes---wsp and ftsz gene were cloned from wild silkworms from different districts and several species of domesticated silkworms. wsp gene fragment are 593bp in length and encode 197 amino acids. The deduced amino acid sequence indicated that it is identified significant domain containing Opacity (Pfam), which may mediate various pathogen-host cell interactions from several pathogenic proteobacteria. The other gene ftsz are with the length of 571bp, encode 189 amino acids with two domain—tubulin and tubulin C, which are related with the division of bacteria. Besides, we find that Wolbachia are almost equably distributed in reproductive and somatic tissues. In addition, wsp and ftsz gene were not detected at mRNA level.2. Phylogenetic analysis and horizontal transfer of Wolbachia Wolbachia can transfer among different hosts, and because of this, Wolbachia Wolbachia are widely distributed in nature. On the ftsz and wsp gene-based phylogenetic tree, Wolbachia in wild silkworm has closer relationship with the Wolbachia affecting parasitic wasp and other Lepidoptera species. Horizontal transmission mechanism of Wolbachia reminds us that Wolbachia in wild silkworm may come from Hymenoptera such as T. confusum, T. bedeguaris, and so on, and Wolbachia in wild silkworm may be passed on to Orius nagaii through predation. We think that the origin of Wolbachia in domesticated silkworm may have two pathways:one is the residual Wolbachia on mulberry leaves; the other is from wild silkworm. Taking into account the domestication process of silkworm and Wolbachia maternal transmission, we tend to support the second one.3. Analysis of horizontal transfer of Wolbachia geneIn order to study whethere Wolbachia genes integrated into the silkworm or the wild silkworm genome because of gene horizontal transfer. Firstly, Wolbachia genes were searched in Silkworm Genome Database and there are not any homologous sequences, so we consider that the gene horizontal transfer do not occur between silkworm Dazao and Wolbachia. Furthermore, using southern blot of wsp gene, we did not find any positive belts about Dazao genome, which showed that wsp gene did not integrate into Dazao genome and Wolbachia density in Dazao is lower. We speculated wsp gene integrated into Wild silkworm from Yunnan, Xichang and Huzhou because different hybridization bands were showed.4. The effection of Wolbachia on development of silkworm eggsBy feeding and injection of different concentrations of different antibiotics for different periods of the silkworm, tetracycline and ciprofloxacin can eliminate a large of Wolbachia in Dazao.and cause abnormal development of silkworm eggs:shorter tubal, significantly less number of eggs, messy arranged of eggs in oviduct and it is difficult to separate egg tubes. All these abnormal individuals do not become eclosion. Some parental generation with Wolbachia partly eliminated, they may have offer springs. Among Fl, it also appears that abnormal development of silkworm eggs are like their treated female parents. As to treated males, we did not find any abnormal phenomena, and the F1 generation(treated males mates with untreated females) are normal. So we consider that Wolbachia elimination similarly did not affect the males. To further study the interaction between Wolbachia and silkworm, we examined some genes related with testes and ovary development. The selected candidate genes respectively are BmVg, BmVgR, Bmdsx, Bmtub, Bm44, BmTST and BmAIB. The results showed that there are no differences about all the genes after Wolbachia elimination.