Effects On The Intestinal Microbial Communities With Virus Infection And High Temperature Treatment In Silkworm,Bombyx Mori

The silkworm Bombyx mori is an ideal model organism for Lepidoptera. Mechanisms researches have made great progress in silkworm functional genomics, development and abnormal condition, gender determination, efficient synthesis of silk fibroin and native immunity since completed silkworm genome sequence. Large numbers of microorganism inhabit in anminal gut and they are important to digest and absorpt nutrients. The traditional culture method has used to investigate the microbiota in the silkworm gut. With the development of molecular biological technique some researchres studied silkworm intestinal microbiota by DGGE method according to 16 S rRNA gene sequence. Due to the vast majority of bacteria were difficult to grow through artificial culture and DGGE method also has defects for they cannot detect bacteria comprehensive. Nowdays, high-throughout sequencing technology has been key techniques to research intestinal bacteria, so this study was to investigate the abundance and diversity of silkworm gut bacteria and provide new datas to understand correlation of silkworm and their gut bacteria. The gut microbiota was affected by gender, development, food and environment and so on. Imblance of them can lead to intestinal bacterial diseases and induce to immune responses. Associated genes of immune pathways and peptide genes also changes, and affected gut bacterial community conversely. To discuss variation responses of silkworm intestinal microbiota to virus and high temperature and then analyse interaction of intestinal microbiota, virus and immune responses, this study has been investigated silkworm intestinal microbiota diversity after stimulate Bm CPV, BmBDV and high temperature. Studied interaction of dynamics changes of silkworm intestinal bacterial community with virus infected, high temperature stimulated and genes express primarily. Meanwhile, we had researched the difference of Bombyx mandarina Leech and Bombyx mori. Correlation researches results provided new datas for fully understanding of gut microbiota.1. Changes of silkworm Bombyx mori gut microbiota after infected by BmCPVThe objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus(BmCPV). Intestinal contents of the BmCPV-infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16 S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female(male) larvae and BmCPV-infected female(male) larvae, respectively. In general, bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera.2. Changes of silkworm Bombyx mori gut microbiota after infection by BmBDVThe objective of our work was to discuss interactive mechanism of silkworm Bombyx mori gut microbiota after the infection of B. mori bidensovirus(BmBDV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 48, 96 and 144 h post infection with BmBDV. The intestinal microbia were analyzed by Illumina miseq sequencing of the 16 S rRNA gene. Result showing visible difference intestinal microbial communities had between healthy and infected B. mori. Microbial diversities were similar after infected by BmBDV, but lower than control group obviously. In Bm BDV-infected samples the relative abundance of Enterococcus was increased gradually, abundance of Incertae_Sedis was most in BDV-96, Lactococcus was decrased. Relevance analysis of plenty of the predominant genera showed the relative abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. The increased of Anderseniella, Incertae_Sedis, Simplicispira and Enterococcus were likely related to BmBDV infection. Real-time quantitative PCR analysis showed that expression level of Toll, IMD and JAK/STAT immune pathways associated genes were increased at late stage. Meanwhile, intestinal antimicrobial peptides(attacins, defensin and cecropins) expression levels were induced. Abundance of Enterococcus was positive coorelation with Bmspz, BmPGRP-LE and BmPGRP-LB expression levels and speculated that BmBDV-infected lead to Enterococcus increased and then stimulate Toll and IMD pathways.3. Changes of silkworm Bombyx mori gut microbiota after high temperature treatmentThere are few researches of influence of high temperature to Bombyx mori gut microbiota and gene expression. Our research was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after high temperature treatment(37?C, 8h). The Intestinal contents of the high temperature treated and healthy larvae of B. mori of fifth instar were collected at 48, 96 and 144 h after high temperature treatment. The intestinal microbia were analyzed by Illumina miseq sequencing of the 16 S rRNA gene. Meanwile, expression level of immune system genes were analysed by real-time quantitative PCR. Results showing intestinal microbial diversity increased after high temperature treatment, and abundance of Enterococcus, Pantoea, Incertae_Sedis, Escherichia-Shigella, Anderseniella and Acinetobacter changed obviously indicating they were sensitive to high temperature. Relevance analysis of plenty of the predominant genera showed the relative abundance of Enterococcus and staphylococcus were in negative correlation with the abundance of the most predominant genera. Canono analysis showed in healthy silkworm Bmspz expression level was in negative correlation with abundance of Enterococcus and staphylococcus, and in positive correlation with other dominant genera(except Variovorax). Bmdrc-2 was in positive correlation with abundance of Enterococcus and staphylococcus. After high temperature treatment abundance of Variovorax was in positive coorelation with Bmspz expression level. Abundance of Enterococcus, Escherichia-Shigella, Incertae_Sedis, Anderseniella and Acinetobacter were in negative with Bmspz expression level and in positive with Bmdrc-2, BmPGRP-LE and BmPGRP-LB expression level. These results provided insight into the relationship of high temperature-microbia-gene expression level.4. Intestinal microbia of Bombyx mandarina LeechBombyx mandarina Leech was Bombyx mori ancestor. To analyse the intestinal microbia changes after domesticated, this research explored intestinal microbia in late stage of fifth instar of Bombyx mandarina Leech and Bombyx mori. In the result, the relative abundance of Enterococcus in Bombyx mandarina Leech(69.73%) was higher than in Bombyx mori(48.99%), the percentage of Advenella in Bombyx mandarina Leech was 11.54%, but without being detected in Bombyx mori. Relative abundances of Lactococcus, Bacillus and Pseudomonas in Bombyx mori were higher than in Bombyx mandarina Leech showed that significant difference of intestinal microbial community existed between Bombyx mandarina Leech and Bombyx mori. Domesticated also can influence gut bacterial community, and these differences maybe related with adaptability to the environment and feeding ability.