Core Bacterial Community Associated With The Intestinal Tract Of Chinese Mitten Crab(Eriocheir Sinensis)

Chinese mitten crab(CMC, Eriocheir sinensis) is an economically valuable species in South-East Asia that has been widely farmed in China. Characterization of the intestinal bacterial diversity of CMC will provide insights into the aquaculturing of CMCs.V3 region pair of primers(341F/518R) is one of the most widely used primer. Sterile DNA extracted from the muscle of Chinese mitten crab,Penaeus vannamei,Portunus trituberculatus, Piaractus mesopotamicus and prostate cancer cell of Homo sapiens. These DNA was amplyfied by V3 region primer and PCR product was visualized by agar gel. Strong bands were detected and sequencing results show that these bands have high identity to animal 18 S r RNA gene. Geneious alignment shows that 518 R match to animal 18 S r RNA gene and also 341 F except 2-3 base pair mismatch at the near 3′end. By searching against the Genbank, lots of sequencing which belong to uncultured bacterium have high identity to the hosts’ 18 S r RNA gene. 6 pairs of primers were tested by Text Primer progress on Silva, results show that 27F/338 R and 27F/518 R have high coverage and 100% specificity to bacteria.Based on the analysis of cloned 16 S r RNA genes from culture-independent methods DGGE and Metagenomic sequencing most of the sequences have high nucleotide similarity to the species belonging to the four phyla of Tenericutes, Bacteroidetes, Firmicutes and Proteobacteria. Both 16 S r RNA gene denaturing gel gradient electrophoresis and metagenome library indicate that the CMC Mollicutes group 2 appears to be present in both the midgut and hindgut with no significant difference in abundance. The CMC Mollicutes group 1, however, was found mostly in the midgut of CMCs. The CMC gut Mollicutes phylotypes appear to be most closely related to Mollicutes symbionts detected in the gut of isopods(Crustacea: Isopoda).Quantity PCR was used to check the universality and source of core bacteria Bacteroides, Marinifilum, Sunxiuqinia, Mollicutes group 1 and Mollicutes group 2 from more than 30 crabs from different ponds in China. Results show that Bacteroides, Sunxiuqinia, Mollicutes group 1 and Mollicutes group 2 are core bacteria that can be found in most of the samples. Marinifilum may be not a resident bacteria in crab gut but come from the water or food, while Sunxiuqinia which comes from the water must be a resident bacteria who can adapt the environment in crab gutOverall, the results suggest that CMCs harbor diverse, novel and specific gut bacteria, which are likely to live in close relationships with the CMC host.