Bacterial Community Associated With The Intestinal Tract Of Chinese Mitten Crab (Eriocheir Sinensis) Farmed In Lake Tai, China

Chinese mitten crab (Eriocheir sinensis) is an economically important species inSouth-East Asia, and presently has been widely farmed in China. Characterization ofthe intestinal bacterial diversity of Chinese mitten crab will give insight into itsimmunity, disease resistance, nutrition, and aquaculture. Thus far, however, little isknown about the intestinal bacterial community of the crab in the commercialaquaculture settings. To get close to the intestinal bacteria diversity and further find outthe potectial probiotics, microscopic observations, quantitative PCR,16S rDNA genecloning and sequencing (culture-independent and-dependent), and fluorescence in situhybridization (FISH) were performed.The number of intestinal bacteria collected from Chinese mitten crab is（1.8±0.8)×10~7/μl with quantity PCR and (2.5±0.9)×10~6/μl with fluorescencestaining enumeration. It is reasonable that different methods lead to different results.The little difference suggest that the abudance of intestinal bacteria is stable and notheavily affected by culture environment and culture season.Based on sequence analysis of culture-independent16S rDNA genes, thedominated intestinal bacteria are assigned to three phyla Proteobacteria（35.5%）,Bacteroidetes (32.6%) and Firmicutes (31.2%). Proteobacteria(49clones) are dividedinto four classes, Alphaproteobacteria (7clones), Betaproteobacteria (2clones),Gammaproteobacteria (14clones) and Epsilonproteobacteria (26clones). Obviously,the two latter classes are the predominated population. On genus level,138clones aredivided into14phylotypes, including five unknown genera and9known genera. The most dominated genera are affiliated with Arcobacter, uncultured（Sphingobacteria）,Marinifilum and uncultured（Firmicutes）. Besides, Seven clones shared91%sequencesimilarity to members of Firmicutes; one clone had91%similarity to a bacteriumclone belonging to the candidate phylum TM7, for which no cultured representativeshave been found. These two phylotypes appeared to be new bacterial species in thecrab gut.Compared with the published results about intestinal bacteria community ofChinese mitten crab, uncultured（Sphingobacteria）and Marinifilum are the indigenousbacteria with large numbers, which probably represents the potential probiotics.Meanwhile, in the phylogenic tree, Marinifilum is clustered with Osedax endosymbiont.The function of this genus for the host is difficult to predict, but the presence of thesymbionts might decrease the risk of infection of the crab intestine by more harmfulparasitic or pathogenic microorganisms. One uncultured bacterium is assigned toSphingobacteria and its function is unknown. Arcobacter is likely the potentialprobiotic as the most predominated genus.Based on sequence analysis of culture-dependent16S rDNA genes,61of62strains were affiliated with ten genera of the class Gammaproteobacteria. Out of them,12(19.4%) and11(17.7%) colonies are assigned to Vibrio and Shewanella,respectively. One colony is a close relative of Chryseobacterium (Bacteroidetes). Andarcobacter is not isolated, suggesting that it is probably not intestinal indigenousbacteria of Chinese mitten crab.Fluorescence in-situ hybridization results discover that Arcobacter indeed don`tstably exists and Bacteroidetes stably dominats in the intestinal bacteria of Chinesemitten crab.Electron microscopy obsereation revealed that spherical and filamentous bacteriawere closely associated with the microvillus brush boarder of the midgut epitheliumand filaments often inserted into the space between the microvilli independently andsphaerita using a stalk-like cell appendage. By contrast, the predominant rod shaped bacteria in the hindgut were tightly attached to the epithelium surface with an unusualpili-like structure. All these special structure might contribute to adherence to intestinalepithelial cells of crab hosts.All the results indicate that unclassified（Sphingobacteria）and Marinifilum arelikely the potential probiotics in intestinal bacteria isolated from Chinese mitten crab.However, numerous investigations are supposed to be developed before they can beused as probiotics.