Diversity And Function Analysis Of Symbiotic Microbes In Small Intestine Of Japanese Eel, Anguilla Japonica

Japanese eel with high economic value, is an important species of eel culture in china. The high density production in the process of aquaculture is easy to cause the malnutrition, deterioration of water quality and environmental pollution. The disadvantage of the aquaculture also cause many diseases such as enteritis wich lead to a significance decrease of the output of A. japonica. The disease will results in enormous economic loss. There are a lot of bacteria living in the intestinal of A. japonica which play an important function on the host's development and immunity. The community composition of A. japonica gut microbes is important for the study of the mechanism of the interaction between symbiotic bacteria and host. The main results were as follows:Based on the method of pure culture of microorganisms,33 strains from the intestinal of A. japonica were screened according to shapes, colors and sizes.16 different strains (GenBank Accession Number:KP716690, KP716697, KP716703, KP716701, KP716691, KP716698, KP716692, KP716699, KP716694, KP716702, KP716695, KP716700, KP716704, KP716689, KP716696, KP716693) were identified. The results suggested that the Proteobacteria was the most highest abundant bacteria in small intestine of A. japonica, which accounted for 75%, while the Firmicutes and the Bacteroidetes were 25% and 6% respectively. We obtained a probiotics strain and a fish pathogenic strain that named Lactococcus lactis M2-4 and Lactococcus garvieae L1-5, respectively.The V3 region from 16S rRNA of the intestinal bacteria of A. japonica was amplified. Based on the method of denatured gradient gel electrophoresis, we also obtained the DGGE fingerprint of small bowel bacterial of A. japonica and cloned 12 different sequences. The nine sequences included AJ-1, AJ-2, AJ-3, AJ-4, AJ-5, AJ-6, AJ-7, AJ-10 and AJ-12 were all belong to Uncultured bacterium. AJ-11, AJ-8 and AJ-9 were confirmed to Mycoplasma sp. of Tenericutes, Aeromonas sp. of Proteobacteria and Cetobacterium sp. of Fusobacteria respectively. The result showed that the the major ingredient was Uncultured bacterium. The reason was that the V3 fragment of 16S rRNA is only 200 bp, it is too short for homology search at NCBI website. What's more, based on the V3 region of 16S rRNA, the cultured and uncultured bacterium were amplified so that the abundance of the uncultured bacterium would increase.The V4 region from 16S rRNA of the intestinal bacteria of A. japonica was amplified. Based on the method of high-throughput sequencing, the diversity of the A. japonica small bowel microbial symbionts were examined. The result suggested that the major phylum were Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria, Bacteroidetes, Tenericutes, Verrucomicrobia, Fusobacteria, Chloroflexi, Gemmatimonadetes, Planctomycetes, Cyanobacteria and Nitrospirae. The classes of Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria dominated the phylum of Proteobacteria, while the class of Bacilli and Clostridia dominated the phylum of Firmicutes. The result of high-throughput sequencing were consistent with pure culture, and the major phylum also were Proteobacteria by fluorescent quantitative PCR. Besides that, the taxonomic classifications were similar in the AJ1 and AJ2 at the orders level, but the proportion and abundance were different. The major orders of the intestinal bacteria of A. japonica were Enterobacteriales, Fusobacteriales, Rhodocyclales, Actinomycetales and Lactobacillales.By observation of the physicochemical factors of strain L. lactis M2-4 and L. garvieae L1-5 were screened from the intestinal of A. japonica including temperature, pH and livability in the feed, it was clear that they could acid-resisting which play a role on standing up all kinds of bad environment in the digestive tract for arriving at intestinal. They had a high livability for just a short period of time in the fodders so that it could ensure effectiveness of the application, and their survival rate can reach more than 87% when the pH=2. The strain of L. garvieae LI-5 was sensitive to antibiotics including cefalexin and ciprofloxacin, so it was easy to treatment. The crude extracts from metabolism of L. lactis M2-4 strain has bacteriostasis which could secrete a variety of antimicrobial substances to pathogens. But the strain couldn't survive under high temperature so that the stability was poor for machine and transportation, we would explore production condition to fit it. The blood biochemical parameters of A. japonica in two groups fed respectively with M2-4 cell and L1-5 cell that were all mixed with basic food respectively, were analyzed. The results showed that the measure of the glucose in the pathogenic groups were reduced by 45% in comparison with the control group, it suggested that A. japonica were damaged in the kidney area under stress factor. The ability of resistance to diseases would declined when the concentration of glucose had dropped through behaviour of trembling. The measure of the acid phosphatase and alkaline phosphatase in the pathogenic groups were increased by 29.48% and 34.81% respectively. If their concentration is increased, the host should damage in the liver area. The activity of superoxide dismutase in the probiotic groups enhanced two times in comparison with the control group, it suggested that the ability of antioxidant and nonspecific immune of A. japonica was improved.Intestinal flora play an important influence on growth, nutrition, health and resistance to diseases. Based on the methods of 16S rRNA sequencing, PCR-DGGE and the method of pure culture of microorganisms, we examined the diversity of the A. japonica microbial symbionts and obtained the intestinal bacteria. Then we analyzed the blood biochemical parameters of A. japonica after feeding with the intestinal bacteria that were all mixed with basic food, and its immunity and resistance to diseases were clarified. Our results suggested that the role of the intestinal bactetia in aquaculture, and provided a theoretical basis and application prospects for the development of probioticsin in the high-yield cultivation of A. japonica.