Pond-cultured Japanese Flounder, Paralichthys Olivaceus Intestinal Bacterial Communities And The Influence Of Probiotics

In order to explore the relationship between healthy growth of pond-cultured Japanese flounder, Paralichthys olivaceus and culture environment, the composition and diversity of intestinal bacterial community in pond-cultured Japanese flounder and its possible influencing factors was investigated from a point of microecology. Furthermore, the probiotics was added to the pond-culture environment to study its effects on water quality, composition and diversity of microbiota communication from environmental samples and Japanese flounder intestine. The results from our study would be benefit for manufacture of high-efficient probiotics and development of practical regulation technique for pond culture of Japanese flounder. The results are listed as follow:1. Composition and diversity of intestinal bacterial community in pond-and indoor tank-cultured Japanese flounderIntestine samples were collected from pond-and indoor tank-cultured Japanese flounder and the composition and diversity of bacterial community were investigated based on high throughput sequencing of 16S rRNA gene using MiSeq platform. In addition, six other samples including culture water, feeds and pond sediment were also collected and their microbiota composition were also investigated using sequencing of 16S rRNA gene, and the comparisons of the intestinal bacterial communities and those from culture-associated environmental factors were performed using bioinformatical analysis. The sequencing data showed that the dominant bacterial communities harboring in pond-culture Japanese flounder intestine were Firmicutes (30.49%), Proteobacteria (19.16%), Fusobacteria (11.11%), wherein the most abundant bacteria was Bacillus (27.66%) and the least abundant was Vibrio based on genus level; whereas for pond-culture Japanese flounder, its intestine holds a core microbiota composed of Proteobacteria (44.31%), Firmicutes (11.57%), Actinobacteria (4.79%) were dominant, and in which the most abundant bacteria was Acinetobacter and Vibrio based on genus level. Difference and phylogenetic analysis revealed that the composition, diversity and origin of Japanese flounder intestinal bacterial communities were closely related with feeds followed by culture water.2. Separation of two probiotic bacterial strains and assessment of its effects on water quality regualtionA kind of probiotics has been selected for its good ettects controlling water quality in outdoor pond. In order to explore the possible underlying mechanisms, two dominant bacterial strains has been isolated and purified, their physiological and biochemical characteristics, phylogenetical roles, suitable culture conditions have been determined. Moreover, the composition of bacterial community in probiotics has also been determined. The results showed that the probiotics consists of Enterobacter (31.99%), Bacillus Lactobacillus (31.81%), Lactobacillus (15.8%), Bacillus (1.44%) and unclassified bacteria (18.96%). We got two bacterial strains including Lactobacillus plantarum A and Bacillus B by using traditional culture medium selection, and the most suitable pH values for growth of Lactobacillus plantarum A, Bacillus B was 6.5 and 6.0 respectively under lab conditions, meanwhile the optimum growth temperatures were 30℃ and 36℃ for which respectively. The effects of the two bacterial strains on pond-culture water quality was investigated by adopting salicylate method, diazo into usage, PhodVer3 ascorbic acid method. The results showed that:the mixed Lactobacillus plantarum A and Bacillus B at maximum density with a 10:1 ratio could get the best effect on the degradation of water ammonia, nitrite and phosphate, the degradation rate was up to 73.1%,57.8% and 52.4%, respectively.3. Responses of pond-culture environment and pond-cultured Japanese flounder intestinal bacterial community to probiotics regulationTo assess the effects of a probiotics on pond-culture environment and intestinal bacterial community from pond-cultured Japanese flounder, a total of 22 samples including intestine, culture water, feed and pond sediment from four batches was sampled, the changes in composition and diversity of bacterial communities form which were analyzed based on high throughput sequencing of 16S rRNA gene using MiSeq platform. And the comparisons of intestinal bacterial communities and other samples from environment-associated factors were performed using bioinformatical analysis. During the experiment, the water quality including temperature and salinity maintained relatively stable levels. The water quality control effects was obvious, the degradation rate of pond water ammonia, nitrite and phosphate was up to 88.9%,67.9%,69%, respectively. On the other hand, the water chlorophyll content increased to 19.05%. For the bacterial communities, the pond water bacterial abundance of Bacteroidetes increased whereas the bacterial communities of Proteobacteria decreased in abundance. As in pond sediment, the bacillus abundance increased from 3.78% to 33.64%. The Enterobacter, Legionella and Acinetobacter abundance increased in intestine, whereas the Vibrio decreased from 0.16% to 0.05%. It is interesting that the lactobacillus plantarum was detected in intestine of pond-cultured Japanese flounder. Biodiversity and phylogenetic analysis showed that:artificially added probiotics could effectively improve the pond water environment quality, meanwhile, it also could influence the composition and diversity of bacterial community in culture water, pond sediment and Japanese flounder intestine. The dominant bacterial communities harboring in Japanese flounder intestine are identified to related with nutrition metabolism, the present study indicated that the growth health of pond-cultured Japanese flounder could be attained by using probiotics treatment.