| Along with rigorous regulations on the use of antibiotics in aquaculture worldwide, probiotics have been promoted as new alternative biotherapeutic agents to replace antibiotics. In recent years, the focus of probiotics has been changed from feeding experiments to the mechanisms underlying predominant strains. The main purpose of this study is to investigate the microbial community adherent to the gastrointestinal (GI) wall of grass carp (Ctenopharyngodon idella), bluntnose black bream (Megalobrama amblycephala) and gibel carp (Carassius gibelio) and the effects of environmental factors. Predominant Lactobacillus spp. adhered to the GI walls of zebrafish (Danio rerio) and tilapia were identified. The effects of the adherent capacity of lactobacilli, fructo-oligosaccharides and antibiotics on the lactobacilli and total bacteria colonized in tilapia intestine were assessed.16S/18S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial community adhered to the gills, skins and the intestines of grass carp, bluntnose black bream and gibel carp raised in a poly-culture pond in the lower reaches of the Yangtze river. These microbes, including bacteria, fungi, and actinomycetes, were compared with that detected in the rearing environment, such as water, feed, reed and pond sediment. Various phyla of microorganisms were identified, such as Actinobacteria, Cyanobacteria, Firmicutes, Proteobacteria, Ascomycota, Basidiomycota, Zygomycota, Haplosporidia, Apicomplexa, Choanozoa and some unclassified bacteria. The adherent microbial communities on the gills and skins were different among these three fish species, and the diversity of that on skin was higher than that on gill. The autochthonous and allochthonous gut microbiota varied, but were not so remarkable as those among species. This result indicated that gut microbiota was influenced by environmental factors, but the dominant bacteria depended on the intestinal microenvironment.Capacity to adhere to the GI wall is considered as one of the most important criteria for probiotic in aquaculture. In this study, 10 lactobacilli were selected to feed zebrafish. A primer set specific for single-copy rpoB gene was designed and combined with PCR-DGGE technique to identify and semi-quantify the lactobacilli colonized on the GI wall of zebrafish. Five lactobacilli including L. buchneri BU, L. brevis CGMCC 1.2028, L. plantarum subsp. plantarum JCM 1149, L. rhamnosus CICC 6141 and L. rhamnosus 20300 were found to be predominant among LAB group. The direct immersion bath test showed that L. plantarum subsp. plantarum JCM 1149T conferred more significant protection to fish against the challenge with Aeromonas hydrophila. The best survival rate was observed when 107 cells of L. plantarum JCM 1149T were supplemented to per gram of feed (mortality 40%).After two weeks feeding trial, 5 lactobacilli were found to adhere to the tilapia intestine. This result varied from that in zebrafish, indicating the host-specific capacity of lactobacilli. Fructo-oligosaccharide was selective to promote the proliferation of lactobacilli, and the efficiencies were L. brevis CGMCC 1.2028>L. rhamnosus CICC 6141>L. plantarum subsp. plantarum JCM 1149T.In summary, this study investigated the microbiota structure of three poly-cultured freshwater fish species and related environment factors. PCR-DGGE technique in combination with microbial quantification based on rpoB gene is proved to be efficient for identification of predominant Lactobacillus spp. adherent to the fish GI wall. Feeding L. plantarum subsp. plantarum JCM 1149T could significantly reduce the mortality caused by A. hydrophila. L. brevis CGMCC 1.2028 was promoted by fructo-oligosaccharide. Whether it is suitable for synbiotics merits further investigations.
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