Diversity Of The Intestinal Bacterial Community Of Diseased Apostichopus Japonicus As Well As Selection And Application Of The Probiotics

The culture of Apostichopus japonicus has become a new hot spot of the nationaleconomic industry with the highest production value and20%production growth rateeach year. However, the expansion of the Apostichopus japonicus aquaculture led tothe deteirorative environment and frequent outburst of disease, which have been be aseirous threat for the sustainable development of sea cucumber (Apostichopusjaponicus) culture in China. Traditional use of antibiotics and disinfectants haveachieved some successes, but excessive and frequent use of them lead to a lot ofproblems, such as the emergence of drug resistance strains, environment pollution andfood safety. Probiotics has gradually been accepted and widely used in aquaculturewith its advantages of the antagonism to pathogenic bacteria, the intestinal bacterialbalance of animals, the immune enhancement of farmed animals, and so on. In thisstudy, Roche GS FLX System was used to study the diversity of the intestinalbacteiral community of diseased Apostichopus japonicus and sea mud in the culturalarea. The potential probiotics which have significant antagonism to Vibrio splendiduswere isolated from the sea mud. At the same time, a probiotics DLOl isolated from thecommercialized EM was optimized to the fermentation and applied in the cultivationof Apostichopus japonicus in large scale. The results provide theoretical basis for thedevelopment and application of probiotics in the cultivation of Apostichopusjaponicus. The results obtained are as follows:1. Using FLX16S rDNA sequence analysis, diversity of the intestinal bacteiralcommunity of diseased Apostichopus japonicus are mainly Proteobacteira whichaccount for74.20%. There are also other seven phylums, such as Fusobacteria,Bacteroidetes, Firmicutes, Actinobacteira, Cyanobacteira, Planctomycetes. Only onestrain is detected from Cyanobacteria and Planctomycetes. Analysis of thecomposition of Proteobacteria bacteria shows y-Proteobacteria and s-Proteobacteira account for71.83%and26.98%respectively, y-proteobacteira include22genus, suchas Vibrios，Leucothrix, Pseudomonas, Shewanella, Colwellia, Moritella, etc. Vibriosaccount for64%which have the highest proportion among y-Proteobacteria. AndVibrio tapetis account for56.09%among Vibrios. Among s-Proteobacteira only onestrain belongs to Sulfuro vum, the others all belong to Arcobacter. Firmicutes whichinclude Bacillus spp. only account for0.57%. And only one strain of Bacillus spp. isfound in the intestine of diseased Apostichopus japonicus.2. Using FLX16S rDNA sequence analysis, eighteen kinds of phylums are detectedin sea mud of the cultural area of Apostichopus japonicus. Proteobacteria accounts for44.49%which have the highest proportion, among them a-Proteobacteria,P-Proteobacteria, y-Proteobacteria,5-Proteobacteira, s-Proteobacteira account for11.78%,0.62%,25.33%,44.64%,16.11%respectively. y-Proteobacteria have thehighest proportion which contains34genus of bacteira, such as Desulfobacter,Desulfobulbus, Desulfosarcina, Desulfobacca, etc. The quantity of Vibrios andBacillus is less, only3strains of Vibrios and1strain of Bacillus were found.3. Twenty-seven culturable predominant bacterial strains were isolated with differentcultural medium and heating treatment from the sea cucumber {Apostichopusjaponicus) culture area in Hongdao. The16S rDNA sequencing showed that y-Proteobacteria was the predominant bacterial group (71.4%of the total bacterialisolates), including Pseudoalteromonas, Vibrio，Shewanella, etc. Bacillus strainsdominated the isolates with heating treatment (80°C,20min). V. splendidus LJ08isolated from the sea cucumber area was virulent to sea cucumber by immersionchallenge. Three strains (LJ03, LJ04and LJ06), which had the antagonistic effects onV. splendidus LJ08in vitro, were investigated for the protection eiffciency to infectedsea cucumber larvae. LJ03and LJ06displayed significant protective effects againstV. splendidus LJ08(p <0.05). Especially, LJ06(belongs to Agarivorans) showed ahigher level of protection rate (94.74%). As a probiotical candidate against V.splendidus, LJ06has great potential in the culture of Apostichopus japonicus. 4. Probiotics DLOl isolated from the commercialized EM was identified as Bacillussubstilis through16S rDNA sequence. Its fermentation conditi°on was:37C,170rpm,17-20h,1%o defoamer and pH at7.0-7.2. DLOl could degrade ammonia-nitrogenand inhibit Vibrio sp. significantly both in the small-scale water treatment expeirmentsand the hatchery tank of Apostichopus japonicas (p <0.01).