Application And Preliminary Study Of The Mechanism Of Autochthonous Probiotic Bacillus In Grouper Epinephelus Coioides Nursery

We used two strains of probiotics which isolated from the gut of grouper Epinepheluscoioides，alone or in combination, to enrich rotifer or copepods, then fed to grouper larvae, toevaluate the effect of probiotics on growth，survival，non-specific immunity of grouper larvae,and the water quality and intestinal flora, providing the basis for the selection and application ofprobiotics in marine fish larvae. In this thesis, we divided the experiment into the following fourparts.1Effect of autochthonous probiotic Bacillus on the growth and non-specific immunefunction of grouper larvalNine day after feeding the probiotic-enriched rotifers, the survival rate of DE5（Bacillusclausii） group and mixed group were higher than the control group. The larvae body length in theprobiotic groups were longer than the control group. The lysozyme activity in the body of theSE5（B. pumilus） group larvae was significantly higher than the control group. The totalsuperoxide dismutase （T-SOD） activity in DE5group and the mixed group were significantlyhigher than the control group. The alkaline phosphatase （AKP） activity in larvae body of the SE5group was higher than the control group. The malondialdehyde（MDA） content in larvae bodyin the SE5and the DE5groups were significantly lower than the control group. The resultsabove suggested that Bacillus SE5and DE5can promote the growth of larval fish and improvethe survival rate and immune function.2Effect of autochthonous probiotic Bacillus on the gut microbiota of grouper larvalNine day after the application of the probiotics, the total number of bacteria and the numberof Vibrio in the probiotic groups of larvae were significantly lower than the control group. TheVibrio number in the rearing water of the probiotic groups were lower than the control group.The total number of Vibrio in the SE5group and the mixed group were lower than the controlgroup. After9d of application of probiotics, some common dominant bands were observed inlarvae gut of the probiotic groups and the control group, where some different bands wereexisted by PCR-DGGE analysis. At the same time, intestine flora of larvae had some similaritywith rearing water and live bait. Bands Z5and Z7, the predominant bands of the larval samplesin the SE5group, and their most similar bacteria were uncultured bacterium clone T1₃₄7016S ribosomal RNA gene and uncultured bacterium clone F1Q32TO03C9LWK16S ribosomal RNAgene, the similarity were93%and96%, respectively. Dominant band Z3was observed in thelarval gut of the DE5’s control group, the most similar bacteria was uncultured bacterium isolateDGGE gel band216S ribosomal RNA gene, with the similarity was98%. Band L14onlyexisted in the rotifers of the combination’s control group, which was also the dominant band,disappeared in larval gut and water, and showed100%similarity to uncultured bacterium isolateDGGE gel band19-716S ribosomal RNA gene.In summary, Bacillus SE5and DE5application alone or in combination can control thenumber of the potentially harmful bacteria in the gut of grouper larvae and modulate the balanceof the gut microbiota.3Effect of autochthonous probiotic Bacillus on the growth and non-specific immunefunction of grouper juvenilesAfter feeding Bacillus SE5and DE5enriched copepods, alone or in combination for14d,the body length and weight in probiotic groups were significantly higher than the control group.When feeding for28d, the body length and weight in the probiotic groups were higher than thecontrol group. At the same time, the survival rate in the SE5group and the DE5group werehigher than the control group. After14d and28d of the application of the Bacillus, comparedwith the control group, the body lysozyme activity in the DE5group and the mixed group wereincreased. After14d and28d of the application of the Bacillus, the body SOD activity in theprobiotic groups were higher than the control group. This suggested that the probiotic canimprove the immune function of juveniles, and this may be the important reason improving thesurvival rate of the juveniles.4Effect of autochthonous probiotic Bacillus on the gut microbiota of grouper juvenilesAt the end of the trial, increased total bacterial number in the rearing water was observed ineach treatment, while the Vibrio count of the SE5group and the DE5group were higher than thecontrol group. After28d of application of probiotics, some common dominant bands wereobserved in juveniles’ gut of the probiotic groups and the control group, where some differentbands were existed by PCR-DGGE analysis. At the same time, intestine flora of juveniles hadsome similarity with rearing water. Bands H5and H12were predominant bands of the juvenilesgut in the SE5group, and showed93%and92%similarity to Vibrio sp.5Bb1S1316S ribosomalRNA gene and uncultured bacterium clone F776O8Q01AUACS16S ribosomal RNA gene,respectively. Dominant band H20was only observed in the juveniles gut in the DE5group, andshowed86%similarity to uncultured bacterium clone ncd1505e06c116S ribosomal RNA gene.Bands H5, H23and H24were dominant bands in juveniles gut of the combination’s group. Bands H23and H24were most similar to Vibrio scophthalmi strain D72516S ribosomal RNAgene and Vibrio sp.5Bb1S1316S ribosomal RNA gene, respectively, with the similarity of98%and94%, respectively. In conclusion, SE5and DE5application alone or in combination are ableto modulate the gut microbiota and promote the balance of intestinal bacterial community. Andthere were some correlation between juveniles’ intestinal flora and rearing water’s microflora.In summer, the application of autochthonous probiotic Bacillus SE5and DE5to the grouperE. coioides larvae and juveniles, either alone or in combination, will benefit the growth andimprove the non-specific immunity function and survival of both larvae and juveniles E. coioides.Probiotic Bacillus SE5and DE5could control over the number of the deleterious bacteria in thelarvae gut and prompt the balance of the gut microbiota. In brief, the result of the experiment hasproved that the application of Bacillus had positive effect on marine fish larvae production.