Effects Of Bacillus Megateium H1 On Growth, Digestion, Immunity And Intestinal Bacterial Flora Of Regeneration Juvenile Sea Cucumber Apostichopus Japonicas

The changes of the intestinal bacterial flora during the regeneration of Apostichopus japonicas after treatment of Bacillus megaterium were studied via plates count method and HiSeq2000 16S rDNA sequence analysis. To analysis immunity response of regeneration intestine, the acidic phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) activities were measured, and the change of digestive enzymes such as protease, amylase and lipase were analysed too. The body weight and special growth rate were also determined. In order to reduce the evisceration rate of Apostichopus japonicas caused by pathogenic vibrios, Bacillus megateium H1 was used to against Vibrio splendidus LJ08. The significant reduction of evisceration rate was observation and the protection rate increased up to 92.31% with the improved growth rate 3.00 ± 0.34 times during the intestinal regeneration. The results provide scientific basis for the development and application of Bacillus megateium HI in the cultivation of Apostichopus japonicas. The majo results obtained are as follows:1. In this study, HiSeq2000 16S rDNA sequence analysis and hetrotrophic bacteria agar plate counting method were used to study the changes of intestinal bacterial flora during the intenstinal regeneration of Apostichopus japonicas. Sequence analysis revealed the diversity of the intestinal bacterial communities of the different stages of regeneration Apostichopus japonicas. The dominant group is Proteobacteria, which accounts more than 70% at all the stages. There are also other several phylums, such as Planctomycetes, Actinobacteria, Verruconicrobia, Chloroflexi, Acetobacter. During the intestinal regeneration of Apostichopus japonicas, the changes of bacterial flora of intestine are fluctuation, but predominate bacterial community is y-Proteobacteria at all the regenerated stages with the increasing proportion of numbers. The intestinal bacterial flora of regeneration group own lower bacterial diversity than that of controls.2. Proteobacteria and Bacteroidetes take major proportion of bacterial flora of regenerated intestine. In which γ-Proteobacteria take more than 70% of the number of bacteria at all regeneration stages. Adding B. megaterium H1 can divert the intestinal bacterial communities structure with the increased counting nmbers of bacillus compared to un-regenearted group and regenerated group without H1 (P<0.01). On the day 25 of post-re gene rated stage, compared to total hetertrophic bacterial plate counting, the precentages of countings of culturable vibrios are 79.15%,91.52% and 71.17% in the H1 treated regenerated group, untreated regenerated group and un-regeneated group repectively. The results indicate that B. megaterium H1 can colonize the rengerated intestine at 107CFU/g and inhibit the growth of vibiros.3. The changes of intestinal immunity responses and digestive enzymes activities during the regeneration of Apostichopus japonicas were studied. In the early stage of intenstinal regeneration, signicant lower ACP and AKP activities were found, and recovered with the intestinal regeneration. Treatment of H1 resulted a lower ACP and AKP activities in the early regeneration but improved ACP and AKP activities signiciantly at the post-regeneration (P< 0.05). The SOD activities of early regenerated intenstine were signiciantly higher that the control, and trended to decrease to normal level with the regeneration of intestine. And treatment of H1 inhited the SOD activities. Protease and amylase activities were significantly increased with the treatment of H1 (P<0.05) and lipase activities were improved signicantly only at the early intestinal regeneration (P< 0.05).4. The significant lower body weight was found to the intestinal regeneration juveniles. Feces were observated from day 10 after evisceration, and the gaining the body weight started from day 12 after evisceration. Fresh bacterial cells of H1 at dose of 105 CFU/ml increased significantly the body weight and specific growth rates of Apostichopus japonicas juvenlies (P<0.05). The result indicated the treatment of H1 can improve significantly the growth of juvenlies at the intestinal regeneration.5. In vivo antagonistic test showed that the protective effects of B. megaterium H1 (5 X 106CFU/ml) against V. splendidus LJ08 (107CFU/ml). The results showed a high level of protection rate of 92.31%. The evisceration rate of B. megaterium treated group was only (3.33±2.89)%, which is significant (P< 0.01) different from the result of positive control (43.33±5.77)%. Thus H1 has a great potential to apply in the culture of Apostichopus japonicas.