| Poly-β-hydroxybutyrate(PHB)is a carbon and energy storage in prokaryotic cells.It can be partially or completely degraded into short chain fatty acids(SCFAs)in the intestine of aquatic animals.Studies have shown that amorphous bacterial PHB is easier to be degraded and utilized by animals than crystalline particle PHB.In this study,Halomonas with and without PHB were obtained by fermentation and the content of PHB was 46.5%cell dry weight.The concentrated Halomonas and Halomonas-PHB were added into pellet feed.The effect of Halomonas-PHB on growth performance,muscle nutritional composition,intestinal microbiota composition and immune-related gene expression of grouper(E.fuscoguttatus × E.Lanceolatus)were investigated.The groupers were cultured in the circulating aquaculture system(RAS).Two experiments were conducted in this thesis,in which the 1.4%and 3%PHB were supplemented in the feed in experiment Ⅰ and experiment Ⅱ,respectively.In experiment Ⅰ,the groupers were fed commercial feed(control),and feeds with dietary supplement of 3%Halomonas containing no PHB(3%HM)and 3%Halomonas containing PHB(3%HM-PHB)The stocking density were 30 fishes per 75 L in each tank connected with recirculating aquaculture system(RAS).The groupers were reared at temperature 28±1℃ with satiation feeding for 7 weeks.In Experiment II,the content of PHB was improved and the groupers were fed with commercial feed(control),and feeds with dietary supplement of 6.5%Halomonas containing no PHB(6.5%HM)and 6.5%Halomonas containing PHB(6.5%HM-PHB).Feeding was conducted at 3%～10%of body weight daily.The rearing condition were the same with experiment Ⅰ.After 7 weeks,36 fishes were randomly selected from each treatment and were challenged with Vibrio anguillarum,and survival rates were measured every 2 h within 48 h.Immune-related gene analysis was performed on the intestinal tract,liver,spleen,head-kidney and blood of groupers after being challenged for 48h,including antioxidant genes CAT and SOD,innate immunity genes TLR3,TLR2,IL-1,IL-10 and adaptive immunity genes MHCI and MHCII.And intestinal bacteria analysis was conducted on healthy grouper and challenged for 24h and 48h through high-throughput sequencing.The results of experiment I show that the survival rate was over 93%.The final weight of grouper in 3%HM-PHB,3%HM and control were 35.23±1.96 g,37.80±2.4 g and 32.71±1.86 g,respectively.The weight of 3%HM group is significantly higher than the control group(p<0.05),3%HM-PHB and the control group had no significant difference(p>0.05).Crude fat content of 3%HM-PHB,3%HM and control was 1.96±0.40%dw,2.04±0.40%dw and 1.63±0.28%dw,respectively and crude protein content of 3%HM-PHB,3%HM and control was 21.17±1.14%,21.17±0.39%and 22.33±0.78%,respectively.The content of seven fatty acids was significantly increased(C14:0、C16:0、C16:1、C17:0、C18:0、C18:1 and C20:1)in 3%HM-PHB than control(p<0.05).ACP and AKP enzyme activity and T-AOC in liver,head-kidney,spleen and serum of grouper were tissue specificity.Among them,the AKP activity of grouper head-kidney in 3%HM-PHB was significantly higher than that in the control(p<0.05),and 3%HM-PHB significantly increased the T-AOC of grouper serum(p<0.05).Halomonas-PHB has no significant promoting effect on the growth of grouper,but it can improve the content of fatty acids by affecting the lipid metabolism of grouper,and improve the activity of immune enzyme and the total antioxidant capacity.The results of experiment Ⅱ showed that the overall fish survival rate was more than 93%.After challenged with V.anguillarum,the survival rate of control,6.5%HM and 6.5%HM-PHB group was 9.1%,16.7%and 25.7%,respectively.The increasing of survival rate after V.anguillarum challenge in 6.5%HM-PHB indicated that Halomonas-PHB improved the resistance of grouper to Vibrio.The qRT-PCR analysis of immune-related genes in different tissues of grouper showed that 6.5%HM-PHB supplementation increased the antioxidant gene expression of CAT and SOD in the blood when challenged for 48 h(p<0.05).And 6.5%HM-PHB supplementation increased the gene expression of IL-1 in the intestines,liver,spleen,head kidney and blood(p<0.05).The expression of IL-10 was similar with IL-1.In addition,6.5%HM-PHB supplementation could improve the expression of MHCI in the blood and MHCII in the head-kidney(p<0.05).Therefore Halomonas-PHB could improve the immune ability of grouper and hence improved resistance to Vibrio.The high-throughput analysis on intestinal microbiota showed that Proteobacteria was the dominate phylum in healthy and challenged groupers(82.6%%～98.7%).Photobacterium was the dominate genus in the healthy grouper gut(41.8%～52.0%)and Vibrio was second dominate bacteria(9.7%～24.7%).Vibrio was the dominate genus in V.anguillarum challenged fish gut(32.8%～80.7%)and the abundance of V.anguillarum ranged 29.0%～79.1%.The abundance of Thauera(6.9%)increased,while those of Nitrosomonas and Arenimonas(0.7582%and 0.0039%,respectively)reduced in 6.5%HM-PHB group.Halomonas-PHB could affect the microbial composition of grouper gut,but it had no effect on the intestinal microbial diversity.In summary,although the application of Halomonas-PHB had no significantly effect on survival,growth,feed conversion rate and muscle nutrition composition of grouper juveniles,it could significantly improve the immunity of grouper and the expression of the immune related genes.Hence Halomonas-PHB can be considered as a potential biological control agent.This study provides data reference for the application of Halomonas-PHB in grouper.The mechanism on how PHB improves the immunity of aquatic animals will be studied in the future. |
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