Mechanisms Of Bacillus Cereus On Regulating Immunity And Lipid Metabolism In Nile Tilapia

With the rapid development of aquaculture industry,high carbohydrate diets have been used in large scales in aquaculture because of their protein sparing effect and lower cost.High carbohydrate diet may cause negative impacts on the growth condition of fish due to their poor utilization ability of carbohydrate.How to reduce the adverse effects caused by a high-carbohydrate diet is important for the development of aquaculture.Regulating intestinal microbiota is considered as a better way to alleviate the adverse effects of high-carbohydrate diet.In this study,a strain affiliated to Bacillus cereus Su1,was isolated from the intestine of Nile tilapia.Su1 could degrade starch and produce acetic acid and have bile salt hydrolase activity to catalyze bile acids in vitro.Therefore,in this study,high carbohydrate diet supplemented with Su1 was used to feed tilapia for 8 weeks and the beneficial effects of Su1 were identified.The aim of this study is to investigate the effects of Su1 on immune reaction and the hepatic lipid accumulation caused by high carbohydrate diet in tilapia.Microbiome analysis was performed by 16 S r RNA sequencing and the results revealed that Su1 enriched acetateproducing bacteria and bile acid metabolism related bacteria in fish intestines.The main results include:1.Regulation of Bacillus cereus addition on the intestinal microbiota of tilapiaShannon,Simpson,Ace and Chao1 indices showed that addition of strain Su1 significantly reduced the richness but increased the diversity of the intestinal microbiota;Beta diversity analysis-PCA analysis showed that the addition of strain Su1 significantly changed the intestinal microbiota composition;16S r RNA sequencing analysis revealed that Su1 enriched acetate-producing bacteria and bile acid metabolism related bacteria in fish intestines.2.Bacillus cereus enhanced resistance to pathogen by restoration acetic acid concentrationIn order to detect the relationship between the increased concentration of acetate and enhanced resistance to pathogen in high-carbohydrate diet（HCS group）,45% of carbohydrate diets（HC）supplemented with different concentrations of sodium acetate（HC + LA,100 mmol/L;HC + MA,200 mmol/L;HC + HA,400 mmol/L）were used to raise Nile Tilapia for eight weeks.The results indicated that addition of 200 mmol/L sodium acetate（HC + MA）reduced the mortality when fish were challenged with Aeromonas hydrophila.Furthermore,we also found that addition of 200 mmol/L sodium acetate mainly inhibited p38 mitogen-activated protein kinase（p38MAPK）and nuclear factor-κB（NF-κB）phosphorylation to decrease the expression level of inflammation cytokines(interleukin-8（il-8）,interleukin-12（il-12）,tumor necrosis factor-α（tnf-α）and interleukin-IL-1β（il-1β）in the intestine.The present study indicated that certain concentration of sodium acetate could alleviate high-carbohydrate induced immune damage mainly by suppressing MAPK activation and NF-κB phosphorylation.3.Bacillus cereus alleviated liver fat accumulation by regulating bile acid metabolismIn order to confirm the effect of Su1 on bile acid levels,this study detected the intestinal and liver bile acid components in various parts of tilapia,and confirmed that the addition of strain Su1 increased the total amount of bile acids circulating in the body.Levels of bile acids in serum,fecal,intestinal,and liver were significantly elevated.Further analysis found that strain Su1 regulated liver bile acid components,increased conjugated bile acids levels,promoted the activation of tilapia liver bile acid receptor farnesoid X receptor（FXR）,and then inhibited sterol regulatory element-binding protein 1,a key gene for lipid synthesis.Sterol-regulatory element binding proteins 1（srebp1）,acetyl Co A carboxylase α（accα）,peroxisome proliferators-activated receptorγ（peroxisome proliferators-activated receptor γ,pparγ）expression.Su1 alleviated liver fat accumulation by inhibiting fatty acid synthesis.This result supplemented the mechanism of probiotics regulating bile acid lipid-lowering,indicating that the Bacillus cereus Su1 has the application prospects in aquaculture.4.Effects of the conjugated bile acid salt addition on tilapiaBile acid was approved as a new feed additive in aquaculture.Most study have found that dietary bile acids could enhance the growth performance and alleviate the hepatic health induced by high starch diet in aquaculture.However,the effect and mechanism of sodium taurocholate were not clear.Conjugated bile acid salt-sodium taurocholate was added in the high carbohydrate diet to feed tilapia for eight weeks.The results showed that the addition of sodium taurocholate（600 mg/kg）caused adverse symptoms such as liver fat accumulation and liver damage in tilapia.600 mg/kg of sodium taurocholate inhibited the expression of FXR.The results provided the information for the application effect and mechanism of sodium taurocholate,it also suggested that hepatic farnesoid X receptor（FXR）could be a crucial target for fish lipid metabolism regulation.In conclusion,this study reveals the molecular mechanism of the protective effect of Bacillus cereus Su1 on pathogen resistance and liver lipid accumulation in Nile tilapia induced by high carbohydrate diet.In addition,this study also showed that intestinal microbiota and their metabolites could alleviate the adverse metabolic syndroms induced by high carbohydrate diet in Nile tilapia.This study analyzed the improvement effect and possible mechanism of probiotics in alleviating the adverse effects of high carbohydrate diet,providing a reference for solving the application of high carbohydrate diet.This study investigated the components,metabolic patterns and action mechanisms of short-chain fatty acid and bile acid metabolites in tilapia,providing a reference for the addition and application of single metabolites.