Microbiome And Metabolome Analysis On The Effect Of Three Beneficial Bacteria On Gut Microbiota Of Litopenaeus Vannamei Postlarvae

Litopenaeus vannamei is one of the most important shrimp aquatic foods in the world,and it is also an important breeding species in China.However,due to the frequent occurrence of diseases,the success rate of shrimp breeding is only maintained at 20%-30%.The control of diseases by chemical drugs can lead to drug residues and drug resistance.Therefore,it is inevitable to seek a green and environmentally friendly method.Lactic acid bacteria(LAB),BALOs,photosynthetic bacteria are good probiotics,which can promote growth and improve water quality.In this study,Litopenaeus vannamei larval were used as the research object,and LAB,BALOs and photosynthetic bacteria were used as test strains.By using traditional bacteriology,high-throughput sequencing and metabolomics research methods,the effects of three probiotics on the growth of Litopenaeus vannamei larval were compared.(1)Comparing the effects of LAB,BALOs and photosynthetic bacteria on the growth performance,water quality,water body and intestinal flora of Litopenaeus vannamei larvae,it was found that BALOs and photosynthetic bacteria could purify water quality and reduce NO₂-N,NO₃-N content in the water.Both LAB and BALOs could improve the survival rate and growth performance of shrimp larvae,and the effect of BALOs on the weight gain of shrimp larvae was stronger than that of LAB,but photosynthetic bacteria had no such effect;when combined application,the compound effect of LAB and BALOs on survival rate and growth rate were both worse than that of BALOs alone,but slightly higher than that of the LAB group.The compound effect of LAB and photosynthetic bacteria was better than that of LAB.(2)Using high-throughput sequencing technology to compare the effects of LAB,BALOs,and photosynthetic bacteria on the structure of shrimp intestinal flora.Alpha diversity results showed that LAB,BALOs and photosynthetic bacteria could adjust the structure of shrimp intestinal flora,increase the richness and diversity of intestinal flora,and the ability of BALOs to regulate the intestinal flora was stronger than in turn for LAB and photosynthetic bacteria.The results of the analysis of the flora structure at the phylum classification level showed that LAB and BALOs could promote the weight gain of shrimps by affecting the ratio between the Bacteroides and Firmicutes.The results of the analysis of the flora structure at the genus classification level showed that BALOs had a good control effect on Vibrio.Further statistical analysis of the ratio of probiotics/functional bacteria and pathogenic bacteria revealed that LAB and BALOs had the control effect on pathogenic bacteria and the promotion of probiotics,and the effect of BALOs was stronger than that of LAB.Predictive function analysis showed that LAB and BALOs regulated the abundance of functions related to life activities,such as energy production and conversion[C],amino acid transport and metabolism[E],carbohydrate transport and metabolism[G],lipid transport and Metabolism[I],and functions related to disease resistance,such as the cytoskeleton[Z],promoting the growth of shrimp and increase survival rate.The Pearson correlation analysis of intestinal flora and shrimp growth parameters revealed that there was a certain correlation between certain specific flora and shrimp growth performance.For example,Alphaproteobacteria had a significant positive correlation with BW,PTWG,and TWG(p<0.05);Saccharimonadales and Sphingobacteriales were significantly positively correlated related to BW and BL,respectively(p<0.05);Vibrionales and Vibrio were significantly negatively correlated with SR(p<0.05).(3)Using LC-MS metabonomics research methods,we compared the effects of LAB,BALOs and photosynthetic bacteria on the intestinal metabolites of shrimps(PL4-5).It was found that the differential metabolites significantly enriched in the experimental group with the control group were different,such as in LAB group compared with the control,Dihydroceramide was up-regulated;L-tyrosine,L-phenylalanine,L-tryptophan,etc.were down-regulated.In BALOs group compared with the control,L-Arginine,stearic acid,and Acetylcarnitine were all up-regulated.In the photosynthetic bacteria group compared with the control,5-Hydroxy-L-tryptophan was up-regulated,and Acetylcarnitine and Xanthosine were down-regulated.Some of the differential metabolites were related to the survival rate and growth performance of shrimps.For example,L-tyrosine and L-phenylalanine were involved in the pathways related to shrimp growth,including Protein digestion and absorption,Aminoacyl-t RNA biosynthesis,Dihydroceramide was involved in the pathway related to shrimp growth was the Sphingolipid signaling pathway and L-Arginine was involved in the Salmonella infection pathway.Photosynthetic bacteria had no effect on the growth-related differential metabolites.The correlation analysis between metabolomics and microbiome showed that there was a significant correlation between different metabolites and bacterial genera.For example,L-Glutamic gamma-semialdehyde had a significant positive correlation with Erythrobacter(p<0.05),and L-Phenylalamine had a significant negative correlation with Pseudomonas(p<0.05).Stearic acid and Xanthosine both had a significant negative correlation with Ruegeria(p<0.05).In summary,it can be seen that both LAB and BALOs could adjust the structure of the intestinal flora,thereby affecting metabolites,and ultimately affecting the growth performance of the body.Among the three kinds of probiotics,only BALOs had a better control effect on vibrio,which suggested that BALOs may be an alternative to chemical drugs and become a green and environmentally friendly method for disease control.At the same time,BALOs had the most obvious effect on the growth of shrimp,especially weight gain,and the effect was best when used alone;LAB had a stronger effect on the growth of shrimp body length,and combined use with photosynthetic bacteria could improve water quality.The results of this study provided a certain theoretical basis for the green production of shrimp farming and food safety"from farmland to table",and had certain practical significance.