Screening of intestinal probiotics and the effects of feeding probiotics on the digestive enzyme activity, immune, intestinal flora and WSSV resistance of Procambarus clarkii

In this experiment, a potential probiotic strain A23 with high antimicrobial activity against common aquatic pathogens and extracellular enzyme production was isolated from the intestine of healthy Procambarus clarkii. Biochemical and 16S rDNA sequencing analysis identified A23 as Bacillus amyloliquefaciens, and a haemolytic test and antibiotic susceptibility test were carried out on strain A23 to determine its safety. First, the whole genome of B. amyloliquefaciens A23 was determined to explore its mechanism of action. The results showed that the total length of the genome was 3,974,398 bp, the GC content was 46.56%, and 3843 genes were encoded, including 27 rRNA operons and 86 transfer RNA genes. Three bacilysin-related genes and 10 antimicrobial peptide (AMP)-related genes were found through manual inspection. Second, we evaluated the beneficial effects of strain A23 in P. clarkii by performing in vivo feeding experiments. The results showed that probiotic supplementation could effectively enhance the intestinal digestive enzyme activities, innate immune enzyme activities and white spot syndrome virus (WSSV) resistance of P. clarkii. Finally, the intestinal microbial community and diversity of crayfish were compared and analyzed by high-throughput sequencing. At the phylum level, the abundance of Proteobacteria decreased with 108 CFU/g probiotics supplement, while the abundance of Firmicutes and Bacteroides increased. The Shannon index of the intestinal microorganism community of the 108 CFU/g probiotic supplementation group was significantly higher than that of the control group, indicateding that 108 CFU/g probiotic supplementation enhanced intestinal microbial diversity. The results from this study demonstrated that B. amyloliquefaciens A23 is a promising probiotic candidate that provides multiple benefits for crayfish cultivation.