Integrated Multiple-Omics Reveals the Regulatory Mechanism Underlying the Effects of Homologous Bacillus Tequilensis (GCB-3) on Growth Performance of Grass Carp (ctenopharyngodon Idellus)

The purpose of this study was to investigate the regulatory mechanism of Bacillus tequilensis (GCB-3) in improving the growth performance of grass carp by microbiome and targeted metabolomics analysis. Fish were fed with different amounts of GCB-3 including B0 (0 CFU/g), B6 (1×106 CFU/g), B7 (1×107 CFU/g), B8 (1×108 CFU/g), and B9 (1×109 CFU/g) for 8 weeks. The results indicate that with the increase of dietary GCB-3 concentration, grass carp showed a significant linear trend in growth performance, and appetite factors showed a significant linear and quadratic trend, the growth performance and appetite-promoting factors in the B8 group reach their maximum value in the B8 group. Microbiome analysis found that Proteobacteria and Actinobacteria were the most abundant phylum in the gut microbiota of grass carp. The functional potential predicted that GCB-3 affected the synthesis and degradation of carbohydrates, amino acids, fatty acids and lipids. It found that GCB-3 also affected the tricarboxylic acid (TCA) cycle VII (acetate-producers) pathway. The results of targeted metabolomics showed a significant linear and quadratic increase in intestinal acetate with GCB-3 concentration and a maximum in the B8 group. The results of the correlation analysis showed a significant positive correlation between Actinobacteria abundance and acetic acid content, and also between TCA cycle VII (acetate-producers) pathway abundance and Actinobacteria abundance and acetic acid content. In B8 group, grass carp showed a faster regulation speed of blood glucose levels after glucose injection, and the GLP-1 content remained stable. The activities of glycolytic enzymes, lipolytic enzymes, protein metabolism enzymes, and their related mRNA expression levels in grass carp showed a linear or quadratic increasing trend. However, the expression levels of gluconeogenic enzymes and fatty acid synthesis enzyme related mRNA showed an opposite trend. In summary, the addition of 1×108 CFU/g GCB-3 in grass carp feed can improve the abundance of dominant bacterial phyla in the intestine, especially Actinobacteria, increase the concentration of intestinal acetate, provide substrate for the TCA cycle VII (acetate-producers) pathway, thereby regulating the nutrient metabolism of grass carp and ultimately promoting growth.