β-glucan is widely used in aquaculture due to its immunostimulatory effects, but the specific effect and potential regulatory mechanism on largemouth bass (Micropterus salmoides) are still unclear. Here, we evaluated the effects of β-glucan on growth, resistance to Aeromonas schubertii, intestinal health, and transcriptome of largemouth bass to reveal the potential regulators, metabolic pathways, and altered differential microbiota. Four experimental diets were designed with β-glucan supplementation levels of 0 (control), 100 (LA-100), 200 (MA-200), and 300 (HA-300) mg kg^-1, and each diet was fed to largemouth bass (79.30 ± 0.50 g) in triplicate for 70 days, followed by a 3-day challenge experiment. Results showed that different β-glucan supplementations had no significant effects on growth performance and whole-body composition. Fish fed a diet with 300 mg kg^-1 β-glucan significantly increased the activity of lysozyme than those fed diets with 0 and 100 mg kg^-1 β-glucan. In addition, the survival rate of largemouth bass in β-glucan supplementation groups was significantly higher than the control group at 12- and 24-h challenge by Aeromonas schubertii. Transcriptome analysis showed that a total of 1,245 genes were differentially expressed [|log₂(fold change)| ≥1, q-value ≤0.05], including 109 immune-related differentially expressed genes (DEGs). Further analysis revealed that significantly upregulated and downregulated DEGs associated with immunity were mapped into 12 and 24 pathways, respectively. Results of intestinal microflora indicated that fish fed a diet with 300 mg kg^-1 β-glucan had higher bacterial richness and diversity as evaluated by Sobs, Chao, Ace, and Simpson indices, but no significant differences were found in the comparison groups. Furthermore, 300 mg kg^-1 β-glucan significantly increased the relative abundance of Mycoplasma and decreased Proteobacteria (mainly Escherichia-Shigella and Escherichia coli) and Bacillus anthracis in largemouth bass intestinal microflora. The findings of this study provided new insights that will be valuable in future studies to elucidate the mechanism of immunity enhancement by β-glucan.

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