Host gut-derived Bacillus probiotics supplementation improves growth performance, serum and liver immunity, gut health, and resistive capacity against Vibrio harveyi infection in hybrid grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus)

Several reports have revealed the vital role that probiotics play in fish growth and health. However, few works are available for host gut-derived probiotics on the growth, immunity, and gut microbiota of fish, especially in hybrid grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus) due to their isolation difficulty and functional verification. This study aimed at assessing 3 host gut-derived Bacillus species' effects on the growth, immune and antioxidant-biochemical responses, haematological parameters, intestinal morphology, immune-related gene expression, gut microbiota, and disease resistance against Vibrio harveyi in hybrid grouper. A total of 480 hybrid grouper (initial weight = 9.03 ± 0.02 g) were randomly allotted into 4 groups, namely, the group fed a basal diet without probiotic inclusion (control, B0), the group fed the basal diet with Bacillus velezensis GPSAK4 (BV), the group fed the basal diet with Bacillus subtilis GPSAK9 (BS), and the group fed the basal diet with Bacillus tequilensis GPSAK2 (BT) strains at 1.0 × 10⁹ CFU/g. After a 6-week feeding trial, the results revealed significant improvements (P < 0.05) in the growth performance, whole fish-body proximate composition, blood haematological parameters, serum, liver, and intestinal biochemical indexes, intestinal morphology, and protection against V. harveyi pathogen in the probiotic-treated groups compared with the untreated. Additionally, the expressions of intestinal tight junction genes (occludin and ZO1), pro- and anti-inflammatory genes, including IL1β, IL6, IL8, TNFα, MyD88, IL10, and TGFβ, were upregulated (P < 0.05) after Bacillus species administration. Host gut-derived Bacillus supplementation shaped the gut microbiota by significantly increasing (P < 0.05) the relative abundance of Proteobacteria, Bacteroidetes, Actinobacteria (except the BS group), Acidobacteria (except the BT group), Cyanobacteria (except the BV and BT groups), and Verrucomicrobia phyla, as well as known beneficial genera (Romboutsia, Turicibacter, Epulopiscium, Clostridium_sensu_stricto 1 and 13, Lactobacillus, and Bacillus), but significantly decreased (P < 0.05) the abundance of Firmicutes, Chloroflexi, and Fusobacteria phyla, and purported pathogenic genera (Staphylococcus and Photobacterium) compared with the control group. Collectively, the results suggest that B. velezensis GPSAK4, B. subtilis GPSAK9 (especially this strain), B. tequilensis GPSAK2 dietary supplementation at 1.0 × 10⁹ CFU/g has positive effects on the intestinal health of hybrid grouper via microbial composition modulation, thus enhancing the assimilation and absorption of nutrients to boost fish growth, immunity, and disease resistance.