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.

To study the effects of acute ammonia stress on the poisoning reaction of the hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu), 300 healthy grouper juveniles with an initial body weight of 51.4 ± 2.57 g were selected for an acute ammonia stress experiment using a half-lethal concentration of ammonia for 24, 48, 72, and 96 h with triplicate. The results show that (1) The half-lethal concentrations of ammonia for a hybrid grouper were 39.5 mg/L for 24 h, 27.3 mg/L for 48 h, 26.5 mg/L for 72 h, and 25.0 mg/L for 96 h, and the safe concentration was 2.50 mg/L. (2) The biochemical indices of the serum and livers of the groupers fluctuated under acute ammonia stress (P < 0.05), and the contents of serum cortisol and lactate reached the maximum value in 72 h (P < 0.05). (3) 24 h ammonia stress increased the activities of alkaline phosphatase, acid phosphatase, catalase, glutathione peroxidase, superoxide dismutase, lysozyme, and the content of immunoglobulin M in the liver (P < 0.05). Meanwhile, all ammonia-stressed fish groups had an increased amount of malondialdehyde in the liver (P < 0.05). (4) The ammonia poisoning caused significantly up-regulation of antioxidant genes, inflammatory cytokines, and apoptosis genes (P < 0.05), and the expression of inflammatory cytokines and apoptosis genes were the highest in 24 h ammonia stress group. (5) The ammonia content in the water changed the abundance and evenness of intestinal microbes in grouper, mainly in that ammonia stress significantly increased the relative abundance of fusobacteria, but significantly decreased the relative abundance of gemmatimonadetes (P < 0.05). It was speculated that apoptosis induced by ammonia poisoning in grouper may be related to oxidative stress and the activation of inflammatory factors, and excessive inflammatory stress may be one of the causes of ammonia poisoning in the hybrid grouper.