Background: Aquaculture increasingly seeks sustainable alternatives to fishmeal, a key protein source in fish diets. Black Soldier Fly Larvae (BSFL) meal is a promising substitute, but its effects on fish growth, immunity, and gut health need further investigation. This study aimed to evaluate the impact of varying BSFL inclusion levels on juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂), a widely farmed species in tropical aquaculture. Methods: Juvenile hybrid grouper were fed diets with four levels of BSFL substitution (0%, 10%, 30%, and 50%) over 56 days. Key metrics such as growth performance, immune function, antioxidant capacity, and gut transcriptome were analyzed. Results: Replacing fish meal with BSFL meal had no significant effect on the survival rate of hybrid grouper (p > 0.05) but significantly affected growth performance, immune function, and antioxidant capacity (p < 0.05). BSFL10 and BSFL30 groups showed good growth and elevated immune enzyme activity, with significantly higher HIS levels (p < 0.05); the Wf of the BSFL10 group was comparable to the control. However, excessive replacement (BSFL50) led to reduced growth (Wf significantly lower, p < 0.05) and increased oxidative stress, as indicated by higher CAT activity (p < 0.05). Transcriptomic analysis revealed upregulation of immune- and metabolism-related genes with increasing BSFL levels, with immune pathways notably activated in the BSFL50 group. Conclusions: BSFL meal is a promising alternative to fishmeal in juvenile hybrid grouper diets, with moderate inclusion (10–30%) being most beneficial. Excessive BSFL substitution (50%) may impair fish health, highlighting the need for careful formulation in aquaculture diets.

Intestinal health is critical for nutrient absorption and disease resistance in cultured fish. Yet, the effects of dietary Eucalyptus-derived biochar on the gut of largemouth bass (Micropterus salmoides) remain largely unexplored. This study evaluated whether supplementing diets with Eucalyptus biochar c profiles. In a 56-day feeding trial, M. salmoides were offered a standard diet containing either 0% (control) or graded levels of biochar. Juvenile fish (initial body weight 13.34 g) were randomly distributed into six groups with three replicates each (30 fish per replicate). Six extruded diets were formulated with 0, 2.5, 5.0, 10.0, 20.0, or 40.0 g kg⁻¹ of biochar, designated G0 through G5. Biochar had no significant effects on villus length, muscle layer thickness, villus width, or the activities of trypsin, amylase, and lipase, though goblet cell number was significantly higher in G5. mRNA expression of Claudin-3 and IL-10 was significantly upregulated in G1–G4, while IL-1β was significantly downregulated in G4 and G5, and TNF-α expression was reduced in G2 and G3. 16S rDNA sequencing showed increasing trends in the relative abundance of Firmicutes (43% to 49.17%) and Lactococcus (0% to 1.10%) in G3, accompanied by decreases in Proteobacteria and Klebsiella. Metabolomic analysis indicated significant upregulation of taurochenodeoxycholic acid-7-sulfate, apigenin, genistein, baicalein, taurocholic acid-3-sulfate, taurochenodeoxycholic acid-3-sulfate, and arginylmethionine in G3, whereas etoxazole and soyasaponin were significantly reduced. Dietary inclusion of 10 g kg⁻¹ Eucalyptus biochar improved intestinal health in largemouth bass by shaping the gut microbiota, promoting isoflavone biosynthesis and bile acid and amino acid metabolism, inhibiting the NF-κB pathway, and reinforcing the intestinal barrier.