Assessment of the Probiotic Potential of Three Host-Associated Bacillus Species on Growth Performance, Health Status, and Immune Responses of Tomato Clownfish (Amphiprion frenatus)

Antibiotic use in marine ornamental fish farming has raised concerns about resistance and tissue damage, motivating the search for safe probiotic alternatives. In this study, three Bacillus strains—B. subtilis (BS), B. megaterium (BM), and B. velezensis (BV)—that were isolated from healthy clownfish intestines were evaluated as dietary supplements for Amphiprion frenatus over a four-week feeding trial. The growth performance of the fish, the antioxidant enzyme activity in the liver and intestine, and the expression of pro-inflammatory and antimicrobial peptide genes in four tissues were examined. The intestinal barrier genes and intestinal morphology were also measured. The obtained data indicate that BM elicited the broadest immune activation, upregulated antimicrobial peptides and all four barrier genes, and increased the hepatosomatic index and MDA. BV has the best promotional effects on growth without elevating the hepatosomatic index; it selectively induced lyz and leap2 while suppressing gill il1b, and it reduced intestinal MDA. BS showed comparatively mild effects. These findings demonstrate strain-specific probiotic actions in clownfish, with B. velezensis offering the most favorable balance of growth promotion and immunomodulation for aquaculture applications.