Liver-expressed antimicrobial peptide 2 (LEAP2) is a cationic peptide that is integral to the innate immune system. However, its regulatory function in the immunity of fish against pathogens remains largely unexplored. This study identifies a LEAP2 homolog (AoLEAP2) in clownfish, Amphiprion ocellaris. Our phylogenetic analysis places AoLEAP2 within the fish LEAP2 cluster, closely related to Tachinotus anak LEAP2. AoLEAP2 mRNA is present across various tissues, with the highest expression in the spleen. Following Vibrio harveyi infection, the AoLEAP2 mRNA levels significantly increased in the liver, spleen, gills, and intestine. The chemically synthesized AoLEAP2 mature peptide exhibits broad-spectrum antibacterial activity without displaying toxicity to FHM cells in vitro. The intraperitoneal injection of AoLEAP2 significantly suppresses the mRNA expression of the pro-inflammatory factors IL1β and TNFα induced by V. harveyi infection, demonstrating its anti-inflammatory and immune-regulatory role.

In previous studies, natural killer lysin (NK-lysin) emerged as a crucial antimicrobial peptide (AMP) discharged by NK cells and CTLs. The sequence of NK-lysin was cloned and discovered in some fishes, but its function remains unclear. In our study, we obtained a copy of NK-lysin from the spleen of the healthy clownfish (Amphiprion ocellaris; AoNK-lysin) through cloning and proceeded to investigate its potential functions and activities. The findings showed that the AoNK-lysin gene’s open reading frame (ORF) had a length of 465 base pairs (bp) and encoded 154 amino acids (aa), which included a saposin B domain and six cysteine residues that are highly conserved, forming three intrachain disulfide bonds to carry out antimicrobial activity. The AoNK-lysin gene was widely present in different tissues, with the skin showing the highest expression, followed by the eye, intestine, and muscle. Additionally, the expression of AoNK-lysin was significantly upregulated in the immune organs (spleen, gill, intestine, and head kidney) of A. ocellaris after being challenged by Singapore group iridovirus (SGIV). Furthermore, a 399 base pair cDNA sequence that encodes the fully developed peptide of AoNK-lysin was successfully inserted into a secretion plasmid called pPIC9K. Subsequently, a significant amount of the recombinant AoNK-lysin protein was efficiently manufactured using the Pichia pastoris expression system. The antibacterial test demonstrated that the AoNK-lysin protein significantly suppressed the growth of various pathogens, particularly Streptococcus agalactiae, Streptococcus iniae, Salmonella typhi, Shigella sonnei, Pseudomonas aeruginosa, and Aeromonas caviae. The minimal inhibitory concentration (MIC) was found to be 7.81 μg/mL. Further analysis of antiviral assays showed all the viral mRNA of SGIV to be significantly reduced after AoNK-lysin protein stimuli in FHM cells. Collectively, these discoveries indicate that AoNK-lysin exhibits features of both direct pathogen-killing abilities and inhibited virus replication.

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.

Trachinotus ovatus (golden pompano) represents one of the dominant high-value mariculture fish species farmed across coastal regions of China. In intensive farming modes, intestinal damage and immune suppression are frequent issues in golden pompano; the widespread application of antibiotics in disease control has also triggered increasing worries about ecological risks and aquatic product safety. Host-origin probiotics have been widely recognized as a green and safe strategy to mitigate health risks and promote growth in farmed fish. Nevertheless, the regulatory roles and mechanisms of autochthonous E. faecalis strains on the intestinal health of golden pompano are still poorly understood. In this study, a 42-day feeding trial was conducted using golden pompano reared in a large-capacity floating net-cage system. The experimental fish were fed diets supplemented with different concentrations of E. faecalis SZ07 (1 × 10⁵ to 1 × 10⁹ CFU/g feed). Dietary supplementation with SZ07 at suitable doses (1 × 10⁷ to 1 × 10⁸ CFU/g feed) notably improved intestinal tissue structure, elevated the activities of digestive, antioxidant, and immune enzymes, and modulated the mRNA levels of key immune genes in the spleen. Analysis of the gut microbiota revealed that dietary E. faecalis SZ07 reshaped the intestinal microbial community, with an increased abundance of beneficial taxa and a decreased proportion of opportunistic pathogens. Taken together, our results indicate that autochthonous E. faecalis SZ07 acts as an effective probiotic to enhance intestinal health and immune function of golden pompano and offers technical support for the healthy development of its aquaculture industry.