Study of the Antimicrobial Activity of Tilapia Piscidin 3 (TP3) and TP4 and Their Effects on Immune Functions in Hybrid Tilapia (Oreochromis spp.)

To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from Oreochromis niloticus, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (Oreochromis spp.) with Vibrio vulnificus and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. In vivo experiments showed that co-treatment with V. vulnificus and TP3 (20 μg/fish) or TP4 (20 μg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and V. vulnificus into tilapia and then re-challenged the fish with V. vulnificus after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 μg/fish) or TP4 (20 μg/fish) for 30 minutes prior to V. vulnificus infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 μg/fish or 30 μg/fish) or TP4 (20 μg/fish) 30 minutes after V. vulnificus infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of V. vulnificus bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with V. vulnificus were 7.8 and 62.5 μg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 μg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed that V. vulnificus disrupted the outer membranes of cells, resulting in the loss of cell shape and integrity. We examined whether TP3 and TP4 increased the membrane permeability of V. vulnificus by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). Treating fish with TP3 and TP4 under different pH and temperature conditions did not significantly increase MIC values, suggesting that temperature and the acid-base environment do not affect AMP function. In addition, the qPCR results showed that TP3 and TP4 influence the expression of immune-responsive genes, including interleukin (IL)-1β, IL-6, and IL-8. In this study, we demonstrate that TP3 and TP4 show potential for development as drugs to combat fish bacterial infections in aquaculture.