Dietary nucleotides can directly stimulate the immunity of zebrafish independent of the intestinal microbiota.

In this study, we firstly tested the effects of dietary nucleotides on the disease resistance and innate immunity of zebrafish. Further, we investigated the role of intestinal microbiota in the nucleotides-induced immunostimulatory effect by using a germ-free zebrafish model and microbiota transfer technique. Fish were fed control or nucleotides (NT)-supplemented diets (at 0.05%,0.1%, 0.15% or 0.2%, m/m) for 4 weeks, followed by immersion challenge with Aeromonas hydrophila NJ-1. The results showed that 0.1% NT group enhanced the resistance of zebrafish against A. hydrophila infection. We further observed that the relative expressions of mucin, claudin16, occlusin1, hepcidin, defensin beta-like, myeloperoxidase (Mpo), and serum amyloid A (Saa) increased in the 0.1% NT group compared with control (P < 0.05), indicating that dietary nucleotides enhanced the physical barrier and mucosal immunity in the intestine of zebrafish. Moreover, ROS level in the head kidney was significantly increased in NT fed zebrafish versus control (P < 0.05), indicating enhanced systematic immunity. Furthermore, dietary NT significantly elevated the relative expressions of mpo, saa and the ROS activity in germ-free zebrafish, while germ-free zebrafish colonized with NT-altered microbiota had no significant difference in the relative expressions of mpo, saa and the ROS activity compared with the control microbiota-colonized fish, suggesting that the immunostimulatory effect of dietary NT is mediated by direct action of NT and does not involve the microbiota. Consistently, dietary NT can protect germ-free zebrafish from pathogenic infection, whereas germ-free zebrafish colonized with NT microbiota showed no difference in disease resistance compared with control microbiota colonized counterparts. Together, these results indicated that the immunostimulatory and disease protection effect of dietary nucleotides in zebrafish was mediated by direct action of the nucleotides, and does not involve the intestinal microbiota.