CHARACTERIZATION AND EXPRESSION OF NADPH OXIDASE GENES IN STIMULATED SPLENIC NEUTROPHILS OF TILAPIA, OREOCHROMIS NILOTICUS BY A MIX OF TRADITIONAL CHINESE MEDICINE AND BACILLUS SPECIES (TCMBS)

Abstract

Traditional Chinese medicine and Bacillus species (TCMBS) mixture is an immunostimulant with considerable promise as an alternative in improving fish health. However, nothing is known on its effects on the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes and production of reactive oxygen species (ROS) in the neutrophils of fish. The full lengths of tilapia phagocytic NADPH oxidase genes gp91phox, p22phox, p40phox, p47phox, and p67phox were cloned and their expression profiles after TCMBS stimulus investigated. The cDNAs of tilapia gp91phox, p22phox, p40phox, p47phox, and p67phox contained open reading frames of 1698 bp, 561 bp, 1053 bp, 1584 bp, and 1470 bp respectively, encoding 561, 186, 350, 527, and 489 amino acids respectively. Comparison of the deduced amino acid sequences showed that tilapia NADPH oxidase genes shared 58%–91% and 21%–67% identity with those of other teleost and mammals respectively. Besides, tilapia NADPH oxidase genes contain conserved domains and motifs required for ROS generation. Phylogenetic analysis suggested tilapia NADPH oxidase genes were close to those of Fundulus heteroclitus. After 2 weeks of TCMBS application showed significant upregulation in expression of NADPH oxidase genes, antioxidant genes (i.e., superoxide dismutase, catalase, and glutathione‐ disulphide reductase), and an increase in the production of ROS compared to the control in splenic neutrophils of tilapia. Collectively, our study provides evidence of the structure of tilapia NADPH oxidase genes and demonstrate that TCMBS application could modulate their activity in neutrophils to improve immunity in tilapia.