Phenoloxidases from black tiger shrimp (Penaeus monodon): gene expression and activity distribution in different tissues

In this study, the expression of four reference genes (beta-actin, EF1-alpha, GAPDH, and SubF0), two prophenoloxidase genes (proPO1 and proPO2), and the distribution of phenoloxidase (PO) activity in eight tissues (carapace, cuticle, hemolymph, hepatopancreas, muscle, pereiopods, pleopods, and uropods) of black tiger shrimp (Penaeus monodon) were evaluated. According to the geNorm and NormFinder algorithms, the most stable reference gene or the best combinations of reference genes for the evaluation of proPO1 and proPO2 expression are SubF0 for cuticle, muscle, and uropods, EF1-alpha for hemolymph and pereiopods, EF1-alpha + SubF0 for carapace, beta-actin + SubF0 for hepatopancreas, and beta-actin + GAPDH for pleopods. In the hemolymph and uropods, proPO1 expression was significantly stronger than that of proPO2 (433 and 5 times higher, respectively). However, in hepatopancreas, proPO2 expression was significantly stronger than proPO1 (36 times higher). Trypsin cleaves the proPO zymogen into active PO in most of the studied tissues, except hepatopancreas. PO activity (units/mg protein) following trypsin activation varied among the tissues in the following order: uropods > hemolymph > pleopods > pereiopods > hepatopancreas > carapace > cuticle > muscle. The results indicate that proPO1 is dominant in hemolymph, whereas proPO2 is dominant in hepatopancreas. Altogether, these results contribute to the elucidation of the phenoloxidase function in P. monodon.