Nonspecific effect of double-stranded (ds) RNA on prophenoloxidase (proPO) expression in Penaeus monodo n

Abstract

RNA interference-mediated silencing is an effective way of controlling white spot syndrome virus (WSSV). However, the effect of RNAi on the innate immune mechanism is not well understood. Prophenoloxidase (proPO) is an important component of the shrimp innate immunity. In the present study, nonspecific effect of two double-stranded (ds)RNA-expressing constructs, one targeting vp28 gene of WSSV (pCMV-VP28-LH) and another targeting green fluorescent protein (GFP) (pCMV-GFP-LH) on proPO2 gene expression, is investigated. mRNA expression levels of proPO2 in hemocytes of DNA construct-injected shrimp were estimated using real-time PCR with elongation factor 1-α as internal control. Empty vector (pcDNA)-injected shrimp were used as experimental control. In pCMV-VP28-LH-injected shrimp, proPO2 showed significant upregulation until 48 h post-injection (p.i.). Similarly, pCMV-GFP-LH-injected animals showed high levels of expression until 72 h p.i. WSSV-challenged animals, compared to pcDNA-injected control group, showed no significant change in expression of the gene until 24 h. However, an increased expression was noticed at 48 h p.i. Our results suggest that neither the plasmids nor the long hairpin RNA expressed by the constructs has any nonspecific silencing effect on the proPO2 expression. On the contrary, the consistent upregulation of proPO2 observed in shrimp injected with dsRNA at early time-points indicates the possibility of nonspecific protection against WSSV infection.