RNA interference confirmed a close association of CYP, ApD, and UCH with the function of CpG ODNs in Litopenaeus vannamei

Abstract

CpG oligonucleotides (CpG ODNs) have been demonstrated to enhance the innate immune response in various organisms, including Litopenaeus vannamei, a species of significant economic importance. However, the precise mechanism or the genetic factors of CpG ODNs underlying function in L. vannamei remained elusive. Our previous experiment has identified five potential marker genes, including cytochrome P450 (CYP), gamma-butyrobetaine dioxygenase (γ-BDO), apolipoprotein D (ApD), prostaglandin reductase 1 (PGR1), and ubiquitin carboxyl-terminal hydrolase (UCH). In this study, RNA interference (RNAi) was employed to validate the relevance of these five genes in responding the stimulation of CpG ODNs. Briefly, shrimps were fed CpG ODNs and subsequently injected dsRNA targeting these five genes for interference. Finally, the non-specific immune parameters of shrimps, including the expression of immune-related genes and antioxidant capacity of hepatopancreas, as well as the mortality of shrimps after pathogen attack were assessed. The tissue distribution analysis revealed a high abundance of CYP in the hepatopancreas, γ-BDO in the hemocytes, ApD in the eyestalk, PGR1 in the hemocytes, and UCH in the gonad. After blocking CYP, ApD, and UCH through RNAi, a significant down-regulation of the mRNA expression levels of immune-related genes, including ALF, TNF-α, Toll, and TOR, was observed. Additionally, a notable decline in the activity of antioxidant enzymes, such as CAT, GPX, GST, and SOD, was also observed. Moreover, the mortality rates of shrimps in the CYP, ApD, and UCH inhibited groups significantly increased compared to other treatment groups following exposure to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND) and white spot syndrome virus (WSSV). These results collectively suggest a close association of CYP, ApD, and UCH with the function of CpG ODNs in shrimps which will provide significant theoretical insights into comprehending the mechanism of CpG ODNs and optimizing their practical application in shrimp's aquaculture.