A formulated double-stranded RNA diet for reducing Penaeus monodon densovirus infection in black tiger shrimp.

Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in the aquaculture industry in Thailand. Significant reductions in levels of PmDNV as assessed by PCR analysis of shrimp hepatopancreas were seen in both prophylactic and curative experiments after feeding shrimp with a formulated diet containing mixed inactivated bacteria harboring dsRNAs corresponding to the PmDNV ns1 and vp genes. Significant reductions of approximately 88% (prophylactic) and 64% (curative) of PmDNV were observed, suggesting that this diet has a high potential for application in commercial aquaculture for reducing PmDNV associated stunted growth of shrimp.

Ingestion of bacteria expressing dsRNA triggers specific RNA silencing in shrimp.

RNAi activation in shrimp through dsRNA injection has been well demonstrated but oral delivery of dsRNA remains controversial. Therefore, this study was conducted to determine whether RNAi was induced in shrimp by ingestion of bacteria expressing dsRNA. We fed shrimp, Penaeus monodon and Litopenaeus vannamei, with inactivated bacteria expressing dsRNA specific to the shrimp genes (Rab7 and STAT). Forty-eight hours after 6 day-continuous feeding, the level of the targeted gene transcript was measured by semi-quantitative RT-PCR. Significant reduction of Rab7 as well as STAT transcript was observed when compared to that of control shrimp fed with bacteria containing the empty vector or bacteria expressing non-related dsRNA (GFP). Moreover, the suppression was detected not only in the hepatopancreas but also in the gills indicating the successful systemic induction of RNAi via oral delivery of dsRNA. Our results suggested that RNAi in shrimp could be triggered by ingestion of dsRNA expressing bacteria. Therefore, oral feeding is a practical approach which can be used to deliver dsRNA for further viral inhibition in farmed shrimp.

Inhibition of Penaeus monodon densovirus replication in shrimp by double-stranded RNA.

Stunted shrimp caused by Penaeus monodon densovirus (PmDNV) infection is one of the main problems leading to a significant economic loss in Thailand. To control this pandemic disease, a double-stranded-RNA-mediated virus-specific gene silencing approach was applied to inhibit viral replication. In this study, two dsRNAs corresponding to the non-structural protein (ns1) and the structural protein (vp) genes of PmDNV were synthesized and introduced into shrimp haemolymph prior to viral challenge. After allowing viral replication for two weeks, the suppression effect by each dsRNA was evaluated by semi-quantitative PCR and compared with the control. A reduction of PmDNV in shrimp treated with each dsRNA was observed. In contrast, a high level of viral infection was detected in the control group (NaCl). Based on a limited sample number, we reached the tentative conclusion that virus-specific dsRNA can inhibit PmDNV replication, in which the dsRNA-ns1 was more effective than the dsRNA-vp.

Inhibition of white spot syndrome virus replication in Penaeus monodon by combined silencing of viral rr2 and shrimp PmRab7.

Although a significant progress has been achieved on dsRNA mediated anti-virus strategy development, there is still no effective means to control the virulent white spot syndrome virus (WSSV). Six double-stranded RNAs specific to different essential genes of WSSV (ie1, ie3, pol (DNA polymerase), rr2 (ribonucleotide reductase small subunit), vp26, and vp28) were employed to suppress viral replication in shrimp. At the condition that non-specific inhibitory effect was overwhelmed, the relative protective degree of these dsRNAs against WSSV infection (rr2>ie3>vp26, vp28>ie1>pol) was observed by semi-quantitative PCR. Besides, more than one injection of dsRNA was needed for an efficient viral inhibition. To improve viral protection in Penaeus monodon, synchronized blocking of viral cellular transport (by dsRNA-PmRab7) and viral essential gene synthesis (by dsRNA-rr2) was first performed in this study. The suppression effects of shrimp mortality by either combined dsRNAs of rr2 and PmRab7 or dsRNA-rr2 alone was monitored for 8 days after viral challenge. Approximately 95% of shrimp survivals were detected from both combined dsRNAs and dsRNA-rr2 alone whereas all shrimp without dsRNA were dead. It revealed that there was no additive inhibitory effect of the combined dsRNAs over dsRNA-rr2 alone.