hemocytes induced by WSSV or Vibrio, showing that the PjCaspase was required in the apoptosis of virus- or bacterium- challenged shrimp. Interestingly, the results indicated that the down-regulation by the siRNA3 of the PjCaspase gene containing fragment 3 led to a 1.5-times decrease (statistically significant, p,0.01) of WSSV- induced apoptotic activities of shrimp hemocytes, with the positive control, but did not influence the Vibrio-induced apoptosis, as compared with controls (Fig. 2c). Taken together, our results suggested that only the PjCaspase gene containing fragment 3 was involved in the virus-induced apoptosis, although there were diverse PjCaspase molecules in shrimp (i. e. gene sequence diversity). To illustrate the effect of overexpression of the PjCaspase gene containing fragment 3 on apoptosis of shrimp hemocytes, the PjCaspase mRNA containing fragment 3 (mRNA3) was synthesized in vitro and injected into shrimp. The Northern blot analysis indicated that the injection increased the levels of the PjCaspase mRNA3 in shrimp hemocytes (Fig. 3a). In this case, the WSSV- induced apoptotic activities were significantly increased or in shrimp hemocytes with high levels of the mRNA3 by 1.2 times (p,0.01), as compared to the controls (p,0.05), whereas the Vibrio-induced apoptosis remained unchanged (Fig. 3b). As a control, the mRNA of glutathione S-transferases (GST) was injected into shrimp. The results showed that the expression of the PjCaspase gene containing fragment 3 and shrimp apoptosis were Gene Sequence Diversity for Antiviral Immunity not affected by the GST mRNA (data not shown). Overall, our results indicated that the PjCaspase molecule containing fragment 3 was specifically responsible for virus-induced apoptosis, and that the PjCaspase gene sequence diversification might contribute to the pathogen-specific immunity in shrimp. The role of virus-specific PjCaspase molecule in immunity against virus infection The preceding data indicated that the PjCaspase gene containing fragment 3 was specifically responsible for the virus-induced apoptosis. How would this virus-specific PjCaspase molecule function in the shrimp’s immunity against virus infection? To address this question, the WSSV replication in shrimp with low or high levels of the PjCaspase gene containing fragment 3 was monitored using quantitative real-time PCR. The quantitative analyses revealed that when the PjCaspase gene containing fragment 3 was silenced by the siRNA3, the frequency of copies of WSSV in shrimp was 6 times higher than that of the control (WSSV only) at 48 h post-infection (p.i.) with WSSV (Fig. 4). On the other hand, the overexpression of the PjCaspase gene containing fragment 3 led to an 11-fold decrease of virus copies, compared to the control (WSSV only) at 36 h after WSSV infection (Fig. 4). These results confirmed that the PjCaspase gene containing fragment 3 could specifically inhibit the proliferation of WSSV. Figure 3. The effects of overexpression of the PjCaspase gene containing fragment 3 on apoptotic activities of shrimp hemocytes. The in vitro–synthesized mRNA of PjCaspase containing fragment 3 (mRNA 3) was injected into the shrimp. The shrimp apoptosis was simultaneously induced by WSSV or pathogenic Vibrio parahaemolyticus. After the mRNA injection, the shrimp hemolymph was collected at 36 h and subjected to Northern blot and apoptotic assays, respectively. a. Detections of PjCaspase mRNA containing fragment 3 (PjCaspase mRNA3) by Northern blots with DIG-labeled gene-specific probes using the total RNAs extracted from shrimp hemolymph. The probes used were indicated on the right. Shrimp b- actin was used as control. Row headings indicated the solutions used for injections. b. Apoptotic activities of shrimp hemocytes, including DAPI staining of hemocytes (left) and detection of caspase 3 activity (right). The relative activity of caspase 3 was indicated by comparison with the negative control (mRNA buffer). All shrimp used were WSSV- sensitive shrimp. All the assays were conducted in triplicate. Each column represented the mean of triplicate assays with standard deviation shown as a bar. Row headings showed the solutions used for injections. The asterisks (*) or double asterisks (**) indicated the significant differences between treatments of interest (p,0.05 or p,0.01, respectively). doi:10.1371/journal.pone.0024955.g003 PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24955