Gene Sequence Diversity for Antiviral Immunity Figure 2. The effects on apoptosis of knocking down a PjCaspase gene containing fragment 3 with RNAi of shrimp hemocytes. a. Schematic diagram of the amino acid and nucleotide sequences of fragment 3 of the PjCaspase gene. The numbers showed the sites of fragment 3 in the PjCaspase gene sequence. The PjCaspase fragment 3 was obtained by comparing the PjCaspase gene sequence diversities of WSSV-susceptible and WSSV-resistant shrimp. The sequence targeted by fragment 3-specific siRNA (siRNA 3) was boxed. The asterisks (*) indicated where the bases or amino acids of fragment 3 differed from those of other PjCaspase molecules. b. Detections of PjCaspase mRNA 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. The sequence of the PjCaspase probe (59-CACCTGGATTAC CTCGTCCAT-39) was identical in all PjCaspase molecules, while the sequence of the PjCaspase fragment 3 probe (59-CCTCTTCACAGGCCGAGGATT-39) was specific for the PjCaspase fragment 3 only. Shrimpb-actin was used as a control. Shrimp were injected with different solutions as shown in the lane headings, respectively. Subsequently, the shrimp hemocytic apoptosis was induced by WSSV or pathogenic Vibrio parahaemolyticus. After injection, the shrimp hemolymph was collected at 36 h and subjected to Northern blot and apoptotic assays. c. Evaluations of shrimp hemocytic apoptotic activities 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 (siRNA buffer). The shrimps used in these assays were WSSV- sensitive. All the assays were biologically repeated three times. Each column indicated the mean of triplicate assays, with the standard deviation shown as a bar. Row headings represented the solutions used for injections. The asterisks (**) indicated the significant differences between treatments of interests (p,0.01). doi:10.1371/journal.pone.0024955.g002 the injection completely suppressed the expression of the PjCaspase mRNA containing fragment 3 in the shrimps treated with the siRNA3 (Fig. 2b). On the other hand, the expression levels of the other PjCaspase mRNAs were not affected by the injection of siRNA3 (Fig. 2b). As control, a single nucleotide mutation was randomly introduced into the sequence of the siRNA3, resulting in a siRNA3- mutation. Surprisingly, the single random mutation abolished the siRNA3’s ability to silence the expression of the PjCaspase mRNA containing fragment 3 (Fig. 2b). Our results indicated that the siRNA3 regulated only the PjCaspase mRNA containing fragment 3, and not the expression of the other genes. Under the condition that the expression of the PjCaspase gene containing fragment 3 was silenced by the siRNA3, we examined the ability of the siRNA3 to affect the apoptosis induced by WSSV or Vibrio parahaemolyticus. As shown in Fig. 2c (lanes labeled ‘‘WSSV+PjCaspase siRNA’’ and ‘‘Vibro+PjCaspase siRNA’’), the silencing of the PjCaspase gene by siRNA caused a statistically significant decrease (p,0.01) in the apoptotic activities of shrimp PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24955