Rimp, qRT-PCR was conducted using isoform-specific primers and probes. The isoform Ago1C was shown to be ubiquitously expressed in all organs or tissues examined (Fig. 4A). However, the mRNA levels of both Ago1A and Ago1B were significantly up-regulated in lymphoid organ (Fig. 4A), suggesting that the two isoforms were involved in shrimp immunity. Considering that Ago proteins represent key components in antiviral RNAi immunity in many species [12,13,14,15], the effects of WSSV infection on the expression of Ago1 isoforms was investigated. Because of the up-regulation of Ago1A and Ago1B 25033180 isoforms in shrimp lymphoid organs, the lymphoid organ was selected to investigate the expression profiles of Ago1 isoforms in response to WSSV challenge. It was showed 
that the expression of Ago1A and Ago1B was significantly increased at 12 and 24 h postinoculation (p,0.05) (Fig. 4B), whereas the expression of Ago1C remained unchanged at all time points compared to the controlResults Identification of Ago1 Isoforms in ShrimpBased on PCR amplification using Autophagy degenerated primers and RACE analysis, full-length cDNA of the shrimp Ago1 gene was obtained. Sequence analysis revealed that the Ago1 gene generated three transcripts: Ago1A (GenBank accession no.: GU265732), Ago1B (GenBank accession no.: JX170715) and Ago1C (GenBank accession no.: JX170716). Sequence comparisons showed that the Ago1 isoforms were different from each other with a 9-nt inserted fragment (Ago1-fragment 1) at their 59 termini and an additional 81-nt fragment of (Ago1-fragment 2) in the PIWI domain (Fig. 1). No amplification was observed when the extracted RNA was used in PCR analyses. These findingsRole of Argonaute-1 Isoforms in Antiviral DefenseRole of Argonaute-1 Isoforms in Antiviral DefenseFigure 6. Roles of Ago1 isoforms in the shrimp immune response against WSSV infection. To characterize the roles of Ago1 isoforms in the antiviral immunity, shrimp were injected with WSSV and isoform-specific siRNAs. Shrimp were injected simultaneously with WSSV and the low or high concentration of Epigenetic Reader Domain Ago1A-siRNA (A), Ago1B-siRNA (B), Ago1A/B-siRNA (C) or Ago1C-siRNA (D), respectively. As control, WSSV+control siRNA and WSSV only were included in the injections. At 48 h post-inoculation, the shrimp from each treatment were subjected to quantitative real-time PCR to quantify the expressions of 
Ago1A, Ago1B, and Ago1C (left). The solutions used for injections were shown in the box. At the same time, the WSSV loads in shrimp were monitored by quantitative real-time PCR (right). The statistically significant differences between treatments were represented with asterisk (*P,0.05). Lane headings showed the solutions used for injections. doi:10.1371/journal.pone.0050581.g(0 h post-inoculation) (Fig. 4B). Taken together, these results indicated that Ago1A and Ago1B isoforms that contained the Ago1-fragment 2 played important roles in shrimp antiviral immunity.Effects of Ago1 Isoforms on Shrimp Antiviral ImmunityTo investigate the roles of Ago1 isoforms in antiviral immunity, the expression of Ago1 isoforms were each silenced in shrimp using isoform-specific siRNAs, followed by WSSV challenge. First, to test the specificities of Ago1 isoform-specific siRNAs, FLAGtagged Ago1 isoform constructs and isoform-specific siRNAs were transfected into S2 cells. Western blot analysis showed that the expression of Ago1A, Ago1B or Ago1C isoforms was inhibited by the corresponding sequence-specific Ago1A-siRNA, Ago1BsiRNA.Rimp, qRT-PCR was conducted using isoform-specific primers and probes. The isoform Ago1C was shown to be ubiquitously expressed in all organs or tissues examined (Fig. 4A). However, the mRNA levels of both Ago1A and Ago1B were significantly up-regulated in lymphoid organ (Fig. 4A), suggesting that the two isoforms were involved in shrimp immunity. Considering that Ago proteins represent key components in antiviral RNAi immunity in many species [12,13,14,15], the effects of WSSV infection on the expression of Ago1 isoforms was investigated. Because of the up-regulation of Ago1A and Ago1B 25033180 isoforms in shrimp lymphoid organs, the lymphoid organ was selected to investigate the expression profiles of Ago1 isoforms in response to WSSV challenge. It was showed that the expression of Ago1A and Ago1B was significantly increased at 12 and 24 h postinoculation (p,0.05) (Fig. 4B), whereas the expression of Ago1C remained unchanged at all time points compared to the controlResults Identification of Ago1 Isoforms in ShrimpBased on PCR amplification using degenerated primers and RACE analysis, full-length cDNA of the shrimp Ago1 gene was obtained. Sequence analysis revealed that the Ago1 gene generated three transcripts: Ago1A (GenBank accession no.: GU265732), Ago1B (GenBank accession no.: JX170715) and Ago1C (GenBank accession no.: JX170716). Sequence comparisons showed that the Ago1 isoforms were different from each other with a 9-nt inserted fragment (Ago1-fragment 1) at their 59 termini and an additional 81-nt fragment of (Ago1-fragment 2) in the PIWI domain (Fig. 1). No amplification was observed when the extracted RNA was used in PCR analyses. These findingsRole of Argonaute-1 Isoforms in Antiviral DefenseRole of Argonaute-1 Isoforms in Antiviral DefenseFigure 6. Roles of Ago1 isoforms in the shrimp immune response against WSSV infection. To characterize the roles of Ago1 isoforms in the antiviral immunity, shrimp were injected with WSSV and isoform-specific siRNAs. Shrimp were injected simultaneously with WSSV and the low or high concentration of Ago1A-siRNA (A), Ago1B-siRNA (B), Ago1A/B-siRNA (C) or Ago1C-siRNA (D), respectively. As control, WSSV+control siRNA and WSSV only were included in the injections. At 48 h post-inoculation, the shrimp from each treatment were subjected to quantitative real-time PCR to quantify the expressions of Ago1A, Ago1B, and Ago1C (left). The solutions used for injections were shown in the box. At the same time, the WSSV loads in shrimp were monitored by quantitative real-time PCR (right). The statistically significant differences between treatments were represented with asterisk (*P,0.05). Lane headings showed the solutions used for injections. doi:10.1371/journal.pone.0050581.g(0 h post-inoculation) (Fig. 4B). Taken together, these results indicated that Ago1A and Ago1B isoforms that contained the Ago1-fragment 2 played important roles in shrimp antiviral immunity.Effects of Ago1 Isoforms on Shrimp Antiviral ImmunityTo investigate the roles of Ago1 isoforms in antiviral immunity, the expression of Ago1 isoforms were each silenced in shrimp using isoform-specific siRNAs, followed by WSSV challenge. First, to test the specificities of Ago1 isoform-specific siRNAs, FLAGtagged Ago1 isoform constructs and isoform-specific siRNAs were transfected into S2 cells. Western blot analysis showed that the expression of Ago1A, Ago1B or Ago1C isoforms was inhibited by the corresponding sequence-specific Ago1A-siRNA, Ago1BsiRNA.
