Could be concluded that Ago1A and Ago1B isoforms were involved in the host immune response against virus infection, suggesting a novel role of Ago isoforms in shrimp antiviral immunity.DiscussionAgo proteins, the effector molecules of siRNA and miRNA pathways, play crucial roles in RNAi and are involved in many physiological processes. In recent years, many Ago proteins and isoforms have been characterized. However, the roles of Ago isoforms are not clear. The present study showed that there were three isoforms of Ago1 (Ago1A, Ago1B and Ago1C) in shrimp. Sequence alignments indicated that Ago1 sequences of M. japonicus displayed higher sequence similarities to Ago1 proteins than Ago2 proteins of other species. Our study, together with a previous report of the identification of the Litopenaeus vannamei Ago1 and Ago2 [20], suggested that shrimp Ago1 protein likely played a role in miRNA-mediated gene silencing, while shrimp Ago 2 protein was potentially involved in siRNA-mediated antiviral 
defense. Our study showed that most sequences of the three isoforms were identical, but differed at their N-terminal region flanking the PAZ and PIWI domains. As reported, Ago proteins play important roles in host innate antiviral immunity [12,13,14,15]. Therefore, the contributions of Ago1 isoforms to the antiviral immunity of shrimp were evaluated. The results indicated that Ago1A and Ago1B, which contained an additional 81-nt fragment (Ago1-fragment 2) in the PIWI domain, affected the shrimp immune response against WSSV infection. Given the key roles of Ago proteins in the host defense against viruses, it is proposed that the isoforms of Ago might be involved in the fine-tuning of host antiviral responses. It is well known that suppressors of RNAi are widely expressed by viruses to counteract host RNAi immunity. Ago proteins, key components of antiviral RNAi pathways, are likely to represent hotspots of host-virus RE 640 web interactions. In this context, the sequence diversification of Ago1 proteins (Ago1 isoforms) might be a consequence of host adaptive evolution in response to viral threats, which was SIS 3 preserved in shrimp during long-term hostpathogen interactions. Similar to our findings, it was revealed thatRole of Argonaute-1 Isoforms in Antiviral DefenseA. gambiae mosquitoes can employ alternative splicing of Down syndrome cell adhesion molecule (Dscam) immunoglobulin to generate an extremely diverse set of more than 31,000 potential alternative splice forms, which enables mosquito-specific recognition and defense against a broad spectrum of pathogens [21]. It was shown that different pathogen 
taxa induce pathogen challenge-specific splice form repertoires in the adult mosquito [21]. Recently, multiple Ago2 mRNA variants with two types of GRRs in the N-terminal portion were demonstrated in Drosophila [16,17], where the GRRs of the long Ago2 isoform were shown to be essential for the normal function of the protein. An altered number of GRRs in long Ago2 isoform causes defects in RNAi and embryonic development that results in disruption of the midblastula transition from membrane growth to microtubulebased organelle transport [16]. However, the short Ago2 isoform with variant GRR copy numbers do not impair RISC assembly and support normal development [17]. The variation observed in N-terminal domain of short Ago2 isoform in Drosophila remains unclear [17]. In this context, the Ago isoforms revealed in this study represent an important strategy of host immu.Could be concluded that Ago1A and Ago1B isoforms were involved in the host immune response against virus infection, suggesting a novel role of Ago isoforms in shrimp antiviral immunity.DiscussionAgo proteins, the effector molecules of siRNA and miRNA pathways, play crucial roles in RNAi and are involved in many physiological processes. In recent years, many Ago proteins and isoforms have been characterized. However, the roles of Ago isoforms are not clear. The present study showed that there were three isoforms of Ago1 (Ago1A, Ago1B and Ago1C) in shrimp. Sequence alignments indicated that Ago1 sequences of M. japonicus displayed higher sequence similarities to Ago1 proteins than Ago2 proteins of other species. Our study, together with a previous report of the identification of the Litopenaeus vannamei Ago1 and Ago2 [20], suggested that shrimp Ago1 protein likely played a role in miRNA-mediated gene silencing, while shrimp Ago 2 protein was potentially involved in siRNA-mediated antiviral defense. Our study showed that most sequences of the three isoforms were identical, but differed at their N-terminal region flanking the PAZ and PIWI domains. As reported, Ago proteins play important roles in host innate antiviral immunity [12,13,14,15]. Therefore, the contributions of Ago1 isoforms to the antiviral immunity of shrimp were evaluated. The results indicated that Ago1A and Ago1B, which contained an additional 81-nt fragment (Ago1-fragment 2) in the PIWI domain, affected the shrimp immune response against WSSV infection. Given the key roles of Ago proteins in the host defense against viruses, it is proposed that the isoforms of Ago might be involved in the fine-tuning of host antiviral responses. It is well known that suppressors of RNAi are widely expressed by viruses to counteract host RNAi immunity. Ago proteins, key components of antiviral RNAi pathways, are likely to represent hotspots of host-virus interactions. In this context, the sequence diversification of Ago1 proteins (Ago1 isoforms) might be a consequence of host adaptive evolution in response to viral threats, which was preserved in shrimp during long-term hostpathogen interactions. Similar to our findings, it was revealed thatRole of Argonaute-1 Isoforms in Antiviral DefenseA. gambiae mosquitoes can employ alternative splicing of Down syndrome cell adhesion molecule (Dscam) immunoglobulin to generate an extremely diverse set of more than 31,000 potential alternative splice forms, which enables mosquito-specific recognition and defense against a broad spectrum of pathogens [21]. It was shown that different pathogen taxa induce pathogen challenge-specific splice form repertoires in the adult mosquito [21]. Recently, multiple Ago2 mRNA variants with two types of GRRs in the N-terminal portion were demonstrated in Drosophila [16,17], where the GRRs of the long Ago2 isoform were shown to be essential for the normal function of the protein. An altered number of GRRs in long Ago2 isoform causes defects in RNAi and embryonic development that results in disruption of the midblastula transition from membrane growth to microtubulebased organelle transport [16]. However, the short Ago2 isoform with variant GRR copy numbers do not impair RISC assembly and support normal development [17]. The variation observed in N-terminal domain of short Ago2 isoform in Drosophila remains unclear [17]. In this context, the Ago isoforms revealed in this study represent an important strategy of host immu.
