caspases (caspase-3, -6 and -7) to initiate the apoptotic process, resulting in an intricate cascade of events including interactions among several families of proteins, i.e., caspases, Bcl-2 family proteins and inhibitors of apoptosis proteins. Among these proteins, caspase-8 is required for all death receptor–mediated apoptotic pathways; caspase-3 has been recognized as the crucial executioner caspase, influencing the impact of a given apoptotic stimulus. The caspase gene PjCaspase of the shrimp Marsupenaeus japonicus, showing a sequence similarity to the caspase-8 of other species, is an initiator gene that is triggered by cell signals during the shrimp’s hemocytic apoptosis to defend against virus infection [16]. The shrimp possesses an open circulatory system, in which the hemocytes (circulating cells) can be analogized functionally to vertebrate leukocytes [19]. Shrimp are economically-important marine invertebrate [20], but in recent years, white spot syndrome virus (WSSV) has become a major pathogen that is devastating the shrimp farming industry worldwide. The WSSV is an enveloped, large, double-stranded DNA virus with an obovate morphology and a single filamentous appendage. Elucidating shrimp’s immunity to WSSV would aid greatly in finding how to treat shrimp against this disease. In this study, our results indicated that the PjCaspase gene possessed sequence diversity. Analysis of the gene sequence diversities of the virus-free and the WSSV-resistant shrimp showed that the PjCaspase gene containing a specific sequence at position 661 bp–687 bp (referred as fragment 3). Fragment 3 was enriched in WSSV-resistant shrimp, modulating virus-induced apoptosis. Furthermore, the results of in vivo experiments showed that the PjCaspase gene containing fragment 3 was specifically responsible for the hemocytic apoptosis induced by the virus and specifically took effect on the innate immune response against virus infection. Overall, our results indicated that the PjCaspase gene sequence diversity protected the shrimp against WSSV infection. This study represented the first report that shrimp employed gene sequence diversification as immunity to defend against virus infection. Results Identification of the PjCaspase gene sequence diversification Two classes of caspases, designated the initiators and the effectors, are the central components of the apoptotic response. PjCaspase, classified as the initiator of apoptosis, affects the immuno-possession in shrimp [16]. Our sequence analysis showed that the PjCaspase cDNAs that derived from the same pool of the same RNA template of a single shrimp presented a much higher frequency of somatic base mutations than the sequence analysis of the actin gene, a house-keeping gene, which we used for comparison (Fig. 1a, Figs. S1 and S2) (GenBank accession numbers GU645234,GU645341). We found a higher frequency of base mutations of the PjCaspase gene in all the tissues and organs we sequenced. In total, the frequency of base mutation was 0.56% for the PjCaspase gene and 0.02% for the actin gene (Fig. 1a, up). When comparing the PjCaspase cDNAs from the same RNA template of an individual shrimp (Shrimp 1), we found that the PjCaspase cDNAs in a tissue or organ were completely different from each other due to base mutations, while most of the actin cDNAs were consistent (Fig. 1a, down). This phenomenon existed in all the tissues or organs we examined (Fig. 1a, down). Further sequence analysis indicated that the somatic mutations in the PjCaspase cDNA sequences yielded no stop codons in the PjCaspase open reading frame (ORF), except for 3 cDNAs (Figs. S1 and S2). The data from control experiments excluded experimental artifacts caused by reverse transcription and PCR amplification Gene Sequence Diversity for Antiviral Immunity (Fig. 1a). Therefore, our results suggested the existence of the cDNA sequence diversity of PjCaspase gene, a phenomenon widely revealed in invertebrates [21]. Cluster analyses using the partial sequences of PjCaspase cDNAs classified these cDNA sequences into 2 and 3 clusters at the nucleotide and amino acid levels, respectively (data not shown). The statistical analyses of these clusters indicated that the sequence diversification of PjCaspase cDNA in the shrimp was not organ- or tissue-specific. To explore the difference of gene sequence diversity between various individuals, the PjCaspase cDNAs from another individual shrimp were sequenced (Shrimp 2) (GenBank accession numbers GU645342,GU645436) (Fig. S2). The cluster analyses yielded essentially similar results as those of Shrimp 1, showing no difference in the cDNA sequence diversity of the PjCaspase gene among these two individual shrimp. Because the sequences analyzed came from an individual shrimp, the sequence diversity revealed in this study should have been derived from somatic mutations, rather than from polymorphism among individuals. The Southern blot analysis of the PjCaspase gene using the shrimp genomic DNA was conducted with a specific DIG-labeled probe (Stringency: 0.03 pg/ml) which could detect all the mutations of the PjCaspase gene. The results showed that the Southern blots yielded only one band (Fig. 1b), suggesting that the gene had a single copy in the shrimp genome. Thus, the sequence diversification did not result from the copy variation of the PjCaspase gene. To determine whether the sequence diversification of the PjCaspase gene occurred in the genomic DNA, 44 clones containing the genomic DNA of the PjCaspase gene were sequenced (Fig. S3). The clones were obtained by PCR from the same pool of the DNA template of a single shrimp. The results showed that the sequences of the 44 genomic DNA clones of the PjCaspase gene were different from each other due to dot mutations (Fig. S3a). On the other hand, the genomic DNA sequences of the actin gene were identical (Fig. S3b). The frequency of base mutation was 1.58% for the PjCaspase gene and 0.01% for the actin gene. These results indicated that the sequence diversification of the PjCaspase gene occurred at the genomic DNA level. Effects of PjCaspase gene sequence diversity on virus- induced apoptosis Gene sequence diversity has been reported in invertebrates [21]. However, the role of this diversity has not yet been addressed. The existence of gene sequence diversity of the key gene (PjCaspase) in antiviral apoptosis led to interest in its role in immunity. When the shrimp was invaded by various pathogens, would the products of the diversified PjCaspase genes play roles in the immune defenses to initiate the apoptosis against different pathogens? To address this issue, the diversities of the PjCaspase gene sequence from WSSV- free shrimp (shrimp 1 and shrimp 2) and WSSV-resistant shrimp (GenBank accession numbers GU645437,GU645531) were compared. The WSSV-resistant shrimp were adapted to the WSSV-rich environment, with special resistance against WSSV infection [2]. Sequence comparisons revealed that some fragments of the ORF of the PjCaspase gene contained base mutations which were present only in the WSSV-resistant shrimp (Figs. S1, S2 and S3). This information suggested the potential involvement of these fragments in the virus-induced apoptosis against virus infection. To reveal the roles of these fragments in apoptosis against virus, the fragment-sequence-specific siRNAs were used to manipulate the expression of the PjCaspase gene separately containing these fragments, followed by WSSV- or Vibrio parahaemolyticus (a pathogen of shrimp)-induced apoptosis assays. The results showed that the PjCaspase gene containing a specific fragment (661–687 nt) (Fig. 2a) (fragment 3) displayed the highest apoptotic activity. At PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24955