Gene Sequence Diversity for Antiviral Immunity Figure 4. The role of PjCaspase gene containing fragment 3 in immunity against virus infection. The PjCaspase gene containing fragment 3 was respectively silenced by PjCaspase fragment 3-specific siRNA (siRNA 3) or overexpressed by injection of the in vitro synthesized PjCaspase fragment 3 mRNA (mRNA 3), followed by infection with WSSV. At different stages after virus infection (24 h, 36 h and 48 h), the shrimp hemolymph was collected and subjected to quantitative real-time PCR to monitor the WSSV replication. The shrimp used in the assays were WSSV-sensitive. All the assays were biologically repeated for three times. Each column indicated the mean of triplicate assays, with the standard deviation shown as a bar. Row headings represent the solutions used for injections. doi:10.1371/journal.pone.0024955.g004 Discussion Vertebrate adaptive immunity is achieved through the vast diversity of somatically rearranged immunological receptors, such as antibodies, to initiate the specific immune response against different invading pathogens [22]. Whether invertebrates are capable of a comparable phenotypic plasticity and specificity has long been a matter of debate. The results of this study on the function of gene sequence diversity in invertebrate immunity shed light on this issue. Based on the results derived from this study, a model for the role of PjCaspase gene sequence diversity in innate immunity was proposed (Fig. 5). In this model, the apoptosis caused by Vibrio was not included. The shrimp’s pre-existing host-defense mutation mechanism produced the PjCaspase genes with different base mutations, resulting in the PjCaspase gene sequence diversity. Among the diversified PjCaspase genes, there was a special PjCaspase molecule containing fragment 3 which existed in a group of cells. When challenged by various pathogens, such as viruses and bacteria, the PjCaspase gene containing fragment 3 responded to the threat of a WSSV virus infection by specifically initiating the apoptosis which inhibited the WSSV infection. The PjCaspase protein, an initiator in the caspase cascade, was required as part of the apoptotic pathway of shrimp [16]. Recent studies revealed that the WSSV perturbed the function of shrimp caspase as part of its infection cycle [23,24]. Therefore, we can infer that the sequence diversity of the PjCaspase gene was employed as a very efficient immune strategy to defend against various invading pathogens. In this system, the diversified PjCaspase molecules (gene sequence diversity) responded to different patho- gens through its death signal receptor. The hypothesized mecha- nism might be similar to the immunoglobulin diversification in vertebrate immunity. Considering the broad existence of sequence diversity of immune-related genes in invertebrates [21, 25 and this study], gene sequence diversity might play very important roles in invertebrate immune reaction against virus infection. The gene sequence diversification in invertebrates could ensure a broad defense to viruses, bacteria and parasites. Finding this immune feature in invertebrates should prompt us to revisit the artificial dichotomy between specific and non-specific immune systems. Unusual genetic mechanisms for diversifying recognition proteins might be a widespread characteristic of animal immunity [26]. The gene sequence diversity employed by invertebrates as an efficient immune strategy against various pathogens might have developed through evolution. Darwin’s theory of evolution tells us that when random genetic mutations occur within an organism, the organism preserves the beneficial mutations because they aid in survival. Among the cells of an individual invertebrate, random genetic mutations (i.e. gene sequence diversity) existed in some key genes, such as the PjCaspase gene revealed in this study. When various pathogens invaded the shrimp, the diversified PjCaspase genes initiated the apoptosis as an immune defense against virus infection. In the absence of acquired immunity, therefore, the shrimp achieved immunity against different pathogens through the vast variety of somatically diversified, pathogen-specific immunological gene se- quences. The gene sequence diversity might not function as a pathogen recognition mechanism like immunoglubin, but instead as a system that avoided pathogen interference in the immune response. Immunity studies in vertebrate and invertebrate species suggest that the invertebrate defense mechanisms may be precursors of vertebrate immunity. The diversity of gene sequences may be an ancient immune response against virus infection. Materials and Methods Shrimp culture, white spot syndrome virus (WSSV) infection of shrimp and WSSV-resistant shrimp Marsupenaeus japonicus shrimps, about 10 g and 10–12 cm in length, were obtained in Xiamen, Fujian province of China and raised in groups of 20 individuals in 80-liter aquariums filled with air- PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24955