Contribution of the Caspase Gene Sequence Diversification to the Specifically Antiviral Defense in Invertebrate Bin Zhi2, Lei Wang2¤, Guangyi Wang 3 , Xiaobo Zhang 1 * 1 Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou, The People’s Republic of China, 2 Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, The People’s Republic of China, 3 Department of Oceanography, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America Abstract Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates’ innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221–229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response. Citation: Zhi B, Wang L, Wang G, Zhang X (2011) Contribution of the Caspase Gene Sequence Diversification to the Specifically Antiviral Defense in Invertebrate. PLoS ONE 6(9): e24955. doi:10.1371/journal.pone.0024955 Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America Received February 20, 2011; Accepted August 24, 2011; Published September 19, 2011 Copyright: 2011 Zhi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was financially supported by National Natural Science Foundation of China (30830084), Hi-Tech Research and Development Program of China (863 program of China) (2010AA09Z403), Project of Ministry of Agriculture, China (201103034) and Chinese Universities Scientific Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zxb0812@zju.edu.cn ¤ Current address: Genomics and Genetics Division, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Introduction The non-specific innate immunity of invertebrates, including their humoral defenses and their cellular defenses such as phagocytosis and apoptosis, is the sole mechanism for them to defend themselves against invading pathogens [1–3]. They achieve these defenses through reorganizing pathogen-associated molecu- lar patterns of the host’s cell-surface proteins [4,5]. Recently, model systems such as Drosophila melanogaster and Caenorhabditis elegans have enabled researchers to learn much about this innate immunity [6–9]. Nevertheless, our understanding of the innate immunity of invertebrates against a variety of noxious microbial pathogens remains largely incomplete. Several lines of evidence indicate that invertebrates exercise some degree of specificity against microbial pathogens and that the specificity likely results from diversifying the sequence of their immune genes [10–13]. The sequence diversification of the somatic genes, which encode recognition molecules, effector-enhancement molecules and other immune molecules, has been postulated as contributing to the immune specificity of invertebrates [14]. The biological signifi- cance of gene sequence diversification in understanding the innate immunity of invertebrates is similar to the significance of diversifying the receptors of the adaptive immunity system in vertebrates. Furthermore, the pre-existing host-defense mutation mechanism is simply co-opted and enhanced in the novel context of obligatory combinatorial diversification [4]. Clearly, under- standing the sequence diversification of immune genes in innate immunity may provide critical information about defending invertebrates against microbial pathogens. Programmed cell death (i.e., apoptosis), as the major component of the cellular defense mechanism, is an essential immune response to protect invertebrates from pathogen infection [15–17]. When pathogen-infected cells undergo apoptosis, the pathogens duplicated in these cells are not able to diffuse and infect other cells. Thus, apoptosis can significantly improve the anti-pathogen response of invertebrates. Caspases, a family of structurally-related cysteine proteases, contain two different subgroups that play essential roles in apoptosis: initiator caspases and effector caspases [18]. The initiator caspases (caspase-8, -9 and -10) can be triggered by either intracellular or extracellular stimuli, and are activated by a complex course of autocatalytic processing or conformational change. The activated initiator caspases specifically activate effector PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24955