This project is directed towards the derivation of longterm in vitro cultured cell lines from the Pacific whiteleg shrimp, Litopenaeus vannamei, a marine crustacean. This is high-risk research because no established cell lines have yet been derived for any marine invertebrate. This proposed task is of high potential value because the availability of even a single cell line would take research on this organism to a different level than is currently possible, where investigators are restricted to experiments either on whole organisms or on short-term primary cell cultures. A novel and previously undescribed general innate anti-viral immune response, induced by double-stranded RNA (dsRNA), has been described in L. vannamei. The occurrence and biological significance of this immunity has been clearly established in vivo, but without a cell line it will not be possible to investigate and understand the molecular mechanisms underpinning this novel phenomenon. The ability to understand this innate anti-viral reaction is important for two reasons 1) it has the potential to fill a major gap in our understanding of the evolution of anti-viral immunity 2) many viral diseases of shrimp are highly virulent, causing significant economic loss to the aquaculture industry and potentially causing significant damage to the coastal marine ecosystem. Given the importance of crustaceans to the food-web in the ocean, the potential environmental threat that emerging viruses have on wild populations should not be underestimated. The development of long-term shrimp hemopoietic cell lines will be explored by a variety of complementary approaches. The PIs have developed a set of tissue culture media formulations that permit the survival of shrimp cells for over 12 months, but which do not support continuous cell division. Development of these media will be continued so that they better support growth and cell division. A major aspect of this work will be the addition to the medium of purified recombinant L. vannamei homologue of astakine, a cytokine recently isolated from a freshwater crayfish and which has been reported to promote crayfish hemopoietic cell proliferation in tissue culture. In parallel with improved media formulations, the chances of obtaining long-term cell cultures will be enhanced by transforming shrimp hemopoietic cell precursors using plasmids constitutively expressing oncogene products, with the initial studies focusing on SV40 T antigen. Broader Impacts: The broadest impact of the project will be on the field of research in marine crustaceans. The availability of a cell line (or cell lines) will enable researchers in the field to extend their investigations to the cellular and molecular level, which to this point has been very difficult because of the necessity to use whole animal models or shortterm primary cell cultures. The PIs on this application consider both graduate student education and the exposure of undergraduate students to research important components of their research program. Additionally, the PIs will participate in NSF-funded programs for undergraduate students including the NSF-REU program and the MIMES program run by the South Carolina Department of Natural Resources. The MIMES program provides an introduction to research for minority and underrepresented students, and funds summer research internships to undergraduate students from these groups.
