Assessing genomic difference in virally infected and healthy Penaeus stylirostris shrimp using representational difference analysis

The global aim ofthis research is to elucidate the role of retrotransposons in stress and immune response. As cloning and sequencing of genes has become commonplace, researchers are finding that the insertion and activity of transposable elements including retrotransposons may directly influence phenotype. There is now evidence that external as well as internal stress, such as that caused by pathogens, will activate the movement of retrotransposons within the genome and this movement may play a role in the organism's immune response (Wessler 1996; Grandbastien 1998). An understanding of the mechanisms behind this movement and determining the identity of the genes that are disrupted due to retrotransposon insertion will lead us to an understanding of the genes involved in the immune pathway. Preliminary data indicates that stress may also induce retrotransposon expression in shrimp (Penaeus stylirostris) making this an informative system for further investigation. In a previous study, genetic markers were identified for infectious hypodermal and hematopoietic virus (IHHNV) resistant and susceptible shrimp populations (Hizer et al. 2002). Sequence characterization of two of the markers revealed partial coding regions of the reverse transcriptase gene for retrotransposable elements. In addition, increased expression activity and movement of the retrotransposon in the virally infected shrimp was observed using real-time PCR. Therefore, the overall goal of this project is to isolate the full-length retrotransposable element and to determine its genomic and expression activity in response to viral stress. The specific objectives of this study are: to identify the full non-long terminal repeat (L TR) element; to identify differences in number of elements present between healthy and virally challenged shrimp; to determine if these elements lie within important genes; and to determine if activation of these elements is important in viral pathogenesis. These studies will increase the limited body ofknowledge ofnon-LTR retrotransposons role in response to stress. Specifically, it will begin to address whether transposition events are consequential in stress immune response to viral pathogenesis. Key Words: Representational Difference Analysis, Penaeus stylirostris, IHHNV, Retrotransposon