ISOLATION OF EXPRESSED SEQUENCE TAGS (ESTs) FROM TAURA SYNDROME VIRUS (TSV)-CHALLENGED SHRIMP (Litopenaeus vannamei) BY mRNA DIFFERENTIAL DISPLAY

¹ Department of Environmental and Population Health, Tufts University School of Veterinary Medicine, North Grafton, MA 01536

² Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan

³ National Institute of Agrobiological Resources, Tsukuba 305-8602, Japan

⁴ The Oceanic Institute, Makapuu Point, 41-202, Kalanianaole Highway, Waimanalo, Hawaii 96795

⁵ Gulf Coast Research Laboratory, Institute of Marine Sciences, University of Southern Mississippi, Ocean Springs, MS 39566-7000

Cultured juvenile L. vannamei shrimp (~1.5g) from 18 family crosses were challenged with TSV and showed variable survival rate (4.2%-87.5%). The mRNA differential display (mRNADD) technique was used to display the differences in gene expression between the animals from the highest and lowest percent survival crosses (Cross #10 and #2, respectively). These differences were examined at 0h, 6h, 12h, 24h and 36h with the hope of obtaining expressed sequence tags (ESTs) that could be used to map the QTL responsible for TSV. Forty-one bands representing potential differentially expressed cDNAs were cloned, 27 from Cross #10 and 14 from #2. A total of 84 plasmid clones representing 41 bands were sequenced and 49 distinct cDNAs were obtained. The cDNA sizes ranged from 43 to 294 bp. Thirty eight out of 49 sequences were weakly (<70%) similar to genes of various organisms, eight were weakly similar with unidentified genes, two were moderately (70-90%) similar to known genes and one was highly (>90%) similar to a known sequence. Fourteen (35%) out of the 40 cDNAs with similarity to known genes were structural genes, 10% were potential immune response genes, 7.5% were other virus-related genes, 5% were mitochondrial genes, and 47.5% were potential regulatory factors (oncogenes, growth factors, DNA binding elements, etc.). Thirteen out of the 49 sequences contained microsatellites, 12 of them contained enough flanking sequences to design primers, making them useful for genome mapping. The results suggest mRNADD is a useful method to rapidly isolate differentially expressed genes after viral infection in shrimp.