TRANT, J.M.; PLACE, A.R.*; Univ. of Maryland Biotechnology Institute, Baltimore; Univ. of Maryland Biotechnology Institute, Baltimore: Production and Analysis of Normalized cDNA Libraries from Blue Crab (Callinectes sapidus) Endocrine Tissues

In order to protect and propagate economically important species of crustaceans (such as the Blue Crab), an understanding of the physiology of molting and reproduction is critical. Our ultimate goal is to devise molecular methods to predict and manipulate these processes in an aquaculture environment. Towards this end, we have generated normalized, unidirectional cDNA libraries of the Y-organ (produces ecdysone which regulates molting), neuroendocrine tissues (X-organ, brain, and thoracic ganglion), and the hepatopancreas (digestive gland where vitellogenesis and xenobiotic metabolism occurs) in order to provide a resource for molecular probes and for gene discovery. Normalization (Trimmer-Direct; Evrogen) of the cDNA libraries (Creator SMART; Clontech) reduced the complexity of each library (826,000, 618,000, and 154,000 cfu, respectively) but the analysis of the first 1,000 clones from the Y-organ shows a redundancy of only 10%. Over 62% of the clones were tentatively identified in GenBank and the remaining 17% are potential new gene products. Only 10% of the sequence data were considered unusable (empty vector, bad sequence, or short insert). These first 1,000 clones yielded 4 cDNAs representing 3 cytochromes P450 putatively responsible for ecdysone synthesis and 3 different �7-pass� transmembrane receptors. Plasmids from over 11,000 colonies (at least 5,000, 5,000, and 1,000 respectively) are being sequenced and subjected to BLAST analysis. Current efforts are focused on the clones representing steroidogenic enzymes and membrane receptors of the Y-organ and members of the CHH family of hormones from the neuroendocrine library. The hepatopancreas will be fully explored in the near future. The results will be posted on the World Wide Web.