BUDA, E/S*; SHAFER, T/H; University of North Carolina at Wilmington; University of North Calorina at Wilmington: The expression of two genes possibly involved in tanning and calcification of the cuticle in the blue crab, Callinectes sapidus.

Decapod crustaceans, such as the blue crab, Callinectes sapidus, are covered and protected from their environment and potential predators by a hard exoskeleton or cuticle, necessitating the ability to molt in order to grow. The molt cycle and the hardening of the new cuticle are under the control of underlying epithelial cells of the hypodermis that synthesize, release, and activate cuticular proteins. RNA was extracted from the hypodermis of C. sapidus in early premolt. Fragments of cDNA were amplified based on at least partial in-frame homology with a peptide sequence derived from a cuticular protein previously described in our laboratory. This cuticular protein has been hypothesized to be a calcification inhibitor, controlling the timing of mineral deposition by blocking nucleation sites within the cuticle. Two separate coding sequences were identified from the cloned fragments. One showed high sequence homology to an insect prophenoloxidase activating factor (PPOAF). PPOAF is involved in a serine protease cascade needed to activate phenoloxidase (PO). PO then produces quinones that cross link proteins with the chitin matrix to form a reinforced cuticle, thereby functioning in sclerotization. The second sequence shows no significant homology with any accession in GenBank, and, therefore, could code for a novel protein functioning in the control of calcification. Full-length sequences for both genes are being obtained. Expression patterns are being determined from RNA extracted at different molt cycle stages and from hypodermis producing both calcified and uncalcified cuticle.