Expression of a serine proteinase homolog prophenoloxidase-activating factor from the blue crab, Callinectes sapidus

Tanning, or sclerotization, of crustacean cuticle provides initial reinforcement by cross linking cuticular proteins attached to the cuticle chitin-fiber matrix. This process is catalyzed in part by phenoloxidase, which is under the control of a serine protease activation cascade. The cDNA of a prophenoloxidase-activating factor (PPAF) was cloned and sequenced from the hypodermal tissue of the blue crab, Callinectes sapidus. It codes for a serine proteinase homolog containing a single clip domain. If it is involved in sclerotization, its transcription might be expected to be molt-cycle related. Expression patterns were determined by quantitative PCR and Northern blotting in hypodermis underlying both arthrodial and dorsal (calcifying) cuticles. Transcript levels in pre-molt RNA from both hypodermis types were high, suggesting that the PPAF produced may be incorporated into the pre-ecdysial cuticle layers and then activated at ecdysis to regulate tanning. After a decrease at ecdysis, a second increase in PPAF mRNA occurred at three to four hours post-molt in arthrodial membrane hypodermis but not dorsal hypodermis. This suggests that cuticle deposited after ecdysis may tan in the non-calcifying regions but may not tan where calcification occurs. The PPAF gene is also transcribed at low levels in the hemocytes of intermolt crabs, but not in the hepatopancreas. (c) 2004 Elsevier Inc. All rights reserved.