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.