Molting in crustaceans is induced by ecdysteroids as in insects. The ecdysteroid titre in hemolymph is negatively regulated by molt-inhibiting hormone (MIH) that inhibits the secretion of ecdysteroids from the Y-organ, an ecdysteroid-producing gland of crustaceans, whereas little is known about the molecular mechanism of inhibition by MIH. Recently, the Halloween genes encoding cytochrome P450 monooxygenases were characterized as the steroidogenic enzymes in insects. To elucidate whether the ecdysteroidogenesis in the Y-organ is regulated by molt-inhibiting hormone (MIH), we analyzed the expression level of an orthologue of a member of the Halloween genes, phantom (Cyp306a1, phm), in the Y-organ of a decapod crustacean, Marsupenaeus japonicus. A cDNA encoding phm (Mj-phm) was cloned by degenerate PCR and 5'- and 3'-RACEs. The deduced amino acid sequence of Mj-phm showed about 40% identity to those of insect phm. The six motif sequences and the four substrate recognition sites were well conserved between Mj-PHM and other PHM. RT-PCR showed the specific expression of Mj-phm mRNA in the Y-organ. In addition, quantitative real-time PCR verified that the expression level of Mj-phm was significantly increased at the pre-molt stage and decreased after ecdysis. Furthermore, exposure of the Y-organ to MIH significantly decreased the Mj-phm expression level in vitro. These results indicate that the transcription of Mj-phm in the Y-organ may be regulated by the inhibitory mechanism of MIH of M. japonicus, which involves the consequent negative regulation of ecdysteroidogenesis at the transcriptional level.
(c) 2009 Wiley Periodicals, Inc.