Inhibitory effect of molt‐inhibiting hormone on phantom expression in the Y‐organ of the kuruma prawn, Marsupenaeus japonicus

Abstract

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.

Crustacean Molting: Regulation and Effects of Environmental Toxicants

“…The expression of phm was confined to YO at all stages of molting though minor expression was observed in ovaries at mature stages which was the basis for selecting the phm gene expression as the limitation in the above studies. The results clearly proposed that the levels of phm expression were high during pre-molt stages and expression was suppressed during the intermolt stages, which was due to the inhibitory action of MIH mediated by binding to the receptors on YO [23]. On the other hand the expression of phm in insects was observed in ovary also suggesting that on degradation of pro thoracic gland during metamorphosis the ovary adopts the function of ecdysteroidogenesis [59].…”

“…The 5-reductase that converts 4-diketol to 5-diketol is a cytosolic enzyme in YO cells that requires NADPH for activity [19]. Orthologs of nvd, nmg/sro, spo, phm, dib, sad, and shd have been identified in the Daphnia pulex genome [20][21][22], and a cDNA encoding Phm has been cloned from Kuruma prawn, Marsupenaeus japonicus [23]. Nvd and spo are located adjacent to each other in the D. pulex genome [20].…”

“…The M. japonicus Phm and D. pulex Phm have ExxR, PxxFxPE/DRF, and PFxxGxRxCxG/A heme-binding motif), five of six substrate recognition sites (SRS 1, 2/3, 4, and 5), and N-terminal ER-targeting and Pro/Gly-rich sequences [23]. Mj-Phm is a target of eyestalk neuropeptides, as its expression in the YO is increased as much as 7-fold during premolt and is decreased about 2.5-fold by sinus gland extract and recombinant MIH [23]. None of the crustacean Halloween enzymes have been characterized biochemically.…”

“…Limited results persist regarding the involvement of Halloween genes and their regulation in molting [23]. YO ecdysteroid synthetic pathway suggest that the P450 monooxygenases, encoded by the Halloween genes Phantom (phm), Disembodied (dib), Shadow (sad), and Shade (shd) can bind multiple substrates.…”

“…Further the identification of Halloween gene orthologues in Daphnia ecdysteroidogenic pathway [21] represents the involvement of Halloween genes in molting of crustaceans. Based on the above studies the involvement of phm genes (Member of Halloween gene family) in molting and their expression levels at different molt stages were studied and were observed to regulate in stage specific manner [23]. The expression of phm was confined to YO at all stages of molting though minor expression was observed in ovaries at mature stages which was the basis for selecting the phm gene expression as the limitation in the above studies.…”