Dual function of the moult-inhibiting hormone (MIH) in the blue crab gives new perspective for the established role of MIH action

The neuroendocrine processes that regulate crustacean moulting and reproduction are multifactorial and dependent on the interaction of several endocrine organs and their products. Identified initially as a neuropeptide belonging to the crustacean hyperglycaemic hormone superfamily, the moult-inhibiting hormone (MIH) is a key member of the subset Type-II hormones playing an active role in the regulation of moulting. However more recently, additional roles for MIH have been suggested. The purpose of the present study was to ascertain the functional significance of the putative MIH gene and determine if MIH has a divergent role other than inhibiting the activity of ecdysteroids. We employ methodology central to functional genomics ̶ RNA interference (RNAi), and protein expression as determined by RT-PCR, reverse phase- HPLC, MALDI-TOF mass spectroscopy, SDS-PAGE, western blot analysis and immunohistochemistry to answer these questions, and use the blue manna crab Portunus pelagicus as our model. This species which has significant commercial value to aquaculture and emerging Asian markets is typical of other crab species and is ideal for determining the established mode of MIH action. A MIH (PpelMIH) gene encoding for an 8.57kDa protein was isolated. Subsequent characterization of PpelMIH by dsRNA mediated gene silencing over a 5 day period found that there was a significant reduction of the number of PpelMIH transcripts. A direct correlation to PpelMIH gene silencing to Ppel-MIH protein production and its effect on gonad maturation was also assessed through the detection of vitellin in ELISA and immunofluroscent assays. Clearly as a consequence of dsRNA mediated silencing, Ppel-MIH has a direct involvement in regulating both MIH and vitellin levels. This suggests that MIH could obtain both a functional role on ecdysteroid production, but also could be a putative gonad stimulating hormone, due to its involvement in upregulating vitellogenin levels in non-treated crabs, but decreases post dsRNA mediated silencing