Molecular Cloning, Expression, and Tissue Distribution of Crustacean Molt-Inhibiting Hormone

In crustaceans, secretion of ecdysteroid molting hormones by Y-organs is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by the X-organ/sinus gland complex of the eyestalks. The current review considers recent research on MIH, with a primary focus on MIH of brachyurans (crabs). New data on the production of recombinant MIH (rMIH) are also included. Available data indicate the MIH gene of brachyurans encodes a 113 amino acid prohormone composed of a 35 residue signal peptide and a 78 residue mature MIH. The primary structure of MIH is highly conserved among brachyurans. The MIH transcript is detectable in eyestalk neural ganglia throughout the molt cycle of the blue crab, Callinectes sapidus. Stage-specific changes in the abundance of MIH mRNA in C. sapidus eyestalks are generally consistent with the hypothesis that MIH negatively regulates ecdysteroid production during the molt cycle. MIH transcripts have also been detected in the brain of two species. Recombinant MIH was produced using prokaryotic (pET vector/Escherichia coli) and eukaryotic (baculovirus/insect cells) expression systems. Recombinant MIH produced in E. coli was of the predicted size and was MIH immunoreactive; it did not have MIH bioactivity. Polyclonal antisera raised against the prokaryotically expressed rMIH bound specifically to neurosecretory cells in the X-organ, their associated axons, and axon terminals in the sinus gland. Recombinant MIH expressed using the baculovirus system was of the predicted size, was MIH immunoreactive, and inhibited ecdysteroid production by Y-organs in vitro.