Neuroendocrine regulation of crustacean molting: Studies of the molt-inhibiting hormone of the blue crab Callinectes sapidus

The physiological mechanisms regulating crustacean growth and molting are not well understood. Molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk neural ganglia, negatively regulates the secretion of ecdysteroids by crustacean Y-organs. The overall goal of this study was to further examine the role of MIH in the neuroendocrine regulation of the molt cycle of the blue crab, Callinectes sapidus. The objectives were (1) to quantify MIH mRNA levels and hemolymph ecdysteroid titers during a molt cycle of C. sapidus, (2) to generate a functional recombinant MIH protein using a baculovirus/insect cell expression system, and (3) to develop antibody probes for use in an immunoassay and for identifying sites of MIH synthesis and secretion. Steady-state MIH mRNA levels and hemolymph ecdysteroid levels were determined by Northern blot and radioimmunoassay, respectively, during various stages of the molt cycle. Stage-specific changes in MIH mRNA levels were accompanied by significant fluctuations in the hemolymph ecdysteroid titer. The results were generally consistent with the hypothesis that changes in MIH levels occur during the molt cycle to negatively regulate hemolymph ecdysteroid titers. A recombinant blue crab MIH was generated using a baculovirus/insect cell expression system. Electrophoretic separation and Western blotting of proteins from infected cells revealed time-dependent expression of two MIH-immunoreactive proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature protein. N-terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and mass. The recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. Synthetic peptides designed from specific regions of blue crab MIH were used to generate anti-MIH immune sera. The immune sera were MIH-specific as determined by Western blot and immunocytochemistry. Characterization of changes in the levels of MIH mRNA and circulating protein and the relation of these changes to the developmental condition of the crab should lead to a more detailed understanding of the specific role of MIH in regulation of crustacean molting.