Demonstration of the cellular expression of genes encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in the eyestalk of the shore crab Carcinus maenas

Based on the amino acid sequence of the molt-inhibiting hormone of Carcinus maenas, two degenerated oligonucleotide primers were synthesized and used in the polymerase chain reaction. By use of complementary DNA of a library constructed from medulla terminalis-X-organ RNA of C. maenas as template, the specific complementary DNA between the primers was amplified, cloned and sequenced. This strategy revealed a DNA sequence for which the deduced amino acid sequence is identical to the recently published C. maenas molt-inhibiting hormone sequence as determined by Edman degradation. Visualization of messenger RNAs encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in different perikarya of the X-organ was obtained using digoxigenin-labelled complementary RNA probes. Combination of immunocytochemical staining using polyclonal antisera against the native C. maenas neuropeptides and in situ hybridization performed on alternating sections confirmed the specificity of the reaction. The results show that there is no co-localization of molt-inhibiting hormone and crustacean hyperglycemic hormone at the messenger RNA and the protein level.