Molecular cloning of crustacean hyperglycemic hormone (CHH) family members (CHH, molt-inhibiting hormone and mandibular organ-inhibiting hormone) and their expression levels in the Jonah crab, Cancer borealis

Abstract

The crustacean hyperglycemic hormone (CHH) neuropeptide family has multiple functions in the regulation of hemolymph glucose levels, molting, ion, and water balance and reproduction. In crab species, three neuroendocrine tissues: the eyestalk ganglia (medulla terminalis X-organ and -sinus gland = ES), the pericardial organ (PO), and guts synthesize a tissue-specific isoforms of CHH neuropeptides. Recently the presence of the mandibular organ-inhibiting hormone (MOIH) was reported in the stomatogastric nervous system (STNS) that regulates the rhythmic muscle movements in esophagus, cardiac sac, gastric and pyloric ports of the foregut. In this study, we aimed to determine the presence of a tissue-specific CHH isoform in the Jonah crab, Cancer borealis using PCR with degenerate primers and 5′, 3′ rapid amplification of cDNA ends (RACE) in the ES. PO, and STNS. The analysis of CHH sequences shows that C. borealis has one type of CHH isoform, unlike other crab species. We also isolated the cDNA sequence of molt-inhibiting hormone (MIH) in the ES and MOIH in the ES and STNS. The presence of CHH, MOIH and MIH in the sinus gland of adult females and males is confirmed by using a dot-blot assay with the putative peaks collected from RP-HPLC and anti-Cancer sera for CHH, MIH, and MOIH. The present of crustacean female sex hormone (CFSH) in the sinus gland of adult females was examined with a dot-blot assay with anti-Callinectes CFSH serum. Levels of CHH, MOIH, and MIH in the sinus gland and their expressions in the eyestalk ganglia are estimated in the adult males, where CHH is the predominant form among these neuropeptides.