Molecular cloning of the crustacean hyperglycemic hormone (CHH) precursor from the X-organ and the identification of the neuropeptide from sinus gland of the Alaskan Tanner crab, Chionoecetes bairdi

摘要

Crustacean hyperglycemic hormone (CHH) secreted from sinus glands primarily elicits hyperglycaemia in crustaceans. CHH is particularly important for energy metabolism during environmental and physiological stress as animals switch to anaerobiosis. CHH has been purified from multiple brachyuran crab species to date, but not from the cold water Tanner crab, Chionoecetes bairdi, a species found in Alaskan coastal waters. The purpose of molecular cloning the C. bairdi CHH precursor and identification of its neuropeptide form in sinus glands is to establish tools to further study cold water crab metabolic physiology. Cold water crabs such as those in the genus Chionoecetes are a good model for understanding the role that climate change and associated water temperature changes might have on metabolic physiology. CHHs in sinus glands of C. bairdi were purified using reverse-phase HPLC and were identified as CHH with an enzyme-linked immunosorbent assay (ELISA) using cross-reacting Callinectes sapidus and Carcinus maenas CHH antisera. The bioactivity of CHH was further assessed using a homologous assay by injecting CHH into eyestalk ablated C. bairdi and measuring subsequent rise in circulating glucose. The full length cDNA (1944 bp) of C. bairdi CHH was determined by PCR using degenerate primers cloning and 5′, 3′ rapid amplification of cDNA ends (RACE). A phylogenetic analysis of deduced amino acid sequences from six brachyuran crab species showed C. bairdi CHH most closely related to the majid crab, Libinia emarginata (P55688). Future studies will enable us to compare metabolic physiology and requirements of cold water C. bairdi with the warm water crab C. sapidus.