Cloning Crustacean Hyperglycaemic Hormone (CHH) in the Dungeness Crab, Metacarcinus magister

The focus of this experiment is to clone the Crustacean Hyperglycemic Hormone (CHH) gene in Dungeness crab, Metacarcinus magister, and learn how CHH regulates molting in response to temperature. CHH is a neuropeptide synthesized in the X-organ/sinus gland complex (XO/SG) that regulates molting, carbohydrate metabolism, water and ion flux, as well as gonad development. The CHH family motif is highly conserved across crustaceans and includes a 28 amino acid signal peptide, a 42 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP), and a 72-74 amino acid mature hormone. The mature CHH hormone includes six cysteines, two arginines, one aspartate, one phenylalanine, and one asparagine. Juvenile Dungeness crab were exposed to temperatures ranging from 5-30° C. cDNA was created by extracting RNA from eye stalks and using reverse transcriptase enzyme to create cDNA from the RNA. The cDNA was then amplified using Polymerase Chain Reaction (PCR) and extracted after gel electrophoresis was run. Most of the mature CHH nucleotide sequence has been deduced and shows high sequence identity with the shore crab, Carcinus maenas. 54 amino acids have been identified based on the nucleotide sequence.