Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Abstract

Heptapeptide (YANAVQV-NH 2=T−) and octapeptide (YANAVQṮV-NH 2=T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T− antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37–39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T− antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24±5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34±4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T− antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.