Production of monoclonal antibody against eyestalk neuropeptide hormones in the black tiger prawn neuropeptide hormones in the black tiger prawn Penaeus monodon

Abstract

Identification of various neuropeptide hormone in the eyestalk of Penaeus monodon were performed by two approaches. Firstly a study of a known neuropeptide family found in various insect the allatostatin (AST) family. Immunohistochemical study on the eyestalk of P. monodon using 4 kinds of anti-allatostatin antisera revealed that AST-like immunoreactivity in various regions; neuronal bodies in the lamina ganglionalis, anterior to the medulla externa (ME) and in the anterior and posterior of the medulla terminalis (MT). Neuronal processes in neuropiles of the ME, MT, sinus gland and nerve fibers in the optic nerve were also recognized. No immunoreactivity in cell bodies or in nerve fibers was found in the medulla interna (MI). Strong AST-like immunoreactivity was found in hundreds of cells of the X- organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X-organ cells of MT in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk. In order to identify unknown neuropeptides in the eyestalk the second approach was performed. Monoclonal antibodies directed against sinus gland neuropeptides were generated from mice immunized with sinus glands dissected from paraffin sections of eyestalks from Penaeus monodon. Selection of peptide specific monoclonal antibodies was done by immunohistochemistry against eyestalk sections. Characterization and specificity of monoclonal antibodies were identified by immunohistochemistey and by dot-ELISA of sinus gland extract separated by one step RPHPLC. Twelve monoclonal antibodies bound to sinus gland and neurons in the eyestalk were isolated. All of these antibodies bound to putative peptides in different fractions of eyestalk extract separated by RP-HPLC. The antibodies were used to monitor the presence of the peptides during purification of sinus gland extract by RP-HPLC using dot-ELISA method. CHHs (Sgp I, III and IV) were isolated and identified with SG 24, 26 and 293 monoclonal antibodies respectively. An unknown peptide with molecular mass of 9127.56 daltons was identified with SG 782 antibody. The N-terminal sequencing of the first 20 residues revealed that the sequence of this peptide is a novel peptide sharing no sequence identity to any known crustacean or vertebrate. Moreover several antibodied bound to the PDH-prepro-related peptide of various peptide were also isolated