|Nachman, Ronald - Ron|
Submitted to: Journal of Peptide Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/14/2014
Publication Date: 8/20/2014
Citation: Nachman, R.J., Kaczmarek, K., Jiang, H., Wei, Z., Zabrocki, J., Park, Y. 2014. A selective CAP2b neuropeptide antagonist for an expressed receptor from the red flour beetle, Tribolium castaneum. Journal of Peptide Science. 20:S162-S163. Interpretive Summary: Insect pests have developed resistance to several conventional pesticides, and new approaches are needed for pest management. Although neuropeptides (short chains of amino acids) serve as potent messengers in insects to regulate vital functions, the neuropeptides hold little promise as pest control agents because they can be degraded in the target pest. New, selective control agents may be developed by designing mimics of these neuropeptides that resist degradation and either inhibit or over-stimulate critical neuropeptide-regulated life functions. We report on the isolation, identification and characterization of an active site for the diapause hormone in the corn earworm, an important agricultural pest. In moths, this hormone regulates the entry into a protective state of dormancy that is critical for winter survival. The work also identifies which life stages of the insect exhibit the largest quantities of this active site. This discovery, and the new information gained from it, will aid in the design and/or discovery of stable neuropeptide-like compounds that can block or over-stimulate this active site and prove capable of disrupting the survival mechanisms of these and related moths. This may eventually lead to development of practical neuropeptide-like substances that can effectively control crop pest insects in an environmentally friendly fashion.
Technical Abstract: Diapause hormone (DH) is an insect neuropeptide that is highly effective in terminating the overwintering pupal diapause in members of the Helicoverpa/Heliothis complex of agricultural pests, thus DH and related compounds have promise as tools for pest management. To augment our development of effective DH analogs and antagonists that could be used as diapause disruptors this study focuses on the cloning and identification of the DH receptor (DHR) in the corn earworm, Helicoverpa zea. The full-length dhr cDNA contains 2153 nucleotides encoding 511 amino acids. Our results suggest there are at least two splicing variants of Hezea-DHR. Hydrophobicity analysis and sequence alignment indicate that Hezea-DHR has 7 transmembrane regions and a highly conserved C-terminal region that is also present in related receptors. Hezea-DHR has 95, 82 and 79% identity to a partial DHR sequence from Heliothis virescens, a full-length DHR in Orgyia thyellina, and DHR-1 in Bombyx mori, but only 45-49% identity to pheromone biosynthesis activating neuropeptide receptor (PBANR). Expression of dhr mRNA remained low in whole body extracts throughout diapause and in young nondiapausing pupae, but was distinctly elevated as development ensued in pharate adults 7 days after pupation. The highest expression we noted was in the ovary. Dhr and dhr-egfp were successfully expressed in X. laevis oocytes, as verified by fluorescent imaging and Western blots, but an electrophysiological assay failed to detect receptor-ligand binding activity, which suggests that an essential cofactor and/or accessory protein is required for functional activity of the DHR.