Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae

Authors: FODOR, JOZSEF - Plant Protection Institute - Hungary; Hull, Joe; KOBLOS, GABRIELLA - Plant Protection Institute - Hungary; JACQUIN-JOLY, EMMANUELLE - Institut National De La Recherche Agronomique (INRA); SZLANKA, TAMAS - Hungarian Academy Of Sciences; FONAGY, ADRIEN - Plant Protection Institute - Hungary

Submitted to: General and Comparative Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2017
Publication Date: 6/1/2018
Citation: Fodor, J., Hull, J.J., Koblos, G., Jacquin-Joly, E., Szlanka, T., Fonagy, A. 2018. Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae. General and Comparative Endocrinology. 258:60-69. https://doi.org/10.1016/j.ygcen.2017.05.024.
DOI: https://doi.org/10.1016/j.ygcen.2017.05.024

Interpretive Summary: For most moth species, adult females attract males by releasing compounds known as sex pheromones. Biosynthesis of these compounds in female pheromone glands is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN), which controls pheromone production by interacting with a specific receptor in pheromone gland cells. Identification and characterization of this receptor’s function in diverse agricultural pests is of great interest as targeted disruption of its functionality has great potential for pest management. In this study, an ARS scientist in Maricopa, AZ collaborated with researchers in Hungary and France to identify and characterize three forms of the PBAN receptor in the cabbage moth, a pest of cruciferous plants as well as other economically important crops. These results provide the first information of this receptor system in this species and will facilitate the development of improved biologically-based management strategies.

Technical Abstract: In most moth species, including Mamestra brassicae, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone production. PBAN acts directly on the pheromone gland (PG) cells via specific G-protein-coupled receptors with calcium as a second messenger. In this study, we have identified cDNAs encoding three isoforms of the M. brassicae PBAN-receptor (Mambr-PBANR) from PG tissue using 5' and 3' RACE. The deduced amino acid sequences of the Mambr-PBANR isoforms have high homology (97-99%) to other members of the PBANR family in heliothine moths. For functional characterization, the full-length coding sequences of the Mambr-PBANR A, B and C isoforms were transiently expressed in cultured Trichoplusia ni cells and Sf9 cells. All three variants dose-dependently mobilized extracellular Ca2+ in response to phermonotropic peptides with Mambr-PBANR-C exhibiting the greatest sensitivity to PBAN with an EC50 value of 26 nM. Fluorescent confocal microscopy imaging studies demonstrated binding of a rhodamine red-labelled pheromonotropin (PT) (RR10CPBAN) ligand to all three Mambr-PBANR isoforms. RR10CPBAN binding did not trigger ligand-induced internalization in cells expressing PBANR-A, but did in cells expressing the PBANR-B and -C isoforms. When co-expressed with a Drosophila melanogaster arrestin homolog (DromeKurtz-mCherry), fluorescent chimeras of the PBANR-B and -C isoforms co-localized with the fly protein following stimulation with Mambr-PT (18 amino acid long fragment) but not with an unrelated peptide, indicating that Mambr-PT is an active ligand. To gain insights into the potential biological functions mediated by the Mambr-PBANRs, we analyzed their tissue expression patterns. In conclusion, all three isoforms were amplified from the M. brassicae PG, with the PBANR-C transcript being the most abundant form.