Functional characterization of CAPA receptors and their ligand specificity in the corn earworm, Helicoverpa zea

CAPA peptides play a crucial role in regulating water balance in insects, with their receptors typically expressed in the Malpighian tubules or midgut of various insect species. However, CAPA-mediated signaling remains poorly characterized in Helicoverpa zea, one of the most destructive crop pests in the United States. We first cloned two CAPA receptor genes from H. zea and investigated their tissue expression patterns. Among eight different larval tissues, CAPA-R1 predominantly expressed in the foregut and larval testis, while CAPA-R2 showed higher expression in the midgut and hindgut, with CAPA-R2 exhibiting overall higher expression levels compared to CAPA-R1. Using a calcium-influx assay on Sf9 cells expressing each of these receptors, we tested their binding affinities with over a dozen different PRXamide peptides. Our results demonstrate that CAPA-R1 is specifically activated by CAPA1 (DGVLNLYPFPRVa), whereas CAPA-R2 responds strongly to both CAPA1 and CAPA2 (QLYAFPRVa). Notably, N-terminal truncated variants of CAPA2 peptide, including the hexapeptide (YAFPRVa) and heptapeptide (LYAFPRVa), activated CAPA-R2 with comparable efficiency to the full-length octapeptide. In contrast, pyrokinin peptides (PBAN, PGN-7, PGN-18, PGN-8, and DH) failed to significantly activate either receptor, while homologous CAPA peptides from Drosophila showed substantial activation. This functional characterization of CAPA receptors and their corresponding ligands in H. zea provides valuable insights into neuropeptide signaling in this pest species and identifies potential molecular targets for the development of novel pest management strategies.