Functional characterization of chemosensory proteins in Pharsalia antennata with special emphasis on the roles of CSP2 orthologs in pheromone detection.

Behaviorally active compounds of longhorned beetles, including sex/aggregation pheromones and plant odorants, have been extensively applied to the integrated pest management. It is largely unknown, however, how beetle's chemosensory proteins (CSPs) mediate the detection of semiochemicals. Here, we characterized six antenna-enriched PantCSPs in Pharsalia antennata, revealing the strong binding to two floral scents syringaldehyde and β-ionone (dissociation constant, Ki < 5.5 μM). In particular, PantCSP11 presented the binding preference to 11-12C aliphatic alcohols/aldehydes (Ki = 0.96 ± 0.02 μM-5.55 ± 0.11 μM) and 6C green leaf volatiles (Ki = 5.07 ± 0.24 μM-11.60 ± 0.07 μM). It was worth noting that a male-antenna-biased PantCSP2 and its 10 orthologs from other 10 cerambycid species played a crucial role in the detection of sex/aggregation pheromones. This pheromone analogue pseudoionone tuned to by 11 CSP2 proteins (Ki < 9.6 μM) may be developed as the component or synergist of cerambycid generic lures. Further, six PantCSPs and 11 CSP2 orthologs could interact strongly with broad-spectrum insecticides, particularly chlorpyrifos and phoxim (Ki < 4.8 μM). We identified 10 key residues involved in the interactions of MaltCSP3 in Monochamus alternatus and chemicals, and meanwhile demonstrated an essential role of two conserved binding sites (Gln61 and Thr65/Ser65) among CSP2 orthologs in the specific recognition of key chemicals. This study enhances our knowledge in understanding CSP-mediated odorant and pheromone detection in Cerambycidae and provides insights into adaptive evolution of beetles in the context of insecticide use.