A Female-Biased Chemosensory Protein PxutCSP19 in the Antennae of Papilio xuthus Tuned to Host Volatiles and Insecticides

Simple SummaryThe swallowtail butterflyPapilio xuthusis a specialized herbivorous insect, with its larvae solely utilizing the Rutaceae plants as hosts. The host seeking and orientation of this butterfly mainly rely on chemosensory-related genes expressed in the antennae, including chemosensory proteins (CSPs). Here, we characterized the putative roles of a female-antenna-biased PxutCSP19 inP. xuthusassociated with the perception of host volatiles and the sequestering of insecticides. This study examined the key functions of PxutCSP19 in the interactions of six host-derived odorants and eight insecticides. Notably, an extended N-terminus of PxutCSP19 did not significantly affect the binding specificities of this protein to ligands with high affinities. Our findings provide insight into the binding mechanisms of PxutCSP19 inP. xuthusto host volatiles and insecticides.AbstractChemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 inPapilio xuthusto 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 otherPapiliobutterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus ofPapilioCSP19s (an average of 154 residues) compared to insects’ typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki< 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae ofP. xuthusin the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance.