Identification and Functional Characterization of the Leg-Enriched Chemosensory Protein PxylCSP9 in Plutella xylostella (Lepidoptera: Plutellidae)

Phytophagous insects rely on highly evolved chemosensory systems to recognize plant cues, which guide essential behaviors such as feeding and oviposition. While the antennal chemosensory functions of Plutella xylostella are well-studied, little is known about the role of its legs in detecting host plant chemicals. This study systematically identified and characterized chemosensory-related genes expressed in the legs of P. xylostella using transcriptome data. A total of 114 leg-expressed chemosensory genes were identified, with PxylCSP9 showing high expression in legs and strong, broad-spectrum binding to key cruciferous plant volatiles. Leg chemosensory proteins, particularly PxylCSP9, play an important role in the chemical perception. Plutella xylostella , a major pest of cruciferous vegetables, depends predominantly on chemoreception to locate host plants. Legs are crucial in insect chemical perception, particularly during close-range and contact chemoreception. However, the molecular basis underlying the chemosensory repertoire in P. xylostella legs remains elusive. To address this, we sequenced chemosensory-related genes in diamondback moth legs. Sequencing identified 32 odorant binding protein (OBP), 18 chemosensory protein (CSP), 26 odorant receptor (OR), 20 gustatory receptor (GR), 15 ionotropic receptor (IR), and 3 sensory neuron membrane protein (SNMP) genes. Comparative analysis with antennal transcriptome data revealed three CSPs, seven ORs, and two GRs newly identified in the legs. Transcriptome analysis showed higher fragments per kilobase of transcript per million mapped reads values for CSPs than for other chemosensory-related gene families. Furthermore, qRT-PCR confirmed the highest expression of PxylCSP9 in the legs, suggesting its role in perceiving external compounds. Fluorescent binding assays revealed high binding affinity of PxylCSP9 for several host plant semiochemicals. Molecular docking predicted a hydrophobic binding pocket in PxylCSP9 with Met11, Leu13, and Leu43 frequently participating in ligand interactions. Our findings indicate that leg-enriched PxylCSP9 is pivotal for host plant recognition during close-range chemoreception, suggesting its potential as a molecular target for precision management through behavior-based strategies.