Molecular and functional characterization of pheromone-binding proteins in Leucinodes orbonalis Guenée (Lepidoptera:...

Pheromone perception is crucial for mating behavior in insects, and its manipulation can significantly influence the reproductive outcomes. In this study, a total of 156 chemosensory genes were identified from the transcriptome of the heads along with antennae of both male and female Leucinodes orbonalis. These included 35 sequences belonging to odorant-binding proteins (OBPs) including four pheromone binding proteins (PBPs) and three general odorant binding proteins (GOBPs), 73 odorant receptors (ORs), 20 ionotropic receptors (IRs), 25 chemosensory proteins (CSPs), and 3 sensory neuron membrane proteins (SNMPs). Expression analysis revealed male-biased expression of PBP1, PBP2, and PBP3, whereas PBP4 showed predominant expression in females. To investigate the molecular interaction with the pheromone compound, three-dimensional (3D) structures of the four identified PBPs were modelled. Molecular docking followed by 100 ns Molecular Dynamics (MD) simulations with the species-specific sex pheromone revealed that hydrophobic and hydrogen-bond interactions between the ligand and conserved residues play a crucial role in stabilizing PBP-ligand binding. Functional validation using RNA interference (RNAi) demonstrated that silencing of PBPs using dsRNA significantly reduced the pheromone sensitivity, as confirmed by electroantennogram (EAG) recordings performed after dsRNA-induction. Further, silencing of individual PBPs led to the overexpression of others, suggesting compensatory functional adaptation in L. orbonalis. In addition, simultaneous silencing of different combinations of PBP genes followed by behavioral assays using a Y-tube olfactometer showed a marked reduction in pheromone response. These findings highlight the crucial role of PBPs in pheromone-mediated mate recognition and suggest potential target for pest management strategies.