Functional characteristics of pheromone binding proteins from Mythimna separata

Olfactory plays an important role in insect behaviors. Pheromone binding proteins (PBPs) are thought to play a certain role in the transport of pheromone molecules in the olfactory recognition process for courtship and mating. Mythimna separata is one of the most serious cereal pests in Asia. The sexual pheromone components of M. separata were clarified; however, to date, little evidence in vivo or in vitro has disclosed the binding properties of PBPs toward the pheromone components of M. separata. To address this research gap, the functional characterization of PBPs in M. separata, spectroscopic investigations were conducted by using recombinant MsepPBPs. Subsequently, MsepPBP1 and MsepPBP3 were selected for RNA interference to assess changes in behavioral responses of male mutants toward normal females. Fluorescence displacement binding assays, combined with fluorescence quenching assays, revealed that MsepPBP3, among the 3 MsepPBPs, exhibited the strongest affinity for Z11-16:Ald, the primary component of sex pheromone in M. separata. Static quenching was observed only between MsepPBP1 and Z9-16:Ald, as well as between MsepPBP3 and Z11-16:Ald or Z9-16:Ald. Transcript levels of MsepPBP1 or MsepPBP3 of male adults were significantly reduced compared to the control when injected with dsMsepPBPs. Both dsPBP1- and dsPBP3-treated males displayed a notable decrease in successful calling behaviors, with this reduction being more pronounced in dsMsepPBP3 injected groups than in dsMsepPBP1 injected groups. These experiments indicated the specificity of MsepPBP1 and MsepPBP3, with both contributing to the sensitivity of female detection. MsepPBP3 appeared to be a key protein for recognizing the sex pheromones of M. separata.