MaltEms-MaltOBP9 axis mediates sexually dimorphic olfactory behavior in Monochamus alternatus via recognition of pheromone 2-methoxy-4-vinylphenol.

The olfactory system is essential for insect survival and behavior, with odorant-binding proteins (OBPs) playing a key role by binding and transporting chemical signals to olfactory receptors. Although MaltOBP9 from Monochamus alternatus, a primary vector of pine wilt disease, has been linked to olfactory functions in M. alternatus, its regulatory mechanisms and specific roles are not well understood. In this study, we show that MaltOBP9 is highly expressed in the antennae of both male and female adults after sexual maturation. Using fluorescence spectroscopy, molecular docking, and site-directed mutagenesis, we identified the amino acid Phe54 as essential for MaltOBP9's ability to bind 18 different volatile compounds. RNA interference (RNAi) and behavioral tests revealed that MaltOBP9 is necessary for sexually mature adults to detect the pheromone 2-methoxy-4-vinylphenol (2M4VP). Additionally, dual-luciferase reporter assays, electrophoretic mobility shift assays, and RNAi experiments demonstrated that the transcription factor MaltEms positively regulates MaltOBP9 expression, thereby influencing the detection of 2M4VP. By combining insights into transcriptional regulation and protein-ligand interactions, this study clarifies the MaltEms-MaltOBP9-2M4VP axis as a mediator of sex-specific olfactory behavior-where females avoid and males are attracted to the pheromone-in sexually mature M. alternatus. These findings provide a theoretical basis for developing pest control methods that target MaltOBP9 and its critical residue Phe54 through behavior-based strategies.