Multi-Omics Integration Reveals Fatty Acid Metabolism and Functional Roles of Fatty Acyl-CoA Reductases in Pheromone Biosynthesis of Monochamus saltuarius.

Monochamus saltuarius, a major vector of Bursaphelenchus xylophilus, relies on pheromone-mediated communication for successful reproduction, yet the metabolic and molecular basis of pheromone biosynthesis remains poorly understood. Here, we generated a comprehensive metabolomic dataset from male adults across five developmental stages and integrated it with previously obtained transcriptomic data to investigate pathways associated with pheromone biosynthesis. Multi-omics analyses consistently identified fatty acid metabolism as a central pathway and highlighted strong associations between FAR genes and lipid-derived metabolites. Comparative metabolomic analyses of males and females further revealed both conserved and sex-biased metabolic features, suggesting that pheromone biosynthesis in the two sexes involves partially shared but differentially regulated metabolic networks. In addition, two independent machine learning approaches convergently identified lipid- and fatty acid-related metabolites, including PG (18:1/13-HODE), as important discriminatory features across developmental stages. Transcriptome-based screening identified 12 putative MsalFARs, of which 8 were selected for further characterization based on their expression patterns, sequence features, and potential relevance to pheromone biosynthesis. RNAi analyses showed that four genes were involved in pheromone biosynthesis in a sex-specific manner: MsalFAR1 and MsalFAR4 affected female pheromone-related biosynthesis, whereas MsalFAR6 and MsalFAR7 affected male pheromone biosynthesis. Their silencing led to reduced pheromone production or pheromone-related responses, weakened antennal electrophysiological responses, and, in some cases, decreased mating success. Together, these findings indicate that pheromone biosynthesis in M. saltuarius is coordinately regulated by multiple pathways, with fatty acid metabolism playing a central role, and identify specific FAR enzymes as important contributors to this process.