Screening of ester-forming and hydrolyzing enzymes linked to pheromone production in Ips typographus (Linnaeus, 1758)

The bark beetle Ips typographus (Coleoptera: Curculionidae: Scolytinae) is a major pest of spruce trees in Central Europe. Its ecological success is mediated by a male-produced aggregation pheromone, which includes the monoterpene cis-verbenol. Cis-verbenol is biosynthesized from host-derived α-pinene but can also be released through enzymatic cleavage of verbenyl-fatty acyl esters, which are initially produced by young beetles during maturation feeding and stored in their fat bodies. The primary objective of this study was to identify the rarely studied ester-forming and hydrolyzing enzymes in I. typographus and to suggest their potential roles in beetle metabolism. By blasting a reference gene set against a newly assembled I. typographus transcriptome and performing phylogenetic analyses, we identified 27 novel ester-modifying genes: 23 carboxylesterases, two (phospho)lipases, one notum-like gene, and one neurolactin-like gene. Full gene structures were described. Based on GC–MS measured production profiles of verbenyl oleate and cis-verbenol across beetle life stages and phenotypes, transcriptome pairs were selected for differential expression analysis. Eight genes were chosen for detailed RT-qPCR expression profiling across sexes, developmental stages, and tissues. Based on these findings, we propose possible roles of genes encoding enzymes in verbenyl-fatty acyl ester metabolism or broader lipid metabolic processes in bark beetles. However, functional validation through enzyme assays and gene silencing will be necessary to confirm their specific roles. Although the functions of these candidate genes remain hypothetical, the identification and structural description of 27 new ester-modifying enzymes, eight of which were further validated by RT-qPCR, provide important insight into this poorly characterized enzyme group in insects. Furthermore, understanding the genetic basis of cis-verbenol biosynthesis in I. typographus may support the development of novel, pheromone-based pest management strategies.