RESUMO
Eurasian spruce bark beetle, Ips typographus is a destructive pest of the Norway spruce (Picea abies). Recent outbreaks in Europe have been attributed to global warming and other anthropogenic impacts. Bark beetles are guided by multiple complex olfactory cues throughout their life cycle. Male-produced aggregation pheromones, comprising 2-methyl-3-buten-2-ol and cis-verbenol, have been identified as the most powerful attractants for dispersing conspecifics. In addition to host trees, bark beetles interact with multiple organisms, including symbiotic ophiostomatoid fungi, which may promote beetle colonization success and offspring development. Previously, in a short-distance laboratory assay, we demonstrated that I. typographus adults are attracted to the volatile organic compounds (VOCs) produced by three symbiotic fungi: Grosmannia penicillata, Endoconidiophora polonica, and Leptographium europhioides. Furthermore, the abundant fusel alcohols and their acetates were found to be the most attractive odorants in the fungal VOC profile. In this study, using a long-distance field-trapping experiment, we analyzed the role of fungal VOCs as attractants for dispersing I. typographus. Two types of fungal lures were tested in combination with pheromones in traps: (1) live cultures of fungi grown on potato dextrose agar (PDA) and (2) dispensers containing synthetic fusel alcohols and their acetates in equal proportions. Subsequently, the composition of VOCs emitted from live fungal lures were analyzed. We found that the symbiotic fungi synergistically increased the attraction of beetles to pheromones in field traps and the attractiveness of live fungal lures depended on the fungal load. While one Petri dish with E. polonica, when combined with pheromones synergistically increased trapping efficiency, three Petri dishes with L. europhioides were required to achieve the same. The synthetic mix of fungal fusel alcohols and acetates improved the catch efficiency of pheromones only at a low tested dose. VOC analysis of fungal cultures revealed that all the three fungi produced fusel alcohols and acetates but in variable composition and amounts. Collectively, the results of this study show that, in addition to pheromones, bark beetles might also use volatile cues from their symbiotic fungi to improve tree colonization and reproductive success in their breeding and feeding sites.
RESUMO
Eurasian spruce bark beetle, Ips typographus, is an aggressive pest among spruce vegetation. I. typographus host trees colonization is mediated by aggregation pheromone, consisting of 2-methyl-3-buten-2-ol and cis-verbenol produced in the beetle gut. Other biologically active compounds such as ipsdienol and verbenone have also been detected. 2-Methyl-3-buten-2-ol and ipsdienol are produced de-novo in the mevalonate pathway and cis-verbenol is oxidized from α-pinene sequestrated from the host. The pheromone production is presumably connected with further changes in the primary and secondary metabolisms in the beetle. To evaluate such possibilities, we obtained qualitative metabolomic data from the analysis of beetle guts in different life stages. We used Ultra-high-performance liquid chromatography-electrospray ionization-high resolution tandem mass spectrometry (UHPLC-ESI-HRMS/MS). The data were dereplicated using metabolomic software (XCMS, Camera, and Bio-Conductor) and approximately 3000 features were extracted. The metabolite was identified using GNPS databases and de-novo annotation in Sirius program followed by manual curation. Further, we obtained differential gene expression (DGE) of RNA sequencing data for mevalonate pathway genes and CytochromeP450 (CyP450) genes from the gut tissue of the beetle to delineate their role on life stage-specific pheromone biosynthesis. CyP450 gene families were classified according to subclasses and given individual expression patterns as heat maps. Three mevalonate pathway genes and five CyP450 gene relative expressions were analyzed using quantitative real-time (qRT) PCR, from the gut tissue of different life stage male/female beetles, as extended knowledge of related research article (Ramakrishnan et al., 2022). This data provides essential information on pheromone biosynthesis at the molecular level and supports further research on pheromone biosynthesis and detoxification in conifer bark beetles.
RESUMO
Eurasian spruce bark beetle, Ips typographus, is a destructive pest in spruce forests. The ability of I. typographus to colonise host trees depends on its massive aggregation behaviour mediated by aggregation pheromones, consisting of 2-methyl-3-buten-2-ol and cis-verbenol. Other biologically active compounds such as ipsdienol and verbenone have also been detected in the beetle. Biosynthesis of 2-methyl-3-buten-2-ol and ipsdienol de novo from mevalonate and that of cis-verbenol from α-pinene sequestrated from the host have been reported in preliminary studies. However, knowledge on the molecular mechanisms underlying pheromone biosynthesis in this pest is currently limited. In this study, we performed metabolomic and differential gene expression (DGE) analysis for the pheromone-producing life stages of I. typographus. The highest amounts of 2-methyl-3-buten-2-ol (238 ng/gut) and cis-verbenol (23 ng/gut) were found in the fed male gut (colonisation stage) and the immature male gut (early stage), respectively. We also determined the amount of verbenyl oleate (the possible storage form of cis-verbenol), a monoterpenyl fatty acid ester, to be approximately 1604 ng/mg in the immature stage in the beetle body. DGE analysis revealed possible candidate genes involved in the biosynthesis of the quantified pheromones and related compounds. A novel hemiterpene-synthesising candidate isoprenyl-di-phosphate synthase Ityp09271 gene proposed for 2-methyl-3-buten-2-ol synthesis was found to be highly expressed only in the fed male beetle gut. Putative cytochrome P450 genes involved in cis/trans-verbenol synthesis and an esterase gene Ityp11977, which could regulate verbenyl oleate synthesis, were identified in the immature male gut. Our findings from the molecular analysis of pheromone-producing gene families are the first such results reported for I. typographus. With further characterisation of the identified genes, we can develop novel strategies to disrupt the aggregation behaviour of I. typographus and thereby prevent vegetation loss.
