Bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes

Abstract

Outbreaks of bark beetles have decimated millions of hectares of conifer forest worldwide in recent years. The ability of these tiny 3-6 mm long insects to kill mature trees over a short period has been ascribed to two factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. We previously demonstrated that Eurasian spruce bark beetles (Ips typographus) can recognize beneficial fungal symbionts of the genera Grosmannia, Endoconidiophora and Ophiostoma by their de novo synthesized volatile compounds. We hypothesized that the fungal symbionts of the bark beetles might metabolize spruce resin monoterpenes of the beetle host tree, Norway spruce (Picea abies), and that the volatile products could be used as cues by beetles for locating breeding sites with beneficial symbionts. Grosmannia penicillata and other fungal symbionts altered the profile of spruce bark volatiles by converting the major monoterpenes to oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans-4-thujanol and other oxygenated products. Extensive electrophysiological measurements showed that bark beetles possess olfactory sensory neurons that are selective for these oxygenated symbiont metabolites. Compounds such as camphor and trans-4-thujanol attracted beetles at specific doses in olfactory experiments and stimulated the response of female beetles to a mixture of pheromones. Finally, the fungal symbiont was found to stimulate bark beetle tunneling on diets. Collectively, our results show that oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by bark beetles as cues to find these essential microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

Odorant receptor orthologues in conifer‐feeding beetles display conserved responses to ecologically relevant odours

“…In contrast, 2-PE appears to have no effect on pheromone attraction in I. typographus (Schlyter et al, 1987); however, it is part of the odour blends released by several species of ophiostomatoid symbiotic fungi, which are attractive to beetles in laboratory bioassays (Kandasamy et al, 2019). These fungi, inoculated by beetles inside their galleries under the tree bark, are likely to benefit beetles by providing nutrients and through metabolism of the tree's chemical defences (Kandasamy et al, 2019(Kandasamy et al, , 2021.…”

“…Since the ORs underlie the responses of the OSNs in the insect antennae, it is of interest to compare OR responses from in vitro heterologous systems with those of putatively corresponding OSN classes. Among the study species, I. typographus provides the best example due to the extensive electrophysiological studies that have been conducted (Andersson et al, 2009;Kandasamy et al, 2019Kandasamy et al, , 2021Schiebe et al, 2019;Tømmerås, 1985). In relation to the GLV alcohols, one OSN class (named "GLV-OH") that responds most strongly and with similar sensitivity to E2-hexenol, Z3-hexenol and 1-hexanol has been identified in I. typographus (Andersson et al, 2009;Kandasamy et al, 2019).…”

Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odors

“…In contrast, 2-PE appears to have no effect on pheromone attraction in I. typographus (Schlyter, et al 1987); however, it is part of the odor blends released by several species of ophiostomatoid symbiotic fungi, which are attractive to beetles in laboratory bioassays (Kandasamy, et al 2019). These fungi, inoculated by beetles inside their galleries under the tree bark, are likely to benefit beetles by providing nutrients and through metabolism of the tree’s chemical defenses (Kandasamy, et al 2019; Kandasamy, et al 2021). I. typographus has an OSN class that primarily responds to 2-PE and secondarily to phenethyl acetate, 3-methyl-1-butanol and a few more compounds (including 1-hexanol) with weaker activity (Kandasamy, et al 2019).…”

“…Since the ORs underlie the responses of the OSNs in the insect antennae, it is of interest to compare OR responses from in vitro heterologous systems with those of putatively corresponding OSN classes. Among the study species, I. typographus provides the best example due to the extensive electrophysiological studies that have been conducted (Andersson et al 2009; Kandasamy et al 2019; Kandasamy et al 2021; Schiebe et al 2019; Tømmerås 1985). In relation to the GLV alcohols, one OSN class (named ‘GLV-OH’) that responds most strongly and with similar sensitivity to E 2-hexenol, Z 3-hexenol, and 1-hexanol has been identified in I. typographus (Andersson et al 2009; Kandasamy et al 2019).…”

“…The test odor panel included 62 compounds (Supplementary Table 3) that are ecologically relevant for conifer-feeding curculionids, including pheromone compounds, volatiles from conifers and angiosperms as well as odorants produced by bark beetle fungal symbionts (Kandasamy, et al 2019; Kandasamy, et al 2021; Yuvaraj, et al 2021). The Orco agonist VUAA1 was tested (at 50 μM) on each cell line as a positive control for functional Orco expression.…”