Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main 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. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle’s host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial 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.

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.

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Bark beetles maintain symbiotic associations with a diversity of microbial organisms, including ophiostomatoid fungi. Studies have frequently reported the role of ophiostomatoid fungi in bark beetle biology, but how fungal symbionts interact with host chemical defenses over time is needed. We first investigated how inoculations by three fungal symbionts of mountain pine beetle affect the terpene chemistry of live lodgepole pine trees. We then conducted a complimentary laboratory experiment specifically measuring the host metabolite degradation by fungi and collected the fungal organic volatiles following inoculations with the same fungal species on lodgepole pine logs. In both experiments, we analyzed the infected tissues for their terpene chemistry. Additionally, we conducted an olfactometer assay to determine whether adult beetles respond to the volatile organic chemicals emitted from each of the three fungal species. We found that all fungi upregulated terpenes as early as two weeks after inoculations. Similarly, oxygenated monoterpene concentrations also increased by several folds (only in logs). A large majority of beetles tested showed a strong attraction to two fungal species, whereas the other fungus repelled the beetles. Together this study shows that fungal symbionts can alter host defense chemistry, assist beetles in overcoming metabolite toxicity, and provide possible chemical cues for bark beetle attraction.

In North America, lodgepole pine is frequently subjected to attacks by various biotic disturbances that compromise its ability to defend against subsequent attacks by insect herbivores. We investigated whether infections of lodgepole pine by different pathogenic fungal species have varying effects on its defense chemistry. We selected two common pathogens: Atropellis canker and western gall rust, affecting mature lodgepole pine trees in western Canada. We also included three ophiostomoid fungi associated with the mountain pine beetle, Grosmannia clavigera, Ophiostoma montium, and Leptographium longiclavatum because symbiotic fungi are commonly used to investigate induced defenses of host trees of bark beetles. We collected phloem samples from lodgepole pine trees infected with the rust or the canker, and healthy lodgepole pine trees in the same stand. We also inoculated mature lodgepole pine trees with the three fungal symbionts and collected phloem two weeks later when the defense chemistry at its highest level. All samples were analyzed for their terpene composition in gas chromatograph/mass spectrometry. Different pathogenic fungal species differentially altered the terpene chemistry of lodgepole pine trees. Western gall rust and the beetle-fungal symbionts altered the tree terpene chemistry in a similar fashion while trees responded to the infection by the Atropellis canker differently. Our study highlights the importance of considering speci c biotic stress agents in tree susceptibility or resistance to the subsequent biotic attacks by insect herbivores, such as mountain pine beetle.