Unraveling the multifaceted effects of climatic factors on mountain pine beetle and its interaction with fungal symbionts.

Mountain pine beetles (MPBs) pose a substantial threat to North American pine forests, causing extensive tree mortality over large areas. Their tree-killing ability is closely linked to mass aggregation on host trees triggered via pheromones and dependence on their symbiotic fungi. However, the influence of a changing climate on the biology of MPBs and their co-evolved interactions with their fungal symbionts remains uncertain. To investigate this, male and female pairs of beetles were introduced into freshly cut logs from lodgepole pine trees and placed in controlled climate chambers with manipulated environmental conditions, including two levels of CO2 (ambient vs. 1000 ppm), O3 (ambient vs. 100 ppb) and humidity (33% vs. 65%). The beetle-infested logs were left in these chambers for 1 month and then returned to ambient conditions until brood emergence. Emerging broods were collected for further analysis. Additionally, three species of fungal symbionts (Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum) were subjected to the same CO2 , O3 and humidity conditions for 5 days. Lower humidity promoted MPB reproduction and fungal growth. Elevated CO2 accelerated larval growth and emergence while improving brood pheromone production. Elevated O3 had a negative impact on MPB reproduction and brood fitness while improving its immune responses to an entomopathogenic fungus (Beauveria bassiana). It also inhibited fungal growth and reproduction, whereas elevated CO2 had varied (positive or negative) effects on fungal growth and ergosterol (proxy to fungal mass) production depending on the fungal species. Together, these findings suggest that climate change can potentially alter the interactions between MPBs and their fungal symbionts, highlighting the importance of understanding how climate change affects forest pests and their symbiotic relationships to develop effective management strategies in the future.

A Pine in Distress: How Infection by Different Pathogenic Fungi Affect Lodgepole Pine Chemical Defenses.

In North America, lodgepole pine is frequently subjected to attacks by various biotic agents 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, Atropellis piniphila, and western gall rust, Endocronartium harknessii, affecting mature lodgepole pine trees in western Canada. We also included three ophiostomatoid fungi Grosmannia clavigera, Ophiostoma montium, and Leptographium longiclavatum associated with the mountain pine beetle (Dendroctonus ponderosae), because they are commonly used to investigate induced defenses of host trees of bark beetles. We collected phloem samples from lodgepole pines infected with the rust or the canker and healthy lodgepole pines in the same stand. We also inoculated mature lodgepole pines with the three fungal symbionts and collected phloem samples 2 weeks later when the defense chemistry was at its highest level. Different fungal species differentially altered the terpene chemistry of lodgepole pine trees. E. harknessii and the fungal symbionts altered the terpene chemistry in a similar pattern while trees responded to the infection by the A. piniphila differently. Our study highlights the importance of considering specific biotic stress agents in tree susceptibility or resistance to the subsequent attacks by insect herbivores, such as mountain pine beetle.

Bark Beetles Utilize Ophiostomatoid Fungi to Circumvent Host Tree Defenses.

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.

Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes.

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 ß-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.

Host Defense Metabolites Alter the Interactions between a Bark Beetle and its Symbiotic Fungi.

Successful host plant colonization by tree-killing bark beetle-symbiotic fungal complexes depends on host suitability, which is largely determined by host defense metabolites such as monoterpenes. Studies have shown the ability of specific blends of host monoterpenes to influence bark beetles or their fungal symbionts, but how biologically relevant blends of host monoterpenes influence bark beetle-symbiotic fungal interaction is unknown. We tested how interactions between two host species (lodgepole pine or jack pine) and two fungal symbionts of mountain pine beetle (Grosmannia clavigera or Ophiostoma montium) affect the performance of adult female beetles in vitro. Beetles treated with the propagules of G. clavigera or O. montium or not treated (natural fungal load) were introduced into media amended with a blend of the entire monoterpene profile of either host species and beetle performance was compared. Overall, host blends altered beetle performance depending on the fungal species used in the beetle amendment. When beetles were amended with G. clavigera, their performance was superior over beetles amended with O. montium in either host blend. Furthermore, G. clavigera-amended beetles performed better in media amended with host blends than without a host blend; in contrast, O. montium-amended beetles performed better in media without a host blend than with a host blend. Overall, this study showed that host defense metabolites affect host suitability to bark beetles through influencing their fungal symbionts and that different species of fungal symbionts respond differentlly to host defense metabolites.