The Legacy Effect of Mountain Pine Beetle Outbreaks on the Chemical and Anatomical Defences of Surviving Lodgepole Pine Trees.

The recent mountain pine beetle outbreaks have caused widespread mortality among lodgepole pine trees in western North America, resulting in a reduced population of surviving trees. While previous studies have focused on the cascading impacts of these outbreaks on the physiology and growth of the surviving trees, there remains a need for a comprehensive study into the interactions among various physiological traits and the growth in post-outbreak stands. Specifically, the relationship between chemical (primarily terpenes) and anatomical (mainly resin ducts) defences, as well as the allocation of non-structural carbohydrates (NSCs) to support these defence modalities, is poorly understood. To address these gaps, we conducted a field survey of surviving lodgepole pine trees in post-mountain pine beetle outbreak stands in western Canada. Our retrospective analysis aimed at determining correlations between the post-outbreak concentrations of monoterpenes, diterpenes, and NSCs in the phloem and the historical resin duct characteristics and growth traits before and after the outbreak. We detected strong correlations between the post-outbreak concentrations of monoterpenes and historical resin duct characteristics, suggesting a possible link between these two defence modalities. Additionally, we found a positive relationship between the NSCs and the total concentrations of monoterpenes and diterpenes, suggesting that NSCs likely influence the production of these terpenes in lodgepole pine. Furthermore, historical tree growth patterns showed strong positive correlations with many individual monoterpenes and diterpenes. Interestingly, while surviving trees had enhanced anatomical defences after the outbreak, their growth patterns did not vary before and after the outbreak conditions. The complexity of these relationships emphasizes the dynamics of post-outbreak stand dynamics and resource allocations in lodgepole pine forests, highlighting the need for further research. These findings contribute to a broader understanding of conifer defences and their coordinated responses to forest insect outbreaks, with implications for forest management and conservation strategies.

Exploring behavioural and physiological adaptations in mountain pine beetle in response to elevated ozone concentrations.

Elevated ozone (eO3) concentrations pose a threat to insect populations by potentially altering their behaviour and physiology. This study investigates the effects of eO3 concentrations on the mountain pine beetle which is a major tree-killing species of conifers in northwestern North America. We are particularly interested in understanding the effects of eO3 concentrations on beetle behaviour and physiology and possible transgenerational impacts on bark beetle broods. We conducted O3-enrichment experiments in a controlled laboratory setting using different O3 concentrations (100-200 ppb; projected for 2050-2100) and assessed various beetle responses, including CO2 respiration, mating behaviour, survival probability, locomotion, and attraction behaviour. Transgenerational impacts on the first and second generations were also analyzed by studying brood morphology, mating behaviour, survival, and pheromone production. We found that beetles exposed to eO3 concentrations had shorter oviposition galleries and reduced brood production. Beetle pheromones were also degraded by eO3 exposure. However, exposure to eO3 also prompted various adaptive responses in beetles. Despite reduced respiration, eO3 improved locomotor activity and the olfactory response of beetles. Surprisingly, beetle survival probability was also improved both in the parents and their broods. We also observed transgenerational plasticity in the broods of eO3-exposed parents, suggesting potential stress resistance mechanisms. This was evident by similar mating success, oviposition gallery length, and brood numbers produced in both control and eO3 concentration treatments. This study demonstrates the sensitivity of mountain pine beetles to increased O3 concentrations, contributing crucial insights into the ecological implications of eO3 concentrations on their populations. Overall, the outcome of this study contributes to informed climate change mitigation strategies and adaptive management practices for the development of resilient forests in response to emerging forest insect pests worldwide.

Terpenoids are involved in the expression of systemic-induced resistance in Austrian pine.

Terpenoids are defense metabolites that are induced upon infection or wounding. However, their role in systemic-induced resistance (SIR) is not known. Here, we explored the role of terpenoids in this phenomenon at a very early stage in the interaction between Austrian pine and the tip blight and canker pathogen Diplodia pinea. We induced Austrian pine saplings by either wounding or inoculating the lower stems with D. pinea. The seedlings were then challenged after 12 h, 72 h, or 10 days with D. pinea on the stem 15 cm above the induction. Lesion lengths and terpenoids were quantified at both induction and challenge locations. Key terpenoids were assayed for antifungal activity in in vitro bioassays. SIR increased with time and was correlated with the inducibility of several compounds. α-Pinene and a cluster of ß-pinene, limonene, benzaldehyde, dodecanol, and n-dodecyl acrylate were positively correlated with SIR and were fungistatic in vitro, while other compounds were negatively correlated with SIR and appeared to serve as a carbon source for D. pinea. This study shows that, overall, terpenoids are involved in SIR in this system, but their role is nuanced, depending on the type of induction and time of incubation. We hypothesize that some, such as α-pinene, could serve in SIR signaling.

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.

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.

Combined drought and bark beetle attacks deplete non-structural carbohydrates and promote death of mature pine trees.

How carbohydrate reserves in conifers respond to drought and bark beetle attacks are poorly understood. We investigated changes in carbohydrate reserves and carbon-dependent diterpene defences in ponderosa pine trees that were experimentally subjected to two levels of drought stress (via root trenching) and two types of biotic challenge treatments (pheromone-induced bark beetle attacks or inoculations with crushed beetles that include beetle-associated fungi) for two consecutive years. Our results showed that trenching did not influence carbohydrates, whereas both biotic challenges reduced amounts of starch and sugars of trees. However, only the combined trenched-bark beetle attacked trees depleted carbohydrates and died during the first year of attacks. While live trees contained higher carbohydrates than dying trees, amounts of constitutive and induced diterpenes produced did not vary between live and beetle-attacked dying trees, respectively. Based on these results we propose that reallocation of carbohydrates to diterpenes during the early stages of beetle attacks is limited in drought-stricken trees, and that the combination of biotic and abiotic stress leads to tree death. The process of tree death is subsequently aggravated by beetle girdling of phloem, occlusion of vascular tissue by bark beetle-vectored fungi, and potential exploitation of host carbohydrates by bark beetle symbionts as nutrients.

Mutualistic Ophiostomatoid Fungi Equally Benefit from Both a Bark Beetle Pheromone and Host Tree Volatiles as Nutrient Sources.

Interactions between mutualistic bark beetles and ophiostomatoid fungi have received considerable attention in recent years. Studies have shown how volatile organic compounds emitted from mutualist fungi affect the behaviors of several bark beetle species. However, we currently lack sufficient knowledge regarding whether bark beetle pheromones can influence mutualist fungi. Here, we measured growth and biomass of two mutualistic fungi of the mountain pine beetle in response to headspace of a beetle pheromone (trans-verbenol), a blend of host tree volatiles, the combination of both, or control (no volatile source) in vitro experiments consisting of a nitrogen-based medium. The surface area and ergosterol content of the mycelia were used as surrogates for fungal growth and biomass respectively. We found that both growth and biomass of Grosmannia clavigera and Ophiostoma montium were greater in medium exposed to any type of volatile sources than the control. While growth and ergosterol content of G. clavigera were highest in the combination treatment, there were no differences in growth or biomass among the types of volatiles introduced for O. montium. These results suggest that both mutualistic fungi can utilize both bark beetle pheromone and host tree volatiles as nutrient sources. Overall, these results support the on-going studies on the role of volatile organic compounds mediating mutualistic bark beetle-fungi interactions.

Mechanisms and consequences of flight polyphenisms in an outbreaking bark beetle species.

Flight polyphenisms naturally occur as discrete or continuous traits in insects. Discrete flight polyphenisms include winged and wingless morphs, whereas continuous flight polyphenisms can take the form of short- or long-distance fliers. The mountain pine beetle (Dendroctonus ponderosae) exhibits polyphenic variation in flight distance but the consequences of this flight variation on life history strategies of beetles is unknown. This study assessed the effect of flight on two particular aspects of beetle biology: (1) an energetic trade-off between flight distance and host colonisation capacity; and (2) the relationship between flight distance and pheromone production. A 23 h flight treatment was applied to a subset of beetles using computer-linked flight mills. After flight treatment, both flown and unflown (control) beetles were given the opportunity to colonise bolts of host trees, and beetles that entered hosts were aerated to collect pheromone. A trade-off occurred between initiation of host colonisation and percentage body mass lost during flight, which indicates energy use during flight affects host acceptance in female mountain pine beetles. Furthermore, production of the aggregation pheromone trans-verbenol by female beetles was influenced by both percentage body mass lost during flight and flight distance. Male production of exo-brevicomin was affected by beetle condition following flight but not by the energy used during flight. These novel results give new insight into the polyphenic flight behaviour of mountain pine beetles. Flight variation is adaptive by acting to maintain population levels through safe and risky host colonisation strategies. These findings suggest mechanisms that facilitate the extremities of the continuous flight polyphenism spectrum. These opposing mechanisms appear to maintain the high variation in flight exhibited by this species.

Rapid monoterpene induction promotes the susceptibility of a novel host pine to mountain pine beetle colonization but not to beetle-vectored fungi.

Chemical induction can drive tree susceptibility to and host range expansions of attacking insects and fungi. Recently, mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) has expanded its host range from its historic host lodgepole pine (Pinus contorta var. latifolia Douglas ex Loudon) to jack pine (Pinus banksiana Lamb) in western Canada. Beetle success in jack pine forests likely depends upon the suitability of tree chemistry to MPB and its symbiotic phytopathogenic fungi. In particular, how rapid induced defenses of jack pine affect MPB colonization and the beetle's symbionts is unknown. In the field, we characterized and compared differences in rapid induced phloem monoterpenes between lodgepole and jack pines in response to various densities of Grosmannia clavigera (Robinson-Jeffery and Davidson)-a MPB symbiotic fungus used to simulate beetle attack-inoculations. Overall, lodgepole pine had higher limonene and myrcene, but lower α-pinene, concentrations than jack pine. However, myrcene concentrations in jack pine increased with inoculation density, while that in lodgepole pine did not respond to density treatments. We compared the growth and reproduction of MPB's symbiotic fungi, G. clavigera, Ophiostoma montium (Rumford) von Arx and Leptographium longiclavatum Lee, Kim and Breuil, grown on media amended with myrcene, α-pinene and limonene at concentrations reflecting two induction levels from each pine species. Myrcene and α-pinene amendments inhibited the growth but stimulated the reproduction of G. clavigera, whereas limonene stimulated its growth while inhibiting its reproduction. However, the growth and reproduction of the other fungi were generally stimulated by monoterpene amendments. Overall, our results suggest that jack pine rapid induction could promote MPB aggregation due to high levels of α-pinene (pheromone precursor), a positive feedback of myrcene (pheromone synergist) and low levels of limonene (resistance). Jack pine is likely as susceptible to MPB-vectored fungi as lodgepole pine, indicating that jack pine induction will likely not adversely affect symbiont activities enough to inhibit the invasion of MPB into jack pine forests.

Weathering the storm: how lodgepole pine trees survive mountain pine beetle outbreaks.

Recent mountain pine beetle outbreaks in western North America killed millions of lodgepole pine trees, leaving few survivors. However, the mechanism underlying the ability of trees to survive bark beetle outbreaks is unknown, but likely involve phytochemicals such as monoterpenes and fatty acids that can drive beetle aggregation and colonization on their hosts. Thus, we conducted a field survey of beetle-resistant lodgepole pine (Pinus contorta) trees to retrospectively deduce whether these phytochemicals underlie their survival by comparing their chemistry to that of non-attacked trees in the same stands. We also compared beetle attack characteristics between resistant and beetle-killed trees. Beetle-killed trees had more beetle attacks and longer ovipositional galleries than resistant trees, which also lacked the larval establishment found in beetle-killed trees. Resistant trees contained high amounts of toxic and attraction-inhibitive compounds and low amounts of pheromone-precursor and synergist compounds. During beetle host aggregation and colonization, these compounds likely served three critical roles in tree survival. First, low amounts of pheromone-precursor (α-pinene) and synergist (mycrene, terpinolene) compounds reduced or prevented beetles from attracting conspecifics to residual trees. Second, high amounts of 4-allyanisole further inhibited beetle attraction to its pheromone. Finally, high amounts of toxic limonene, 3-carene, 4-allyanisole, α-linolenic acid, and linoleic acid inhibited beetle gallery establishment and oviposition. We conclude that the variation of chemotypic expression of local plant populations can have profound ecological consequences including survival during insect outbreaks.

Fatty Acid Composition of Novel Host Jack Pine Do Not Prevent Host Acceptance and Colonization by the Invasive Mountain Pine Beetle and Its Symbiotic Fungus.

Fatty acids are major components of plant lipids and can affect growth and development of insect herbivores. Despite a large literature examining the roles of fatty acids in conifers, relatively few studies have tested the effects of fatty acids on insect herbivores and their microbial symbionts. Particularly, whether fatty acids can affect the suitability of conifers for insect herbivores has never been studied before. Thus, we evaluated if composition of fatty acids impede or facilitate colonization of jack pine (Pinus banksiana) by the invasive mountain pine beetle (Dendroctonus ponderosae) and its symbiotic fungus (Grosmannia clavigera). This is the first study to examine the effects of tree fatty acids on any bark beetle species and its symbiotic fungus. In a novel bioassay, we found that plant tissues (hosts and non-host) amended with synthetic fatty acids at concentrations representative of jack pine were compatible with beetle larvae. Likewise, G. clavigera grew in media amended with lipid fractions or synthetic fatty acids at concentrations present in jack pine. In contrast, fatty acids and lipid composition of a non-host were not suitable for the beetle larvae or the fungus. Apparently, concentrations of individual, rather than total, fatty acids determined the suitability of jack pine. Furthermore, sampling of host and non-host tree species across Canada demonstrated that the composition of jack pine fatty acids was similar to the different populations of beetle's historical hosts. These results demonstrate that fatty acids composition compatible with insect herbivores and their microbial symbionts can be important factor defining host suitability to invasive insects.