Divergent Response of Two Bark Beetle-Fungal Symbiotic Systems to Host Monoterpenes Reflects Niche Partitioning Strategies.

The successful establishment of bark beetle-fungus symbionts on plants is required to overcome host defenses. However, little is known about how different bark beetle-fungus symbionts adapt to different niches on the same host plant. Here, we investigated the niche partitioning mechanism of two co-occurring bark beetle-fungus symbiotic systems, Ips nitidus-Ophiostoma bicolor and Dendroctonus micans-Endoconidiophora laricicola, on Qinghai spruce (Picea crassifolia) tree. The lower niche of the spruce trunk inhabited by D. micans showed a higher content of monoterpenes than the upper niche of the trunk inhabited by I. nitidus. Dendroctonus micans showed greater tolerance and higher metabolic efficiency toward monoterpenes than I. nitidus. However, both beetle species showed a similar metabolic profile toward α-pinene, albeit with different levels of metabolites. Additionally, O. bicolor, transmitted by I. nitidus, showed a significantly higher tolerance to monoterpenes and pathogenicity to spruce trees than E. laricicola, transmitted by D. micans. In particular, monoterpenoid metabolites were observed to attenuate the inhibitory effect of high-dose α-pinene on E. laricicola, thus increasing its fitness in a high-dose monoterpene microhabitat. These results show that these two bark beetle-fungus symbionts have adapted to different niches, leading to fitness differences in niche distribution that are at least partly related to the different distribution of monoterpene concentration in the spruce trunk. This research provides a novel perspective for understanding the coevolution between bark beetle-fungus symbionts and their host plants.

A mountain pine beetle (Coleoptera: Curculionidae) adult development rate model confirms evolved geographic differences.

Insects live in a wide range of thermal environments and have evolved species- and location-specific physiological processes for survival in hot and cold extremes. Thermally driven dormancy strategies, development rates and thresholds are important for synchronizing cohorts within a population and to local climates and often vary among populations within a species. Mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae, Scolytinae), is a widely distributed forest insect native to North America with clinal genetic differentiation in thermally dependent traits. MPB development occurs in Pinus phloem beneath the bark, and its cryptic habitat makes experimentation difficult, particularly for the adult stage. We describe a novel method for modeling MPB adult development following pupation and terminating in emergence from a brood tree. We focus on an Arizona (southern) MPB population with previously described preadult development rates. Field-observed tree attack, adult emergence, and phloem temperature data are combined in a parameterized cohort model and candidate rate curves are evaluated to describe adult emergence timing. Model competition indicates that the Brière rate curve provided the best fit to field data and performed well under cross-validation. Results confirm that the development of Arizona MPB adults is slower than the previously described development rate of more northern Utah adults. Using the estimated adult rate curve in a scenario of increasing mean temperatures, we show that the timing of second-generation adult emergence in the same year would result in cold-intolerant lifestages during winter, limiting the success of bivoltine populations.

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.

An enhanced lure for eastern populations of the North American spruce beetle, Dendroctonus rufipennis Kirby (Coleoptera: Curculionidae).

Regional variation in pheromone production and response has practical implications for the use of semiochemical lures to monitor and control bark beetle populations. We tested 4 lure formulations including 2 new formulations that reflect the pheromone production profiles of western and eastern populations of spruce beetles, Dendroctonus rufipennis Kirby (Coleoptera: Curculionidae), as well as 2 commercially available formulations (current Rocky Mountain lure and current Atlantic lure), in 2 locations in New Brunswick, Canada. In 2 separate years, the new eastern lure containing seudenol, MCOL, and spruce terpenes captured 4 times (2021) and 11 times (2022) more spruce beetles than the current Atlantic lure that consisted of frontalin, seudenol, and spruce terpenes. In 2021, we also captured more eastern larch beetles, Dendroctonus simplex LeConte (Coleoptera: Curculionidae), with the new eastern lure, whereas in 2022, we captured the most D. simplex with the current Atlantic lure, suggesting that more research is needed on D. simplex pheromone production and response across its range. The bark beetle predator, Thanasimus dubius (Fabr.; Coleoptera: Cleridae), did not respond well to the new eastern blend that lacks frontalin, suggesting that response to frontalin is important in finding prey and might be conserved in predator populations. The reduced trap catch of T. dubius to the enhanced lure is beneficial because it does not inhibit natural population control by removing predators from the community. Our study reveals an improved trap lure for eastern populations of spruce beetles and highlights gaps and research needs in bark beetle pheromone ecology.

Modeling the pest-pathogen threats in a warming world for the red turpentine beetle (Dendroctonus valens) and its symbiotic fungus (Leptographium procerum).

BACKGROUND: Dendroctonus valens along with its symbiotic fungi have caused unprecedented damage to pines in China. Leptographium procerum, its primary symbiotic fungus, facilitates the invasion and colonization of the pest, thereby aggravating ecological threats. Assessing shifts in the niches and ranges of D. valens and its symbiotic fungus could provide a valuable basis for pest control. Here, we conducted niche comparisons between native and invasive populations of D. valens. Then, we employed standard ecological niche models and ensembles of small models to predict the potential distributions of D. valens and L. procerum under climate change conditions and to estimate areas of overlap. RESULTS: The niche of invasive population of D. valens in Chinese mainland only occupied a limited portion of the niche of native population in North America, leaving a substantial native niche unfilled and without any niche expansion. The suitable regions for D. valens are predicted in central and southern North America and central and northeastern Chinese mainland. The overlap with the suitable regions of L. procerum included eastern North America and the central and northeastern Chinese mainland under historical climatic scenarios. The regions susceptible to their symbiotic damage will shift northward in response to future climate change. CONCLUSIONS: Projected distributions of D. valens and its symbiotic fungus, along with areas vulnerable to their symbiotic damage, provide essential insights for devising strategies against this association. Additionally, our study contributes to comprehending how biogeographic approaches aid in estimating potential risks of pest-pathogen interactions in forests within a warming world. © 2024 Society of Chemical Industry.

Evidence that Ophiostomatoid Fungal Symbionts of Mountain Pine Beetle Do Not Play a Role in Overcoming Lodgepole Pine Defenses During Mass Attack.

Mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins) is a devastating forest insect pest that has killed millions of hectares of pines in western North America over the past two decades. Like other bark beetles, MPB vectors ophiostomatoid fungal species, some of which are pathogenic to host pine species. The phytopathogenicity of these fungal symbionts has sparked considerable debate regarding their role in facilitating MPB attack success. We tested the hypothesis that MPB ophiostomatoid fungal associates like Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield contribute to overwhelming host defenses during MPB mass attack. We compared responses of mature lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) trees growing in natural stands that were mass attacked by MPB with those inoculated with G. clavigera by examining host defense hormones, secondary metabolites, and gene expression profiles. The jasmonate and ethylene signatures of necrotrophic pathogen-triggered response were identified in G. clavigera-inoculated trees, but only the jasmonate signature of a herbivore-triggered response was measured in MPB-attacked trees. Several G. clavigera-induced changes in pine phenolic metabolite profiles and phenolic biosynthesis gene expression patterns were absent in MPB-attacked pines. These findings indicate that ophiostomatoid fungi like G. clavigera are not a major factor in overwhelming host defenses during MPB mass attack. Instead, fungal pathogenicity likely is more important in aiding MPB colonization and development within the host tree. Phenolics appear to play a larger role in the host response to G. clavigera than to MPB, although phenolics may also influence MPB feeding and behavior. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Southern pine beetle (Coleoptera: Curculionidae) and its associated insect community: similarities and key differences between northeastern and southeastern pine forests.

We examine consequences of climate-induced range expansion on community composition and diversity within trees attacked by the southern pine beetle (Dendroctonus frontalis Zimmermann). At the northernmost limit of the southern pine beetle range where populations have persisted for multiple years (currently Long Island, NY), we collected and reared bark samples and placed emergence traps on southern pine beetle-attacked pitch pine, Pinus rigida Mill. (Pinales: Pinaceae). From these samples, we quantified southern pine beetle gallery length and emergence as well as the diversity and abundance of all associated insects including known and suspected competitors, predators, and parasitoids. We compared our results to that of historic sampling data (1975-1997) in the core of southern pine beetle's range in the southern United States. Key community members were present in both the northern and southern regions; composition and relative abundances differed markedly. A key predator, the clerid beetle Thanasimus dubius (Fabricius) (Coleoptera: Cleridae), was present in similar densities in both regions. Southern pine beetle infested a greater proportion of the length of the tree bole in the North. This increased tree utilization may be a consequence of a lack of resource competition by Ips De Geer (Coleoptera: Curculionidae) engraver beetles, which we found only in very low abundance in the northern sites. We discuss the implications of these results in the context of southern pine beetle range expansion. Continued study of the southern pine beetle community and temporal southern pine beetle dynamics in the North will add to our current knowledge base and aid preservation of rare and ecologically valuable pine barrens of New England.

Effects of simulated gut pH environment on bacterial composition and pheromone production of Dendroctonus valens.

Bark beetles are an economically and ecologically important insect group, with aggregation behavior and thus host colonization success depends on pheromone-mediated communication. For some species, such as the major invasive forest pest in China, red turpentine beetle (Dendroctonus valens), gut microbiota participates in pheromone production by converting tree monoterpenes into pheromone products. However, how variation in gut microenvironment, such as pH, affects the gut microbial composition, and consequently pheromone production, is unknown. In this study, we fed wild caught D. valens with 3 different pH media (main host diet with natural pH of 4.7; a mildly acidic diet with pH 6 mimicking the beetle gut pH; and highly acidic diet with pH 4), and measured their effects on the gut pH, bacterial community and production of the main aggregation and anti-aggregation pheromone (verbenone). We further tested the verbenone production capacity of 2 gut bacterial isolates in different pH environments (pH 6 and 4). Compared to natural state or main host diet, feeding on less acidic diet (pH 6) diluted the acidity of the gut, whereas feeding on highly acidic diet (pH 4) enhanced it. Both changes in gut pH reduced the abundance of dominant bacterial genera, resulting in decreased verbenone production. Similarly, the highest pheromone conversion rate of the bacterial isolates was observed in pH mimicking the acidity in beetle gut. Taken together, these results indicate that changes in gut pH can affect gut microbiota composition and pheromone production, and may therefore have the potential to affect host colonization behavior.

How Effective Are Push-Pull Semiochemicals as Deterrents for Bark Beetles? A Global Meta-Analysis of Thirty Years of Research.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are among the most damaging tree pests globally. Rising temperatures, drought, fire, storms, cyclones, and poor forest management cause stress and loss of vigour in trees, and these conditions favour bark beetle outbreaks. While research has been conducted on push-pull strategies to deter bark beetles, using attractive and deterrent semiochemicals, the potential of this strategy to reduce bark beetle populations, particularly in the genera Dendroctonus and Ips, remains uncertain. Here, we conducted a global meta-analysis of 52 research articles to quantify the effects of semiochemical treatments on managing different species of Dendroctonus and Ips for forest protection. Based on this analysis, we found that push-pull semiochemicals can significantly reduce Dendroctonus and Ips populations measured by a reduction in the attraction to lure/trap catches, tree mortality, and attacks on trees. The overall efficacy of the push-pull semiochemical treatment shows a 66% reduction for Ips compared to control and a 54% reduction compared to control for Dendroctonus, while, at the species level, there was a 69% reduction for Dendroctonus ponderosae (Hopkins) and a 94% reduction in Ips perturbatus (Eichhoff), and a 93% reduction in Ips latidens (LeConte). Interestingly, among different treatment sources, the efficacy of conspecific semiochemicals in combination with heterospecific semiochemicals and non-host volatiles showed a 92% reduction in Dendroctonus spp., and conspecific semiochemicals in combination with non-host volatiles showed a 77% significant reduction in Ips spp., while the efficacy of heterospecific semiochemicals in reducing Ips population was about 69%, and 20% in Dendroctonus. Among different ecological regions, the use of a push-pull strategy showed a 70% reduction in Dendroctonus in central-west North America, and Ips showed a 75% reduction in southwest North America. Our results demonstrate that semiochemical-based push-pull techniques have the potential to reduce Dendroctonus and Ips bark beetle populations. Furthermore, based on our analysis, the efficacy of such eco-friendly interventions could be further improved and provide a good tool for forest managers to control these pests, at least under some circumstances.

Evolution and functional role prediction of the CYP6DE and CYP6DJ subfamilies in Dendroctonus (Curculionidae: Scolytinae) bark beetles.

Dendroctonus-bark beetles are natural components and key ecological agents of coniferous forests. They spend most of their lives under the bark, where they are exposed to highly toxic terpenes present in the oleoresin. Cytochrome P450 (CYP) is a multigene family involved in the detoxification of these compounds. It has been demonstrated that CYP6DE and CYP6DJ subfamilies hydroxylate monoterpenes, whose derivatives can act as pheromone synergist compounds or be pheromones themselves in these insects. Given the diversity and functional role of CYPs, we investigated whether these cytochromes have retained their function throughout the evolution of these insects. To test this hypothesis, we performed a Bayesian phylogenetic analysis to determine phylogenetic subgroups of cytochromes in these subfamilies. Subgroups were mapped and reconciled with the Dendroctonus phylogeny. Molecular docking analyses were performed with the cytochromes of each subgroup and enantiomers of α-pinene and ß-pinene, (+)-3-carene, ß-myrcene and R-(+)-limonene. In addition, functional divergence analysis was performed to identify critical amino acid sites that influence changes in catalytic site conformation and/or protein folding. Three and two phylogenetic subgroups were recovered for the CYP6DE and CYP6DJ subfamilies, respectively. Mapping and reconciliation analysis showed different gain and loss patterns for cytochromes of each subgroup. Functional predictions indicated that the cytochromes analyzed are able to hydroxylate all monoterpenes; however, they showed preferential affinities to different monoterpenes. Functional divergence analyses indicated that the CYP6DE subfamily has experimented type I and II divergence, whereas the CYP6DJ subfamily has evolved under strong functional constraints. Results suggest cytochromes of the CYP6DE subfamily evolve to reinforce their detoxifying capacity hydroxylating mainly α- and ß-pinene to (+) and (-)-trans-verbenol, being the negative enantiomer used as a pheromone by several Dendroctonus species; whereas cytochromes of the CYP6DJ subfamily appear to retain their original function related to the detoxification of these compounds.

Coupling Mountain Pine Beetle and Forest Population Dynamics Predicts Transient Outbreaks that are Likely to Increase in Number with Climate Change.

Mountain pine beetle (MPB) in Canada have spread well beyond their historical range. Accurate modelling of the long-term dynamics of MPB is critical for assessing the risk of further expansion and informing management strategies, particularly in the context of climate change and variable forest resilience. Most previous models have focused on capturing a single outbreak without tree replacement. While these models are useful for understanding MPB biology and outbreak dynamics, they cannot accurately model long-term forest dynamics. Past models that incorporate forest growth tend to simplify beetle dynamics. We present a new model that couples forest growth to MPB population dynamics and accurately captures key aspects of MPB biology, including a threshold for the number of beetles needed to overcome tree defenses and beetle aggregation that facilitates mass attacks. These mechanisms lead to a demographic Allee effect, which is known to be important in beetle population dynamics. We show that as forest resilience decreases, a fold bifurcation emerges and there is a stable fixed point with a non-zero MPB population. We derive conditions for the existence of this equilibrium. We then simulate biologically relevant scenarios and show that the beetle population approaches this equilibrium with transient boom and bust cycles with period related to the time of forest recovery. As forest resilience decreases, the Allee threshold also decreases. Thus, if host resilience decreases under climate change, for example under increased stress from drought, then the lower Allee threshold makes transient outbreaks more likely to occur in the future.

A polyphasic taxonomy analysis reveals the presence of an ecotype of Rahnella contaminans associated with the gut of Dendroctonus-bark beetles.

Species belonging to the genus Rahnella are dominant members of the core gut bacteriome of Dendroctonus-bark beetles, a group of insects that includes the most destructive agents of pine forest in North and Central America, and Eurasia. From 300 isolates recovered from the gut of these beetles, 10 were selected to describe an ecotype of Rahnella contaminans. The polyphasic approach conducted with these isolates included phenotypic characteristics, fatty acid analysis, 16S rRNA gene, multilocus sequence analyses (gyrB, rpoB, infB, and atpD genes), and complete genome sequencing of two isolates, ChDrAdgB13 and JaDmexAd06, representative of the studied set. Phenotypic characterization, chemotaxonomic analysis, phylogenetic analyses of the 16S rRNA gene, and multilocus sequence analysis showed that these isolates belonged to Rahnella contaminans. The G + C content of the genome of ChDrAdgB13 (52.8%) and JaDmexAd06 (52.9%) was similar to those from other Rahnella species. The ANI between ChdrAdgB13 and JaDmexAd06 and Rahnella species including R. contaminans, varied from 84.02 to 99.18%. The phylogenomic analysis showed that both strains integrated a consistent and well-defined cluster, together with R. contaminans. A noteworthy observation is the presence of peritrichous flagella and fimbriae in the strains ChDrAdgB13 and JaDmexAd06. The in silico analysis of genes encoding the flagellar system of these strains and Rahnella species showed the presence of flag-1 primary system encoding peritrichous flagella, as well as fimbriae genes from the families type 1, α, ß and σ mainly encoding chaperone/usher fimbriae and other uncharacterized families. All this evidence indicates that isolates from the gut of Dendroctonus-bark beetles are an ecotype of R. contaminans, which is dominant and persistent in all developmental stages of these bark beetles and one of the main members of their core gut bacteriome.

Identification and functional characterization of insulin-like peptides in a pine beetle.

Insulin - including insulin-like peptides (ILPs), relaxins and insulin-like growth factors (IGFs) - is an evolutionarily conserved hormone in all metazoans. It is involved in various physiological processes, such as metabolism, growth, reproduction, lifespan and stress resistance. However, there are no reports on the functional role of ILPs in the Chinese white pine beetle, Dendroctonus armandi. In this study, we have cloned and identified two ILP cDNAs in D. armandi. The expression levels of DaILP1 and DaILP2 were significantly changed in different developmental stages. Both ILPs were expressed mostly in the head and fat body. Moreover, starvation induces the reduction of ILP1 mRNA level in adults and larvae, while ILP2 only in larvae of D. armandi, respectively. Additionally, RNA-interference (RNAi) using double stranded RNA to knock down ILP1 and ILP2 reduced the mRNA levels of the target genes, and caused a significant reduction in body weight of D. armandi. Moreover, silencing ILP1 led to an increase of trehalose and glycogen and significantly enhanced starvation resistance in both adults and larvae. The results show that the ILP signaling pathway plays a significant role in growth and carbohydrate metabolism of D. armandi and may provide a potential molecular target for pest control.

Naïve Pine Terpene Response to the Mountain Pine Beetle (Dendroctonus ponderosae) through the Seasons.

Insect herbivores must contend with constitutive and induced plant defenses. The mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae, Scolytinae) has expanded its range east of the Rocky Mountains into the western boreal forest and is encountering evolutionarily naïve lodgepole pines (Pinus contorta) and jack pines (Pinus banksiana). Pinus contorta and P. banksiana in the expanded range have different constitutive and induced defenses in response to wounding and inoculation with fungal associates of D. ponderosae. In the historic range, previous studies have examined phloem terpene content prior to and just after D. ponderosae mass attack, but the terpene profile of attacked trees post-overwintering is unknown. We examined the response of mature P. contorta and P. banksiana trees to experimentally-applied mass attack by D. ponderosae and quantified phloem terpenes at three time points, pre-attack, post-attack (same season), and the following spring, post-overwintering. Phloem content of total terpenes as well as many individual terpenes increased after D. ponderosae attack but were only significantly higher than pre-attack levels at the post-overwintering time point in both P. contorta and P. banksiana. The absence of a significant increase in phloem terpenes in the month following attack in naïve pines is a potential cause for increased D. ponderosae offspring production reported in naïve P. contorta. Beetle attack density did not influence the phloem terpene profiles of either species and there was no significant interaction between attack density and sampling time on terpene content. High phloem terpenes in trees that are attacked at low densities could prime these trees for defense against attacks in the following season but it could also make these trees more apparent to early-foraging beetles and facilitate efficient mass attack at low D. ponderosae population densities in the expanded range.

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.

Decoupling of height growth and drought or pest resistance tradeoffs is revealed through multiple common-garden experiments of lodgepole pine.

The environment could alter growth and resistance tradeoffs in plants by affecting the ratio of resource allocation to various competing traits. Yet, how and why functional tradeoffs change over time and space is poorly understood particularly in long-lived conifer species. By establishing four common-garden test sites for five lodgepole pine populations in western Canada, combined with genomic sequencing, we revealed the decoupling pattern and genetic underpinnings of tradeoffs between height growth, drought resistance based on δ13C and dendrochronology, and metrics of pest resistance based on pest suitability ratings. Height and δ13C correlation displayed a gradient change in magnitude and/or direction along warm-to-cold test sites. All cold test sites across populations showed a positive height and δ13C relationship. However, we did not observe such a clinal correlation pattern between height or δ13C and pest suitability. Further, we found that the study populations exhibiting functional tradeoffs or synergies to various degrees in test sites were driven by non-adaptive evolutionary processes rather than adaptive evolution or plasticity. Finally, we found positive genetic relationships between height and drought or pest resistance metrics and probed five loci showing potential genetic tradeoffs between northernmost and the other populations. Our findings have implications for deciphering the ecological, evolutionary, and genetic bases of the decoupling of functional tradeoffs due to environmental change.

The effect of inactivation of aldehyde dehydrogenase on pheromone production by a gut bacterium of an invasive bark beetle, Dendroctonus valens.

Semiochemical-based management strategies are important for controlling bark beetles, such as invasive Red Turpentine Beetle (Denroctonus valens), the causal agent for mass mortality of pine trees (Pinus spp.) in China. It has been previously shown that the pheromone verbenone regulates the attack density of this beetle in a dose-dependent manner and that the gut bacteria of D. valens are involved in verbenone production. However, molecular functional verification of the role of gut bacteria in the pheromone production of D. valens is still lacking. To better understand the molecular function of gut bacterial verbenone production, we chose a facultative anaerobic gut bacterium (Enterobacter xiangfangensis) of D. valens based on its strong ability to convert cis-verbenol to verbenone, as shown in our previous study, and investigated its transcriptomics in the presence or absence of cis-verbenol under anaerobic conditions (simulating the anoxic environment in the beetle's gut). Based on this transcriptome analysis, aldehyde dehydrogenase (ALDH1) was identified as a putative key gene responsible for verbenone production and was knocked-down by homologous recombination to obtain a mutant E. xiangfangensis strain. Our results show that these mutants had significantly decreased the ability to convert the monoterpene precursor to verbenone compared with the wild-type bacteria, indicating that ALDH1 is primarily responsible for verbenone conversion for this bacterium species. These findings provide further mechanistic evidence of bacterially mediated pheromone production by D. valens, add new perspective for functional studies of gut bacteria in general, and may aid the development of new gene silencing-based pest management strategies.

Disruption of CYP6DF1 and CYP6DJ2 increases the susceptibility of Dendroctonus armandi to (+)-α-pinene.

Bark beetles rely on detoxifying enzymes to resist the defensive oleoresin terpenes of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of plant allelochemicals and pesticides. CYP6 family is unique to Insecta, and its biochemical function is basically related to catabolize heterologous substances. In this study, two Dendroctonus armandi CYP6 genes, CYP6DF1 and CYP6DJ2, were characterized. Spatiotemporal expression profiling revealed that CYP6DF1 and CYP6DJ2 expressions were higher in larvae and adult stages of D. armandi than in egg and pupae stages, and that two genes predominantly expressed in brain, midgut, fat body, or Malpighian tubules. Moreover, CYP6DF1 and CYP6DJ2 expressions were significantly induced after exposure to (+)-α-pinene. Importantly, silencing CYP6DF1 and CYP6DJ2 significantly inhibited the CYP activity and increased the mortality in the adults fumigated with (+)-α-pinene. Additionally, piperonyl butoxide exposure to adults also increase the sensitivity after treatment with (+)-α-pinene, which led to a significant reduction of the CYP activity, resulting a significant increase in adult mortality. These results suggest that the CYP6 family plays a key role in determining the susceptibility of D. armandi to (+)-α-pinene, which may have implications for the development of novel therapeutics to control this important pest.

Identification and Functional Characterization of the Transcription Factors AhR/ARNT in Dendroctonus armandi.

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the bHLH-PAS (basic Helix-Loop-Helix-Period/ARNT/Single-minded) family of transcription factors, which participate in the sensing and transmitting stimuli of exogenous and endogenous chemical substances, and subsequently activates genes transcription involved in various detoxification and physiological functions. However, they have not been identified in Dendroctonus armandi, and their roles in the detoxification metabolism are unclear. In the present study, AhR and ARNT of D. armandi were characterized. Spatiotemporal expression profiling indicated that DaAhR and DaARNT were highly expressed in the adult and larval stages of D. armandi and mainly expressed in the midgut and Malpighian tubules of adults. Additionally, the expression of DaAhR and DaARNT significantly increased after exposure to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene. Silencing DaAhR and DaARNT increased the susceptibility of D. armandi to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene, and the activities of detoxification enzyme were also remarkably reduced. Moreover, DaCYP6DF1 and DaGSTs2 were significantly down-regulated after injections of dsAhR and dsARNT in the male and female adults, with the expression of DaCYP6DF1 decreasing by higher than 70%. The present study revealed that the transcription factors AhR and ARNT of D. armandi were induced by terpenoids and participated in the regulation of DaCYP6DF1 expression, which was associated with D. armandi's susceptibility to (-)-𝛽-pinene and (±)-limonene. These results may provide a theoretical basis for the integrated control of D. armandi and improve our comprehension of insect toxicology.

Metabarcoding of mycetangia from the Dendroctonus frontalis species complex (Curculionidae: Scolytinae) reveals diverse and functionally redundant fungal assemblages.

Dendroctonus-bark beetles are associated with microbes that can detoxify terpenes, degrade complex molecules, supplement and recycle nutrients, fix nitrogen, produce semiochemicals, and regulate ecological interactions between microbes. Females of some Dendroctonus species harbor microbes in specialized organs called mycetangia; yet little is known about the microbial diversity contained in these structures. Here, we use metabarcoding to characterize mycetangial fungi from beetle species in the Dendroctonus frontalis complex, and analyze variation in biodiversity of microbial assemblages between beetle species. Overall fungal diversity was represented by 4 phyla, 13 classes, 25 orders, 39 families, and 48 genera, including 33 filamentous fungi, and 15 yeasts. The most abundant genera were Entomocorticium, Candida, Ophiostoma-Sporothrix, Ogataea, Nakazawaea, Yamadazyma, Ceratocystiopsis, Grosmannia-Leptographium, Absidia, and Cyberlindnera. Analysis of α-diversity indicated that fungal assemblages of D. vitei showed the highest richness and diversity, whereas those associated with D. brevicomis and D. barberi had the lowest richness and diversity, respectively. Analysis of ß-diversity showed clear differentiation in the assemblages associated with D. adjunctus, D. barberi, and D. brevicomis, but not between closely related species, including D. frontalis and D. mesoamericanus and D. mexicanus and D. vitei. A core mycobiome was not statistically identified; however, the genus Ceratocystiopsis was shared among seven beetle species. Interpretation of a tanglegram suggests evolutionary congruence between fungal assemblages and species of the D. frontalis complex. The presence of different amplicon sequence variants (ASVs) of the same genus in assemblages from species of the D. frontalis complex outlines the complexity of molecular networks, with the most complex assemblages identified from D. vitei, D. mesoamericanus, D. adjunctus, and D. frontalis. Analysis of functional variation of fungal assemblages indicated multiple trophic groupings, symbiotroph/saprotroph guilds represented with the highest frequency (∼31% of identified genera). These findings improve our knowledge about the diversity of mycetangial communities in species of the D. frontalis complex and suggest that minimal apparently specific assemblages are maintained and regulated within mycetangia.