Mutualisms between symbiotic microbes and animals have been well documented, and nutritional relationships provide the foundation for maintaining beneficial associations. The well-studied mutualism between bark beetles and their fungi has become a classic model system in the study of symbioses. Despite the nutritional competition between bark beetles and beneficial fungi in the same niche due to poor nutritional feeding substrates, bark beetles still maintain mutualistic associations with beneficial fungi over time. The mechanism behind this phenomenon, however, remains largely unknown. Here, we demonstrated the bark beetle Dendroctonus valens LeConte relies on the symbiotic bacterial volatile ammonia, as a nitrogen source, to regulate carbohydrate metabolism of its mutualistic fungus Leptographium procerum to alleviate nutritional competition, thereby maintaining the stability of the bark beetle–fungus mutualism. Ammonia significantly reduces competition of L. procerum for carbon resources for D. valens larval growth and increases fungal growth. Using stable isotope analysis, we show the fungus breakdown of phloem starch into d-glucose by switching on amylase genes only in the presence of ammonia. Deletion of amylase genes interferes with the conversion of starch to glucose. The acceleration of carbohydrate consumption and the conversion of starch into glucose benefit this invasive beetle–fungus complex. The nutrient consumption–compensation strategy mediated by tripartite beetle–fungus–bacterium aids the maintenance of this invasive mutualism under limited nutritional conditions, exacerbating its invasiveness with this competitive nutritional edge.

Since its introduction from North America, Dendroctonus valens LeConte has become a destructive forest pest in China. Although gut aerobic bacteria have been investigated and some are implicated in beetle pheromone production, little is known about the abundance and significance of facultative anaerobic bacteria in beetle gut, especially with regards to effects of oxygen on their role in pheromone production. In this study, we isolated and identified gut bacteria of D. valens adults in an anaerobic environment, and further compared their ability to convert cis-verbenol into verbenone (a multi-functional pheromone of D. valens) under different O2 concentrations. Pantoea conspicua, Enterobacter xiangfangensis, Staphylococcus warneri were the most frequently isolated species among the total of 10 species identified from beetle gut in anaerobic conditions. Among all isolated species, nine were capable of cis-verbenol to verbenone conversion, and the conversion efficiency increased with increased oxygen concentration. This O2-mediated conversion of cis-verbenol to verbenone suggests that gut facultative anaerobes of D. valens might play an important role in the frass, where there is higher exposure to oxygen, hence the higher verbenone production. This claim is further supported by distinctly differential oxygen concentrations between gut and frass of D. valens females.

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 dosedependent 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 wildtype 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.

No abstract

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.

Insect gut microbiota have been widely reported to help the insects to overcome host tree defense. Streltzoviella insularis (Lepidoptera: Cossidae) is one of the most common wood borers in China, attacking various hosts, including ash trees (Fraxinus sp.), but little is known about its gut microbial associates and their involvement in host tree defense. We isolated gut bacteria of S. insularis larvae, analyzed their ability to degrade pinoresinol (a defense compound of ash trees) and cellulose, and identified pinoresinol degradation products. Larval mortality increased with increasing pinoresinol concentration (reflecting natural variation observed in the host trees). All the five detected gut bacteria isolates were able to degrade pinoresinol, two of which were also capable of cellulose degradation. Furthermore, gut bacteria were also shown to degrade pinoresinol via the gluconeogenesis pathway. These results suggest that S. insularis-associated microorganisms help to overcome host pinoresinol defense and possibly contribute to insects or gut microbial nutrition via carbohydrate synthesis.

Bark beetles are economically and ecologically important insect group, which aggregation behavior and thus host colonization success depended on pheromone-mediated communication. For some species, such as for 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 three different pH mediums (main host diet with natural pH of 4.7; a mildly acidic diet with pH 6 mimicking the beetle’s gut pH; and highly acidic diet with pH 4), and measured their effects on the gut pH, gut bacterial community and production of the main aggregation and anti-aggregation pheromone (verbenone). We further tested the verbenone production capacity of two 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 gut’s acidity, whereas feeding on highly acidic diet (pH 4) enhanced it. Both changes in gut pH reduced the abundance of dominant bacterial genera, resulting in degrease in 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.

Semiochemical-based strategies are important for bark beetle management worldwide. One of the most destructive invasive bark beetles, the causal agent of mass pine (Pinus sp.) mortality in China, is the red turpentine beetle (RTB) Dendroctonus valens (LeConte), originating from North America. For this species, verbenone pheromone regulates the beetles’ attack density in a dosage dependent manner. In addition, we have previously shown that RTB’s gut bacteria is involved in the pheromone production under both anaerobic and micro-oxygen environments. However, although some investigations of anaerobic gut bacteria of bark beetles have been made, functional verification of their role at molecular level is still lacking. To clarify the function of gut bacteria in verbenone production, we investigated the activity of key genes of the primary gut bacteria involved in verbenone production in D. valens under anaerobic environment. These key genes (aldehyde dehydrogenase) identified by transcriptome analysis were then knocked out by homologous recombination to obtain mutant bacteria strains. Our results show that these mutants had significantly decreased ability to convert the monoterpene precursor to verbenone compared to the wild type bacteria. Our findings provide further evidence of the mechanism of pheromone production of D. valens and a new perspective for functional studies of gut bacteria in general.