Insect-microbe interaction is a key area of research in multiplayer symbiosis, yet little is known about the role of microbe-microbe interactions in insect-microbe symbioses. The red turpentine beetle (RTB) has destroyed millions of healthy pines in China and forms context-dependent relationships with associated fungi. The adult-associated fungusLeptographium procerumhave played key roles in RTB colonization. However, common fungal associates (L. procerumandOphiostoma minus) with RTB larvae compete for carbohydrates. Here, we report that dominant bacteria associated with RTB larvae buffer the competition by inhibiting the growth and D-glucose consumption ofO. minus. However, they didn’t inhibit the growth ofL. procerumand forced this fungus to consume D-pinitol before consuming D-glucose, even though D-glucose was available and a better carbon source not only forL. procerumbut also for RTB larvae and associated bacteria. This suggests the most frequently isolated bacteria associated with RTB larvae could affect fungal growth and the sequence of carbohydrate consumption. Thus, this regulates carbohydrate allocation in the RTB larva-microbe community, which may in turn benefit RTB larvae development. We also discuss the mechanism of carbohydrate allocation in the RTB larva-microbe community and its potential contribution to the maintenance of a symbiotic community.

The exposure to plant defense chemicals has negative effects on insect feeding activity and modifies insect gut microbial community composition. Dendroctonus valens is a very destructive forest pest in China, and harbors a large diversity and abundance of gut microorganisms. Host pine defensive chemicals can protect the pines from attack by the holobiont. In this study, boring length of D. valens feeding on 0 mg/g α-pinene and 9 mg/g α-pinene concentration in phloem media for 6 and 48 h were recorded, and their gut bacterial communities were analyzed in parallel. Nine milligram per gram α-pinene concentration significantly inhibited boring length of D. valens and altered its gut microbial community structure after 6 h. The inhibition of boring length from 9 mg/g α-pinene in diets ceased after 48 h. No significant differences of the bacterial communities were observed between the beetles in 0 and 9 mg/g α-pinene concentration in phloem media after 48 h. Our results showed that the inhibition of the feeding behavior of D. valens and the disturbance to its gut bacterial communities in 9 mg/g α-pinene concentration in phloem media after 6 h were eliminated after 48 h. The resilience of gut bacterial community of D. valens may help the beetle catabolize pine defense chemical.

Bark beetles are among the most destructive of pine forest pests and they form close symbiotic relationships with ophiostomatoid fungi. Although some fungi are considered to be mutualistic symbionts of bark beetles with respect to the supply of nutrients, detrimental effects of fungal symbionts on larval growth have also been frequently reported. The mechanisms of such antagonistic effects are hypothesized to be a decrease in nutritional resources caused by competition for saccharides by the fungi. Here, we provide experimental evidence that three beetle-associated fungi modify the nutritional content of an artificial phloem diet, leading to a detrimental effect on the growth of Dendroctonus valens larvae. When larvae were fed a diet of pine phloem in agar medium colonized with any of these fungi, feeding activity was not affected but weight significantly decreased. Additional analysis showed that fungi depleted the fructose and glucose concentrations in the phloem media. Furthermore, these detrimental effects were neutralized by supplementing the media with fructose or glucose, suggesting that fungi may affect larval growth by modifying diet saccharide contents. These data indicate that fungus-induced nutritional changes in bark beetle diet can affect larval growth, and that the mechanism involves fungus-induced saccharide depletion from the larval diet.

Interactions among microbial symbionts have multiple roles in the maintenance of insect–microbe symbiosis. However, signals mediating microbial interactions have been scarcely studied. In the classical model system of bark beetles and fungal associates, fungi increase the fitness of insects. However, not all interactions are mutualistic, some of these fungal symbionts compete for sugars with beetle larvae. How this antagonistic effect is alleviated is unknown, and recent research suggests potential roles of bacterial symbionts. Red turpentine beetle (RTB), Dendroctonus valens LeConte, is an invasive pest in China, and it leads to wide spread, catastrophic mortality to Chinese pines. In the symbiotic system formed by RTB, fungi and bacteria, volatiles from predominant bacteria regulate the consumption sequence of carbon sources d-pinitol and d-glucose in the fungal symbiont Leptographium procerum, and appear to alleviate the antagonistic effect from the fungus against RTB larvae. However, active components of these volatiles are unknown. We detected 67 volatiles by Gas Chromatography-Mass Spectrometer (GC-MS). Seven of them were identified as candidate chemicals mediating bacteria-fungus interactions, among which ammonia made L. procerum consume its secondary carbon source D-pinitol instead of its preferred carbohydrate D-glucose. In conclusion, ammonia regulated the consumption sequence of these two carbon sources in the fungal symbiont.

Interactions between invasive insects and their fungal associates have important effects on the behavior, reproductive success, population dynamics and evolution of the organisms involved. The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Scolytinae), an invasive forest pest in China, is closely associated with fungi. By carrying fungi on specialized structures in the exoskeleton, RTB inoculates fungi in the phloem of pines (when females dig galleries for egg laying and when males join them for mating). After eggs hatch, larvae gregariously feed on the phloem colonized by the fungi. We examined the effects of five isolates of RTB associated fungi (two from North America, Leptographium terebrantis and L. procerum, and three from China, Ophiostoma minus, L. sinoprocerum and L. procerum) on larval feeding activity, development and mortality. We also studied the effects of volatile chemicals produced in the beetle hindgut on fungal growth. Ophiostoma minus impaired feeding activity and reduced weight in RTB larvae. Leptographium sinoprocerum, L. terebrantis and L. procerum did not dramatically influence larval feeding and development compared to fungi-free controls. Larval mortality was not influenced by any of the tested fungi. Hindgut volatiles of RTB larvae, verbenol, myrtenol and myrtenal, inhibited growth rate of all the fungi. Our results not only show that D. valens associated fungus, O. minus, can be detrimental to its larvae; but, most importantly, they also show that these notorious beetles have an outstanding adaptive response evidenced by the ability to produce volatiles that inhibit growth of harmful fungus.