Bacterial volatile ammonia regulates the consumption sequence of d-pinitol and d-glucose in a fungus associated with an invasive bark beetle.

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.

Does cryptic microbiota mitigate pine resistance to an invasive beetle-fungus complex? Implications for invasion potential.

Microbial symbionts are known to assist exotic pests in their colonization of new host plants. However, there has been little evidence linking symbiotic invasion success to mechanisms for mitigation of native plant resistance. The red turpentine beetle (RTB) was introduced with a fungus, Leptographium procerum, to China from the United States and became a destructively invasive symbiotic complex in natural Pinus tabuliformis forests. Here, we report that three Chinese-resident fungi, newly acquired by RTB in China, induce high levels of a phenolic defensive chemical, naringenin, in pines. This invasive beetle-fungus complex is suppressed by elevated levels of naringenin. However, cryptic microbiotas in RTB galleries strongly degrade naringenin, and pinitol, the main soluble carbohydrate of P. tabuliformis, is retained in L. procerum-infected phloem and facilitate naringenin biodegradation by the microbiotas. These results demonstrate that cryptic microbiota mitigates native host plant phenolic resistance to an invasive symbiotic complex, suggesting a putative mechanism for reduced biotic resistance to symbiotic invasion.

Altered Carbohydrates Allocation by Associated Bacteria-fungi Interactions in a Bark Beetle-microbe Symbiosis.

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 fungus Leptographium procerum have played key roles in RTB colonization. However, common fungal associates (L. procerum and Ophiostoma 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 of O. minus. However, they didn't inhibit the growth of L. procerum and forced this fungus to consume D-pinitol before consuming D-glucose, even though D-glucose was available and a better carbon source not only for L. procerum but 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.

Gut-Associated Bacteria of Dendroctonus valens and their Involvement in Verbenone Production.

Bark beetles are the most important mortality agent in coniferous forests, and pheromones play important roles in their management. Dendroctonus valens LeConte was introduced from North America to China and has killed millions of healthy pines there. Trapping with semiochemicals and pheromones was deployed in D. valens management in the last decade, but little is known about the ability of gut bacteria to produce the pheromone. In this study, we analyzed the volatiles in D. valens guts and frass after antibiotic treatment versus control. Then, we isolated and identified the bacteria in D. valens guts and frass, examined verbenone (a multifunctional pheromone of D. valens) production by 16 gut bacterial isolates from the precursor cis-verbenol at three concentrations, and further compared the cytotoxicities between the cis-verbenol and verbenone to the bacterial isolates. cis-Verbenol was not detected in the frass in the control group, but it was in the antibiotic treatment. The amount of verbenone was significantly suppressed in D. valens guts after antibiotic treatment versus control. Thirteen out of 16 gut bacterial isolates were capable of cis-verbenol to verbenone conversion, and cis-verbenol had stronger cytotoxicities than verbenone to all tested gut bacterial isolates. The bacterial species capable of verbenone production largely exists in D. valens guts and frass, suggesting that gut-associated bacteria may help the bark beetle produce the pheromone verbenone in guts and frass. The bacteria may benefit from the conversion due to the reduced cytotoxicity from the precursor to the beetle pheromone.

Yeast diversity associated with invasive Dendroctonus valens killing Pinus tabuliformis in China using culturing and molecular methods.

Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are discussed.