Convergent bacterial microbiotas in the fungal agricultural systems of insects.

UNLABELLED: The ability to cultivate food is an innovation that has produced some of the most successful ecological strategies on the planet. Although most well recognized in humans, where agriculture represents a defining feature of civilization, species of ants, beetles, and termites have also independently evolved symbioses with fungi that they cultivate for food. Despite occurring across divergent insect and fungal lineages, the fungivorous niches of these insects are remarkably similar, indicating convergent evolution toward this successful ecological strategy. Here, we characterize the microbiota of ants, beetles, and termites engaged in nutritional symbioses with fungi to define the bacterial groups associated with these prominent herbivores and forest pests. Using culture-independent techniques and the in silico reconstruction of 37 composite genomes of dominant community members, we demonstrate that different insect-fungal symbioses that collectively shape ecosystems worldwide have highly similar bacterial microbiotas comprised primarily of the genera Enterobacter, Rahnella, and Pseudomonas. Although these symbioses span three orders of insects and two phyla of fungi, we show that they are associated with bacteria sharing high whole-genome nucleotide identity. Due to the fine-scale correspondence of the bacterial microbiotas of insects engaged in fungal symbioses, our findings indicate that this represents an example of convergence of entire host-microbe complexes. IMPORTANCE: The cultivation of fungi for food is a behavior that has evolved independently in ants, beetles, and termites and has enabled many species of these insects to become ecologically important and widely distributed herbivores and forest pests. Although the primary fungal cultivars of these insects have been studied for decades, comparatively little is known of their bacterial microbiota. In this study, we show that diverse fungus-growing insects are associated with a common bacterial community composed of the same dominant members. Furthermore, by demonstrating that many of these bacteria have high whole-genome similarity across distantly related insect hosts that reside thousands of miles apart, we show that these bacteria are an important and underappreciated feature of diverse fungus-growing insects. Because of the similarities in the agricultural lifestyles of these insects, this is an example of convergence between both the life histories of the host insects and their symbiotic microbiota.

Cellulolytic Streptomyces strains associated with herbivorous insects share a phylogenetically linked capacity to degrade lignocellulose.

Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.

Bacteria associated with a tree-killing insect reduce concentrations of plant defense compounds.

Bark beetles encounter a diverse array of constitutive and rapidly induced terpenes when attempting to colonize living conifers. Concentrations of these compounds at entry sites can rapidly reach levels toxic to beetles, their brood, and fungal symbionts. Large numbers of beetles can overwhelm tree defenses via pheromone-mediated mass attacks, but the mechanisms are poorly understood. We show that bacteria associated with mountain pine beetles can metabolize monoterpenes and diterpene acids. The abilities of different symbionts to reduce concentrations of different terpenes appear complementary. Serratia reduced concentrations of all monoterpenes applied to media by 55-75 %, except for α-pinene. Beetle-associated Rahnella reduced (-)- and (+)-α-pinene by 40 % and 45 %, respectively. Serratia and Brevundimonas reduced diterpene abietic acid levels by 100 % at low concentrations. However, high concentrations exhausted this ability, suggesting that opposing rates of bacterial metabolism and plant induction of terpenes are critical. The two major fungal symbionts of mountain pine beetle, Grosmannia clavigera and Ophiostoma montium were highly susceptible to abietic acid. Grosmannia clavigera did not reduce total monoterpene concentrations in lodgepole pine turpentine. We propose the ability of bark beetles to exert landscape-scale impacts may arise partly from micro-scale processes driven by bacterial symbionts.

Mountain pine beetles colonizing historical and naive host trees are associated with a bacterial community highly enriched in genes contributing to terpene metabolism.

The mountain pine beetle, Dendroctonus ponderosae, is a subcortical herbivore native to western North America that can kill healthy conifers by overcoming host tree defenses, which consist largely of high terpene concentrations. The mechanisms by which these beetles contend with toxic compounds are not well understood. Here, we explore a component of the hypothesis that beetle-associated bacterial symbionts contribute to the ability of D. ponderosae to overcome tree defenses by assisting with terpene detoxification. Such symbionts may facilitate host tree transitions during range expansions currently being driven by climate change. For example, this insect has recently breached the historical geophysical barrier of the Canadian Rocky Mountains, providing access to näive tree hosts and unprecedented connectivity to eastern forests. We use culture-independent techniques to describe the bacterial community associated with D. ponderosae beetles and their galleries from their historical host, Pinus contorta, and their more recent host, hybrid P. contorta-Pinus banksiana. We show that these communities are enriched with genes involved in terpene degradation compared with other plant biomass-processing microbial communities. These pine beetle microbial communities are dominated by members of the genera Pseudomonas, Rahnella, Serratia, and Burkholderia, and the majority of genes involved in terpene degradation belong to these genera. Our work provides the first metagenome of bacterial communities associated with a bark beetle and is consistent with a potential microbial contribution to detoxification of tree defenses needed to survive the subcortical environment.

Responses of bark beetle-associated bacteria to host monoterpenes and their relationship to insect life histories.

Bark beetles that colonize living conifers and their microbial associates encounter constitutive and induced chemical defenses of their host. Monoterpene hydrocarbons comprise a major component of these allelochemicals, and many are antibiotic to insects, fungi, and bacteria. Some bark beetle species exhaust these defenses by killing their host through mass attacks mediated by aggregation pheromones. Others lack adult aggregation pheromones and do not engage in pheromone-mediated mass attacks, but rather have the ability to complete development within live hosts. In the former species, the larvae develop in tissue largely depleted of host terpenes, whereas in the latter exposure to these compounds persists throughout development. A substantial literature exists on how monoterpenes affect bark beetles and their associated fungi, but little is known of how they affect bacteria, which in turn can influence beetle performance in various manners. We tested several bacteria from two bark beetle species for their ability to grow in the presence of a diversity of host monoterpenes. Bacteria were isolated from the mountain pine beetle, Dendroctonus ponderosae Hopkins, which typically kills trees during colonization, and the red turpentine beetle, Dendroctonus valens LeConte, which often lives in their host without causing mortality. Bacteria from D. ponderosae were gram-positive Actinobacteria and Bacilli; one yeast also was tested. Bacteria from D. valens were Actinobacteria, Bacilli, and γ-Proteobacteria. Bacteria from D. valens were more tolerant of monoterpenes than were those from D. ponderosae. Bacteria from D. ponderosae did not grow in the presence of α-pinene and 3-carene, and grew in, but were inhibited by, ß-pinene and ß-phellandrene. Limonene and myrcene had little inhibitory effect on bacteria from either beetle species. Tolerance to these antibiotic compounds appears to have resulted from adaptation to living in a terpene-rich environment.

Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio.

Sirex noctilio is an invasive wood-feeding wasp that threatens the world's commercial and natural pine forests. Successful tree colonization by this insect is contingent on the decline of host defenses and the ability to utilize the woody substrate as a source of energy. We explored its potential association with bacterial symbionts that may assist in nutrient acquisition via plant biomass deconstruction using growth assays, culture-dependent and -independent analysis of bacterial frequency of association and whole-genome analysis. We identified Streptomyces and γ-Proteobacteria that were each associated with 94% and 88% of wasps, respectively. Streptomyces isolates grew on all three cellulose substrates tested and across a range of pH 5.6 to 9. On the basis of whole-genome sequencing, three Streptomyces isolates have some of the highest proportions of genes predicted to encode for carbohydrate-active enzymes (CAZyme) of sequenced Actinobacteria. γ-Proteobacteria isolates grew on a cellulose derivative and a structurally diverse substrate, ammonia fiber explosion-treated corn stover, but not on microcrystalline cellulose. Analysis of the genome of a Pantoea isolate detected genes putatively encoding for CAZymes, the majority predicted to be active on hemicellulose and more simple sugars. We propose that a consortium of microorganisms, including the described bacteria and the fungal symbiont Amylostereum areolatum, has complementary functions for degrading woody substrates and that such degradation may assist in nutrient acquisition by S. noctilio, thus contributing to its ability to be established in forested habitats worldwide.

Presence and diversity of Streptomyces in Dendroctonus and sympatric bark beetle galleries across North America.

Recent studies have revealed several examples of intimate associations between insects and Actinobacteria, including the Southern Pine Beetle Dendroctonus frontalis and the Spruce Beetle Dendroctonus rufipennis. Here, we surveyed Streptomyces Actinobacteria co-occurring with 10 species of Dendroctonus bark beetles across the United States, using both phylogenetic and community ecology approaches. From these 10 species, and 19 other scolytine beetles that occur in the same trees, we obtained 154 Streptomyces-like isolates and generated 16S sequences from 134 of those. Confirmed 16S sequences of Streptomyces were binned into 36 distinct strains using a threshold of 0.2% sequence divergence. The 16S rDNA phylogeny of all isolates does not correlate with the distribution of strains among beetle species, localities, or parts of the beetles or their galleries. However, we identified three Streptomyces strains occurring repeatedly on Dendroctonus beetles and in their galleries. Identity of these isolates was corroborated using a house-keeping gene sequence (efTu). These strains are not confined to a certain species of beetle, locality, or part of the beetle or their galleries. However, their role as residents in the woodboring insect niche is supported by the repeated association of their 16S and efTu from across the continent, and also having been reported in studies of other subcortical insects.

Geographic variation in bacterial communities associated with the red turpentine beetle (Coleoptera: Curculionidae).

Bacterial communities are known to play important roles in insect life histories, yet their consistency or variation across populations is poorly understood. Bacteria associated with the bark beetle Dendroctonus valens LeConte from eight populations, ranging from Wisconsin to Oregon, were evaluated and compared. We used the culture-independent technique of denaturing gradient gel electrophoresis to visualize bacterial diversity, or individual operational taxonomic units (OTUs), from individual beetles. One-way analysis of similarities was used to test for differences of bacterial communities between sites. Analysis of community profiles showed that individual beetles on average contained 10 OTUs, with frequency of association from 2 to 100% of beetles. OTU sequences most closely matched beta- and gamma-proteobacteria, and one each matched Bacilli and Actinobacteria. Several OTUs were particularly abundant, most notably an Actinobacterium from 100% and two Proteobacteria from 60% of beetles sampled. Some OTUs were similar to previously described bacteria with known biochemical capabilities and ecological functions, suggesting that some bacterial associates of D. valens may contribute to its ability to exploit a resource low in nutrients and high in defensive compounds. There were significant differences of bacterial communities between sites. The strength of these differences was positively correlated with distance between sites, although additional unexplained factors also contribute to the variation.

Tri-trophic linkages in disease: pathogen transmission to rainbow trout through stonefly prey.

Relationships between macroinvertebrates and microorganisms in aquatic environments are only poorly understood despite the fact that many aquatic macroinvertebrates feed on microbial biofilms during some life stage. Better understanding of trophic interactions between microbial biofilms, macroinvertebrates, and fish may also help control fish diseases and loss of natural resources. It has also been suggested that pollution, habitat fragmentation, and poor water quality may contribute to increased pathogenesis and mortality in fish. Increased disease incidence is difficult to assess, however, in part because of the complexity of pathogen transmission dynamics. Several environmental pathogens exist whose reservoir(s) and means of transmission remain poorly understood, highlighting the need to study pathogen ecology and interactions with organisms other than susceptible hosts. Aeromonas salmonicida is rarely isolated from freshwater sediments. However, stonefly nymphs were found to frequently harbor A. salmonicida and were shown to preferentially feed on the bacterium. Rainbow trout juveniles were presented with different feeding regimes to determine the transmission capacity of nymphs, and all fish fed stoneflies harboring A. salmonicida expressed symptoms of disease. Although current rates of furunculosis in freshwater ecosystems are unknown, trout primarily feed on stoneflies when water oxygen levels are high and temperatures are low (winter months), which is presumed to correspond to high resistance to the pathogen. Given that furunculosis is associated with physiological stress and higher water temperatures, its natural incidence may change in response to global or regional climatological effects.

In vitro interactions between yeasts and bacteria and the fungal symbionts of the mountain pine beetle (Dendroctonus ponderosae).

Multi-trophic interactions between prokaryotes, unicellular eukaryotes, and ecologically intertwined metazoans are presumably common in nature, yet rarely described. The mountain pine beetle, Dendroctonus ponderosae, is associated with two filamentous fungi, Grosmannia clavigera and Ophiostoma montium. Other microbes, including yeasts and bacteria, are also present in the phloem, but it is not known whether they interact with the symbiotic fungi or the host beetle. To test whether such interactions occur, we performed a suite of in vitro assays. Overall, relative yield of O. montium grown with microbes isolated from larval galleries was significantly greater than when the fungus was grown alone. Conversely, the yield of G. clavigera grown with these same microbes was less than or equal to when it was grown alone, suggesting that O. montium, and at least some microbes in larval galleries, have a mutualistic or commensal relationship, while G. clavigera and those same microbes have an antagonistic relationship. A bacterium isolated from phloem not colonized by beetles was found to inhibit growth of both G. clavigera and O. montium and appears to be an antagonist to both fungi. Our results suggest that bacteria and yeasts likely influence the distribution of mycangial fungi in the host tree, which, in turn, may affect the fitness of D. ponderosae.

Temporal variation in mycophagy and prevalence of fungi associated with developmental stages of Dendroctonus ponderosae (Coleoptera: Curculionidae).

Mycophagy by bark beetles is widespread. However, little is known regarding which developmental stages of bark beetles actually feed on fungi. To study this question, we sampled fungi associated with Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae) throughout development in naturally attacked trees. Isolations of fungi were made from phloem adjacent to brood and from brood exoskeletons and guts. Overall, the incidence of fungi with individual brood increased as brood development progressed. Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingf. and Ophiostoma montium (Rumbold) von Arx exhibited generally opposing trends in prevalence. G. clavigera was most likely to be found in phloem adjacent to prewintering third- and postwintering fourth-instar larvae. O. montium was most likely to be found in phloem adjacent to eggs, first-instar larvae, pupae, and teneral adults. In contrast to isolations made from phloem, fungi isolated from brood guts and exoskeletons were not observed to shift in prevalence. First- and third-instar larvae were often observed migrating to older portions of their galleries, indicating that they do not spend all of their time feeding at, and extending, the apex of the gallery. Our results suggest that not only are D. ponderosae brood in contact with and feeding on fungi throughout development, but also, that during development, contact of brood with a particular fungus is likely to change. Such temporal shifts in fungal symbionts may be environmentally driven and have important implications in how these fungi interact with their hosts within and across generations.