Charles J. Mason

Plant-associated bacteria degrade defense chemicals and reduce their adverse effects on an insect defoliator.

Phytophagous insects must contend with numerous secondary defense compounds that can adversely affect their growth and development. The gypsy moth (Lymantria dispar) is a polyphagous herbivore that encounters an extensive range of hosts and chemicals. We used this folivore and a primary component of aspen chemical defenses, namely, phenolic glycosides, to investigate if bacteria detoxify phytochemicals and benefit larvae. We conducted insect bioassays using bacteria enriched from environmental samples, analyses of the microbial community in the midguts of bioassay larvae, and in vitro phenolic glycoside metabolism assays. Inoculation with bacteria enhanced larval growth in the presence, but not absence, of phenolic glycosides in the artificial diet. This effect of bacteria on growth was observed only in larvae administered bacteria from aspen foliage. The resulting midgut community composition varied among the bacterial treatments. When phenolic glycosides were included in diet, the composition of midguts in larvae fed aspen bacteria was significantly altered. Phenolic glycosides increased population responses by bacteria that we found able to metabolize these compounds in liquid growth cultures. Several aspects of these results suggest that vectoring or pairwise symbiosis models are inadequate for understanding microbial mediation of plant–herbivore interactions in some systems. First, bacteria that most benefitted larvae were initially foliar residents, suggesting that toxin-degrading abilities of phyllosphere inhabitants indirectly benefit herbivores upon ingestion. Second, assays with single bacteria did not confer the benefits to larvae obtained with consortia, suggesting multi- and inter-microbial interactions are also involved. Our results show that bacteria mediate insect interactions with plant defenses but that these interactions are community specific and highly complex.

Acquisition and Structuring of Midgut Bacterial Communities in Gypsy Moth (Lepidoptera: Erebidae) Larvae

ABSTRACT Insects are associated with a diversity of bacteria that colonize their midguts. The extent to which these communities reflect maternal transmission, environmental acquisition, and subsequent structuring by the extreme conditions within the insect gut are poorly understood in many species. We used gypsy moth (Lymantria dispar L.) as a model to investigate interactions between egg mass and environmental sources of bacteria on larval midgut communities. Egg masses were collected from several wild and laboratory populations, and the effects of diet, initial egg mass community, and internal host environment were evaluated using 454 16S-rRNA gene pyrosequencing. Wild populations were highly diverse, while laboratory-maintained egg masses were associated with few operational taxonomic units. As larvae developed, their midgut bacterial communities became more similar to each other and the consumed diet despite initial differences in egg mass-associated bacteria. Subsequent experiments revealed that while midgut membership was more similar to bacteria associated with diet than with egg mass-associated bacteria, we were unable to detect distinct, persistent differences attributable to specific host plants. The differences between foliar communities and midgut communities of larvae that ingested them were owing to relative changes in populations of several bacteria phylotypes. We conclude that gypsy moth has a relatively characteristic midgut bacterial community that is reflective of, but ultimately distinct from, its foliar diet. This work demonstrates that environmental acquisition of diverse microbes can lead to similar midgut bacterial assemblages, underscoring the importance of host physiological environment in structuring bacterial communities.

Aspen Defense Chemicals Influence Midgut Bacterial Community Composition of Gypsy Moth

Microbial symbionts are becoming increasingly recognized as mediators of many aspects of plant – herbivore interactions. However, the influence of plant chemical defenses on gut associates of insect herbivores is less well understood. We used gypsy moth (Lymantria dispar L.), and differing trembling aspen (Populus tremuloides Michx.) genotypes that vary in chemical defenses, to assess the influence of foliar chemistry on bacterial communities of larval midguts. We evaluated the bacterial community composition of foliage, and of midguts of larvae feeding on those leaves, using next-generation high-throughput sequencing. Plant defense chemicals did not influence the composition of foliar communities. In contrast, both phenolic glycosides and condensed tannins affected the bacterial consortia of gypsy moth midguts. The two most abundant operational taxonomic units were classified as Ralstonia and Acinetobacter. The relative abundance of Ralstonia was higher in midguts than in foliage when phenolic glycoside concentrations were low, but lower in midguts when phenolic glycosides were high. In contrast, the relative abundance of Ralstonia was lower in midguts than in foliage when condensed tannin concentrations were low, but higher in midguts when condensed tannins were high. Acinetobacter showed a different relationship with host chemistry, being relatively more abundant in midguts than with foliage when condensed tannin concentrations were low, but lower in midguts when condensed tannins were high. Acinetobacter tended to have a greater relative abundance in midguts of insects feeding on genotypes with high phenolic glycoside concentrations. These results show that plant defense chemicals influence herbivore midgut communities, which may in turn influence host utilization.

Contributions by Host Trees and Insect Activity to Bacterial Communities in Dendroctonus valens (Coleoptera: Curculionidae) Galleries, and Their High Overlap With Other Microbial Assemblages of Bark Beetles

Abstract Bark beetles are associated with a diversity of symbiotic microbiota that can mediate interactions with their host plants. Dendroctonus valens LeConte is a widely distributed bark beetle in North and Central America, and initiates solitary attacks on several species of Pinus in the Great Lakes region. In this study, we aimed to further characterize the bacterial community associated with D. valens feeding galleries using next-generation sequencing, and the possible contributions of both tree-resident and insect-associated bacteria to these consortia. We found that D. valens galleries harbor a diversity of microbial associates. Many of these associates were classified into a few taxonomic groups, of which Gammaproteobacteria were the most abundant class. Of the Gammaproteobacteria detected, many formed clades with 16S-rRNA sequences of bacteria previously associated with D. valens. Many of the bacteria sequences detected in the galleries were similar to bacteria that function in detoxification, kairomone metabolism, and nitrogen fixation and cycling. The abundance of bacteria in galleries were 7× and 44× higher than in the surrounding uninfested tissues, and that were not attacked by D. valens, respectively. This suggests that the bacteria present in beetle galleries are largely introduced by D. valens and proliferate in this environment.

Defence syndromes in lodgepole – whitebark pine ecosystems relate to degree of historical exposure to mountain pine beetles

Warming climate is allowing tree-killing bark beetles to expand their ranges and access naïve and semi-naïve conifers. Conifers respond to attack using complex mixtures of chemical defences that can impede beetle success, but beetles exploit some compounds for host location and communication. Outcomes of changing relationships will depend on concentrations and compositions of multiple host compounds, which are largely unknown. We analysed constitutive and induced chemistries of Dendroctonus ponderosaes primary historical host, Pinus contorta, and Pinus albicaulis, a high-elevation species whose encounters with this beetle are transitioning from intermittent to continuous. We quantified multiple classes of terpenes, phenolics, carbohydrates and minerals. Pinus contorta had higher constitutive allocation to, and generally stronger inducibility of, compounds that resist these beetle-fungal complexes. Pinus albicaulis contained higher proportions of specific monoterpenes that enhance pheromone communication, and lower induction of pheromone inhibitors. Induced P. contorta increased insecticidal and fungicidal compounds simultaneously, whereas P. albicaulis responses against these agents were inverse. Induced terpene accumulation was accompanied by decreased non-structural carbohydrates, primarily sugars, in P. contorta, but not P. albicaulis, which contained primarily starches. These results show some host species with continuous exposure to bark beetles have more thoroughly integrated defence syndromes than less-continuously exposed host species.

Contrasting Patterns of Diterpene Acid Induction by Red Pine and White Spruce to Simulated Bark Beetle Attack, and Interspecific Differences in Sensitivity Among Fungal Associates

Conifers possess a suite of physiochemical defenses that protect their subcortical tissues from bark beetle - fungal complexes. These defenses include rapid induction of terpenoids and phenolics at the site of attack. Studies of the distribution, induction, and bioactivity of conifer terpenoids have focused heavily on monoterpenes. We assessed induction of diterpene acids in white spruce (Picea glauca) and red pine (Pinus resinosa) to fungal associates of two bark beetles, and the responses of four spruce beetle (Dendroctonus rufipennis)—associated fungi to three diterpene acids. Constitutive phloem contents differed between species, in that red pine had extremely low concentrations of diterpene acids, whereas white spruce had substantial constitutive levels. Induction differed quantitatively. Both red pine and white spruce exhibited marked increases, but red pine underwent greater increases and achieved higher concentrations than white spruce. Induction also differed qualitatively in that red pine showed lower diversity and fewer compositional changes during induction than white spruce. In red pine,fungal inoculation accompanying wounding elicited greater increases than wounding alone, but in white spruce total concentrations were higher following wounding alone. Spruce beetle fungal symbiont growth varied among species and compounds. Some diterpenes elicited both stimulatory and inhibitory effects on fungi, depending on concentration. All four fungi exhibited higher tolerances compared to those associated with pine bark beetles in previous studies. Variation in tolerances to, and potentially metabolism of, diterpene acids by symbionts may reflect differences in constitutive levels between spruce and pine, and partially explain differences in concentrations achieved during induction.

Contrasting diets reveal metabolic plasticity in the tree-killing beetle, Anoplophora glabripennis (Cerambycidae: Lamiinae)

Wood-feeding insects encounter challenging diets containing low protein quantities, recalcitrant carbohydrate sources, and plant defensive compounds. The Asian longhorned beetle (Anoplophora glabripennis) is a wood-feeding insect that attacks and kills a diversity of hardwood tree species. We compared gene expression of midguts collected from larvae feeding in a preferred tree, sugar maple, to those consuming a nutrient-rich artificial diet, to identify genes putatively involved in host plant utilization. Anoplophora glabripennis larvae exhibited differential expression of ~3600 genes in response to different diets. Genes with predicted capacity for plant and microbial carbohydrate usage, detoxification, nutrient recycling, and immune-related genes relevant for facilitating interactions with microbial symbionts were upregulated in wood-feeding larvae compared to larvae feeding in artificial diet. Upregulation of genes involved in protein degradation and synthesis was also observed, suggesting that proteins incur more rapid turnover in insects consuming wood. Additionally, wood-feeding individuals exhibited elevated expression of several mitochondrial cytochrome C oxidase genes, suggesting increased aerobic respiration compared to diet-fed larvae. These results indicate that A. glabripennis modulates digestive and basal gene expression when larvae are feeding in a nutrient-poor, yet suitable host plant compared to a tractable and nutrient-rich diet that is free of plant defensive compounds.

Interactions between Bacteria And Aspen Defense Chemicals at the Phyllosphere – Herbivore Interface

Plant- and insect-associated microorganisms encounter a diversity of allelochemicals, and require mechanisms for contending with these often deleterious and broadly-acting compounds. Trembling aspen, Populus tremuloides, contains two principal groups of defenses, phenolic glycosides (salicinoids) and condensed tannins, which differentially affect the folivorous gypsy moth, Lymantria dispar, and its gut symbionts. The bacteria genus Acinetobacter is frequently associated with both aspen foliage and gypsy moth consuming that tissue, and one isolate, Acinetobacter sp. R7-1, previously has been shown to metabolize phenolic glycosides. In this study, we aimed to characterize further interactions between this Acinetobacter isolate and aspen secondary metabolites. We assessed bacterial carbon utilization and growth in response to different concentrations of phenolic glycosides and condensed tannins. We also tested if enzyme inhibitors reduce bacterial growth and catabolism of phenolic glycosides. Acinetobacter sp. R7-1 utilized condensed tannins but not phenolic glycosides or glucose as carbon sources. Growth in nutrient-rich medium was increased by condensed tannins, but reduced by phenolic glycosides. Addition of the P450 enzyme inhibitor piperonyl butoxide increased the effects of phenolic glycosides on Acinetobacter sp. R7-1. In contrast, the esterase inhibitor S,S,S,-tributyl-phosphorotrithioate did not affect phenolic glycoside inhibition of bacterial growth. Degradation of phenolic glycosides by Acinetobacter sp. R7-1 appears to alleviate the cytotoxicity of these compounds, rather than provide an energy source. Our results further suggest this bacterium utilizes additional, complementary mechanisms to degrade antimicrobial phytochemicals. Collectively, these results provide insight into mechanisms by which microorganisms contend with their environment within the context of plant-herbivore interactions.

Foliar bacterial communities of trembling aspen in a common garden.

Microbial associations with plants are widely distributed and are structured by a number of biotic and physical factors. Among biotic factors, the host plant genotype may be integral to these plant-microbe interactions. Trees in the genus Populus have become models for studies in scaling effects of host plant genetics and in plant-microbe interactions. Using 454 pyrosequencing of the 16S rRNA gene, we assessed the foliar bacterial community of 7 genotypes of mature trembling aspen trees (Populus tremuloides Michx.) grown in a common garden. Trees were selected based on prior analyses showing clonal variation in their concentration of chemicals conferring resistance against insect herbivores. At broad taxonomic designations, the bacterial community of trembling aspen was similar across all plant genotypes. At a finer taxonomic scale, the foliage of these trees varied in their community composition, but there was no distinct pattern to colonization or abundance related to plant genotype. The most abundant operational taxonomic units (OTUs) were classified as Ralstonia, Bradyrhizobium, Pseudomonas, and Brucella. These OTUs varied across the common garden, but there was no significant effect of host plant genotype or spatial position on the abundance of these members. Our results suggest that aspen genotype is less important in the structuring of its foliar bacterial communities than are other, poorly understood processes.

Spatial and temporal components of induced plant responses in the context of herbivore life history and impact on host

Plants defend against herbivores and pathogens through integrated constitutive and induced defenses. Induced responses may be expressed locally or tissue/plant-wide, i.e. systemically, and may also be primed for subsequent attack. Although the elicitation and efficacy of induced responses are increasingly well-characterized, we have little understanding of how timing and within-plant spatial patterns of induced defenses relate to different herbivore behaviors and selective pressures. We used interactions between pines and their major mortality agents, native bark beetle-fungal complexes, to explore this dimension. We analyzed concentrations of multiple terpenoid and phenolic classes, and lesion formation, to provide a comprehensive profile of specialized metabolites and histological responses in red pine (Pinus resinosa) phloem. We examined these profiles in constitutive tissue and following simulated attack, sampling both at the point of challenge and away from the attack site, and following a second simulated attack. Terpenoid concentrations increased by > 100-fold at the site of simulated attack. In contrast, systemic induction of terpenoids was absent or weak, with most exhibiting no change and others increasing only 1.5–2 fold. Previous elicitation did not influence terpenoid concentrations, either locally or tissue-wide, when trees were challenged a second time. Phenolics had mixed responses in localized tissues, with some compounds increasing and others decreasing. Like terpenoids, phenolics did not show substantial systemic, tissue-wide changes, and likewise showed no evidence of priming. Collectively, these results indicate that red pine employs a strategy of maximizing its response at each point of attack by bark beetles. Pines have been shown to express systemic induced resistance against several canker fungi, so the absence of these responses suggests agent-specific reactions, rather than inherent incapability. Rapid local induction seems to be a better strategy in this instance because when a bark beetle succeeds in entering and producing pheromones from a host, the resulting mass attack rapidly covers the entire circumference and kills the tree. These results highlight how plant defense syndromes can modulate the spatial and temporal dynamics of induced responses, in addition to the chemical and morphological traits deployed. This article is protected by copyright. All rights reserved.