Upper respiratory microbial communities of healthy populations are shaped by niche and age.

Background: Alterations in upper respiratory microbiomes have been implicated in shaping host health trajectories, including by limiting mucosal pathogen colonization. However, limited comparative studies of respiratory microbiome development and functioning across age groups have been performed. Herein, we perform shotgun metagenomic sequencing paired with pathogen inhibition assays to elucidate differences in nasal and oral microbiome composition and functioning across healthy 24-month-old infant (n=229) and adult (n=100) populations. Results: We find that beta diversity of nasal and oral microbiomes varies with age, with nasal microbiomes showing greater population-level variation compared to oral microbiomes. Infant microbiome alpha diversity was significantly lower across nasal samples and higher in oral samples, relative to adults. Accordingly, we demonstrate significant differences in genus- and species-level composition of microbiomes between sites and age groups. Antimicrobial resistome patterns likewise varied across body sites, with oral microbiomes showing higher resistance gene abundance compared to nasal microbiomes. Biosynthetic gene clusters encoding specialized metabolite production were found in higher abundance across infant oral microbiomes, relative to adults. Investigation of pathogen inhibition revealed greater inhibition of gram-negative and gram-positive bacteria by oral commensals, while nasal isolates had higher antifungal activity. Conclusions: In summary, we identify significant differences in the microbial communities inhabiting nasal and oral cavities of healthy infants relative to adults. These findings inform our understanding of the interactions impacting respiratory microbiome composition and functioning, with important implications for host health across the lifespan.

Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

Disturbance-diversity relationships of microbial communities change based on growth substrate.

Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances and how those responses can vary is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that the community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the genera Cellvibrio, Lacunisphaera, and Asticcacaulis are the most abundant microbes. However, Lacunisphaera is only abundant in the lower disturbance frequency treatments, while Asticcacaulis is more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are two Pseudomonas sequence variants and a Cohnella sequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (1.95-7.33 Hill 1 diversity) that peaks at the intermediate disturbance frequency treatment or one disturbance every 3 days. Communities grown on glucose, however, ranged from 1.63 to 5.19 Hill 1 diversity with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency and may potentially explain the variety of diversity-disturbance relationships observed in microbial systems.IMPORTANCEA generalizable diversity-disturbance relationship (DDR) of microbial communities remains a contentious topic. Various microbial systems have different DDRs. Rather than finding support or refuting specific DDRs, we investigated the underlying factors that lead to different DDRs. In this study, we measured a cellulose-enriched microbial community's response to a range of disturbance frequencies from high to low, across two different substrates: cellulose and glucose. We demonstrate that the community displays a unimodal DDR when grown on cellulose and a monotonically increasing DDR when grown on glucose. Our findings suggest that the same community can display different DDRs. These results suggest that the range of DDRs we observe across different microbial systems may be due to the nutritional resources microbial communities can access and the interactions between bacteria and their environment.

Disturbance-Diversity Relationships of Microbial Communities Change Based on Growth Substrate.

Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances, and how those responses can vary, is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source, and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the genera Cellvibrio, Lacunisphaera, and Asticaccacaulis are the most abundant microbes. However, Lacunisphaera is only abundant in the lower disturbance frequency treatments, while Asticaccaulis is more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are two Pseudomonas sequence variants, and a Cohnella sequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (0.67-1.99 Shannon diversity and 1.38-5.25 Inverse Simpson diversity) that peak at the intermediate disturbance frequency treatment, or 1 disturbance every 3 days. Communities grown on glucose, however, ranged from 0.49-1.43 Shannon diversity and 1.37- 3.52 Inverse Simpson with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency, and may potentially explain the variety of diversity-disturbance relationships observed in microbial ecosystems.

Molecular insights into the evolution of woody plant decay in the gut of termites.

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism's having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of "lower" termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

Rothia from the Human Nose Inhibit Moraxella catarrhalis Colonization with a Secreted Peptidoglycan Endopeptidase.

Moraxella catarrhalis is found almost exclusively within the human respiratory tract. This pathobiont is associated with ear infections and the development of respiratory illnesses, including allergies and asthma. Given the limited ecological distribution of M. catarrhalis, we hypothesized that we could leverage the nasal microbiomes of healthy children without M. catarrhalis to identify bacteria that may represent potential sources of therapeutics. Rothia was more abundant in the noses of healthy children compared to children with cold symptoms and M. catarrhalis. We cultured Rothia from nasal samples and determined that most isolates of Rothia dentocariosa and "Rothia similmucilaginosa" were able to fully inhibit the growth of M. catarrhalis in vitro, whereas isolates of Rothia aeria varied in their ability to inhibit M. catarrhalis. Using comparative genomics and proteomics, we identified a putative peptidoglycan hydrolase called secreted antigen A (SagA). This protein was present at higher relative abundance in the secreted proteomes of R. dentocariosa and R. similmucilaginosa than in those from non-inhibitory R. aeria, suggesting that it may be involved in M. catarrhalis inhibition. We produced SagA from R. similmucilaginosa in Escherichia coli and confirmed its ability to degrade M. catarrhalis peptidoglycan and inhibit its growth. We then demonstrated that R. aeria and R. similmucilaginosa reduced M. catarrhalis levels in an air-liquid interface culture model of the respiratory epithelium. Together, our results suggest that Rothia restricts M. catarrhalis colonization of the human respiratory tract in vivo. IMPORTANCE Moraxella catarrhalis is a pathobiont of the respiratory tract, responsible for ear infections in children and wheezing illnesses in children and adults with chronic respiratory diseases. Detection of M. catarrhalis during wheezing episodes in early life is associated with the development of persistent asthma. There are currently no effective vaccines for M. catarrhalis, and most clinical isolates are resistant to the commonly prescribed antibiotics amoxicillin and penicillin. Given the limited niche of M. catarrhalis, we hypothesized that other nasal bacteria have evolved mechanisms to compete against M. catarrhalis. We found that Rothia are associated with the nasal microbiomes of healthy children without Moraxella. Next, we demonstrated that Rothia inhibit M. catarrhalis in vitro and on airway cells. We identified an enzyme produced by Rothia called SagA that degrades M. catarrhalis peptidoglycan and inhibits its growth. We suggest that Rothia or SagA could be developed as highly specific therapeutics against M. catarrhalis.

Genomic diversification of the specialized parasite of the fungus-growing ant symbiosis.

Fungi shape the diversity of life. Characterizing the evolution of fungi is critical to understanding symbiotic associations across kingdoms. In this study, we investigate the genomic and metabolomic diversity of the genus Escovopsis, a specialized parasite of fungus-growing ant gardens. Based on 25 high-quality draft genomes, we show that Escovopsis forms a monophyletic group arising from a mycoparasitic fungal ancestor 61.82 million years ago (Mya). Across the evolutionary history of fungus-growing ants, the dates of origin of most clades of Escovopsis correspond to the dates of origin of the fungus-growing ants whose gardens they parasitize. We reveal that genome reduction, determined by both genomic sequencing and flow cytometry, is a consistent feature across the genus Escovopsis, largely occurring in coding regions, specifically in the form of gene loss and reductions in copy numbers of genes. All functional gene categories have reduced copy numbers, but resistance and virulence genes maintain functional diversity. Biosynthetic gene clusters (BGCs) contribute to phylogenetic differences among Escovopsis spp., and sister taxa in the Hypocreaceae. The phylogenetic patterns of co-diversification among BGCs are similarly exhibited across mass spectrometry analyses of the metabolomes of Escovopsis and their sister taxa. Taken together, our results indicate that Escovopsis spp. evolved unique genomic repertoires to specialize on the fungus-growing ant-microbe symbiosis.

The future of fungi: threats and opportunities.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Utilization of lignocellulosic biofuel conversion residue by diverse microorganisms.

BACKGROUND: Lignocellulosic conversion residue (LCR) is the material remaining after deconstructed lignocellulosic biomass is subjected to microbial fermentation and treated to remove the biofuel. Technoeconomic analyses of biofuel refineries have shown that further microbial processing of this LCR into other bioproducts may help offset the costs of biofuel generation. Identifying organisms able to metabolize LCR is an important first step for harnessing the full chemical and economic potential of this material. In this study, we investigated the aerobic LCR utilization capabilities of 71 Streptomyces and 163 yeast species that could be engineered to produce valuable bioproducts. The LCR utilization by these individual microbes was compared to that of an aerobic mixed microbial consortium derived from a wastewater treatment plant as representative of a consortium with the highest potential for degrading the LCR components and a source of genetic material for future engineering efforts. RESULTS: We analyzed several batches of a model LCR by chemical oxygen demand (COD) and chromatography-based assays and determined that the major components of LCR were oligomeric and monomeric sugars and other organic compounds. Many of the Streptomyces and yeast species tested were able to grow in LCR, with some individual microbes capable of utilizing over 40% of the soluble COD. For comparison, the maximum total soluble COD utilized by the mixed microbial consortium was about 70%. This represents an upper limit on how much of the LCR could be valorized by engineered Streptomyces or yeasts into bioproducts. To investigate the utilization of specific components in LCR and have a defined media for future experiments, we developed a synthetic conversion residue (SynCR) to mimic our model LCR and used it to show lignocellulose-derived inhibitors (LDIs) had little effect on the ability of the Streptomyces species to metabolize SynCR. CONCLUSIONS: We found that LCR is rich in carbon sources for microbial utilization and has vitamins, minerals, amino acids and other trace metabolites necessary to support growth. Testing diverse collections of Streptomyces and yeast species confirmed that these microorganisms were capable of growth on LCR and revealed a phylogenetic correlation between those able to best utilize LCR. Identification and quantification of the components of LCR enabled us to develop a synthetic LCR (SynCR) that will be a useful tool for examining how individual components of LCR contribute to microbial growth and as a substrate for future engineering efforts to use these microorganisms to generate valuable bioproducts.

Long-Term Cellulose Enrichment Selects for Highly Cellulolytic Consortia and Competition for Public Goods.

The complexity of microbial communities hinders our understanding of how microbial diversity and microbe-microbe interactions impact community functions. Here, using six independent communities originating from the refuse dumps of leaf-cutter ants and enriched using the plant polymer cellulose as the sole source of carbon, we examine how changes in bacterial diversity and interactions impact plant biomass decomposition. Over up to 60 serial transfers (∼8 months) using Whatman cellulose filter paper, cellulolytic ability increased and then stabilized in four enrichment lines and was variable in two lines. Bacterial community characterization using 16S rRNA gene amplicon sequencing showed community succession differed between the highly cellulolytic enrichment lines and those that had slower and more variable cellulose degradation rates. Metagenomic and metatranscriptomic analyses revealed that Cellvibrio and/or Cellulomonas dominated each enrichment line and produced the majority of cellulase enzymes, while diverse taxa were retained within these communities over the duration of transfers. Interestingly, the less cellulolytic communities had a higher diversity of organisms competing for the cellulose breakdown product cellobiose, suggesting that cheating slowed cellulose degradation. In addition, we found competitive exclusion as an important factor shaping all of the communities, with a negative correlation of Cellvibrio and Cellulomonas abundance within individual enrichment lines and the expression of genes associated with the production of secondary metabolites, toxins, and other antagonistic compounds. Our results provide insights into how microbial diversity and competition affect the stability and function of cellulose-degrading communities. IMPORTANCE Microbial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut. However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define. Here, we performed serial enrichment on cellulose using communities inoculated from leaf-cutter ant refuse dumps, a cellulose-rich environment. By concurrently tracking cellulolytic ability and community composition and through metagenomic and metatranscriptomic sequencing, we analyzed the ecological dynamics of the enrichment lines. Our data suggest that antagonism is prevalent in these communities and that competition for soluble sugars may slow degradation and lead to community instability. Together, these results help reveal the relationships between competition and polysaccharide decomposition, with implications in diverse areas ranging from microbial community ecology to cellulosic biofuels production.

Bacillimidazoles A-F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus.

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A-F (1-6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1-6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1-6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1-6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

Rhinovirus increases Moraxella catarrhalis adhesion to the respiratory epithelium.

Rhinovirus causes many types of respiratory illnesses, ranging from minor colds to exacerbations of asthma. Moraxella catarrhalis is an opportunistic pathogen that is increased in abundance during rhinovirus illnesses and asthma exacerbations and is associated with increased severity of illness through mechanisms that are ill-defined. We used a co-infection model of human airway epithelium differentiated at the air-liquid interface to test the hypothesis that rhinovirus infection promotes M. catarrhalis adhesion and survival on the respiratory epithelium. Initial experiments showed that infection with M. catarrhalis alone did not damage the epithelium or induce cytokine production, but increased trans-epithelial electrical resistance, indicative of increased barrier function. In a co-infection model, infection with the more virulent rhinovirus-A and rhinovirus-C, but not the less virulent rhinovirus-B types, increased cell-associated M. catarrhalis. Immunofluorescent staining demonstrated that M. catarrhalis adhered to rhinovirus-infected ciliated epithelial cells and infected cells being extruded from the epithelium. Rhinovirus induced pronounced changes in gene expression and secretion of inflammatory cytokines. In contrast, M. catarrhalis caused minimal effects and did not enhance RV-induced responses. Our results indicate that rhinovirus-A or C infection increases M. catarrhalis survival and cell association while M. catarrhalis infection alone does not cause cytopathology or epithelial inflammation. Our findings suggest that rhinovirus and M. catarrhalis co-infection could promote epithelial damage and more severe illness by amplifying leukocyte inflammatory responses at the epithelial surface.

Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae.

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants' fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. IMPORTANCE In some plants and animals, beneficial microbes mediate host immune response against pathogens, including by serving as defensive symbionts that produce antimicrobial compounds. Defensive symbionts are known in several insects, including some leaf-cutter ants where antifungal-producing Actinobacteria help protect the fungal mutualist of the ants from specialized mycoparasites. In many defensive symbioses, the extent and specificity of defensive benefits received by the host are poorly understood. Here, using "aposymbiotic" rearing, symbiont switching experiments, and imaging mass spectrometry, we explore the ecological and chemical dynamics of the model defensive symbiosis between Acromyrmex ants and their defensive symbiotic bacterium Pseudonocardia. We show that the defensive symbiont not only protects the fungal crop of Acromyrmex but also provides protection from fungal pathogens that infect the ant workers themselves. Furthermore, we reveal that the increased immunity to pathogen infection differs among strains of defensive symbionts and that the degree of reliance on a defensive symbiont for protection varies across congeneric ant species. Taken together, our results suggest that Acromyrmex-associated Pseudonocardia have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.

Biogeography of Bacterial Communities and Specialized Metabolism in Human Aerodigestive Tract Microbiomes.

The aerodigestive tract (ADT) is the primary portal through which pathogens and other invading microbes enter the body. As the direct interface with the environment, we hypothesize that the ADT microbiota possess biosynthetic gene clusters (BGCs) for antibiotics and other specialized metabolites to compete with both endogenous and exogenous microbes. From 1,214 bacterial genomes, representing 136 genera and 387 species that colonize the ADT, we identified 3,895 BGCs. To determine the distribution of BGCs and bacteria in different ADT sites, we aligned 1,424 metagenomes, from nine different ADT sites, onto the predicted BGCs. We show that alpha diversity varies across the ADT and that each site is associated with distinct bacterial communities and BGCs. We identify specific BGC families enriched in the buccal mucosa, external naris, gingiva, and tongue dorsum despite these sites harboring closely related bacteria. We reveal BGC enrichment patterns indicative of the ecology at each site. For instance, aryl polyene and resorcinol BGCs are enriched in the gingiva and tongue, which are colonized by many anaerobes. In addition, we find that streptococci colonizing the tongue and cheek possess different ribosomally synthesized and posttranslationally modified peptide BGCs. Finally, we highlight bacterial genera with BGCs but are underexplored for specialized metabolism and demonstrate the bioactivity of Actinomyces against other bacteria, including human pathogens. Together, our results demonstrate that specialized metabolism in the ADT is extensive and that by exploring these microbiomes further, we will better understand the ecology and biogeography of this system and identify new bioactive natural products. IMPORTANCE Bacteria produce specialized metabolites to compete with other microbes. Though the biological activities of many specialized metabolites have been determined, our understanding of their ecology is limited, particularly within the human microbiome. As the aerodigestive tract (ADT) faces the external environment, bacteria colonizing this tract must compete both among themselves and with invading microbes, including human pathogens. We analyzed the genomes of ADT bacteria to identify biosynthetic gene clusters (BGCs) for specialized metabolites. We found that the majority of ADT BGCs are uncharacterized and the metabolites they encode are unknown. We mapped the distribution of BGCs across the ADT and determined that each site is associated with its own distinct bacterial community and BGCs. By further characterizing these BGCs, we will inform our understanding of ecology and biogeography across the ADT, and we may uncover new specialized metabolites, including antibiotics.

Experimental Warming Reduces Survival, Cold Tolerance, and Gut Prokaryotic Diversity of the Eastern Subterranean Termite, Reticulitermes flavipes (Kollar).

Understanding the effects of environmental disturbances on insects is crucial in predicting the impact of climate change on their distribution, abundance, and ecology. As microbial symbionts are known to play an integral role in a diversity of functions within the insect host, research examining how organisms adapt to environmental fluctuations should include their associated microbiota. In this study, subterranean termites [Reticulitermes flavipes (Kollar)] were exposed to three different temperature treatments characterized as low (15°C), medium (27°C), and high (35°C). Results suggested that pre-exposure to cold allowed termites to stay active longer in decreasing temperatures but caused termites to freeze at higher temperatures. High temperature exposure had the most deleterious effects on termites with a significant reduction in termite survival as well as reduced ability to withstand cold stress. The microbial community of high temperature exposed termites also showed a reduction in bacterial richness and decreased relative abundance of Spirochaetes, Elusimicrobia, and methanogenic Euryarchaeota. Our results indicate a potential link between gut bacterial symbionts and termite's physiological response to environmental changes and highlight the need to consider microbial symbionts in studies relating to insect thermosensitivity.

A high-quality carabid genome assembly provides insights into beetle genome evolution and cold adaptation.

The hyperdiverse order Coleoptera comprises a staggering ~25% of known species on Earth. Despite recent breakthroughs in next generation sequencing, there remains a limited representation of beetle diversity in assembled genomes. Most notably, the ground beetle family Carabidae, comprising more than 40,000 described species, has not been studied in a comparative genomics framework using whole genome data. Here we generate a high-quality genome assembly for Nebria riversi, to examine sources of novelty in the genome evolution of beetles, as well as genetic changes associated with specialization to high-elevation alpine habitats. In particular, this genome resource provides a foundation for expanding comparative molecular research into mechanisms of insect cold adaptation. Comparison to other beetles shows a strong signature of genome compaction, with N. riversi possessing a relatively small genome (~147 Mb) compared to other beetles, with associated reductions in repeat element content and intron length. Small genome size is not, however, associated with fewer protein-coding genes, and an analysis of gene family diversity shows significant expansions of genes associated with cellular membranes and membrane transport, as well as protein phosphorylation and muscle filament structure. Finally, our genomic analyses show that these high-elevation beetles have endosymbiotic Spiroplasma, with several metabolic pathways (e.g., propanoate biosynthesis) that might complement N. riversi, although its role as a beneficial symbiont or as a reproductive parasite remains equivocal.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

Burkholderia from Fungus Gardens of Fungus-Growing Ants Produces Antifungals That Inhibit the Specialized Parasite Escovopsis.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia and Paraburkholderia from 50% of the 52 colonies sampled, indicating that members of the family Burkholderiaceae are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. IMPORTANCE Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia spp., bacteria that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia, is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates' ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.