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.

Defensive Symbioses in Social Insects Can Inform Human Health and Agriculture.

Social animals are among the most successful organisms on the planet and derive many benefits from living in groups, including facilitating the evolution of agriculture. However, living in groups increases the risk of disease transmission in social animals themselves and the cultivated crops upon which they obligately depend. Social insects offer an interesting model to compare to human societies, in terms of how insects manage disease within their societies and with their agricultural symbionts. As living in large groups can help the spread of beneficial microbes as well as pathogens, we examine the role of defensive microbial symbionts in protecting the host from pathogens. We further explore how beneficial microbes may influence other pathogen defenses including behavioral and immune responses, and how we can use insect systems as models to inform on issues relating to human health and agriculture.

The antimicrobial potential of Streptomyces from insect microbiomes.

Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery.

Convergent evolution of complex structures for ant-bacterial defensive symbiosis in fungus-farming ants.

Evolutionary adaptations for maintaining beneficial microbes are hallmarks of mutualistic evolution. Fungus-farming "attine" ant species have complex cuticular modifications and specialized glands that house and nourish antibiotic-producing Actinobacteria symbionts, which in turn protect their hosts' fungus gardens from pathogens. Here we reconstruct ant-Actinobacteria evolutionary history across the full range of variation within subtribe Attina by combining dated phylogenomic and ultramorphological analyses. Ancestral-state analyses indicate the ant-Actinobacteria symbiosis arose early in attine-ant evolution, a conclusion consistent with direct observations of Actinobacteria on fossil ants in Oligo-Miocene amber. qPCR indicates that the dominant ant-associated Actinobacteria belong to the genus Pseudonocardia Tracing the evolutionary trajectories of Pseudonocardia-maintaining mechanisms across attine ants reveals a continuum of adaptations. In Myrmicocrypta species, which retain many ancestral morphological and behavioral traits, Pseudonocardia occur in specific locations on the legs and antennae, unassociated with any specialized structures. In contrast, specialized cuticular structures, including crypts and tubercles, evolved at least three times in derived attine-ant lineages. Conspicuous caste differences in Pseudonocardia-maintaining structures, in which specialized structures are present in worker ants and queens but reduced or lost in males, are consistent with vertical Pseudonocardia transmission. Although the majority of attine ants are associated with Pseudonocardia, there have been multiple losses of bacterial symbionts and bacteria-maintaining structures in different lineages over evolutionary time. The early origin of ant-Pseudonocardia mutualism and the multiple evolutionary convergences on strikingly similar anatomical adaptations for maintaining bacterial symbionts indicate that Pseudonocardia have played a critical role in the evolution of ant fungiculture.

Imaging with Mass Spectrometry of Bacteria on the Exoskeleton of Fungus-Growing Ants.

Mass spectrometry imaging is a powerful analytical technique for detecting and determining spatial distributions of molecules within a sample. Typically, mass spectrometry imaging is limited to the analysis of thin tissue sections taken from the middle of a sample. In this work, we present a mass spectrometry imaging method for the detection of compounds produced by bacteria on the outside surface of ant exoskeletons in response to pathogen exposure. Fungus-growing ants have a specialized mutualism with Pseudonocardia, a bacterium that lives on the ants' exoskeletons and helps protect their fungal garden food source from harmful pathogens. The developed method allows for visualization of bacterial-derived compounds on the ant exoskeleton. This method demonstrates the capability to detect compounds that are specifically localized to the bacterial patch on ant exoskeletons, shows good reproducibility across individual ants, and achieves accurate mass measurements within 5 ppm error when using a high-resolution, accurate-mass mass spectrometer.

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin's shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin's biosynthesis including a subcluster of genes consistent with selvamicin's 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

Evolution and Ecology of Actinobacteria and Their Bioenergy Applications.

The ancient phylum Actinobacteria is composed of phylogenetically and physiologically diverse bacteria that help Earth's ecosystems function. As free-living organisms and symbionts of herbivorous animals, Actinobacteria contribute to the global carbon cycle through the breakdown of plant biomass. In addition, they mediate community dynamics as producers of small molecules with diverse biological activities. Together, the evolution of high cellulolytic ability and diverse chemistry, shaped by their ecological roles in nature, make Actinobacteria a promising group for the bioenergy industry. Specifically, their enzymes can contribute to industrial-scale breakdown of cellulosic plant biomass into simple sugars that can then be converted into biofuels. Furthermore, harnessing their ability to biosynthesize a range of small molecules has potential for the production of specialty biofuels.

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability.

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.

Microbes are trophic analogs of animals.

In most ecosystems, microbes are the dominant consumers, commandeering much of the heterotrophic biomass circulating through food webs. Characterizing functional diversity within the microbiome, therefore, is critical to understanding ecosystem functioning, particularly in an era of global biodiversity loss. Using isotopic fingerprinting, we investigated the trophic positions of a broad diversity of heterotrophic organisms. Specifically, we examined the naturally occurring stable isotopes of nitrogen ((15)N:(14)N) within amino acids extracted from proteobacteria, actinomycetes, ascomycetes, and basidiomycetes, as well as from vertebrate and invertebrate macrofauna (crustaceans, fish, insects, and mammals). Here, we report that patterns of intertrophic (15)N-discrimination were remarkably similar among bacteria, fungi, and animals, which permitted unambiguous measurement of consumer trophic position, independent of phylogeny or ecosystem type. The observed similarities among bacterial, fungal, and animal consumers suggest that within a trophic hierarchy, microbiota are equivalent to, and can be interdigitated with, macrobiota. To further test the universality of this finding, we examined Neotropical fungus gardens, communities in which bacteria, fungi, and animals are entwined in an ancient, quadripartite symbiosis. We reveal that this symbiosis is a discrete four-level food chain, wherein bacteria function as the apex carnivores, animals and fungi are meso-consumers, and the sole herbivores are fungi. Together, our findings demonstrate that bacteria, fungi, and animals can be integrated within a food chain, effectively uniting the macro- and microbiome in food web ecology and facilitating greater inclusion of the microbiome in studies of functional diversity.

Comparing in situ particle monitoring to microscopic counts of plankton in a drinking water reservoir.

In a one-year study, the multispecies assemblages of phytoplankton (picoplankton to microplankton) within a drinking water reservoir were counted, determined and evaluated in their size fractions using microscope enumeration (MC). The manual counts were compared with the size evaluation obtained by a light obscuring particle counter (PC) in order to evaluate its use for the monitoring practice of a drinking water reservoir. With this multispecies comparison we present a novel approach for the evaluation of automated counting systems. The picoplankton clearly remained uncounted by the PC even though its lower size limits imply an adequate match. The highest and most consistent count numbers of plankton (nano- and microplankton) and particles were obtained during the spring mass development. However, from the middle of the year onwards, the measured particle concentration surpassed the counted plankton abundances by two- to threefold indicating the rise of seston within the water column. This fraction would be missed if counted solely by MC. Further, the PC consistently undersized the biological counts, but not the minerogenic fraction represented by the manganese oxidising bacteria. Consequently, the rise and decline of Metallogenium bacteria was reliably monitored with the PC. The PC provides additional size information compared to other bulk optical sensors (turbidity, chlorophyll-a). The correlation of particles with probe measurement always exceeded the plankton coefficient, but all combinations of plankton, particle and probe measurement revealed significant linear regressions. However, the redundancy of the chlorophyll-a probes was also shown in order to explain plankton abundances. Our results indicate that background knowledge of the monitored system and cautious interpretation of data is required to allocate and understand automated particle counts. Therefore, only in combination with MC, the PC enables phytoplankton or minerogenic particle counts under frequent real-time monitoring conditions. As such it may serve as a helpful tool for example in critical situations in the management of drinking water reservoirs.