Exploiting a mutualism: parasite specialization on cultivars within the fungus-growing ant symbiosis

Fungus-growing ants, their cultivated fungi and the cultivar-attacking parasite Escovopsis coevolve as a complex community. Higher-level phylogenetic congruence of the symbionts suggests specialized long-term associations of host-parasite clades but reveals little about parasite specificity at finer scales of species-species and genotype-genotype interactions. By coupling sequence and amplified fragment length polymorphism genotyping analyses with experimental evidence, we examine (i) the host specificity of Escovopsis strains infecting colonies of three closely related ant species in the genus Cyphomyrmex, and (ii) potential mechanisms constraining the Escovopsis host range. Incongruence of cultivar and ant relationships across the three focal Cyphomyrmex spp. allows us to test whether Escovopsis strains track their cultivar or the ant hosts. Phylogenetic analyses demonstrate that the Escovopsis phylogeny matches the cultivar phylogeny but not the ant phylogeny, indicating that the parasites are cultivar specific. Cross-infection experiments establish that ant gardens can be infected by parasite strains with which they are not typically associated in the field, but that infection is more likely when gardens are inoculated with their typical parasite strains. Thus, Escovopsis specialization is shaped by the parasite's ability to overcome only a narrow range of garden-specific defences, but specialization is probably additionally constrained by ecological factors, including the other symbionts (i.e. ants and their antibiotic-producing bacteria) within the coevolved fungus-growing ant symbiosis.     

Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.     

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.     

Symbiotic complexity: discovery of a fifth symbiont in the attine ant-microbe symbiosis

The fungus-growing ant-microbe mutualism is a classic example of organismal complexity generated through symbiotic association. The ants have an ancient obligate mutualism with fungi they cultivate for food. The success of the mutualism is threatened by specialized fungal parasites (Escovopsis) that consume the cultivated fungus. To defend their nutrient-rich garden against infection, the ants have a second mutualism with bacteria (Pseudonocardia), which produce antibiotics that inhibit the garden parasite Escovopsis. Here we reveal the presence of a fourth microbial symbiont associated with fungus-growing ants: black yeasts (Ascomycota; Phialophora). We show that black yeasts are commonly associated with fungus-growing ants, occurring throughout their geographical distribution. Black yeasts grow on the ants' cuticle, specifically localized to where the mutualistic bacteria are cultured. Molecular phylogenetic analyses reveal that the black yeasts form a derived monophyletic lineage associated with the phylogenetic diversity of fungus growers. The prevalence, distribution, localization and monophyly indicate that the black yeast is a fifth symbiont within the attine ant-microbe association, further exemplifying the complexity of symbiotic associations.     

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.     

Antagonistic bacterial interactions help shape host-symbiont dynamics within the fungus-growing ant-microbe mutualism

Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria (Pseudonocardia). Specifically, we examine conflict between the ants and their Pseudonocardia symbionts maintained to derive antibiotics against parasitic microfungi (Escovopsis) infecting the ants' fungus garden. Symbiont assays pairing isolates of Pseudonocardia spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with Acromyrmex leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of Pseudonocardia symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of Pseudonocardia within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.     

Identifying the transition between single and multiple mating of queens in fungus-growing ants

Obligate mating of females (queens) with multiple males has evolved only rarely in social Hymenoptera (ants, social bees, social wasps) and for reasons that are fundamentally different from those underlying multiple mating in other animals. The monophyletic tribe of ('attine') fungus-growing ants is known to include evolutionarily derived genera with obligate multiple mating (the Acromyrmex and Atta leafcutter ants) as well as phylogenetically basal genera with exclusively single mating (e.g. Apterostigma, Cyphomyrmex, Myrmicocrypta). All attine genera share the unique characteristic of obligate dependence on symbiotic fungus gardens for food, but the sophistication of this symbiosis differs considerably across genera. The lower attine genera generally have small, short-lived colonies and relatively non-specialized fungal symbionts (capable of living independently of their ant hosts), whereas the four evolutionarily derived higher attine genera have highly specialized, long-term clonal symbionts. In this paper, we investigate whether the transition from single to multiple mating occurred relatively recently in the evolution of the attine ants, in conjunction with the novel herbivorous 'leafcutter' niche acquired by the common ancestor of Acromyrmex and Atta, or earlier, at the transition to rearing specialized long-term clonal fungi in the common ancestor of the larger group of higher attines that also includes the genera Trachymyrmex and Sericomyrmex. We use DNA microsatellite analysis to provide unambiguous evidence for a single, late and abrupt evolutionary transition from exclusively single to obligatory multiple mating. This transition is historically correlated with other evolutionary innovations, including the extensive use of fresh vegetation as substrate for the fungus garden, a massive increase in mature colony size and morphological differentiation of the worker caste.     

Weeding and grooming of pathogens in agriculture by ants

The ancient mutualism between fungus-growing ants and the fungi they cultivate for food is a textbook example of symbiosis. Fungus-growing ants' ability to cultivate fungi depends on protection of the garden from the aggressive microbes associated with the substrate added to the garden as well as from the specialized virulent garden parasite Escovopsis. We examined ants' ability to remove alien microbes physically by infecting Atta colombica gardens with the generalist pathogen Trichoderma viride and the specialist pathogen Escovopsis. The ants sanitized the garden using two main behaviours: grooming of alien spores from the garden (fungus grooming) and removal of infected garden substrate (weeding). Unlike previously described hygienic behaviours (e.g. licking and self-grooming), fungus-grooming and garden-removal behaviours are specific responses to the presence of fungal pathogens. In the presence of pathogens, they are the primary activities performed by workers, but they are uncommon in uninfected gardens. In fact, workers rapidly eliminate Trichoderma from their gardens by fungus grooming and weeding, suggesting that these behaviours are the primary method of garden defence against generalist pathogens. The same sanitary behaviours were performed in response to the presence of the specialist pathogen Escovopsis. However, the intensity and duration of these behaviours were much greater in this treatment. Despite the increased effort, the ants were unable to eliminate Escovopsis from their gardens, suggesting that this specialized pathogen has evolved counter-adaptations in order to overcome the sanitary defences of the ants.     

Ecology of microfungal communities in gardens of fungus-growing ants (Hymenoptera: Formicidae): a year-long survey of three species of attine ants in Central Texas

We profiled the microfungal communities in gardens of fungus-growing ants to evaluate possible species-specific ant-microfungal associations and to assess the potential dependencies of microfungal diversity on ant foraging behavior. In a 1-year survey, we isolated microfungi from nests of Cyphomyrmex wheeleri, Trachymyrmex septentrionalis and Atta texana in Central Texas. Microfungal prevalence was higher in gardens of C. wheeleri (57%) than in the gardens of T. septentrionalis (46%) and A. texana (35%). Culture-dependent methods coupled with a polyphasic approach of species identification revealed diverse and changing microfungal communities in all the sampling periods. Diversity analyses showed no obvious correlations between the number of observed microfungal species, ant species, or the ants' changing foraging behavior across the seasons. However, both correspondence analysis and 5.8S-rRNA gene unifrac analyses suggested structuring of microfungal communities by ant host. These host-specific differences may reflect in part the three different environments where ants were collected. Most interestingly, the specialized fungal parasite Escovopsis was not isolated from any attine garden in this study near the northernmost limit of the range of attine ants, contrasting with previous studies that indicated a significant incidence of this parasite in ant gardens from Central and South America. The observed differences of microfungal communities in attine gardens suggest that the ants are continuously in contact with a diverse microfungal species assemblage.     

Symbiont recruitment versus ant-symbiont co-evolution in the attine ant-microbe symbiosis

The symbiosis between fungus-farming ants (Attini, Formicidae), their cultivated fungi, garden-infecting Escovopsis pathogens, and Pseudonocardia bacteria on the ant integument has been popularized as an example of ant-Escovopsis-Pseudonocardia co-evolution. Recent research could not verify earlier conclusions regarding antibiotic-secreting, integumental Pseudonocardia that co-evolve to specifically suppress Escovopsis disease in an ancient co-evolutionary arms-race. Rather than long-term association with a single, co-evolving Pseudonocardia strain, attine ants accumulate complex, dynamic biofilms on their integument and in their gardens. Emerging views are that the integumental biofilms protect the ants primarily against ant diseases, whereas garden biofilms protect primarily against garden diseases; attine ants selectively recruit ('screen in') microbes into their biofilms; and the biofilms of ants and gardens serve diverse functions beyond disease-suppression.     

Experimental evidence of a tripartite mutualism: bacteria protect ant fungus gardens from specialized parasites

Symbioses shape all levels of biological organization. Although symbiotic interactions are typically viewed as bipartite associations, with two organisms interacting largely in isolation from other organisms, the presence and importance of additional symbionts is becoming increasingly more apparent. This study examines the importance of a third mutualist within the ancient symbiosis between leaf-cutting ants and their fungal cultivars. Specifically, we experimentally examine the role of a filamentous bacterium (actinomycete), which is typically carried on the cuticle of fungus-growing ants, in suppressing the growth of a specialized microfungal parasite ( Escovopsis ) of the fungus garden. We conducted two-by-two factorial design experiments crossing the presence/absence of actinomycete with the presence/absence of Escovopsis within small sub-colonies of Acromyrmex octospinosus . In these experiments, infection by Escovopsis became much more extensive within fungus gardens and had a greater impact on the health of gardens in those sub-colonies with the bacterium removed from workers as compared to gardens with the bacterium still present on the ants. We establish that the actinomycete bacterium is most abundant on those major workers tending the garden, providing further support that the bacterium is involved in garden hygiene. We also found a significantly higher abundance of actinomycete on workers in colonies experimentally infected with Escovopsis as compared to uninfected control colonies. We suggest that mutualisms between antibiotic-producing microbes and higher organisms may be common associations that are mostly overlooked and that the role of symbionts in reducing the impact of parasites is likely an important aspect in the cost-benefit assessment of mutualisms.     

Pathogenicity of Escovopsis weberi: The parasite of the attine ant-microbe symbiosis directly consumes the ant-cultivated fungus

Fungi in the genus Escovopsis are known only from the fungus gardens of attine ants. Previous work has established that these anamorphic fungi, allied with the Hypocreales, are specialized and potentially virulent parasites of the ancient mutualism between attine ants and their fungal cultivars. It is unclear whether the primary nutrient source for the pathogen is the mutualist fungal cultivar or the vegetative substrate placed on the gardens by the ants. Here, we determine whether Escovopsis weberi is a parasite of the fungal cultivar, a competitor for the leaf substrate, or both. Bioassays reveal that E. weberi exhibits rapid growth on pure cultivar and negligible growth on sterilized leaf fragments. Light microscopy examination of hyphalhyphal interactions between E. weberi and the ant fungal cultivar indicate that E. weberi, unlike invasive necrotrophs that always penetrate host hyphae, can secrete compounds that break down host mycelium before contact occurs. Thus, E. weberi is a necrotrophic parasite of the fungal cultivar of attine ants.     

Convergent coevolution in the domestication of coral mushrooms by fungus-growing ants

Comparisons of phylogenetic patterns between coevolving symbionts can reveal rich details about the evolutionary history of symbioses. The ancient symbiosis between fungus-growing ants, their fungal cultivars, antibiotic-producing bacteria and cultivar-infecting parasites is dominated by a pattern of parallel coevolution, where the symbionts of each functional group are members of monophyletic groups. However, there is one outstanding exception in the fungus-growing ant system, the unidentified cultivar grown only by ants in the Apterostigma pilosum group. We classify this cultivar in the coral-mushroom family Pterulaceae using phylogenetic reconstructions based on broad taxon sampling, including the first mushroom collected from the garden of an ant species in the A. pilosum group. The domestication of the pterulaceous cultivar is independent from the domestication of the gilled mushrooms cultivated by all other fungus-growing ants. Yet it has the same overall assemblage of coevolved ant-cultivar-parasite-bacterium interactions as the other ant-grown fungal cultivars. This indicates a pattern of convergent coevolution in the fungus-growing ant system, where symbionts with both similar and very different evolutionary histories converge to functionally identical interactions.     

Prevalence and impact of a virulent parasite on a tripartite mutualism

The prevalence and impact of a specialized microfungal parasite (Escovopsis) that infects the fungus gardens of leaf-cutting ants was examined in the laboratory and in the field in Panama. Escovopsis is a common parasite of leaf-cutting ant colonies and is apparently more frequent in Acromyrmex spp. gardens than in gardens of the more phylogenetically derived genus Atta spp. In addition, larger colonies of Atta spp. appear to be less frequently infected with the parasite. In this study, the parasite Escovopsis had a major impact on the success of this mutualism among ants, fungi, and bacteria. Infected colonies had a significantly lower rate of fungus garden accumulation and produced substantially fewer workers. In addition, the extent of the reduction in colony growth rate depended on the isolate, with one isolate having a significantly larger impact than two others, suggesting that Escovopsis has different levels of virulence. Escovopsis is also spatially concentrated within parts of ant fungus gardens, with the younger regions having significantly lower rates of infection as compared to the older regions. The discovery that gardens of fungus-growing ants are host to a virulent pathogen that is not related to any of the three mutualists suggests that unrelated organisms may be important but primarily overlooked components of other mutualistic associations.     

Population genetic signatures of diffuse co-evolution between leaf-cutting ants and their cultivar fungi

Switching of symbiotic partners pervades most mutualisms, despite mechanisms that appear to enforce partner fidelity. To investigate the interplay of forces binding and dissolving mutualistic pairings, we investigated partner fidelity at the population level in the attine ant-fungal cultivar mutualism. The ants and their cultivars exhibit both broad-scale co-evolution, as well as cultivar switching, with short-term symbiont fidelity maintained by vertical transmission of maternal garden inoculates via dispersing queens and by the elimination of alien cultivar strains. Using microsatellite markers, we genotyped cultivar fungi associated with five co-occurring Panamanian attine ant species, representing the two most derived genera, leaf-cutters Atta and Acromyrmex. Despite the presence of mechanisms apparently ensuring the cotransmission of symbiont genotypes, different species and genera of ants sometimes shared identical fungus garden genotypes, indicating widespread cultivar exchange. The cultivar population was largely unstructured with respect to host ant species, with only 10% of the structure in genetic variance being attributable to partitioning among ant species and genera. Furthermore, despite significant genetic and ecological dissimilarity between Atta and Acromyrmex, generic difference accounted for little, if any, variance in cultivar population structure, suggesting that cultivar exchange dwarfs selective forces that may act to create co-adaptive ant-cultivar combinations. Thus, binding forces that appear to enforce host fidelity are relatively weak and pairwise associations between cultivar lineages and ant species have little opportunity for evolutionary persistence. This implicates that mechanisms other than partner fidelity feedback play important roles in stabilizing the leafcutter ant-fungus mutualism over evolutionary time.     

Complex host-pathogen coevolution in the Apterostigma fungus-growing ant-microbe symbiosis

Background  

The fungus-growing ant-microbe symbiosis consists of coevolving microbial mutualists and pathogens. The diverse fungal lineages that these ants cultivate are attacked by parasitic microfungi of the genus Escovopsis. Previous molecular analyses have demonstrated strong phylogenetic congruence between the ants, the ants-cultivated fungi and the garden pathogen Escovopsis at ancient phylogenetic levels, suggesting coevolution of these symbionts. However, few studies have explored cophylogenetic patterns between these symbionts at the recent phylogenetic levels necessary to address whether these parasites are occasionally switching to novel hosts or whether they are diversifying with their hosts as a consequence of long-term host fidelity.     

Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus,the Fungal Symbiont of Leaf-Cutter Ants

Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.     

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.     

Faecal proteinases of the fungus-growing ant, Atta texana: Their fungal origin and ecological significance

The fluid contained within the mycelium of the fungus cultured by the attine ant, Atta texana, contains three proteolytic enzymes. One enzyme is a DFP-sensitive alkaline proteinase; the other two are metal-chelator-sensitive neutral proteinases. These three enzymes are indentical by all criteria examined to the three proteinases previously isolated from the faecal fluid of A. texana. It is concluded that the faecal enzymes of the fungus-growing ants are derived from the mycelial fluid upon which they feed. The basis for the symbiosis between the attine ants and the fungi which they cultivate in their nests is reinterpreted in the context of this finding.     

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ～30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.