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.

Parasites may help stabilize cooperative relationships.

BACKGROUND: The persistence of cooperative relationships is an evolutionary paradox; selection should favor those individuals that exploit their partners (cheating), resulting in the breakdown of cooperation over evolutionary time. Our current understanding of the evolutionary stability of mutualisms (cooperation between species) is strongly shaped by the view that they are often maintained by partners having mechanisms to avoid or retaliate against exploitation by cheaters. In contrast, we empirically and theoretically examine how additional symbionts, specifically specialized parasites, potentially influence the stability of bipartite mutualistic associations. In our empirical work we focus on the obligate mutualism between fungus-growing ants and the fungi they cultivate for food. This mutualism is exploited by specialized microfungal parasites (genus Escovopsis) that infect the ant's fungal gardens. Using sub-colonies of fungus-growing ants, we investigate the interactions between the fungus garden parasite and cooperative and experimentally-enforced uncooperative ("cheating") pairs of ants and fungi. To further examine if parasites have the potential to help stabilize some mutualisms we conduct Iterative Prisoner's Dilemma (IPD) simulations, a common framework for predicting the outcomes of cooperative/non-cooperative interactions, which incorporate parasitism as an additional factor. RESULTS: In our empirical work employing sub-colonies of fungus-growing ants, we found that Escovopsis-infected sub-colonies composed of cheating populations of ants or fungi lost significantly more garden biomass than sub-colonies subjected to infection or cheating (ants or fungi) alone. Since the loss of fungus garden compromises the fitness of both mutualists, our findings suggest that the potential benefit received by the ants or fungi for cheating is outweighed by the increased concomitant cost of parasitism engendered by non-cooperation (cheating). IPD simulations support our empirical results by confirming that a purely cooperative strategy, which is unsuccessful in the classic IPD model, becomes stable when parasites are included. CONCLUSION: Here we suggest, and provide evidence for, parasitism being an external force that has the potential to help stabilize cooperation by aligning the selfish interests of cooperative partners in opposition to a common enemy. Specifically, our empirical results and IPD simulations suggest that when two mutualists share a common enemy selection can favor cooperation over cheating, which may help explain the evolutionary stability of some mutualisms.

Black yeast symbionts compromise the efficiency of antibiotic defenses in fungus-growing ants.

Multiplayer symbioses are common in nature, but our understanding of the ecological dynamics occurring in complex symbioses is limited. The tripartite mutualism between fungus-growing ants, their fungal cultivars, and antibiotic-producing bacteria exemplifies symbiotic complexity. Here we reveal how black yeasts, newly described symbionts of the ant-microbe system, compromise the efficiency of bacteria-derived antibiotic defense in fungus-growing ants. We found that symbiotic black yeasts acquire nutrients from the ants' bacterial mutualist, and suppress bacterial growth. Experimental manipulation of ant colonies and their symbionts shows that ants infected with black yeasts are significantly less effective at defending their fungus garden from Escovopsis, a prevalent and specialized pathogen. The reduction of mutualistic bacterial biomass on ants, likely caused by black yeast symbionts, apparently reduces the quantity of antibiotics available to inhibit the garden pathogen. Success of the ant-fungal mutualism is directly dependent on fungus garden health. Thus our finding that black yeasts compromise the ants' ability to deal with the garden parasite indicates that it is an integral component of the symbiosis. This is further evidence that a full understanding of symbiotic associations requires examining the direct and indirect interactions of symbionts in their ecological community context.

Low host-pathogen specificity in the leaf-cutting ant-microbe symbiosis.

Host-parasite associations are shaped by coevolutionary dynamics. One example is the complex fungus-growing ant-microbe symbiosis, which includes ancient host-parasite coevolution. Fungus-growing ants and the fungi they cultivate for food have an antagonistic symbiosis with Escovopsis, a specialized microfungus that infects the ants' fungus gardens. The evolutionary histories of the ant, cultivar and Escovopsis are highly congruent at the deepest phylogenetic levels, with specific parasite lineages exclusively associating with corresponding groups of ants and cultivar. Here, we examine host-parasite specificity at finer phylogenetic levels, within the most derived clade of fungus-growing ants, the leaf-cutters (Atta spp. and Acromyrmex spp.). Our molecular phylogeny of Escovopsis isolates from the leaf-cutter ant-microbe symbiosis confirms specificity at the broad phylogenetic level, but reveals frequent host-switching events between species and genera of leaf-cutter ants. Escovopsis strains isolated from Acromyrmex and Atta gardens occur together in the same clades, and very closely related strains can even infect the gardens of both ant genera. Experimental evidence supports low host-parasite specificity, with phylogenetically diverse strains of Escovopsis being capable of overgrowing all leaf-cutter cultivars examined. Thus, our findings indicate that this host-pathogen association is shaped by the farming ants having to protect their cultivated fungus from phylogenetically diverse Escovopsis garden pathogens.

Variation in Pseudonocardia antibiotic defence helps govern parasite-induced morbidity in Acromyrmex leaf-cutting ants.

Host-parasite associations are potentially shaped by evolutionary reciprocal selection dynamics, in which parasites evolve to overcome host defences and hosts are selected to counteract these through the evolution of new defences. This is expected to result in variation in parasite-defence interactions, and the evolution of resistant parasites causing increased virulence. Fungus-growing ants maintain antibiotic-producing Pseudonocardia (Actinobacteria) that aid in protection against specialized parasites of the ants' fungal gardens, and current evidence indicates that both symbionts have been associated with the ants for millions of years. Here we examine the extent of variation in the defensive capabilities of the ant-actinobacterial association against Escovopsis (parasite-defence interactions), and evaluate how variation impacts colonies of fungus-growing ants. We focus on five species of Acromyrmex leaf-cutting ants, crossing 12 strains of Pseudonocardia with 12 strains of Escovopsis in a Petri plate bioassay experiment, and subsequently conduct subcolony infection experiments using resistant and non-resistant parasite strains. Diversity in parasite-defence interactions, including pairings where the parasites are resistant, suggests that chemical variation in the antibiotics produced by different actinobacterial strains are responsible for the observed variation in parasite susceptibility. By evaluating the role this variation plays during infection, we show that infection of ant subcolonies with resistant parasite strains results in significantly higher parasite-induced morbidity with respect to garden biomass loss. Our findings thus further establish the role of Pseudonocardia-derived antibiotics in helping defend the ants' fungus garden from the parasite Escovopsis, and provide evidence that small molecules can play important roles as antibiotics in a natural system.

Rules of engagement: interspecies interactions that regulate microbial communities.

Microbial communities comprise an interwoven matrix of biological diversity modified by physical and chemical variation over space and time. Although these communities are the major drivers of biosphere processes, relatively little is known about their structure and function, and predictive modeling is limited by a dearth of comprehensive ecological principles that describe microbial community processes. Here we discuss working definitions of central ecological terms that have been used in various fashions in microbial ecology, provide a framework by focusing on different types of interactions within communities, review the status of the interface between evolutionary and ecological study, and highlight important similarities and differences between macro- and microbial ecology. We describe current approaches to study microbial ecology and progress toward predictive modeling.

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.

Defending against parasites: fungus-growing ants combine specialized behaviours and microbial symbionts to protect their fungus gardens.

Parasites influence host biology and population structure, and thus shape the evolution of their hosts. Parasites often accelerate the evolution of host defences, including direct defences such as evasion and sanitation and indirect defences such as the management of beneficial microbes that aid in the suppression or removal of pathogens. Fungus-growing ants are doubly burdened by parasites, needing to protect their crops as well as themselves from infection. We show that parasite removal from fungus gardens is more complex than previously realized. In response to infection of their fungal gardens by a specialized virulent parasite, ants gather and compress parasitic spores and hyphae in their infrabuccal pockets, then deposit the resulting pellet in piles near their gardens. We reveal that the ants' infrabuccal pocket functions as a specialized sterilization device, killing spores of the garden parasite Escovopsis. This is apparently achieved through a symbiotic association with actinomycetous bacteria in the infrabuccal pocket that produce antibiotics which inhibit Escovopsis. The use of the infrabuccal pocket as a receptacle to sequester Escovopsis, and as a location for antibiotic administration by the ants' bacterial mutualist, illustrates how the combination of behaviour and microbial symbionts can be a successful defence strategy for hosts.

The infrabuccal pellet piles of fungus-growing ants.

Fungus-growing ants (Attini) live in an obligate mutualism with the fungi they cultivate for food. Because of the obligate nature of this relationship, the success of the ants is directly dependent on their ability to grow healthy fungus gardens. Attine ants have evolved complex disease management strategies to reduce their garden's exposure to potential parasitic microbes, to prevent the establishment of infection in their gardens, and to remove infected garden sections. The infrabuccal pocket, a filtering device located in the oral cavity of all ants, is an integral part of the mechanisms that leaf-cutter ants use to prevent the invasion and spread of general microbial parasites and the specific fungal-garden parasite Escovopsis. Fungus-growing ants carefully groom their garden, collecting general debris and pathogenic spores of Escovopsis in their infrabuccal pocket, the contents of which are later expelled in dump chambers inside the nest or externally. In this study we examined how a phylogenetically diverse collection of attine ants treat their infrabuccal pellets. Unlike leaf-cutters that deposit their infrabuccal pellets directly in refuse piles, ants of the more basal attine lineages stack their infrabuccal pellets in piles located close to their gardens, and a separate caste of workers is devoted to the construction, management, and eventual disposal of these piles.