Chemical warfare between fungus-growing ants and their pathogens

Fungus-growing attine ants are under constant threat from fungal pathogens such as the specialized mycoparasite Escovopsis, which uses combined physical and chemical attack strategies to prey on the fungal gardens of the ants. In defence, some species assemble protective microbiomes on their exoskeletons that contain antimicrobial-producing Actinobacteria. Underlying this network of mutualistic and antagonistic interactions are an array of chemical signals. Escovopsis weberi produces the shearinine terpene-indole alkaloids, which affect ant behaviour, diketopiperazines to combat defensive bacteria, and other small molecules that inhibit the fungal cultivar. Pseudonocardia and Streptomyces mutualist bacteria produce depsipeptide and polyene macrolide antifungals active against Escovopsis spp. The ant nest metabolome is further complicated by competition between defensive bacteria, which produce antibacterials active against even closely related species.     

Distinct and enhanced hygienic responses of a leaf‐cutting ant toward repeated fungi exposures

Leaf‐cutting ants and their fungal crops are a textbook example of a long‐term obligatory mutualism. Many microbes continuously enter their nest containing the fungal cultivars, destabilizing the symbiosis and, in some cases, outcompeting the mutualistic partners. Preferably, the ant workers should distinguish between different microorganisms to respond according to their threat level and recurrence in the colony. To address these assumptions, we investigated how workers of Atta sexdens sanitize their fungal crop toward five different fungi commonly isolated from the fungus gardens: Escovopsis sp., Fusarium oxysporum, Metarhizium anisopliae, Trichoderma spirale, and Syncephalastrum sp. Also, to investigate the plasticity of these responses toward recurrences of these fungi, we exposed the colonies with each fungus three times fourteen days apart. As expected, intensities in sanitization differed according to the fungal species. Ants significantly groom their fungal crop more toward F. oxysporum, M. anisopliae, and Syncephalastrum sp. than toward Escovopsis sp. and T. spirale. Weeding, self‐, and allogrooming were observed in less frequency than fungus grooming in all cases. Moreover, we detected a significant increase in the overall responses after repeated exposures for each fungus, except for Escovopsis sp. Our results indicate that A. sexdens workers are able to distinguish between different fungi and apply distinct responses to remove these from the fungus gardens. Our findings also suggest that successive exposures to the same antagonist increase hygiene, indicating plasticity of ant colonies'' defenses to previously encountered pathogens.     

Disease management in two sympatric Apterostigma fungus‐growing ants for controlling the parasitic fungus Escovopsis

Antagonistic interactions between host and parasites are often embedded in networks of interacting species, in which hosts may be attacked by competing parasites species, and parasites may infect more than one host species. To better understand the evolution of host defenses and parasite counterdefenses in the context of a multihost, multiparasite system, we studied two sympatric species, of congeneric fungus‐growing ants (Attini) species and their symbiotic fungal cultivars, which are attacked by multiple morphotypes of parasitic fungi in the genus, Escovopsis. To assess whether closely related ant species and their cultured fungi are evolving defenses against the same or different parasitic strains, we characterized Escovopsis that were isolated from colonies of sympatric Apterostigma dentigerum and A. pilosum. We assessed in vitro and in vivo interactions of these parasites with their hosts. While the ant cultivars are parasitized by similar Escovopsis spp., the frequency of infection by these pathogens differs between the two ant species. The ability of the host fungi to suppress Escovopsis growth, as well as ant defensive responses toward the parasites, differs depending on the parasite strain and on the host ant species.     

Co‐evolutionary patterns and diversification of ant–fungus associations in the asexual fungus‐farming ant Mycocepurus smithii in Panama

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.     

A Brazilian Population of the Asexual Fungus-Growing Ant Mycocepurus smithii (Formicidae,Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50–60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as “higher agriculture” and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called “Clade 1”of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.     

Microfungal “Weeds” in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.     

Ant versus fungus versus mutualism: ant-cultivar conflict and the deconstruction of the attine ant-fungus symbiosis

A century of research on fungus-growing ants (Attini, Formicidae) has ignored the cultivated fungi as passive domesticates and viewed the attine fungicultural symbiosis as an integrated unit dominated by the evolutionary interests of the ant farmers. This article takes a different perspective and explores first the evolutionary interests and leverages of the fungal cultivars, then dissects eight potential evolutionary conflicts between ants and cultivars. Three types of ant-cultivar conflict are examined in depth. First, ant-cultivar conflict over the ant sex ratio is predicted because the cultivars are dispersed by female foundresses but not by males; cultivars thus may be selected to bias the ant sex ratio toward females. Second, ant-cultivar conflict over fungal sexual reproduction exists if the fungi are able to escape from the symbiosis and live independently, as is implied by phylogenetic analyses of the fungi; this conflict is exacerbated in colonies that experience queen death or senescence. A literature review reveals that sexual fruiting of attine cultivars is more common than has been traditionally realized and often occurs in moribund colonies. Third, the routine transplanting of fungal mycelium by ants could generate, through sensory-biased symbiont choice, selection favoring fungal features that increase the likelihood of transplantation within nests (symbiont drive) but that are detrimental to the survival of the whole colony. A balanced perspective incorporating both ant and fungal interests emerges as a more appropriate framework than the traditional myrmicocentric perspective. Indeed, the attine symbiosis offers unique experimental opportunities (cultivar switch experiments) to unravel the evolutionary dynamics of conflict and cooperation between ant and fungal partners.     

Prudent inquilines and proactive hosts: behavioral dynamics between an ant social parasite, Megalomyrmex symmetochus and its fungus-growing ant host, Sericomyrmex amabilis

Ant social parasites evolve adaptive relationships with their hosts. Theoretically, coevolution predicts strong selection to maximize fitness of the parasite that minimizes costs to its host, which potentially leads to the evolution of benign interactions. We studied the demographic and behavioral traits of the ant social parasite Megalomyrmex symmetochus (Solenopsidini), an agro-predator that feeds on larvae and fungal garden products of their host, Sericomyrmex amabilis (Attini). Based on demographic data from 15 parasitized colonies, the proportion of parasitic workers to those of the host is 1:2. Moreover, defensive prophylactic behaviors observed during infections with Metarhizium brunneum, a generalist entomopathogen, and Escovopsis, a specialized fungal garden parasite, showed that S. amabilis works extensively to remove and control fungal infections, in contrast to M. symmetochus. M. symmetochus, however, performed intraspecific allogrooming during infections with Escovopsis and M. brunneum, suggesting that they may recognize fungal pathogens and indirectly limit dispersion of spores. Our results indicate that M. symmetochus did not have a strong role in maintaining a hygienic nest.     

Preliminary In Vitro Insights into the Use of Natural Fungal Pathogens of Leaf-cutting Ants as Biocontrol Agents

Leaf-cutting ants are one of the main herbivores of the Neotropics, where they represent an important agricultural pest. These ants are particularly difficult to control because of the complex network of microbial symbionts. Leaf-cutting ants have traditionally been controlled through pesticide application, but there is a need for alternative, more environmentally friendly, control methods such as biological control. Potential promising biocontrol candidates include the microfungi Escovopsis spp. (anamorphic Hypocreales), which are specialized pathogens of the fungi the ants cultivate for food. These pathogens are suppressed through ant behaviors and ant-associated antibiotic-producing Actinobacteria. In order to be an effective biocontrol agent, Escovopsis has to overcome these defenses. Here, we evaluate, using microbial in vitro assays, whether defenses in the ant-cultivated fungus strain (Leucoagaricus sp.) and Actinobacteria from the ant pest Acromyrmex lundii have the potential to limit the use of Escovopsis in biocontrol. We also explore, for the first time, possible synergistic biocontrol between Escovopsis and the entomopathogenic fungus Lecanicillium lecanii. All strains of Escovopsis proved to overgrow A. lundii cultivar in less than 7 days, with the Escovopsis strain isolated from a different leaf-cutting ant species being the most efficient. Escovopsis challenged with a Streptomyces strain isolated from A. lundii did not exhibit significant growth inhibition. Both results are encouraging for the use of Escovopsis as a biocontrol agent. Although we found that L. lecanii can suppress the growth of the cultivar, it also had a negative impact on Escovopsis, making the success of simultaneous use of these two fungi for biocontrol of A. lundii questionable.     

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Land-cover and climate factors contribute to the prevalence of the ectoparasitic fungus Laboulbenia formicarum in its invasive ant host Lasius neglectus

Understanding the distribution of parasites is crucial for biodiversity conservation. Here, we studied the distribution of the ectoparasitic fungus Laboulbenia formicarum in native and invasive Lasius ants in a 2000 km² area. We screened over 16,000 ant workers in 478 colonies of five different species. We found that Lab. formicarum was rare in native Lasius species but infected 58% of the colonies of the invasive species Las. neglectus. At landscape scale, Lab. formicarum presence could not be explained by geographic and genetic distances between Las. neglectus colonies but was associated with hotter and dryer climatic conditions and its prevalence in colonies increased with urbanization. Within infected colonies, fungal prevalence varied from 0 to 100 percent within meters and was negatively correlated with impervious ground cover. In a changing world, our findings emphasize the importance of land-use and climatic factors in shaping the distribution and prevalence of fungal parasites.     

Planting of fungus onto hibernating workers of the fungus-growing ant Mycetosoritis clorindae (Attini, Formicidae)

We describe a peculiar fungus-coating behavior of the attine ant Mycetosoritis clorindae, where workers plant fungal mycelium on hibernating nestmates. Hibernating nestmates become ultimately enveloped in a live mycelial coat, remain motionless in this coated state, and essentially become integrated into the garden matrix. The shallow nest architecture of M. clorindae (depth of main garden is 15–30 cm) in southern Brazil forces the ants to overwinter at relatively low temperatures in the topmost soil layer. Fungal coating may help the ants to survive the prolonged periods of immobility during winter. Fungus-planting on attine adults is so far unknown from other attine species, but the behavior parallels the planting of mycelium on larvae and pupae occurring in many attine species. Planting of mycelium on adult nestmates may have been overlooked so far in attine ants because this behavior may occur only in dormant nests, which are least frequently collected. The possible adaptive functions of fungus coatings of hibernating adults and developing brood are likely similar, including for example physical protection, prevention of desiccation, shielding against parasites and predators (e.g., army ants), or defense against diseases.     

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.     

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.     

Caste specialization in behavioral defenses against fungus garden parasites in <Emphasis Type="Italic">Acromyrmex octospinosus</Emphasis> leaf-cutting ants

Division of labor and caste specialization plays an important role in many aspects of social insect colony organization, including parasite defense. Within leaf-cutting ant colonies, worker caste specialization permeates colony tasks ranging from foraging, substrate incorporation, brood care, and chemical defenses via glandular secretions and mutualistic bacteria. Leaf-cutting ants rely on protecting a mutualistic fungus they grow for food from microfungi in the genus Escovopsis that parasitizes the ant–fungus relationship. Here, we examine whether Acromyrmex octospinosus leaf-cutter ant castes (minors and majors) display task specialization in two behavioral defenses against Escovopsis: fungus grooming (the removal of Escovopsis spores) and weeding (the removal of large pieces of Escovopsis-infected fungus garden). Using behavioral observations, we show that minors are the primary caste that performs fungus grooming, while weeding is almost exclusively performed by majors. In addition, using a sub-colony infection experimental setup, we show that at the early stages of infection, minors more efficiently remove Escovopsis spores from the fungus garden, thereby restricting Escovopsis spore germination and growth. At later stages of infection, after Escovopsis spore germination, we find that major workers are as efficient as minors in defending the fungus garden, likely due to the increased importance of weeding. Finally, we show, using SEM imaging, that the number of sensory structures is similar between minor and major workers. If these structures are invoked in recognition of the parasites, this finding suggests that both castes are able to sense Escovopsis. Our findings support that leaf-cutter ant behavioral defense tasks against Escovopsis are subject to caste specialization, likely facilitated by worker sizes being optimal for grooming and weeding by minors and majors, respectively, with important consequences for cultivar defense.     

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.