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.

Some lineages of ants, termites, and beetles independently evolved a symbiotic association with lignocellulolytic fungi cultivated for food, in a lifestyle known as fungiculture. Fungus-growing insects' symbiosis also hosts a bacterial community thought to integrate their physiology. Similarities in taxonomic composition support the microbiota of fungus-growing insects as convergent, despite differences in fungus-rearing by these insects. Here, by comparing fungus-growing insects to several hosts ranging diverse dietary patterns, we investigate whether the microbiota taxonomic and functional profiles are characteristic of the fungiculture environment. Compared to other hosts, the microbiota associated with fungus-growing insects presents a distinctive taxonomic profile, dominated by Gammaproteobacteria at class level and by Pseudomonas at genera level. even with a functional profile presenting similarities with the gut microbiota of herbivorous and omnivorous hosts, some differentially abundant features codified by the microbiota of fungus-growing insects suggest these communities occupying microhabitats that are characteristic of fungiculture. these features include metabolic pathways involved in lignocellulose breakdown, detoxification of plant secondary metabolites, metabolism of simple sugars, fungal cell wall deconstruction, biofilm formation, antimicrobials biosynthesis, and metabolism of diverse nutrients. Our results suggest that the microbiota could be functionally adapted to the fungiculture environment, codifying metabolic pathways potentially relevant to the fungus-growing insects' ecosystems functioning. Most of the organic carbon in land plants is stocked as lignocellulose 1 , a recalcitrant mesh constituted by biopolymers including cellulose, hemicellulose, pectin, and lignin 2,3. For feeding on recalcitrant and indigestible lignocellulosic plant tissues, herbivorous animals rely largely on the association with symbiotic microorganisms, which mediates the use of otherwise non-accessible resources 4-7. Besides metabolizing plant biomass components by hydrolysis and fermentation, the host-associated microbiota also assists the detoxification of plant-derived defensive secondary compounds 4,7,8. A fascinating example of insect-microbial symbiosis for exploring recalcitrant plant biomass is observed in fungus-growing insects (FGI), which maintain lignocellulolytic fungi as crops 9. The active maintenance of fungus crops, also known as fungiculture, evolved independently in three insect lineages 9 : ants in the subtribe Attina (Hymenoptera: Formicidae: Myrmicinae, "the attines"), which are strict to the New World 10,11 ; beetles in the subfamilies Scolytinae and Platypodinae (Coleoptera: Curculionidae), which are predominantly found in tropical and subtropical ecosystems 12 ; and termites in the subfamily Macrotermitinae (Isoptera: Termitidae), which occur in the Old-World tropics, mainly in Africa and Asia 13. The fungal lignocellulose-degrading capacity has been fundamental for the evolutionary succes...

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.

Thirty-nine yeast strains were recovered from a field nest of a primitive and putative novel attine ant species in the genus Myrmicocrypta (Hymenoptera: Formicidae: tribe Attini). Yeasts isolated from the fungus garden and waste deposit included Candida dubliniensis, Candida oleophila, Cryptococcus haglerorum and Hanseniaspora uvarum. In addition, one morphological type was isolated overwhelmingly. Sequencing data of partial large-subunit (LSU) rDNA and the internal transcribed spacer (ITS) region coupled with morphological and physiological characterization accommodated this morphotype in a separate taxonomic position in relation to the known species of Trichosporon (Basidiomycota: Trichosporonales). Here, we propose a novel yeast species named Trichosporon chiarellii sp. nov. based on the description of 34 isolates; the type strain is strain FCP 540806T (=CBS 11177T).

Leaf-cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant-altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf-cutting ants in Brazil, were determined and compared with non-nest soils. Culture-dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non-nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non-nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non-nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf-cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non-nest soils.