Attine Ants Research Articles

The Prevalence of Killer Yeasts in the Gardens of Fungus-Growing Ants and the Discovery of Novel Killer Toxin named Ksino.

Attine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. Cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study describes a new toxin named Ksino from the yeast Candida sinolaborantium , a member of a new group of putative toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy.

In macroscale ecosystems, such as rainforests or coral reefs, the spatial localization of organisms is the basis of our understanding of community ecology. In the microbial world, likewise, microscale ecosystems are far from a random and homogeneous mixture of organisms and habitats. Accessing the spatial distribution of microbes is fundamental for understanding the functioning and ecology of the microbiota, as cohabiting species are more likely to interact and influence each other's physiology. An interkingdom microbial ecosystem is at the core of fungus-growing ant colonies, which cultivate basidiomycete fungi as a nutritional resource. Attine ants forage for diverse substrates (mostly plant-based), metabolized by the cultivated fungus while forming a spongy structure, a "microbial garden" that acts as an external gut. The garden is an intertwined mesh of fungal hyphae growing by metabolizing the substrate, opening niches for a characteristic and adapted microbiota to establish. The microbiota is thought to be a contributor to substrate degradation and fungal growth, though its spatial organization is yet to be determined. Here, we describe how we employ Scanning Electron Microscopy (SEM) to investigate, with unprecedented detail, the microbiota and biofilm spatial organization across different fungiculture systems of fungus-growing ants. SEM imaging has provided a description of the microbiota spatial structure and organization. SEM revealed that microbiota commonly assemble in biofilms, a widespread structure of the microbial landscapes in fungiculture. We present the protocols employed to fix, dehydrate, dry, sputter coating, and image such a complex community. These protocols were optimized to deal with delicate and heterogeneous samples, comprising plant and fungal biomass, as well as the microbiota and the biofilm.

From the inside out: Were the cuticular Pseudonocardia bacteria of fungus-farming ants originally domesticated as gut symbionts?

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.

Genomic insights into the evolution of secondary metabolism of Escovopsis and its allies, specialized fungal symbionts of fungus-farming ants.

The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.IMPORTANCEMicrobial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts' potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts.

Fungal Cultivars of Higher Attine Ants Promote Escovopsis Chemotropism.

In varied environments, microorganisms search for partners or nutritional resources using chemical signals. Microbes are drawn (chemotaxis) or grow directionally (chemotropism) towards the chemical source, enabling them to establish and maintain symbiosis. The hypocrealean fungi Escovopsis enhance their growth towards the basidiomycete fungus Leucoagaricus gongylophorus, which is cultivated by leaf-cutting attine ants for food. Although directional growth is well documented in this symbiosis, it is unclear whether non-volatile or volatile organic compounds participate in the interaction between cultivar and Escovopsis, and which specific chemical compounds might attract and induce chemotropism. In this study, we examined the growth responses of Escovopsis isolates to non-volatile and volatile organic compounds produced by fungal cultivars of higher attine ants. We also isolated and identified molecules released by the ant-cultivar and assessed the chemotropism of Escovopsis towards them. Our results indicate that the growth of Escovopsis is stimulated in the presence of both non-volatile and volatile compounds from fungal cultivars. We also identified three isomeric diketopiperazines molecules from crude extracts of the ant cultivar, suggesting that these might play a role in Escovopsis chemotropism. Our findings provide insights into the complex chemical interactions that govern the association between Escovopsis and fungal cultivars.

Insects and their antibiotic-producing bacteria

Many, if not all, plants and animals form mutually beneficial symbioses (mutualisms) with microbes and a subset of these mutualisms are defensive, in which the host provides food and housing in return for defence against disease. These symbioses typically involve antibiotic-producing bacteria, the best known of which are filamentous actinomycetes in the genera Streptomyces and Pseudonocardia and unicellular species in the genus Pseudomonas. Such mutualisms are likely to be widespread in nature, but they are best characterised in insects, which provide experimentally tractable models for studying symbiosis and microbiome formation because they typically host less complex microbial communities. Here, we examine the mutualisms formed between insects and antibiotic-producing bacteria using well-characterised examples, including digger wasps and their endosymbiotic Streptomyces species, attine ants and their mutualist Pseudonocardia species and Paederus beetles with their pederin-producing Pseudomonas species. We also discuss how searching such symbiotic niches can give insights into the evolution and functions of microbial specialised metabolites and provide new platforms for antibiotic discovery.

Unraveling fungal species cultivated by lower attine ants

Unraveling fungal species cultivated by lower attine ants

Retracted: Symbiotic Association Between Ants and Fungus

AbstractAnts are eusocial insects belong to family Formicidae and live in symbiotic association with different species of the fungus. Fungi serving them as food and others relationship between fungus and ants is fungi increase the durability of the nest ants living in carton nests in the trees. Our goal was to investigate through literature obvious interactions between ants and common fungi, protection of ants' colonies from pathogens and uses of fungal isolates in sustainable agriculture. Fungal bank in the ants colony is their source of food and colonies of ants (Hymenoptera: Formicidae) are attacked by numerous parasites, pathogens, and parasitoids that exploit entire colonies and their resources. Symbiotic fungi strains belonging to Penicillium found in ants nests are antibiotic producer. High frequency and high diversity of these colonies suggested that insects preferentially sustained this fungus for their survival. Fungal growing ants evolved a close mutualistic relationship with multiple species of Actinobacteria belonging to the genus Pseudonocardia for protection of colony from various diseases. Fungus-growing ants and Pseudonocardia symbionts are novel and significant source of antibiotic and antifungal agents and used as model system due to experimental tractability and well-studied ecology. Beauveria bassiana, Metarhizium anisopliae and Lecanicillium psalliotae are Entomopathogenic fungi found in mutualistic association with Attine ants used in a much smaller scale. Formulation of entomopathogenic fungi-based pesticides biosynthesis of fungal-based nanoparticles at industrial scale is useful in recent sustainable agriculture.

Uncovering the Yeast Communities in Fungus-Growing Ant Colonies.

Yeast-insect interactions are compelling models to study the evolution, ecology, and diversification of yeasts. Fungus-growing (attine) ants are prominent insects in the Neotropics that evolved an ancient fungiculture of basidiomycete fungi over 55-65 million years, supplying an environment for a hidden yeast diversity. Here we assessed the yeast diversity in the attine ant environment by thoroughly sampling fungus gardens across four out of five ant fungiculture systems: Acromyrmex coronatus and Mycetomoellerius tucumanus standing for leaf-cutting and higher-attine fungicultures, respectively; Apterostigma sp., Mycetophylax sp., and Mycocepurus goeldii as ants from the lower-attine fungiculture. Among the fungus gardens of all fungus-growing ants examined, we found taxonomically unique and diverse microbial yeast communities across the different fungicultures. Ascomycete yeasts were the core taxa in fungus garden samples, with Saccharomycetales as the most frequent order. The genera Aureobasidium, Candida, Papiliotrema, Starmerella, and Sugiyamaella had the highest incidence in fungus gardens. Despite the expected similarity within the same fungiculture system, colonies of the same ant species differed in community structure. Among Saccharomycotina yeasts, few were distinguishable as killer yeasts, with a classical inhibition pattern for the killer phenotype, differing from earlier observations in this environment, which should be further investigated. Yeast mycobiome in fungus gardens is distinct between colonies of the same fungiculture and each ant colony harbors a distinguished and unique yeast community. Fungus gardens of attine ants are emergent environments to study the diversity and ecology of yeasts associated with insects.

The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270–4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511–520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

Escovopsioides nivea is a non-specific antagonistic symbiont of ant-fungal crops

Fungus-growing attine ants maintain a mutualistic relationship with basidiomycete fungi which they cultivate for food. In addition to the fungal partner, attine ant colonies harbor a myriad of microorganisms, including the genus Escovopsis , fungal parasites of the ant crops. Because Escovopsioides nivea is phylogenetically close to Escovopsis , previous studies assumed it has a negative interaction in the ant-fungus association. Here, we present an extended phylogeny of E. nivea based on new collections from different attine ant genera found in different localities. We also carried out co-culture experiments between E. nivea with different fungal cultivars. Our results suggest E. nivea is a symbiont of attine ant colonies, which inhibits the growth of fungal crops, supporting the hypothesis it is antagonistic to the system. However, the patterns of interaction between E. nivea and fungal crops differ from those shown by Escovopsis , suggesting a different evolution from that of the parasite. • Escovopsioides nivea has been repeatedly found in attine ant colonies. • E. nivea inhibited diverse ant fungal cultivars but did not show preference for any of them. • E. nivea growth was inhibited by Apterostigma cultivars. • E. nivea is yet another antagonistic fungal symbiont found in the attine ant colonies.

Yeasts in the attine ant-fungus mutualism: Diversity, functional roles, and putative biotechnological applications.

Insects interact with a wide variety of yeasts, often providing a suitable substrate for their growth. Some yeast-insect interactions are tractable models for understanding the relationships between the symbionts. Attine ants are prominent insects in the Neotropics and have performed an ancient fungiculture of mutualistic basidiomycete fungi for more than 55-65 million years. Yeasts gain access to this sophisticated mutualism, prompting diversity, ecological, and biotechnological studies in this environment. We review half a century research in this field, surveying for recurrent yeast taxa and their putative ecological roles in this environment. We found that previous studies mainly covered the yeast diversity from a small fraction of attine ants, being Saccharomycetales, Tremellales, and Trichosporonales as the most frequent yeast or yeast-like orders found. Apiotrichum, Aureobasidium, Candida, Cutaneotrichosporon, Debaryomyces, Meyerozyma, Papiliotrema, Rhodotorula, Trichomonascus, and Trichosporon are the most frequent recovered genera. On the other hand, studies of yeasts' ecological roles on attine ant-fungus mutualism only tapped the tip of the iceberg. Previous established hypotheses in the literature cover the production of lignocellulosic enzymes, chemical detoxification, and fungus garden protection. Some of these roles have parallels in biotechnological processes. In conclusion, the attine ant environment has a hidden potential for studying yeast biodiversity, ecology, and biotechnology, which has been particularly unexplored considering the vast diversity of fungus-growing ants.

Host Susceptibility Modulates Escovopsis Pathogenic Potential in the Fungiculture of Higher Attine Ants

Health and disease emerge from intricate interactions between genotypes, phenotypes, and environmental features. The outcomes of such interactions are context-dependent, existing as a dynamic continuum ranging from benefits to damage. In host-microbial interactions, both the host and environmental conditions modulate the pathogenic potential of a microorganism. Microbial interactions are the core of the agricultural systems of ants in the subtribe Attina, which cultivate basidiomycete fungi for food. The fungiculture environment harbors a diverse microbial community, including fungi in the genus Escovopsis that has been studied as damage-causing agent. Here, we consider the ant colony as a host and investigate to what extent its health impacts the dynamics and outcomes of host-Escovopsis interactions. We found that different ant fungal cultivars vary in susceptibility to the same Escovopsis strains in plate-assays interactions. In subcolony-Escovopsis interactions, while healthy subcolonies gradually recover from infection with different concentrations of Escovopsis conidia, insecticide-treated subcolonies evidenced traits of infection and died within 7 days. The opportunistic nature of Escovopsis infections indicates that diseases in attine fungiculture are a consequence of host susceptibility, rather than the effect of a single microbial agent. By addressing the host susceptibility as a major modulator of Escovopsis pathogenesis, our findings expand the understanding of disease dynamics within attine colonies.

Male-biased dispersal in a fungus-gardening ant symbiosis.

For nearly all organisms, dispersal is a fundamental life‐history trait that can shape their ecology and evolution. Variation in dispersal capabilities within a species exists and can influence population genetic structure and ecological interactions. In fungus‐gardening (attine) ants, co‐dispersal of ants and mutualistic fungi is crucial to the success of this obligate symbiosis. Female‐biased dispersal (and gene flow) may be favored in attines because virgin queens carry the responsibility of dispersing the fungi, but a paucity of research has made this conclusion difficult. Here, we investigate dispersal of the fungus‐gardening ant Trachymyrmex septentrionalis using a combination of maternally (mitochondrial DNA) and biparentally inherited (microsatellites) markers. We found three distinct, spatially isolated mitochondrial DNA haplotypes; two were found in the Florida panhandle and the other in the Florida peninsula. In contrast, biparental markers illustrated significant gene flow across this region and minimal spatial structure. The differential patterns uncovered from mitochondrial DNA and microsatellite markers suggest that most long‐distance ant dispersal is male‐biased and that females (and concomitantly the fungus) have more limited dispersal capabilities. Consequently, the limited female dispersal is likely an important bottleneck for the fungal symbiont. This bottleneck could slow fungal genetic diversification, which has significant implications for both ant hosts and fungal symbionts regarding population genetics, species distributions, adaptive responses to environmental change, and coevolutionary patterns.

Cooperative Breeding in the Ambrosia Beetle Xyleborus affinis and Management of Its Fungal Symbionts

Fungus-farming is known from attine ants, macrotermites, and ambrosia beetles (Scolytinae, Platypodinae). Farming ant and termite societies are superorganismal and grow fungal cultivars in monocultures. Social organization of ambrosia beetle groups and their farming systems are poorly studied, because of their enigmatic life within tunnel systems inside of wood. Ambrosia beetle-fungus symbioses evolved many times independently in both the beetles and their fungal cultivars. Observations suggest that there is evolutionary convergence between these lineages, but also a high variation in the degree of sociality and the modes of fungiculture. Using a laboratory observation technique, I here tried to give insights into the social system and fungus symbiosis of the sugar-cane borer, Xyleborus affinis Eichhoff (Scolytinae: Curculionidae), a currently poorly studied ambrosia beetle. The study revealed a cooperatively breeding system characterized by delayed dispersal of adult daughters, alloparental brood care by larvae and adults, and about half of the totipotent adult daughters laying eggs within the natal nest. Most interesting, there was a tendency of egg-laying females to engage more commonly in mutually beneficial behaviors than non-egg-layers. Fungus gardens covering gallery walls composed of five different filamentous fungi. A Raffaelea isolate was predominant and together with an unidentified fungus likely served as the main food for adults and larvae. Three isolates, a Mucor, a Fusarium and a Phaeoacremonium isolate were most abundant in the oldest gallery part close to the entrance; Mucor, Fusarium and the Raffaelea isolate in diseased individuals. Additionally, there was correlative evidence for some fungal isoaltes influencing beetle feeding and hygienic behaviors. Overall, X. affinis is now the second ambrosia beetle that can be classified as a cooperative breeder with division of labor among and between adults and larvae.

Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants

During crop domestication, human farmers traded greater productivity for higher crop vulnerability outside specialized cultivation conditions. We found a similar domestication tradeoff across the major co-evolutionary transitions in farming systems of attine ants. First, the fundamental nutritional niches (FNNs) of cultivars narrowed during ~ 60 million years of naturally selected domestication, and laboratory experiments showed that ant farmers representing subsequent domestication stages strictly regulate protein harvest relative to cultivar FNNs. Second, ants with different farming systems differed in their abilities to harvest the resources that best matched the nutritional needs of their fungal cultivars. This was assessed by quantifying realized nutritional niches (RNNs) from analyses of items collected from the mandibles of laden ant foragers in the field. Third, extensive field collections suggest that among-colony genetic diversity of cultivars in small-scale farms may offer population-wide resilience benefits that species with large-scale farming colonies achieve by more elaborate and demanding cultivation practices of less diverse crops. Our results underscore that naturally selected farming systems have potential to shed light on nutritional tradeoffs that shaped the course of culturally evolved human farming.

Resisting Antimicrobial Resistance: Lessons from Fungus Farming Ants

Attine ants use antimicrobials produced by commensal bacteria to inhibit parasites on their fungal gardens. However, in this agricultural system, antimicrobial use does not lead to overwhelming resistance, as is typical in clinical settings. Mixtures of continually evolving antimicrobial variants could support these dynamics.

Antifungal compounds from Streptomyces associated with attine ants also inhibit Leishmania donovani.

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from the exoskeleton of Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 μM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 μM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1–4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants’ extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction.

Garden microbiomes of Apterostigma dentigerum and Apterostigma pilosum fungus-growing ants (Hymenoptera: Formicidae).

Fungus-growing ants share a complex symbiosis with microbes, including fungal mutualists, antibiotic-producing bacteria, and fungal pathogens. The bacterial communities associated with this symbiosis are poorly understood but likely play important roles in maintaining the health and function of fungal gardens. We studied bacterial communities in gardens of two Apterostigma species, A. dentigerum, and A. pilosum, using next-generation sequencing to evaluate differences between the two ant species, their veiled and no-veiled fungal garden types, and across three collection locations. We also compared different parts of nests to test for homogeneity within nests. Enterobacteriaceae dominated gardens of both species and common OTUs were shared across both species and nest types. However, differences in community diversity were detected between ant species, and in the communities of A. dentigerum veiled and no-veiled nests within sites. Apterostigma pilosum had a higher proportion of Phyllobacteriaceae and differed from A. dentigerum in the proportions of members of the order Clostridiales. Within A. dentigerum, nests with veiled and no-veiled fungus gardens had similar taxonomic profiles but differed in the relative abundance of some groups, with veiled gardens having more Rhodospirillaceae and Hyphomicrobiaceae, and no-veiled having more Xanthomonadaceae and certain genera in the Enterobacteriaceae C. However, bacterial communities in Apterostigma fungal gardens are highly conserved and resemble those of the nests of other attine ants with dominant taxa likely playing a role in biomass degradation and defense. Further work is required to understand and explain how bacterial community composition of fungus-growing nests is maintained.

Natural history and ecology of fungus-farming ants: a field study in Atlantic rainforest

Ants in the subtribe Attina belong to a monophyletic group, exclusive to the New World, that contains approximately 250 described species. All attine ants have a mutualistic relationship with the fungus they cultivate as food source. The present study provides a natural history and ecological account of five species of fungus-farming ants in the Brazilian Atlantic rainforest: Mycocepurus smithii, Mycetarotes parallelus, Mycetophylax morschi, Sericomyrmex parvulus, and Sericomyrmex saussurei. Specifically, we investigated nesting and foraging behavior and daily activity rhythms, and identified the substrates collected for fungiculture. Nests of all five ant species studied consist of chambers excavated in the soil, with variation on external appearance from inconspicuous holes in the ground to entrances surrounded by mounds of excavated soil pellets. S. saussurei was mainly nocturnal, M. morschi was active all day, and the other species presented diurnal activity. All species foraged exclusively on the ground and near their nests. All five species collected substrates of animal origin (mainly arthropod feces) and vegetable matter on which to cultivate their fungus gardens. Data on basic biological features of these ants, which represent phylogenetically diverse lineages, contribute to a better understanding of their mutualistic relationships with their fungal symbionts and of the evolutionary processes that produced the derived characteristics in the subtribe Attina. Additionally, this study adds to our knowledge of the natural history of fungus-farming ants in Atlantic rainforest and increases our understanding of their roles in this threatened biome.