Fungiculture in the insect world is practised by ants, termites, beetles and gall midges . The best-characterized examples are the attine ants, which are endemic to South and Central America and to the southern USA. The ancestor of these ants evolved the ability to cultivate fungus as a food source around 50 million years ago, leading to the monophyletic tribe Attini, which number 12 genera with more than 230 species. The genera Acromyrmex and Atta (40 species) evolved 8-12 million years ago and form a branch of the higher attines, also known as leaf-cutting ants, which are characterized by large colonies of up to several million individuals . Like the other leaf-cutting ants, the well-studied species Acromyrmex octospinosus forms a mutualism with a single basidiomycete fungus (Agaricales: Lepiotaceae: Leucocoprineae) Leucoagaricus gongylophorus in which they exchange food as well as protection and transport services .
The mutualistic fungal garden can be parasitized by a variety of other fungi  but the major pathogen of leaf-cutting ant fungal gardens is a necrotrophic fungus (Ascomycota: anamorphic Hypocreales) in the genus Escovopsis . Around 25% of the gardens in Panamanian ant colonies contain Escovopsis which feed on the fungal cultivar and can destroy fungal gardens, leading to the collapse of the colony .
There is evidence that the fungal cultivar produces antibiotics in order to defend itself [7–9] and the ant workers also defend their fungal gardens through a combination of grooming and weeding , production of their own antimicrobials through metapleural gland secretions  and the application of weedkillers. These weedkillers are natural product antimicrobials produced by symbiotic actinomycete bacteria [7, 11–13]. A long-standing theory suggests that bacteria from the genus Pseudonocardia co-evolved with the ants and are transmitted vertically by the gynes (reproductive females) along with the fungal cultivar. However, more recently, evidence has emerged that suggests attine ants are also associated with bacteria from the actinomycete genera Streptomyces and Amycolatopsis and that antibiotic-producing actinomycetes can be horizontally acquired through male dispersal and sampling of actinomycetes from the soil [7, 14].
The identities of the antifungals produced by attine ant-associated actinomycetes remain largely unknown. Only two compounds have been identified so far: a previously unknown antifungal named dentigerumycin that is produced by Pseudonocardia species isolated from the lower attines Apterostigma dentigerum and candicidin, a well known antifungal that is produced by Streptomyces species isolated from the higher attine ants belonging to the genus Acromyrmex [12, 13]. Pseudonocardia isolated from A. octospinosus also inhibit the growth of Escovopsis in bioassays, but the antifungal compounds have not been isolated or identified .
The aims of this work were to isolate and identify actinomycete bacteria from A. octospinosus, identify antifungal compounds produced by these bacteria and thereby gain insights into whether the actinomycetes (i) co-evolved with the ants, as suggested by unusual antifungal compounds produced by Pseudonocardia mutualists, or (ii) were acquired from the environment, as suggested by the presence of well known antifungals that are widely produced by environmental isolates. We isolated actinomycetes from three colonies of A. octospinosus that were collected in Trinidad, identified two Pseudonocardia and nine Streptomyces species and chose single antifungal producing Pseudonocardia and Streptomyces species isolated from the same ant colony for further analysis. The Streptomyces species was found to produce candicidin and is closely related to the candicidin-producing Streptomyces bacteria isolated from A. octospinosus in Panama , supporting the hypothesis that candicidin-producing Streptomyces species are common mutualists of higher attines and are probably acquired via environmental sampling. The Pseudonocardia species produces an unusual antifungal compound that is related to the clinically important polyene antifungal nystatin. The isolation of these species suggests that the diversity of actinomycetes associated with attine ants probably occurs through both co-evolution of Pseudonocardia with the ants and environmental sampling.
This work also takes the total number of known antifungals associated with attine ants to three, two of which are associated with A. octospinosus, and provides the first direct biochemical evidence that a diversity of actinomycete symbionts translates into a diversity of antifungal compounds in attine ant colonies.