Analysis of the bacterial communities associated with two ant-plant symbioses.

Insect fungiculture is practiced by ants, termites, beetles, and gall midges and it has been suggested to be widespread among plant-ants. Some of the insects engaged in fungiculture, including attine ants and bark beetles, are known to use symbiotic antibiotic-producing actinobacteria to protect themselves and their fungal cultivars against infection. In this study, we analyze the bacterial communities on the cuticles of the plant-ant genera Allomerus and Tetraponera using deep sequencing of 16S rRNA. Allomerus ants cultivate fungus as a building material to strengthen traps for prey, while Tetraponera ants cultivate fungus as a food source. We report that Allomerus and Tetraponera microbiomes contain >75% Proteobacteria and remarkably the bacterial phyla that dominate their cuticular microbiomes are very similar despite their geographic separation (South America and Africa, respectively). Notably, antibiotic-producing actinomycete bacteria represent a tiny fraction of the cuticular microbiomes of both Allomerus and Tetraponera spp. and instead they are dominated by γ-proteobacteria Erwinia and Serratia spp. Both these phyla are known to contain antibiotic-producing species which might therefore play a protective role in these ant-plant systems.

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria.

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as 'weedkillers' in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus.

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ∼30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.

A mutualistic microbiome: How do fungus-growing ants select their antibiotic-producing bacteria?

We recently published a paper titled "A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus" showing that attine ants use multidrug therapy to maintain their fungal cultivars. This paper tested two theories that have been put forward to explain how attine ants establish mutualism with actinomycete symbionts: environmental acquisition versus co-evolution. We found good evidence for environmental acquisition, in agreement with other recent studies. We also found evidence that supports (but does not prove) co-evolution. Here we place the environmental acquisition and co-evolution arguments within the framework of general mutualism theory and discuss how this system provides insights into the mechanisms that assemble microbiomes. We conclude by discussing future directions for research into the attine ant-actinomycete mutualism.

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus.

BACKGROUND: Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive. RESULTS: In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus. CONCLUSIONS: Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.