Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants

Abstract

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.

‘Candidatus Streptomyces philanthi’, an endosymbiotic streptomycete in the antennae of Philanthus digger wasps

“…A similar symbiotic relationship for pathogen defence between insects and actinomycetes has been described for leaf-cutter ants (Currie et al, 1999). A species of the family Pseudonocardiaceae protects the ants' fungus gardens against a parasitic fungus by producing antibiotic substances (Cafaro & Currie, 2005;Currie et al, 1999).…”

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

“…This finding is supported by various other studies. Cafaro & Currie [27] determined the generic placement of Pseudonocardia using ultrastructure examination of the bacteria directly on the cuticle of Acromyrmex workers. In addition, three population-level studies, representing 180 colonies from four fungusgrowing ant species, support specific associations between fungus-growing ants and Pseudonocardia (34 colonies of Acromyrmex octospinosus and A. echinatior: [28]; 43 colonies in Trachymyrmex septentrionalis: [29]; and 103 colonies in Apterostigma dentigerum: E. J. Caldera 2010, personal communication).…”

“…Fungus-growing ant taxa sampled in this study included: (i) basal ant genera, designated as 'lower' attines, which include representative members of both the Palaeoattini (genera Mycocepurus and Myrmicocrypta) and the Neoattini (genera Mycetarotes, Mycetosoritis and Cyphomyrmex); (ii) species of 'Coral fungus agriculture', members of the Palaeoattini in the genus Apterostigma that culture fungi in the family Pterulaceae; and (iii) the 'higher' attines, spanning the 'Higher attine' and 'Leafcutter ant' agricultural systems [11]. Actinobacterial isolations were performed using chitin agar plates containing antifungals (electronic supplementary material, figure S1b-d); following described protocols (see [27]). Ant colonies were collected aseptically in the field, using flame-sterilized forceps and spoons.…”

“…Pseudonocardia strains were isolated from 14 colonies spanning the attine ant phylogeny, including the ant species Apterostigma dentigerum, Cyphomyrmex muelleri, Mycetarotes parallelus, Myrmicocrypta sp., Trachymyrmex cornetzi, Acromyrmex niger, Acromyrmex hispidus fallax, Acromyrmex echinatior, Acromyrmex octospinosus and Atta colombica. Pseudonocardia strains were isolated using established methods ( [27]; see above). Free-living Pseudonocardia were obtained from either ARS or DSMZ and non-ant-associated Streptomyces were isolated from environmental samples (soil and different plants) in Madison, WI, USA.…”

Generalized antifungal activity and 454-screening ofPseudonocardiaandAmycolatopsisbacteria in nests of fungus-growing ants

“…Attine ants have evolved defenses against such diseases, such as physical weeding, antibiotic secretion, and management of disease-suppressing auxiliary microbes (1,4,5). The most prominent microbes thought to be involved in disease-suppression in attine gardens are actinomycete bacteria in the genus Pseudonocardia, which accumulate on the ants' bodies mixed into integumental accretions of likely glandular origin (12)(13)(14). Many of the ant-associated Pseudonocardia species show antibiotic activity in vitro against Escovopsis (13)(14)(15).…”