Andre Rodrigues

COEVOLUTION BETWEEN ATTINE ANTS AND ACTINOMYCETE BACTERIA : A REEVALUATION

Abstract We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution—reciprocal modification—therefore remains to be established for the attine ant-actinomycete mutualism.

Microfungal 'Weeds' in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.

Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants.

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.

Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation

Twenty-seven thermophilic and thermotolerant fungal strains were isolated from soil, decaying organic matter and sugarcane piles based on their ability to grow at 45°C on medium containing corn straw and cardboard as carbon sources. These fungi were identified in the genera Aspergillus, Thermomyces, Myceliophthora, Thermomucor and Candida. The majority of the isolated strains produced xylanase and cellulases under solid state fermentation (SSF). The highest cellulase and xylanase productions were obtained by the cultivation of the strains identified as Aspergillus fumigatus M.7.1 and Myceliophthora thermophila M.7.7. The enzymes from these strains exhibited maximum activity at pH 5.0 and at 60 and 70oC. The endo-glucanase from A. fumigatus was stable from 40°C to 65°C and both endo-glucanase and xylanase from M. thermophila were stable in this temperature range when in absence of substrate. The enzymes were stable from pH 4.0 to 9.0.

Variability of non-mutualistic filamentous fungi associated with Atta sexdens rubropilosa nests

A survey of the filamentous fungi other than the symbiotic one found in association withAtta sexdens rubropilosa colonies was carried out. Different fungal species (27 taxa) were isolated a few days after treating the workers with toxic baits (sulfluramid; Mirex-S®), from 40 laboratory and 20 field nests.Syncephalastrum racemosum (54 %) andEscovopsis weberi (21 %),Trichoderma harzianum (38 %) andFusarium oxysporum (23 %) were the prevalent species in laboratory and field nests, respectively.Acremonium kiliense, Acremonium strictum, E. weberi, F. oxysporum, Fusarium solani, Moniliella suaveolens andT. harzianum were found in both nests’ groups. We revealed that many filamentous fungi can co-exist in a dormant state inside the nests of these insects and some of them appear to be tightly associated with this environment.

Yeasts isolated from a fungus-growing ant nest, including the description of Trichosporon chiarellii sp nov., an anamorphic basidiomycetous yeast

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 540806(T) (=CBS 11177(T)).

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification

To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification.

Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

Leaf-cutting ant faecal fluid and mandibular gland secretion: effects on microfungi spore germination

The mandibular gland secretion (MGS) and the faecal fluid (FF) of the leaf-cutting ant Atta sexdens rubropilosa Forel affected the spore germination of selected microfungi isolated from nests of this insect. MGS was more effective than the FF, completely inhibiting the spore germination of four out of six microfungi species.

Generation of Nutrients and Detoxification: Possible Roles of Yeasts in Leaf-Cutting Ant Nests

The possible roles played by yeasts in attine ant nests are mostly unknown. Here we present our investigations on the plant polysaccharide degradation profile of 82 yeasts isolated from fungus gardens of Atta and Acromyrmex species to demonstrate that yeasts found in ant nests may play the role of making nutrients readily available throughout the garden and detoxification of compounds that may be deleterious to the ants and their fungal cultivar. Among the yeasts screened, 65% exhibited cellulolytic enzymes, 44% exhibited pectinolytic activity while 27% and 17% possess enzyme systems for the degradation of protease and amylase, respectively. Galacturonic acid, which had been reported in previous work to be poorly assimilated by the ant fungus and also to have a negative effect on ants’ survival, was assimilated by 64% and 79% of yeasts isolated from nests of A. texana and Acromyrmex respectively. Our results suggest that yeasts found in ant nests may participate in generation of nutrients and removal of potentially toxic compounds, thereby contributing to the stability of the complex microbiota found in the leaf-cutting ant nests.