Ammonia Production by Streptomyces Symbionts of Acromyrmex Leaf-Cutting Ants Strongly Inhibits the Fungal Pathogen Escovopsis

Leaf-cutting ants live in mutualistic symbiosis with their garden fungus Leucoagaricus gongylophorus that can be attacked by the specialized pathogenic fungus Escovopsis. Actinomyces symbionts from Acromyrmex leaf-cutting ants contribute to protect L. gongylophorus against pathogens. The symbiont Streptomyces sp. Av25_4 exhibited strong activity against Escovopsis weberi in co-cultivation assays. Experiments physically separating E. weberi and Streptomyces sp. Av25_4 allowing only exchange of volatiles revealed that Streptomyces sp. Av25_4 produces a volatile antifungal. Volatile compounds from Streptomyces sp. Av25_4 were collected by closed loop stripping. Analysis by NMR revealed that Streptomyces sp. Av25_4 overproduces ammonia (up to 8 mM) which completely inhibited the growth of E. weberi due to its strong basic pH. Additionally, other symbionts from different Acromyrmex ants inhibited E. weberi by production of ammonia. The waste of ca. one third of Acomyrmex and Atta leaf-cutting ant colonies was strongly basic due to ammonia (up to ca. 8 mM) suggesting its role in nest hygiene. Not only complex and metabolically costly secondary metabolites, such as polyketides, but simple ammonia released by symbionts of leaf-cutting ants can contribute to control the growth of Escovopsis that is sensitive to ammonia in contrast to the garden fungus L. gongylophorus.

Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants’ fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.

Taxonomic and nomenclatural novelties in Leucoagaricus (Agaricaceae) from Brazil

Leucoagaricus nzumbae sp. nov. (Agaricaceae) is described based on morphological and molecular data from Teresópolis, Rio de Janeiro, Brazil. This species is characterized by the delicate small basidiomes that turn completely lilac when dried, brownish color of the pileus surface, and trichodermal elements with intracellular and parietal brown pigments of the pileus covering. A lectotype and an epitype are designated for Leucoagaricus gongylophorus, a symbiotic fungus of leaf-cutting ants. Full description, nomenclatural notes, and illustrations of fresh basidiomes in situ and of the main macro-and micromorphological features of both species are provided.

From Plants to Ants: Fungal Modification of Leaf Lipids for Nutrition and Communication in the Leaf-Cutter Ant Fungal Garden Ecosystem

ABSTRACT Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry, we evaluated differences between the molecular species of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens, we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression toward 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an interkingdom communication tool in leaf-cutter ant fungal gardens. IMPORTANCE In this work, we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an interkingdom communication tool, in this case, as an attractant rather than as a repellant, which is more often seen.

From plants to ants: Fungal modification of leaf lipids for nutrition and communication in the leaf-cutter ant fungal garden ecosystem

Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants, and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry we evaluated differences between the molecular speciation of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens, but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression towards 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an inter-kingdom communication tool in leaf-cutter ant fungal gardens.

INSECTICIDAL AND FUNGICIDAL ACTIVITY OF A MAGNESIUM COMPOUND CONTAINING ISOVANILLIC ACID AGAINST LEAF-CUTTING ANT AND ITS SYMBIOTIC FUNGUS

Pesticides offer a simple, cost-effective solution to ensure food supplies for a growing world population. However, their environmental performance needs improvement if pesticides are to continue contributing importantly. This study explores the insecticidal potential of a magnesium (II) metal complex containing 1,10-phenanthroline and isovanillic acid (MgPhenIso) against leaf-cutting ant (Atta sexdens rubropilosa) and its symbiotic fungus (Leucoagaricus gongylophorus). The MgPhenIso complex is easy-to-prepare, stable in solid state and in aqueous solution, and resistant to sunlight irradiation. The insecticidal activity of MgPhenIso-containing bait against A. sexdens rubropilosa and L. gongylophorus showed a delayed action mode at 2 mg g-1 (100% kill, 6th day, S50=2b) and suppressed fungus development and growth. These results, together with earlier investigations on MgPhenIso regarding its absence of toxicity to Zebrafish and Wistar male rats, emphasize the potential of this complex as an environmentally friendly insecticide candidate.