Rainer Wirth

Candicidin-producing Streptomyces support leaf-cutting ants to protect their fungus garden against the pathogenic fungus Escovopsis

Leaf-cutting ants such as Acromyrmex octospinosus live in obligate symbiosis with fungi of the genus Leucoagaricus, which they grow with harvested leaf material. The symbiotic fungi, in turn, serve as a major food source for the ants. This mutualistic relation is disturbed by the specialized pathogenic fungus Escovopsis sp., which can overcome Leucoagaricus sp. and thus destroy the ant colony. Microbial symbionts of leaf-cutting ants have been suggested to protect the fungus garden against Escovopsis by producing antifungal compounds [Currie CR, Scott JA, Summerbell RC, Malloch D (1999) Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398:701–704.]. To date, however, the chemical nature of these compounds has remained elusive. We characterized 19 leaf-cutting ant–associated microorganisms (5 Pseudonocardia, 1 Dermacoccus, and 13 Streptomyces) from 3 Acromyrmex species, A. octospinosus, A. echinatior, and A. volcanus, using 16S-rDNA analysis. Because the strain Streptomyces sp. Ao10 proved highly active against the pathogen Escovopsis, we identified the molecular basis of its antifungal activity. Using bioassay-guided fractionation, high-resolution electrospray mass spectrometry (HR-ESI-MS), and UV spectroscopy, and comparing the results with an authentic standard, we were able identify candicidin macrolides. Candicidin macrolides are highly active against Escovopsis but do not significantly affect the growth of the symbiotic fungus. At least one of the microbial isolates from each of the 3 leaf-cutting ant species analyzed produced candicidin macrolides. This suggests that candicidins play an important role in protecting the fungus gardens of leaf-cutting ants against pathogenic fungi.

Chemical basis of the synergism and antagonism in microbial communities in the nests of leaf-cutting ants.

Leaf-cutting ants cultivate the fungus Leucoagaricus gongylophorus, which serves as a major food source. This symbiosis is threatened by microbial pathogens that can severely infect L. gongylophorus. Microbial symbionts of leaf-cutting ants, mainly Pseudonocardia and Streptomyces, support the ants in defending their fungus gardens against infections by supplying antimicrobial and antifungal compounds. The ecological role of microorganisms in the nests of leaf-cutting ants can only be addressed in detail if their secondary metabolites are known. Here, we use an approach for the rapid identification of established bioactive compounds from microorganisms in ecological contexts by combining phylogenetic data, database searches, and liquid chromatography electrospray ionisation high resolution mass spectrometry (LC-ESI-HR-MS) screening. Antimycins A1–A4, valinomycins, and actinomycins were identified in this manner from Streptomyces symbionts of leaf-cutting ants. Matrix-assisted laser desorption ionization (MALDI) imaging revealed the distribution of valinomycin directly on the integument of Acromyrmex echinatior workers. Valinomycins and actinomycins were also directly identified in samples from the waste of A. echinatior and A. niger leaf-cutting ants, suggesting that the compounds exert their antimicrobial and antifungal potential in the nests of leaf-cutting ants. Strong synergistic effects of the secondary meta-bolites produced by ant-associated Streptomyces were observed in the agar diffusion assay against Escovopsis weberi. Actinomycins strongly inhibit soil bacteria as well as other Streptomyces and Pseudonocardia symbionts. The antifungal antimycins are not only active against pathogenic fungi but also the garden fungus L. gongylophorus itself. In conclusion, secondary metabolites of microbial symbionts of leaf-cutting ants contribute to shaping the microbial communities within the nests of leaf-cutting ants.

Plant Herbivore Interactions at the Forest Edge

An ever-increasing proportion of the global forested landscape is in close proximity to edges and edge effects have been shown to represent key forces affecting both organisms and ecological processes. Despite increasing recognition of edge effects on species interactions, a systematic review devoted to plant- herbivore interactions along forest edges has not yet been performed. Here we syn- thesize published research attempting to detect patterns of herbivore densities and herbivory at forest edges, identify the underlying mechanisms generating these patterns, and explore their potential impacts for the forest edge as an ecosystem. Key conclusions are that herbivores, especially generalists, profoundly benefit from forest edges, often due to favourable microenvironmental conditions, an edge-induced increase in food quantity/quality, and (less well documented) disrupted top-down regulation of herbivores. Finally, we present evidence and causal explanations that edge-associated herbivores, via a range of direct and indirect impacts, may alter species interactions, delay successional processes at the edge, and amplify the often human-induced changes on forest biota.

Non-specific association between filamentous bacteria and fungus-growing ants

Fungus-growing ants and their fungal cultivar form a highly evolved mutualism that is negatively affected by the specialized parasitic fungus Escovopsis. Filamentous Pseudonocardia bacteria occurring on the cuticle of attine ants have been proposed to form a mutualistic interaction with these ants in which they are vertically transmitted (i.e. from parent to offspring colonies). Given a strictly vertical transmission of Pseudonocardia, the evolutionary theory predicts a reduced genetic variability of symbionts among ant lineages. The aim of this study was to verify whether actinomycetes, which occur on Acromyrmex octospinosus leaf-cutting ants, meet this expectation by comparing their genotypic variability with restriction fragment length polymorphisms. Multiple actinomycete strains could be isolated from both individual ant workers and colonies (one to seven strains per colony). The colony specificity of actinomycete communities was high: Only 15% of all strains were isolated from more than one colony, and just 5% were present in both populations investigated. Partial sequencing of 16S ribosomal deoxyribonucleic acid of two of the isolated strains assigned both of them to the genus Streptomyces. Actinomycetes could also be isolated from workers of the two non-attine ant species Myrmica rugulosa and Lasius flavus. Sixty-two percent of the strains derived from attine ants and 80% of the strains isolated from non-attine ants inhibited the growth of Escovopsis. Our data suggest that the association between attine ants and their actinomycete symbionts is less specific then previously thought. Soil-dwelling actinomycetes may have been dynamically recruited from the environment (horizontal transmission), probably reflecting an adaptation to a diverse community of microbial pathogens.

Increasing densities of leaf-cutting ants (Atta spp.) with proximity to the edge in a Brazilian Atlantic forest

Leaf-cutting ants (genera Atta and Acromyrmex) have been denoted key species of American rain-forest ecosystems (Fowler et al . 1989) because of their multifarious effects on the vegetation. Being dominant herbivores, cutting up to 13% of the standing leaf crop in a colonys territory per year, they affect directly and significantly individual plants, plant communities and ecosystems (Wirth et al. 2003). The considerable ecological impact of these ants is paralleled by the well-known fact that some species strongly benefit from human-driven habitat alterations and represent prime pests throughout Latin America (Cherrett 1986). Numerous studies have documented populations of leaf-cutting ant to increase with increasing agricultural land use, deforestation and/or disturbance (Fowler et al . 1986, Jaffe & Vilela 1989, Jonkman 1979). Specifically, elevated colony densities have been recorded in (1) transformed vegetation such as pastures (Fowler 1983) and plantations (Jaffe 1986, Oliveira et al. 1998), (2) early successional forests (Vasconcelos & Cherrett 1995), and recently (3) isolated forest remnants (Terborgh et al . 2001).

Spatio-temporal permanence and plasticity of foraging trails in young and mature leaf-cutting ant colonies (Atta spp.)

The distribution and formation of foraging trails have largely been neglected as factors explaining harvesting patterns of leaf-cutting ants. We applied fractal analysis, circular, and conventional statistics to published and newly recorded trail maps of seven Atta colonies focusing on three aspects: permanence, spatio-temporal plasticity and colony life stage. In the long term, trail patterns of young and mature Atta colonies revealed that foraging activities were focused on distinct, static sectors that made up only parts of their potentially available foraging range. Within these foraging sectors, trails were typically ephemeral and highly variable in space and time. These ephemeral trails were concentrated around permanent trunk trails in mature and around nest entrances in young colonies. Besides these similarities, the comparison of trail systems between the two life stages indicated that young colonies exploited fewer leaf sources, used smaller and less-complex systems of foraging trails, preferred different life forms as host plants, and switched hosts more often compared with mature colonies. Based on these analyses, we propose a general hypothesis which describes the foraging pattern in Atta as a result of initial foraging experiences, spatio-temporal distribution of suitable host plants, energetic constraints, and other factors such as seasonality and interspecific predation.

Annual foraging of the leaf-cutting ant Atta colombica in a semideciduous rain forest in Panama

In a 1-y study of vegetation harvested by the leaf-cutting ants, Atta Colombica Guerin, daily harvesting activity of two nests was observed for 24 h at c. I-wk intervals (colony I: June 1993-June 1994; colony II: February-June 1994) on Barro Colorado Island. The average daily quantity of green leaves harvested by colony I was higher during the wet season (11.4 m(2) d(-1)) than during the dry season (9.0 m(2) d(-1)), but was highly variable between survey days. Total annual herbivory of green leaves was estimated to be 3,855 m(2) foliage area for colony I and 1,707 m(2) for colony II. Total dry weight of biomass harvested was higher in the dry season because most material collected during the wet season consisted of green leaves, while during the dry season, more than 50% of the total collected biomass was non-green plant material (stipules of Ficus sp., fruits, seeds, and flower parts of a variety of other species) which represented c. one third (ill kg y(-1)) of the total annual intake (370 kg y(-1)) of plant material. Total daily biomass intake was negatively correlated with daytime rainfall. The peak of daily foraging was affected by timing and duration of rainfall events. Highest input rates normally occurred between 15:00 and 16:00 h (colony I). Dry weight and surface area of harvested leaf fragments differed between plant species, with thicker leaves generally being cut into smaller pieces. Significant linear correlations were found between total daily harvest of fragments and the respective harvesting rate at the maximum of daily activity. High correspondence was found between estimates using this relationship and the measured daily leaf harvest of four other Atta colonies and of two colonies reported in published literature. The use of this relationship as a research tool is discussed.

Ecosystem engineering by leaf-cutting ants: nests of Atta cephalotes drastically alter forest structure and microclimate

1. The role played by Atta species as ecosystem engineers remains poorly investigated despite previous evidence that their nests can impact plant assemblages.

DISPERSAL OF MICONIA ARGENTEA SEEDS BY THE LEAF-CUTTING ANT ATTA COLOMBICA

While leaf-cutter ants are thought to collect mainly vegetative plant material, they have also been observed collecting seeds or fruit parts on the forest floor (Alvarez-Buylla & Martinez-Ramos 1990, Kaspari 1996). For example, leaf-cutter ants have been observed carrying considerable numbers of Brosimum alicastrum Sw. and Cecropia spp. seeds into their nests (Wirth 1996) and Leal & Oliveira (1998; pers. comm. ) found them foraging on the fruits and seeds of 19 different species of Brazilian cerrado vegetation, including six Miconia species. Under some circumstances, seed removal and relocation by leaf cutter ants might even be sufficient to affect local recruitment patterns of trees. For example, in Costa Rica, Atta cephalotes can remove all fallen fig fruit from beneath a Ficus hondurensis crown in a single night (Roberts & Heithaus 1986), while in Venezuela, seedling recruitment of the savanna tree Tapirira velutinifolia was positively associated with the seed harvesting and seed cleaning activities of the ant Atta laevigata (Farji Brenner & Silva 1996).

Edge‐mediated reduction of phorid parasitism on leaf‐cutting ants in a Brazilian Atlantic forest

Previous studies have shown that leaf‐cutting ant populations benefit greatly from living in or near the edges of the Brazilian Atlantic forest. One of the mechanisms responsible for this rise in population density is an edge‐mediated increase of pioneer plants, resulting in increased food availability for the ants (i.e., less bottom‐up control). Here, we hypothesized that the release from natural enemies (i.e., less top‐down control) may also contribute to the phenomenon. We investigated whether parasitism of phorid flies on leaf‐cutting ants decreases in colonies located along the forest edge vs. the interior of a large tract of Atlantic forest in northeastern Brazil. For this, we assessed abundance and rates of oviposition attack by phorids in bimonthly intervals over a period of 1 year in 10 adult colonies of Atta cephalotes (L.) (Hymenoptera: Formicidae: Myrmicinae), five at the forest edge and five in the forest interior. The number of phorids attracted by ants at edge colonies was 40% lower than that at interior colonies. The temporal variation in phorid attraction was also significant, with approximately 35% fewer flies in the dry months as compared to the rainy months. As a result of lower phorid abundance, ant workers of edge colonies suffered three times fewer oviposition attacks than those of interior colonies. There was a tendency for fewer attacks during dry months, but the difference in the temporal variation was not significant. Our findings suggest that edge creation contributes to increased leaf‐cutting ant abundance, not only via the attenuation of bottom‐up forces, but also through an environmentally triggered depression of parasitoid abundance/efficiency, possibly because of adverse environmental conditions in edge habitats.