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.Acromyrmex ͉ antifungal agent ͉ symbionts ͉ Attini ͉ polyketides

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 A 1 -A 4 , 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 metabolites 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.

ABSTRACT. The suitability of several methods for estimating light conditions in the understorey of tropical forests, and of different sampling schedules was evaluated. Light conditions at 16 understorey sites in a Panamanian lowland forest were continuously measured for 9 mo with quantum sensors and photodiodes. Light conditions at the sites were also assessed indirectly with hemispherical fisheye photographs, plant canopy analysis, 38-mm photographs, 24-mm photographs and a spherical densiometer. Estimates from all indirect methods, except the spherical densiometer, were highly correlated with the direct measurements. Short-term direct light measurements for a day or a week also correlated with long-term light conditions. The indirect measures differed by up to c. 70% from the direct measures relative to single site measurements. Hence, the indirect methods are inadequate where single site light conditions have to be assessed accurately. However, because light conditions encountered in the understorey varied up to 13-fold, the indirect methods were found to be well suited to rank understorey light conditions among a large number of sites. The results from frequent and infrequent sampling schedules differed only slightly, suggesting that taking indirect measures at the beginning and the end of a study offers a reasonable compromise between accuracy and sampling effort.KEY WORDS: fisheye photography, hemispherical photography, light measurement, plant canopy analysis, PPFD, spherical densiometer I NT R OD U C TI O N Light is a crucial factor determining physiological and ecological processes in plants, as well as in animals , Endler 1993. Among different tropical forest formations, and along climatic gradients light conditions in

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