Symbioses shape all levels of biological organization. Although symbiotic interactions are typically viewed as bipartite associations, with two organisms interacting largely in isolation from other organisms, the presence and importance of additional symbionts is becoming increasingly more apparent. This study examines the importance of a third mutualist within the ancient symbiosis between leaf-cutting ants and their fungal cultivars. Specifically, we experimentally examine the role of a filamentous bacterium (actinomycete), which is typically carried on the cuticle of fungusgrowing ants, in suppressing the growth of a specialized microfungal parasite (Esco6opsis) of the fungus garden. We conducted two-by-two factorial design experiments crossing the presence/absence of actinomycete with the presence/absence of Esco6opsis within small sub-colonies of Acromyrmex octospinosus. In these experiments, infection by Esco6opsis became much more extensive within fungus gardens and had a greater impact on the health of gardens in those sub-colonies with the bacterium removed from workers as compared to gardens with the bacterium still present on the ants. We establish that the actinomycete bacterium is most abundant on those major workers tending the garden, providing further support that the bacterium is involved in garden hygiene. We also found a significantly higher abundance of actinomycete on workers in colonies experimentally infected with Esco6opsis as compared to uninfected control colonies. We suggest that mutualisms between antibiotic-producing microbes and higher organisms may be common associations that are mostly overlooked and that the role of symbionts in reducing the impact of parasites is likely an important aspect in the cost-benefit assessment of mutualisms.

The paired exocrine metapleural glands present in the large majority of ant species produce compounds with antibiotic properties. In the leaf-cutting ant, Acromyrmex octospinosus, the secretion consists of more than 20 different compounds and it has generally been assumed that the glands serve as a general defence against various infectious microbes of fungal and bacterial origin. We present results illuminating the direct costs and benefits of these metapleural gland defences in A. octospinosus. We show that major workers of this leaf-cutting ant experience a significant reduction in their respiration rate when the metapleural glands are experimentally closed, indicating that metapleural gland secretion incurs a substantial cost and that the production of compounds from these glands is terminated when the ants are incapable of secreting them. In another set of experiments, we show that the ability to secrete antibiotic compounds from the metapleural glands is of significant importance when ants are exposed to a general but potentially virulent insect pathogen, Metarhizium anisopliae. Infection with this fungus is lethal within a few days when ants have their metapleural glands experimentally closed, but relatively harmless when the metapleural glands are functional. These findings support experimentally the view that the metapleural glands play an important hygienic role in leaf-cutting ants.

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Hygienic behaviour is an important aspect of social organisation because living in aggregations facilitates the spread of disease. Leaf-cutting ants face the additional problem of an obligatory dependency on a fungus. which itself is also susceptible to parasites. In this study we provide evidence for the importance of effective waste management in colonies of several Panamanian species of Atta and Acromyrmex leaf-cutting ants. differing in colony size and typical mode of waste accumulation (external or internal dumps). We show that: (1) waste is dangerous for the ants, which die at a higher rate in the presence of waste; (2) waste is dangerous for the mutualistic fungus because waste in field colonies is infected with the specialised fungal parasite Escovopsis; (3) the ants allocate considerable effort to active management of waste in order to reduce these dangers. This management follows a "conveyer belt" model according to which increasingly dangerous tasks are performed by older workers, who are less valuble to their colony. Our approach is kaleidoscopic, as different species of leafcutting ants are unequally suitable for direct observation and experimental manipulation. and suggests that more in depth studies of waste management in attine ants would be highly rewarding.

Summary1. Stable mutualistic interactions require the long-term alignment of fitness interests of participating species. This condition is fulfilled when the benefits of the relationship exceed the costs for all partners. 2. One apparent stabilizing factor in mutualisms is the vertical (parent to offspring) transmission of symbionts, as this tends to reduce the expression of virulent traits and reproductive conflicts. This study examines the cost and mode of transmission of a mutualistic Streptomyces bacterium that grows on the cuticle of leaf-cutting ants and produces antibiotics against a specialized fungal parasite of the ant fungus gardens. 3. It is shown that ant respiration rates are elevated by 10-20% when the bacterium is present on their cuticle. This increase is due to direct respiration of the bacterium and possible excess respiration by the ants. Although these two factors cannot be separated, it is clear that the total increase gives a reasonable quantification of the metabolic costs incurred by the Streptomyces symbiont. 4. Ants that actively maintain Streptomyces cultures on their cuticle tend to consume more of their mutualistic fungus garden than controls and this excess consumption increases with the amount of Streptomyces bacteria present. 5. Scanning electron microscopy showed that the mutualistic bacterium is not present on major workers immediately following eclosion, indicating that the bacterium is not transferred to callow workers until later. 6. The results of an experiment simulating within-colony transmission to callow workers suggest that the bacterium is predominantly transmitted from older to newly eclosed major workers, but that transmission may also occur via the fungus garden. The presence of Streptomyces bacteria in the fungus garden implies that rare events of horizontal transmission of the fungal cultivar of attine ants may also imply horizontal transmission of strains of the mutualistic bacterium.