Fungus-growing ants (Attini: Formicidae) engage in an obligate mutualism with fungi they cultivate for food. Although biologists have been fascinated with fungus-growing ants since the resurgence of natural history in the modern era, the early stages of research focused mainly on the foraging behavior of the leaf-cutters (the most derived attine lineage). Indeed, the discovery that the ants actually use leaf fragments to manure a fungus did not come until the 1800s. More recently, three additional microbial symbionts have been described, including specialized microfungal parasites of the ant's fungus garden, antibiotic-producing actinobacteria that help protect the fungus garden from the parasite, and a black yeast that parasitizes the ant-actinobacteria mutualism. The fungus-growing ant symbiosis serves as a particularly useful model system for studying insect-microbe symbioses, because, to date, it contains four well-characterized microbial symbionts, including mutualists and parasites that encompass micro-fungi, macro-fungi, yeasts, and bacteria. Here, we discuss approaches for studying insect-microbe symbioses, using the attine ant-microbial symbiosis as our framework. We draw attention to particular challenges in the field of symbiosis, including the establishment of symbiotic associations and symbiont function. Finally, we discuss future directions in insect-microbe research, with particular focus on applying recent advances in DNA sequencing technologies.
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