Symbiont interactions in a tripartite mutualism: Exploring the presence and impact of antagonism between two fungus-growing ant mutualists

Abstract

Mutualistic associations are shaped by the interplay of cooperation and conflict among the partners involved, and it is becoming increasingly clear that within many mutualisms multiple partners simultaneously engage in beneficial interactions. Consequently, a more complete understanding of the dynamics within multipartite mutualism communities is essential for understanding the origin, specificity, and stability of mutualisms. Fungus-growing ants cultivate fungi for food and maintain antibiotic-producing <I>Pseudonocardia</I> actinobacteria on their cuticle that help defend the cultivar fungus from specialized parasites. Within both ant-fungus and ant-bacterium mutualisms, mixing of genetically distinct strains can lead to antagonistic interactions (i.e., competitive conflict), which may prevent the ants from rearing multiple strains of either of the mutualistic symbionts within individual colonies. The success of different ant-cultivar-bacterium combinations could ultimately be governed by antagonistic interactions between the two mutualists, either as inhibition of the cultivar by <I>Pseudonocardia</I> or <I>vice versa</I>. Here we explore cultivar-<I>Pseudonocardia</I> antagonism by evaluating <I>in vitro</I> interactions between strains of the two mutualists, and find frequent antagonistic interactions both from cultivars towards <I>Pseudonocardia</I> and <I>vice versa</I>. To test whether such <I>in vitro</I> antagonistic interactions affect ant colonies <I>in vivo</I>, we performed sub-colony experiments using species of <I>Acromyrmex</I> leaf-cutting ants. We created novel ant-fungus-bacterium pairings in which there was antagonism from one, both, or neither of the ants&#39; microbial mutualists, and evaluated the effect of directional antagonism on cultivar biomass and <I>Pseudonocardia</I> abundance on the cuticle of workers within sub-colonies. Despite the presence of frequent <I>in vitro</I> growth suppression between cultivars and <I>Pseudonocardia</I>, antagonism from <I>Pseudonocardia</I> towards the cultivar did not reduce sub-colony fungus garden biomass, nor did cultivar antagonism towards <I>Pseudonocardia</I> reduce bacteria abundance on the cuticle of sub-colony workers. Our findings suggest that inter-mutualist antagonism does not limit what combinations of cultivar and <I>Pseudonocardia</I> strains <I>Acromyrmex</I> fungus-growing ants can maintain within nests.