Antagonistic bacterial interactions help shape host-symbiont dynamics within the fungus-growing ant-microbe mutualism

Abstract

Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria (Pseudonocardia). Specifically, we examine conflict between the ants and their Pseudonocardia symbionts maintained to derive antibiotics against parasitic microfungi (Escovopsis) infecting the ants' fungus garden. Symbiont assays pairing isolates of Pseudonocardia spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with Acromyrmex leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of Pseudonocardia symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of Pseudonocardia within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.

Figure 1. Schematic diagram of the assessment of actinomycete antagonism.

The resident actinomycete was inoculated in the center of a Petri dish and left at room temperature for three weeks, after which a suspension of the intruder actinomycete was inoculated to the entire unoccupied Petri dish area (top). One week after the intruder strain was applied, the minimum diameter of the resident strain and the minimum zone of inhibition (zoi) imposed on the intruder strains were measured. The bottom section of the figure shows four examples of actinomycete-actinomycete reactions, with no antagonism displayed by the residing clone towards the intruding actinomycete (top left), intermediate levels of antagonism with slight to strong inhibition (top right and bottom left), and to complete inhibition of the intruder by the residing strain (bottom right). Pictures are framed with colors according to the size of the zoi: white = no inhibition, light grey = 0.01–0.29 cm, grey = 0.30–0.59 cm, darker grey = 0.60–0.89 cm, and darkest grey>0.90 cm.

Figure 2. Antagonistic reactions across the phylogeny of Pseudonocardia associated with attine ants.

Average degree of antagonism from the resident towards the intruder actinomycete for the 361 combinations of actinomycetes, spanning the attine phylogeny and including five free-living actinomycetes. Ant-associated Pseudonocardia strains are labeled with the genus and species name of the ants they were isolated from, and all strains are numbered as in Table 1 to distinguish strains originating from the same ant species. Different shades of grey denote strength of inhibition (average size of zone of inhibition, zoi; n = 3): white = no inhibition, light grey = 0.01–0.29cm, grey = 0.30–0.59cm, darker grey = 0.60–0.89cm, and darkest grey = above 0.90cm. A) Shows the bioassay results organized according to the phylogenetic placement of the nineteen strains paired. The phylogeny is based on 1393bp of 16S and 1004bp of EF-Tu. Bootstrap support values after 1000 pseudoreplicates under MP (top, left), ML (top, right), and NJ (bottom) conditions are given for the branches separating the major clades in the phylogeny (see text for details). B) Shows the same bioassay result organized according to the ant origin of the actinomycetes: leaf-cutting ants, Trachymyrmex ants, the lower attines, paleoattines, or free-living.

Figure 3. Antagonistic reactions between Pseudonocardia strains associated with Acromyrmex leaf-cutting ants.

Average degree of antagonism from the resident towards the intruder actinomycetes for the total of the 144 combinations performed within and between actinomycete strains isolated from five species of Acromyrmex. Strains are labeled after the ant species they were isolated from, and strains from the same ant species are distinguished by their labeling (A through L). Different shades of grey denote strength of inhibition (average size of zone of inhibition, zoi; n = 3): white = no inhibition, light grey = 0.01–0.29cm, grey = 0.30–0.59cm, darker grey = 0.60–0.89cm, and darkest grey = above 0.90cm. A) Shows the bioassay results organized according to the phylogenetic placement of the twelve strains paired, based on 1394bp of 16S and 959bp of EF-Tu sequences; bootstrap support values after 1000 pseudoreplicates under MP (top, left), ML (top, right), and NJ (bottom) conditions are given for the branches separating the major clades. B) Shows the bioassay results organized according to the ant species origin of the actinomycetes, with horizontal lines separating reactions displayed by isolates from the five ant species.