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.

Generalized antifungal activity and 454-screening ofPseudonocardiaandAmycolatopsisbacteria in nests of fungus-growing ants

“…The prevailing view of attine actinomycete-Escovopsis antagonism is a coevolutionary arms race between antibiotic-producing Pseudonocardia and Escovopsis parasites (5,(18)(19)(20)(21)(22). Attine ants are thought to use their integumental actinomycetes to specifically combat Escovopsis parasites, which fail to evolve effective resistance against Pseudonocardia because of some unknown disadvantage in the coevolutionary arms race (14,18,20).…”

“…Attine ants are thought to use their integumental actinomycetes to specifically combat Escovopsis parasites, which fail to evolve effective resistance against Pseudonocardia because of some unknown disadvantage in the coevolutionary arms race (14,18,20). This view on specific Pseudonocardia-Escovopsis coevolution was based on very little direct evidence in support of 4 key observations.…”

“…First, in 2 species studied so far using PCR-based bacterial screens (with Pseudonocardia-specific primers), workers of a single attine nest were thought to associate with only one Pseudonocardia lineage (23). Second, in 2 species studied so far for presence/absence of bacterial growth on reproductives, attine queens carried visible growth during their mating flights, but not the males, suggesting vertical transmission from mother to daughter queen (18); this is expected to generate selection for beneficial bacterial traits within a long-term ant-Pseudonocardia partnership (5,18,20,24). Third, one study showed that a single, unidentified actinomycete bacterium isolated from an Apterostigma worker secreted compounds that enhanced the growth of the cultivated fungus, suggesting a derived actinomycete metabolism promoting the ant-cultivar mutualism (18).…”

“…Most importantly, Pseudonocardia of various attine species have nonspecific antibiotic properties that inhibit garden pathogens, endophytes, saprotrophs, arthropod pathogens, and most severely the ant-cultivated fungi. We evaluate hypotheses on nonadaptive and adaptive functions of attine integumental bacteria and develop an alternative conceptual framework to replace the prevailing Pseudonocardia-Escovopsis model (18)(19)(20)(21)(22)(23)(24)27). For those attine ants for which association with Pseudonocardia is adaptive, possible alternate roles of the integumental bacteria could include protection of the ants, control of cultivar growth, or sanitation of the nest environment.…”

Farming of a defensive fungal mutualist by an attelabid weevil

“…Inhibition of fungi and bacteria was measured on PDA and NA, respectively, following the published methods (Poulsen et al, 2007). Bioassays were run for 6 weeks or until the growth of microorganisms ceased.…”