Symbiont-Mediated Protection of <i>Acromyrmex</i> Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae

Abstract

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus <i>Pseudonocardia</i>, which help protect the ants' fungal mutualist from a specialized mycoparasite, <i>Escovopsis</i>. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if <i>Acromyrmex</i> ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their <i>Pseudonocardia</i> symbionts. We further examine the ecological dynamics and defensive capacities of <i>Pseudonocardia</i> against <i>M. anisopliae</i> across seven different <i>Acromyrmex</i> species by controlling <i>Pseudonocardia</i> acquisition using ant-nonnative <i>Pseudonocardia</i> switches, <i>in vitro</i> challenges, and <i>in situ</i> mass spectrometry imaging (MSI). We show that <i>Pseudonocardia</i> protects the ants against <i>M. anisopliae</i> across different <i>Acromyrmex</i> species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative <i>Pseudonocardia</i> symbionts receive protection from <i>M. anisopliae</i> regardless of the strain acquired compared with <i>Pseudonocardia</i>-free conditions, we find significant variation in the degree of protection conferred by different <i>Pseudonocardia</i> strains. Additionally, when ants were reared in <i>Pseudonocardia</i>-free conditions, some species exhibit more susceptibility to <i>M. anisopliae</i> than others, indicating that some ant species depend more on defensive symbionts than others. <i>In vitro</i> challenge experiments indicate that <i>Pseudonocardia</i> reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that <i>Pseudonocardia</i>-carrying ants produce more chemical signals than <i>Pseudonocardia</i>-free treatments, indicating that <i>Pseudonocardia</i> produces bioactive metabolites on the <i>Acromyrmex</i> cuticle. Our results indicate that <i>Pseudonocardia</i> can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. <b>IMPORTANCE</b> In some plants and animals, beneficial microbes mediate host immune response against pathogens, including by serving as defensive symbionts that produce antimicrobial compounds. Defensive symbionts are known in several insects, including some leaf-cutter ants where antifungal-producing <i>Actinobacteria</i> help protect the fungal mutualist of the ants from specialized mycoparasites. In many defensive symbioses, the extent and specificity of defensive benefits received by the host are poorly understood. Here, using "aposymbiotic" rearing, symbiont switching experiments, and imaging mass spectrometry, we explore the ecological and chemical dynamics of the model defensive symbiosis between <i>Acromyrmex</i> ants and their defensive symbiotic bacterium <i>Pseudonocardia</i>. We show that the defensive symbiont not only protects the fungal crop of <i>Acromyrmex</i> but also provides protection from fungal pathogens that infect the ant workers themselves. Furthermore, we reveal that the increased immunity to pathogen infection differs among strains of defensive symbionts and that the degree of reliance on a defensive symbiont for protection varies across congeneric ant species. Taken together, our results suggest that <i>Acromyrmex</i>-associated <i>Pseudonocardia</i> have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.