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Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae - PubMed

  • ️Fri Jan 01 2021

Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae

Gaspar Bruner-Montero et al. mBio. 2021.

Abstract

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants' fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. IMPORTANCE 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 Actinobacteria 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 Acromyrmex ants and their defensive symbiotic bacterium Pseudonocardia. We show that the defensive symbiont not only protects the fungal crop of Acromyrmex 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 Acromyrmex-associated Pseudonocardia have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.

Keywords: antifungal; defensive symbiosis; fungus-growing ants; host-parasite interactions; symbiont acquisition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1

Pseudonocardia as a defensive symbiont in four species of Acromyrmex leaf-cutter ants. Survivorship curves of Acromyrmex workers with A. echinatior (A), A. octospinosus (B), A. laticeps (C), and A. niger (D) carrying Pseudonocardia from their own colony (native) (■ and □) or under a Pseudonocardia-free condition (● and ○), exposed to Metarhizium (solid symbols) or a control solution (open symbols) of sterile deionized water + 0.01% Tween 20. Error bars represent standard error. Letters represent significant differences from one treatment to another at a P value of <0.05 in pairwise comparisons using a Kaplan-Meier pairwise test.

FIG 2
FIG 2

Metapleural glands and Pseudonocardia effects on ant defense. Metapleural glands and Pseudonocardia effects on A. echinatior workers infected with Metarhizium (A) and a control solution (B). Ants were reared under either Pseudonocardia-carrying conditions (+P) or Pseudonocardia-free conditions (−P) with either MGs sealed (−MG) or MGs open (+MG). A control solution was made of sterile deionized water + 0.01% Tween 20. Error bars represent standard error. Letters represent significant differences at a P value of <0.05 in pairwise comparisons using a Kaplan-Meier pairwise test. The inset graph shows the effects of the acrylic solution, which was used to block the MGs, on gaster-painted ants (red bar) and unpainted ants (pink bar).

FIG 3
FIG 3

Nonnative Pseudonocardia acquisition and bacterial coverage effects on ant individual susceptibility. (A) Survivorship curves of A. echinatior workers carrying the Pseudonocardia ectosymbiont from different Acromyrmex species after being exposed to Metarhizium. Species names on the legend denote the ant host species from which each Pseudonocardia strain was derived. The A. echinatior Pseudonocardia is the native strain. A. echinatior ants raised by A. cephalotes ants are Pseudonocardia free. Letters represent significant differences from one treatment to another at a P value of <0.05 in pairwise comparisons using the Kaplan-Meier pairwise test. (B) Effects of different strains of Pseudonocardia isolated from different species of Acromyrmex ants on the conidial germination area of Metarhizium. Streptomyces coelicolor (a common soil-dwelling Actinobacteria) was used for comparative effects. (C) Micrograph illustrating the interactions between Metarhizium (left) and Pseudonocardia (right). Error bars represent standard error.

FIG 4
FIG 4

In situ imaging mass spectrometry of Acromyrmex ants. Heat map shows the 50 top putative metabolites (row) of importance. Metabolites were selected based on an ANOVA test between treatments and clustered by similarities. Ants were reared under either Pseudonocardia-carrying conditions (+P) with MG opened (pink) and MG opened control (yellow) or Pseudonocardia-free conditions (−P) with MGs opened (light blue), MGs sealed (blue), beheaded (red), and MG opened control (green). Metarhizium-treated ants are denoted by the inset green square. Control denotes uninfected treatments. The color key indicates metabolite relative intensity (blue, lowest; red, highest).

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