Targeted treatment of injured nestmates with antimicrobial compounds in an ant society

Abstract

Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.

Fig. 1. Lethal effects and diversity of soil pathogens.

a Relative 16 S rRNA gene copies (bacterial load ΔCq) for individuals whose wounds were exposed to a sterile PBS solution (blue: Sterile), or soil pathogens diluted in PBS (red: Infected, OD = 0.1), 2 and 11 h after exposure (n = 10 per boxplot, see Supplementary Table 1 for statistical results). Significant differences (P < 0.05) are shown with different letters and were calculated using a two-sided least square means with Holm-Bonferroni correction. b Absolute 16 S rRNA gene copy numbers summarized at the genus-level for the 18 amplicon sequence variants (ASV) that had at least 1% relative abundance in five of the 40 analyzed ants across the experiment (n = 10 per boxplot). Significance is shown for pairwise comparisons between sterile (blue) and infected ants (red): ***=P < 0.001; **=P < 0.01; *=P < 0.05; n.s.= not significant (P > 0.05). Detailed statistical results in Supplementary Table 2, significant differences were calculated with a two-sided permutation t-test with Holm-Bonferroni correction. (c) Kaplan – Meier cumulative survival rates of workers in isolation (dotted line) or inside the nest (solid line) whose wounds were exposed to a sterile PBS solution (blue: sterile), or soil pathogens diluted in PBS (red: infected, OD = 0.1). Significant differences (P < 0.05) are indicated with different letters. Detailed statistical results in Supplementary Fig. 2a and Supplementary Table 3, significant differences were calculated using a two-sided least square means with Holm-Bonferroni correction. Boxplots show median (horizontal line), interquartile range (box), distance from upper and lower quartiles times 1.5 inter-quartile range (whiskers), outliers (>1.5x upper or lower quartile). Source data are provided as a Source Data file.

Fig. 2. Survival probability and pathogen load of sterile and infected ants.

a Kaplan–Meier cumulative survival rates of workers in isolation or inside the nest whose wounds were exposed to P. aeruginosa diluted in PBS (Infected, OD = 0.05) or a sterile PBS solution (Sterile). Detailed statistical results in Supplementary Fig. 2c and Supplementary Table 3. b relative bacterial load (ΔCq) of Pseudomonas at two different time points (2 h and 11 h) for ants in isolation or inside the nest with wounds treated the same way as in Fig. 2a (Infected or Sterile). n = 6 per boxplot, significant differences (P < 0.05) are shown with different letters (Supplementary Table 4). Boxplots show median (horizontal line), interquartile range (box), distance from upper and lower quartiles times 1.5 inter-quartile range (whiskers), outliers (>1.5x upper or lower quartile). Significant differences in both panels were calculated using a two-sided least square means with Holm-Bonferroni correction. Colors in both panels show in red: infected ants in isolation, in light blue: sterile ants in isolation, in dark blue: sterile ants inside the nest, in green: infected ants inside the nest. Source data are provided as a Source Data file.

Fig. 3. Use and efficacy of the metapleural gland (MG) secretions during wound care.

a Micro CT scan showing the location of the MG. Blue: secretory cells; yellow: atrium. b Probability of receiving wound care over 24 h fitted with a hierarchical generalized (binomial) additive model (HGAM); shaded bars indicate periods during which the probability of receiving care was significantly (P < 0.05) higher for infected (red; n = 6) than sterile (blue; n = 6) individuals. The line represents the predicted probability by the HGAM, with the colored shaded area representing the 95% confidence interval. c Probability of receiving antimicrobial wound care with metapleuralgland (MG) secretions (the same ants as in Fig. 3b), modeled with identical HGAM specifications. d Bacterial growth assay for P. aeruginosa either in LB broth (positive control, n = 6) or LB broth with MG secretions (Metapleural gland n = 9). Two-sided Mann-Whitney U test: W = 54, P < 0.001. Boxplots show median (horizontal line), interquartile range (box), distance from upper and lower quartiles times 1.5 inter-quartile range (whiskers), outliers (>1.5x upper or lower quartile). Source data are provided as a Source Data file.

Fig. 4. Effect of MG secretions on survival of sterile and infected ants.

Kaplan–Meier cumulative survival rates of workers inside sub-colonies with a plugged metapleuralgland (MG) opening (dotted line) or with an unmanipulated MG opening (solid line) whose wounds were exposed to a sterile PBS solution (blue, sterile n = 12) or P. aeruginosa diluted in PBS (OD = 0.05) (red, infected n = 12). Detailed statistical results in Supplementary Fig. 2d and Supplementary Table 6, significant differences were calculated using a two-sided least square means with Holm-Bonferroni correction. Additional data for ants in isolation can be found in Supplementary Fig 5. Source data are provided as a Source Data file.

Fig. 5. Differential gene expression between sterile and infected ants 2 and 11 h after injury.

Infection triggers changes in the expression of hundreds of genes. Volcano plot illustrates the fold up- and down-regulation of immune-related genes (red triangles, Supplementary Table 11) and lipids and CHC-related genes (yellow squares, Supplementary Table 10), 11 h (a) and 2 h (b) after infection. Positive Log2FoldChange values correspond to genes up-regulated in infected ants when compared to sterile ants, while negative values are down-regulated in infected ants. Significant differences were calculated using a two-sided Wald test and corrected for multiple testing using the Benjamini and Hochberg method (genes with significant differential expression are marked in blue, i.e. adjusted P-value < 0.05). Source data are provided as a Source Data file.