Figure 3. Destructive disinfection by ants prevents pathogen replication.

(A) Destructive disinfection greatly reduced the probability of pupae sporulating compared to pupae that received no destructive disinfection (time point 0), and its effectiveness increased with the length of time ants could perform destructive disinfection (1 vs. 5 days; error bars show ± 95% CI; letters denote groups that differ significantly in Tukey post-hoc comparisons [p<0.05]). (B) The individual components of destructive disinfection (unpacking, biting and poison spraying) interacted to inhibit pathogen replication (% of pupae sporulating in each treatment shown under graph in green). The odds of sporulation for cocooned and unpacked pupae treated with poison were not significantly different to those of control pupae (cocooned pupae treated with water). But when unpacking, biting and poison spraying were combined, the odds of sporulation were significantly reduced (logistic regression; ns = non-significant deviation from control, ***=p<0.001; complete data set of full factorial experiment displayed in Figure 3—figure supplement 3 and all statistics in Table 3).

Figure 3—figure supplement 1. Destructive disinfection of infected pupae in small groups of ants is less efficient.

Destructive disinfection by groups of three ants greatly reduced the probability of pupae sporulating compared to pupae that received no destructive disinfection (time point 0) 5 days after unpacking. However, the proportion of pupae sporulating was equal when ants only had one day to perform destructive disinfection, as compared to those that received no destructive disinfection (time point 0). Error bars show ± 95% CI; letters denote groups that differ significantly in Tukey post-hoc comparisons (p<0.05).

Figure 3—figure supplement 2. Comparison of ant and synthetic poison spraying.

Experimental application of synthetic poison (60% formic acid and 2% acetic acid, in water [Tragust et al., 2013a]) resulted in pupae receiving quantities of poison similar to pupae kept with ants for 1 day after unpacking (determined by measuring pupal pH after spraying them with poison and comparing to data from Figure 1—figure supplement 3; Mann-Whitney U test: U = 303, p=0.38), meaning we were applying poison to pupae in realistic amounts as compared to the ants and inducing an equivalent decrease in pH change. All data points displayed; lines ± shaded boxes show mean ± 95% CI. Treatments were non-significant (ns) in a Mann-Whitney U test (p>0.05).

Figure 3—figure supplement 3. Destructive disinfection by ants prevents pathogen replication.

Unpacking, biting and poison application interact to inhibit fungal sporulation. Only when all three ‘behaviours’ are performed is fungal sporulation completely prevented (grey = poison, white = water). Error bars show ± 95% CI. Uppercase letters denote bars that differ (p<0.05) within the water treatment, lowercase letters show differences within the poison treatment, and asterisks indicate differences within pupal groups: ns = non significant, *=p<0.05, ***=p<0.001 (full post-hoc comparisons given in Table 3).

Figure 3—figure supplement 4. The pupal cocoon blocks the application of poison.

Cocooned pupae treated with poison had a pH equivalent to untreated cocooned pupae, whereas poison-treated unpacked pupae had a significantly decreased pH, revealing that the cocoon blocks the application of poison (KW test: H = 18.22, df = 2, p<0.001; post hoc comparisons: untreated cocooned vs. poison-treated unpacked, p=0.91; all others, p<0.001). All data points displayed; lines ± shaded boxes show mean ± 95% CI. Letters denote groups that differ significantly in post-hoc comparisons (p<0.05).