Social transfer of pathogenic fungus promotes active immunisation in ant colonies

Abstract

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members--that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses ("variolation" or "inoculation").

Figure 1. Antifungal immune assay of nestmates after social contact to treated individuals.

Nestmates of fungus-exposed individuals (light green bars) inhibited fungal growth significantly more than nestmates of control-treated individuals (light grey bars), both after 3 and 5 d of social contact with the exposed ant. Bars indicate mean ± SEM of proportional antifungal activity compared to the growth control (n = 10 samples per treatment consisting of a pool of five individuals each). Different letters indicate statistically significant differences at α = 0.05.

Figure 2. Behavioural interactions among group members.

(A) Cumulative allogrooming frequencies over the 5 experimental days were significantly higher between treated individuals and their nestmates (striped bars, n = 240 per treatment type) than among nestmates (single colour bars, n = 480 per treatment type)—irrespective of treatment type (sham control, grey; fungus treatment, green). (B) Allogrooming frequencies between fungus-exposed individuals and their nestmates were significantly higher in the first 2 d of the experiment (observations 0–5 h and 24–29 h post-treatment) than at later time points (>48 h). (C) Cumulative frequencies of social feeding (trophallaxis behaviour) were not affected by type of group member and fungus versus control treatment. Bars represent average frequency (mean ± SEM) of interactions per individual over the total time (A and C) or periods (B) of observation. Different letters indicate statistically significant differences at α = 0.05; n.s., non-significant.

Figure 3. Fungal infection levels of treated individuals and their nestmates.

Proportion of exposed individuals (dark green) and nestmates (light green) that show fungal growth inside their bodies (left panels) and number of fungal colony forming units in infected ants (right panels), after (A) 3 d and (B) 5 d of social contact. On both days, the proportion of infected individuals was equally high between directly fungus-exposed ants and their nestmates, indicating a high frequency of pathogen transfer between group members. Yet the infection load of infected nestmates was significantly lower on both days (approximately 8 times lower on day 3 and 12 times lower on day 5). Bars give the proportion of infected individuals in the different groups (n = 10 for directly exposed and n = 14 for nestmates per day) and boxplots show median and 25%–75% quartiles of CFUs in infected individuals (day 3: n = 8 directly exposed individuals and n = 9 nestmates; day 5: n = 9 each for directly exposed and nestmate ants). Different letters indicate statistically significant differences at α = 0.05.

Figure 4. Antifungal activity of directly exposed individuals with low-level infections versus high-dose infections.

Individuals directly exposed to a low pathogen dosage (exposure to LD₂; dotted bar) had a significantly higher capacity to inhibit fungal growth than control-treated individuals (grey), whereas individuals exposed to a high dosage (exposure to LD₅₀; green) had a significantly lower antifungal activity than controls and low-dose exposed ants (n = 10 for all groups). Bars show mean ± SEM of proportional antifungal activity compared to the growth control (n = 10 samples per treatment, each consisting of a pool of five individuals each). Different letters indicate statistically significant differences at α = 0.05.

Figure 5. Antifungal activity measures to test for passive transfer of antimicrobial substances.

(A, B) Antifungal activity of “new nestmates” of (A) directly treated ants and (B) early nestmates (n = 10 samples per group, each sample consisting of a pool of five individuals) for sham control (light grey) and fungus treatment (light green). The groups did not differ from one another. Bars show mean ± SEM of proportional antifungal activity compared to the growth control; n.s., non-significant. (C, D) Antifungal properties of the exterior and interior of fungus-exposed individuals compared to control individuals for the directly treated ants (C) and their respective nestmates (D). We found no difference in the potentially transferable substances from the body surface (cuticle of the ant gaster) and the thorax including the antimicrobially active metapleural glands, nor the trophallactic droplet between individuals treated with a sham control, or with the fungus (dark green for directly exposed individuals, light green for their nestmates). The antifungal activity of control-treated individuals (respectively, their nestmates) is given as a dotted line. Boxplots with whiskers represent mean ± SEM proportion and 95% confidence intervals (indicated in grey shading) of fungal growth inhibition of the ants from the fungus treatment, all standardised to the sham control (n = 10 samples per treatment, except for cuticle and thorax samples: n = 6 per group; each sample consisted of a pool of 5 ants); n.s., non-significant.

Figure 6. Immune gene expression in nestmate ants.

Expression of the immune genes (A) defensin, (B) prophenoloxidase (PPO), and (C) cathepsin L normalised to the housekeeping gene 18s rRNA in nestmates of individuals treated with sham control (light grey) and fungus (light green), after 3 d of social contact. Nestmates of fungus-exposed individuals had significantly elevated defensin and PPO expression levels compared to nestmates of controls, whereas there was no difference in cathepsin L expression. Bars show mean ± SEM (n = 7 nestmates of control-treated and 21 nestmates of fungus-exposed individuals for each gene). Different letters indicate statistically significant differences at α = 0.05; n.s., non-significant.

Figure 7. Antibacterial activity of nestmates after social immunisation against the fungal pathogen.

The capacity to inhibit growth of the bacterium Arthrobacter globiformis did not differ between nestmates of individuals treated with sham control (light grey) and fungus (light green). Bars show mean ± SEM of bacterial growth inhibition standardised to the bacterial growth control (n = 10 samples per group, each sample consisting of a pool of five nestmates); n.s., non-significant.

Figure 8. Epidemiological model including two modes of immunisation. Model setup and outcomes.

(A) Illustration of the SIRM (Susceptible-Infectious-Removed-iMmune) model, with (B) corresponding state changes and transition rates under which ants change their states. The dotted line in (A) illustrates the influence of infectious individuals (I) on the state change rate from susceptible (S) to initially immunised (M_i) ants for passive immunisation. (C,D) Model predictions for the proportions of individuals in the different states over time, comparing passive (C) and active (D) immunisation. Passive immunisation allows for a higher number of immune individuals (M_i and entering the M_l state, pale and dark blue dashed lines), whereas active immunisation leads to a faster elimination of the disease (infectious [I, black solid line] individuals go to 0) and a lower death rate in the colony (R, red solid line), despite the fact that disease spread from the first exposed ants can only occur in the active immunisation scenario. Immunisation is transient so that M_l individuals become susceptible (S, green dotted line) over time for both passive and active immunisation.