We found that the presence of a pupal cocoon affects sanitary behaviours and fungal infection in ants. Species from different subfamilies of ants showed consistent patterns depending on pupal type (naked versus cocooned), suggesting that the presence or absence of the cocoon may be a main predictor of the observed effects. This is further corroborated by the fact that similar patterns were found within a single species that simultaneously produces naked and cocooned pupae within the same nest. Still, as we have only covered four subfamilies of ants (one species each of Ponerinae, Myrmicinae, Dolichoderinae, and two species of Formicinae), the generality of this finding across the whole ant phylogeny remains to be tested.

Similar to previous studies [28, 29], the ants in our experiment showed some differences in retrieval of the various brood types, likely as a consequence of chemical, morphological, and behavioural brood recognition cues (for a review see [30]), modulated further by brood developmental stage. Interestingly, with the exception of pupal intake in C. smithi, the ants did not distinguish between brood treatments, and brought in the live pathogen-exposed brood in equally high numbers as dead fungus or sham-treated brood (Table 1). This confirms a similar finding in another ant species [10] and seems to reveal the general pattern that, on one hand, the pathogen cannot manipulate its host in retrieving a higher proportion of contaminated versus healthy brood, and on the other hand, that potential repellent effects of the pathogen are likely overridden by the attractiveness of brood. This is despite the fact that social insects are capable of quickly detecting pathogen presence (ants: [10, 31]; honeybees: [32]; termites: [33–35]). Brood being a strong elicitor for intake behaviour is also exploited, for example, by parasitic Maculinea butterflies, which morphologically and chemically mimic ant larvae and are picked up and brought into the nest [36].

Rapid detection and reaction to the fungal pathogen also occurred in our experiment. Brood grooming frequencies were significantly increased towards live fungus-exposed brood in species with cocooned pupae, La. neglectus and P. punctata, (Figure 1C,D) within the two days post exposure (i.e. before infection), whereas application of dead fungus was not enough to elicit this effect. This finding confirms previous reports of elevated grooming frequencies in other social insects, directed either towards live pathogen-exposed ant larvae [10], or adult nestmates in both ants [37] (but see [11]) and termites [12]. Currently it cannot be resolved, whether the other three species (C. smithi, Li. humile and F. selysi) did not show this adaptive behaviour due to a lack of pathogen detection or response. It seems that these species had an overall high grooming activity towards all brood, including the sham-treatment, which may suggest a constitutively high grooming level, acting as prophylactic defence (similar to [11]). Importantly, the observed upregulation of grooming directed towards pathogen-exposed brood in the two species with cocooned pupae documents that the presence of a silk cocoon around the pupae does not interfere with the ants’ capabilities to detect fungal conidiospores.

Sanitary brood care is not limited to the mechanical removal of infectious particles from the brood surface during allogrooming, and the following disinfection of those particles in the mouth of the cleaning individual and discarding in dump sites [9, 38]. La. neglectus workers were recently found to further apply their antiseptic poison on their brood, thereby efficiently inhibiting germination of Metarhizium conidiospores [9]. The poison of other social Hymenoptera is also known to have antimicrobial properties and to be applied during nest hygiene (ants; subfamily Formicinae: [39], subfamily Myrmicinae: [40, 41]; wasps: [42]; bees: [43]). It is thus likely that also the other ant species in our experiment may employ their poison in chemical brood disinfection. Ants further have evolved a unique gland–the metapleural gland–that produces potent antibiotic secretions, which can serve as antifungal defence [3, 44]. All our study species possess metapleural glands, which may represent a second component of chemical surface disinfection complementing mechanical removal, though in La. neglectus, metapleural gland components do not seem to play a major role in protection of brood against Metarhizium fungus [9].

If these cleaning measures fail to prevent infection of the individual brood items, removal of the diseased brood from the colony is an effective way to limit disease transmission inside the colony. In fact, all ant species in our experiment showed hygienic behaviour, i.e. they removed assumedly infectious or infected brood from the brood chamber, thereby dooming this brood to death. This suggests that hygienic behaviour–so far mostly known from honeybees [2, 10, 14]–is a widespread behaviour also in ants. Species with naked pupae removed consistently more brood items (both larvae and pupae) treated with live fungus than sham control and dead fungus (Figures 2A,B and 3B; exceptions: ns trend in Li. humile larvae and sign. only against sham control in F. selysi). Species with cocooned pupae removed only their larvae in the same pattern, i.e. they also removed more live fungus-exposed larvae than either sham or dead fungus-treated larvae (Figure 2C,D). However, in the three species with obligatory or facultative cocoons, the cocooned pupae were all removed at equally low rates, independent of being exposed to the pathogen or control treatments (Figures 2C,D and 3C).

Differential removal patterns seem to represent an adaptive behaviour by the ants based on infection state (as in [10]). The risk of getting infected after exposure to live fungus, during the course of our experiment, was lowest in cocooned pupae, which were also removed less frequently, as compared to both, naked pupae and larvae (Figures 2, 3). By combining sanitary behaviours and selective removal of diseased brood, the ants successfully reduced the likelihood of the pathogen to self-replicate inside their nest. Only 5% of all fungal outgrowth thus occurred inside the nest, whereas 2/3 of the removed brood showed fungal outgrowth (black bars in Figures 2 and 3B,C).

Fungal outgrowth was not limited to the brood experimentally exposed to live fungal conidiospores, but also occurred on approximately 20% of the previously unexposed brood items, revealing disease transmission among brood (which we could monitor due to the colour marking of brood items). These high disease transmission rates explain, why also previously un-exposed control brood was removed at relatively high rates–particularly in the case of larvae (Figures 2 and 3B,C). It is interesting that the ants do not seem able to contain the transmission to healthy brood, and place all retrieved brood onto a common brood pile enabling disease contraction, but later are highly efficient in removing the infected brood.

In contrast to the high transmission of disease at the brood stage, cross infection of adult workers through contact to contaminated brood was practically absent (< 1%). This may result from both hygiene–including sanitary behaviours [5, 45] and use of exocrine gland-derived antiseptics [3, 9, 39]–and a lower susceptibility due to a melanised and sclerotised cuticle [46]. Low rates of workers contracting the disease after contact to fungus-exposed brood or workers have also been reported for other ants and termites [10, 12, 45], whereas contact with sporulating cadavers led to high infection rates in ants [45]. This emphasizes the importance of hygienic brood removal before any visible fungal outgrowth. Indeed, in our experiment, we found that brood removal anticipated fungal outgrowth, and was performed approximately one to two days before fungal outgrowth (Figure 4), revealing that the ants are able to detect the infection state before infectious stages occur.