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”).

Close social contact facilitates pathogen transmission in societies, often causing epidemics. In contrast to this, we show that limited transmission of a fungal pathogen in ant colonies can be beneficial for the host, because it promotes “social immunisation” of healthy group members. We found that ants exposed to the fungus are heavily groomed by their healthy nestmates. Grooming removes a significant number of fungal conidiospores from the body surface of exposed ants and reduces their risk of falling sick. At the same time, previously healthy nestmates are themselves exposed to a small number of conidiospores, triggering low-level infections. These micro-infections are not deadly, but result in upregulated expression of a specific set of immune genes and pathogen-specific protective immune stimulation. Pathogen transfer by social interactions is therefore the underlying mechanism of social immunisation against fungal infections in ant societies. There is a similarity between such natural social immunisation and human efforts to induce immunity against deadly diseases, such as smallpox. Before vaccination with dead or attenuated strains was invented, immunity in human societies was induced by actively transferring low-level infections (“variolation”), just like in ants.

Funding: Funding for this project was obtained by the German Research Foundation DFG ( http://www.dfg.de/en/index.jsp ) as an Individual Research Grant (CR118/2-1 to SC) and the European Research Council ( http://erc.europa.eu/ ) in form of two ERC Starting Grants (ERC-2009-StG240371-SocialVaccines to SC and ERC-2010-StG259294-LatentCauses to FJT). In addition, the Junge Akademie (Young Academy of the Berlin-Brandenburg Academy of Sciences and Humanities and the National Academy of Sciences Leopoldina ( http://www.diejungeakademie.de/english/index.html ) funded this joint Antnet project of SC and FJT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2012 Konrad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we applied a multi-level approach to determine the functional mechanism of social immunisation of ant colonies against a fungal pathogen. We analysed the behavioural interaction rates between group members and determined whether social contact may lead to exchange of the pathogen or immune effectors, or whether social immunisation may be triggered by social signals. We determined both the physiological immunity of fungus-exposed individuals and their nestmates, as well as their immune gene expression. Lastly, we developed an epidemiological model to explore long-term colony-level effects of social immunisation depending on the underlying mechanisms.

Passive immunisation may result from a social exchange of antimicrobials produced by the exposed individuals and transferred to their nestmates. Possible transfer pathways include the “external route” over the body surface or the “internal route” by exchange of body fluids [16] . The external body surface (cuticle) of ants is covered with antimicrobial substances produced in an ant-specific gland (metapleural gland [36] , [37] ) and nestmates could easily pick up these substances and apply them on their own bodies by allo- and self-grooming. Immune effectors produced inside the body of infected individuals may be exchanged during the common social feeding behaviour of regurgitation and feeding of trophallactic droplets [16] , [38] , as has recently been suggested as a mechanism for social immunisation of ant colonies after bacterial exposure [16] . Whereas bacterial infections are typically orally transmitted [39] , entomopathogenic fungi are externally transmitted, making distinct disease dynamics of these pathogen taxa likely.

Active upregulation of the nestmates' immune system may be caused by perception of a trigger signal elicited from the exposed individual, possibly of behavioural or chemical nature. In humans, mere visual perception of sick individuals was recently shown to cause preventive stimulation of the immune system [31] . Similarly, in plants, herbivory defence was promoted by perception of volatile chemical cues elicited by an attacked neighbouring plant [32] . Active stimulation of the immune system can also be caused by low-level infections [3] , [8] , [33] , [34] , which may result from social transfer of the pathogen from the exposed individual to its nestmates (as suggested by [3] ), occurring during “normal” social interactions, or as a byproduct of collective sanitary behaviour such as allogrooming of the exposed individual by its nestmates [22] , [35] .

The observed protection in nestmates of exposed ants may be caused by the active upregulation of their own immune systems following social contact to the fungus-exposed individual. Alternatively, social transfer of immune mediators produced by colony members may lead to passive protection of nestmates without requiring the activation of their own immune systems (as outlined by [3] , [17] , [27] ). The active and passive route to social immunisation may also act in concert.

The phenomenon of social immunisation occurs broadly in insect societies—in unrelated social host species (ants and termites) and against divergent pathogen taxa (fungi [17] , [18] and bacteria [16] )—yet the mechanisms underlying this effect are largely elusive (but see [16] ) and have only been hypothesised upon for fungal pathogens [3] , [17] , [18] , [27] . In this study, we therefore aimed to determine the underlying causes of social immunisation in colonies of the ant Lasius neglectus after exposure of single individuals to the entomopathogenic fungus Metarhizium anisopliae, a common natural pathogen of ants [28] , [29] . In this system, we have previously described that 5 d of social contact to an individual exposed to fungal conidia (conidiospores; [30] ) led to a lower susceptibility of nestmate ants when challenged with a high fungal dose after this period [18] . It remained open, however, which social interactions may trigger this effect and how they elicit changes in nestmate immunity.

The first encounter of a host with a particular pathogen often leads to the outbreak of the disease, yet a secondary exposure rarely causes illness, due to the immunological memory of the host. Whereas immune memory in vertebrates is well appreciated [1] , the phenomenon of an individual developing specific immunity against a subsequent pathogen exposure—referred to as immune priming—has only recently been described in invertebrates, both within the lifetime of an individual [2] – [8] and in transgenerational protection of offspring ( [8] – [12] , but see [13] ). In contrast to vertebrates, the underlying mechanisms are not yet understood in invertebrates [14] , [15] . In addition to this immunological memory at the level of individuals, a similar phenomenon occurs at the colony level in insect societies [16] – [18] . Society members act collectively, similar to cells in a body, and work as a superorganism [19] , [20] in multiple aspects, including anti-pathogen defence [21] . For instance, an initial pathogen contact of a colony due to the presence of exposed individuals has been shown to lower the susceptibility of their nestmates to infection when they are later exposed to the same pathogen [16] – [18] . In addition to this physiological “social immunisation,” the collectively performed hygiene behaviour that complements individual defences in social insects [22] – [24] is also affected. Allogrooming of exposed individuals by their nestmates occurs more frequently in colonies with previous experience with this pathogen than in naive colonies [25] , [26] . In contrast to individual immune priming, social immunisation thus refers to a protection of naive individuals of a colony after social contact to exposed individuals.

Results and Discussion

Nestmates of Fungus-Exposed Ants Show Increased Antifungal Defence We have previously shown that social contact to a Lasius worker exposed to conidia (dispersal form, conidiospores; [30]) of the entomopathogenic fungus M. anisopliae, but not to control-treated ants, increased the survival of previously naive nestmates when challenged with the same M. anisopliae strain 5 d later [18]. We now directly assessed the immune function of nestmates with a novel and sensitive “antifungal activity assay.” We incubated ant tissue with blastospores (within-host infection form; [30]) of the fungus to measure the ability of ants to inhibit fungal growth. We found a significantly higher antifungal activity in nestmates of fungus-exposed as compared to nestmates of control-treated individuals (Figure 1). This was true not only after 5 d of social contact to an exposed individual, but already after 3 d (GLM, F = 3.859, df = 3, p = 0.017; treatment type [fungus treatment versus sham control]: F = 10.634, df = 1, p = 0.002; time [3 versus 5 d post-treatment]: F = 0.001, df = 1, p = 0.973; interaction [Treatment Type×Time]: F = 0.942, df = 1, p = 0.338). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g001 To understand the mechanism behind increased antifungal defence in nestmates of exposed ants, it is important to study the behaviour of group members. First, behavioural changes of individuals after fungal exposure may be a signal to their nestmates to upregulate their immune system. Second, the social interactions define the routes and opportunities for potential exchange of immune effectors [40],[41] or the pathogen itself [42].

Behaviourial Interactions as Pathways for Pathogen Exchange Among Colony Members Compared to control-treated ants, which did not elicit social immunisation in their nestmates, fungus-exposed ants did not show significantly changed rates of either brood care behaviour [18] or self-grooming activity (LVU, unpublished data). Similarly, other studies found that pathogen exposure had no effect on self-grooming [26] or only when doses present in the colony were very high [25]. This makes it unlikely that nestmates may have perceived a trigger signal by social interaction or potential observation of the individual behaviour of exposed ants. To obtain information on possible pathways for transfer of the pathogen or immune mediators, we analysed the social interactions between colony members in more detail. As in our original experimental setup we grouped five naive nestmates with a single treated Lasius worker that had either received infectious M. anisopliae conidia (fungus treatment) or the same treatment without the pathogen (sham control). We observed three types of social interactions between group members. Antennation behaviour—that is, nestmate recognition behaviour by antennal contact [43]—occurred extremely rarely (6.6% of all interactions). Moreover, rates did not differ between treated and nestmate ants or among nestmates, for both fungus treatment and sham control (Generalised Linear Model [GLM] with negative binomial errors, LR χ2 = 1.969, df = 3, p = 0.579; data not shown). All other social interactions observed between group members consisted of (a) allogrooming (i.e., cleaning the body surface of another ant) and (b) trophallaxis behaviour (i.e., exchange of regurgitated liquid food droplets) [43]. Both may be important pathways for social immunisation [3],[16],[17],[27]. It is well known that nestmates actively contact exposed individuals and remove infectious material with their mouth by allogrooming, which is a very efficient social sanitary behaviour [43],[44] increasing survival of pathogen-exposed individuals, but typically not compromising the survival of the nestmates [25],[35],[45],[46]. Still, the grooming ant may contract the pathogen if it is not able to kill all infectious material in its mouth (infrabuccal pockets; [47],[48]) or gut [49], or if it unintentionally rubs off conidia with other body parts than the mouth during this intimate social interaction. In addition, allogrooming may lead to uptake of antimicrobial substances from the body surface of an exposed individual similar to exchanges of cuticular waxes important for nestmate recognition [50]. In our experiment, allogrooming rates between treated individuals and their nestmates were higher than among nestmates, but independent of the treatment type (fungus versus sham control; Figure 2A; GLM with negative binomial errors, LR χ2 = 15.134, df = 3, p = 0.002; ant pairing [treated-nestmate versus nestmate-nestmate]: Wald χ2 = 14.501, df = 1, p<0.001; treatment type [fungus versus sham control]: Wald χ2 = 0.006, df = 1, p = 0.939). Upregulation of grooming frequency not only against individuals treated with infectious material but also with sham control solutions is known from previous studies [29],[51] and indicates that ants are very sensitive to applications on the bodies of their group members. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g002 Despite the lack of difference between the two treatment types, intensive grooming towards treated individuals provides a potential route for transfer of either the pathogen itself or external immune effectors. One important factor is the timing of allogrooming expression during the infection course of M. anisopliae. Entomopathogenic fungi like M. anisopliae infect their hosts by external adhesion onto and active penetration of the cuticle [52]. After contact to the insect cuticle, the conidia first adhere loosely to the body surface within several hours and then germinate and form a penetration plug to actively enter the host body within approximately 24 to 48 h [46],[53]. Infection of the host and onset of an active immune response therefore occurs with a time delay of 2 to 3 d after exposure [54],[55]. Allogrooming in the first 1 to 2 d would therefore allow for pathogen transfer, whereas after this time exposed ants lose their infectiousness [26]. Intensified allogrooming 3 or 4 d after exposure would instead indicate exchange of external antimicrobial substances. We analysed the time course of allogrooming frequency between treated individuals and their nestmates and found no change over time in the control treatment (GLM with repeated measures, time: F = 0.973, df Huynh-Feldt = 3.648, p = 0.416). Allogrooming between nestmates and fungus-exposed individuals, however, was significantly higher in the first 2 d compared to later phases of the experiment (Figure 2B; time: F = 4.006, df Huynh-Feldt = 3.306, p = 0.006 [day1 versus day2: p = 0.178; day1 versus day3: p = 0.041; day1 versus day4: p = 0.001; day1 versus day5: p = 0.014]). Based on these data we suggest that if a transfer between group members occurs via allogrooming, it more likely involves a transfer of conidia, detachable early after exposure, than immune effectors, which can only be upregulated and transferred to the cuticle after infection of the individual 24–48 h after exposure. Social feeding via regurgitation and transfer of a trophallactic droplet may promote transfer of internal antimicrobial substances [16]. However, we found no differences in the rates of trophallaxis among all four groups, that is, neither between treated ants and their nestmates nor among the nestmates in either the fungus treatment or the control group (Figure 2C; GLM with negative binomial errors, LR χ2 = 2.555, df = 3, p = 0.465). Our data show that fungal exposure does not alter trophallaxis rates between exposed individuals and their nestmates, making passive immunisation by transfer of internally produced antimicrobial substances rather unlikely in our model system. Our findings after fungal exposure contrast with observations that trophallaxis rates between individuals injected with dead bacteria or bacterial cell wall components (but also wounding controls) were increased compared to trophallaxis rates among untreated individuals ([16],[56], but see [57]). Taken together, our behavioural observations strongly suggest exchange of the fungal pathogen between the fungus-exposed ant and its nestmates during intensified, early grooming as the most likely mechanism for the observed anti-fungal protection in the nestmates. We therefore determined if fungal conidia indeed were transferred from the exposed individual to its untreated nestmates by direct tracing of fluorescently labelled conidia.

Pathogen Transfer to Nestmates Occurs After Social Contact to an Exposed Ant We applied conidia of M. anisopliae labelled with red fluorescent protein (RFP) onto the exposed ant and determined their presence or absence on the cuticle of all group members after 2 d of social contact. We expected maximum pathogen transfer to have occurred at this time as (a) grooming activity between exposed ants and their nestmates is most intense in the first 30 h (Figure 2B) and (b) conidia are no longer transferable after this time [26],[53]. As expected we found high amounts of conidia on all directly exposed individuals (15/15) and furthermore detected low numbers of conidia on the cuticles of 37% (17/45) of nestmates (Figure S1; for negative controls see Materials and Methods). Interestingly, not only the quantity but also the location of conidia differed: whereas directly exposed individuals carried them mostly in areas likely difficult to reach by grooming such as joints and the antennal grooves, conidia on nestmates were rather attached to antennae and legs (Figure S1), suggesting that nestmates pick up the pathogen from the fungus-exposed individual during grooming. We can thus confirm pathogen transfer to the nestmates. In a next step we determined if the transferred conidia successfully established an infection in the nestmates.

Fungus Transfer Leads to Sublethal Low-Level Infections in Nestmates To quantitatively determine the infection load of directly fungus-exposed individuals and their nestmates over the course of the experiment, we sterilised their body surface to destroy all remaining conidia, dissected the ants, and plated their body contents on agar plates to count emerging fungal colony forming units (CFUs). We used morphological determination, as well as PCR [58], to confirm that outgrowing CFUs were indeed M. anisopliae, which was the case for all CFUs (see Figure S2 as an example). None of the 30 negative controls (see Materials and Methods) and none of the individuals measured within 24 h after exposure (0/10 fungus-treated, 0/14 nestmates; Figure S3) showed fungal growth, confirming that we effectively sterilised the ants and measured only live fungus from inside the body. Three as well as five days after exposure, CFUs grew from the body content of nearly all directly exposed ants (80% [8/10] and 90% [9/10]) and a similarly high number of nestmates (64% and 64% [each 9/14]; Figures 3, S3; Fisher's exact test; day 3, p = 0.653; day 5, p = 0.341). These data show that fungal infections in nestmates were more common than estimated from external pathogen transfer using labelled conidia. This may either indicate that we did not detect all conidia or that an additional infection route via the infrabuccal pocket in the mouth or the gut system occurred, for instance if groomed-off conidia were not completely prevented from germinating [47]–[49]. Fungal infection load in nestmates revealed that their infections were “low-level infections.” The number of CFUs growing out of their bodies when infected was significantly lower than those growing from directly exposed ants at both day 3 (Figures 3A, S3; Mann-Whitney U-test: n 1 = 8, n 2 = 9, U = 4.0, p = 0.002) and day 5 (Figures 3B, S3: n 1 = 9, n 2 = 9, U = 0.0, p<0.001). On average, the infection load of infected nestmates was 8 (4.4 versus 36.0) and 12 (8.1 versus 102.4) times smaller than that of directly exposed individuals on days 3 or 5, respectively. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g003 Even if low-level infections occurred in the majority of nestmates, only 2% (3/150) died from a M. anisopliae infection after 5 d of social contact with the exposed individuals (who showed death rates of approximately 50% due to application of an LD 50 ). This confirms that the effects of M. anisopliae infections are highly dosage dependent ([35] and MKo and STr, unpublished data).

Low-Level Infections Are Sufficient to Explain the Increased Antifungal Activity of Nestmates To determine if the observed increase in antifungal activity of nestmates was a direct cause of these low-level infections, we established low-level infections in individuals in the absence of social interactions. To this end, we exposed isolated ants with a conidia dose that led to the same death rate (LD 2 ) and infection level as observed in the socially exposed nestmates. We found that low-dose, directly exposed ants had a significantly increased antifungal activity 3 d after exposure compared to control-treated ants (Figure 4). Interestingly, directly exposed individuals with a high dose (LD 50 ; as used for exposure of the single ants in our experiment above) showed a significantly decreased capacity to inhibit fungal growth (Figure 4; ANOVA: F = 10.361, df = 2, p<0.001; post hoc Protected Fisher's LSD tests all pairwise: sham control versus LD 2 : p = 0.046, sham control versus LD 50 : p = 0.021; LD 2 versus LD 50 : p<0.001). This immune-suppressive effect of a high-dose infection is likely caused by the immune-interference and toxicity of M. anisopliae or by the fact that the immune responses had been depleted [41],[59]–[61]. Immune stimulation of low-level infections has previously been described for both vertebrates and invertebrates [3],[8],[33],[34], and its protective effect yielded clinical application in humans [62],[63] and poultry health management [64]. PPT PowerPoint slide

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larger image TIFF original image Download: 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 LD 2 ; 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 LD 50 ; 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. https://doi.org/10.1371/journal.pbio.1001300.g004 We have established that low-level infections, caused by social contact or direct low-dose exposure, lead to increased antifungal activity. Yet this does not exclude that nestmates with social contact to an exposed individual may also obtain signals that could actively trigger their antifungal immunity (similar to [31],[32]). To test this, we performed a “spatial-separation experiment” in which body contact and pathogen transfer to the exposed individual were prevented, whereas exchange of visual signals or volatile chemicals was still possible. The antifungal activity of nestmates of fungus-exposed individuals did not differ from that of nestmates of control-treated ants after 3 d of this constrained contact (t test: t = −0.376, df = 18, p = 0.711). These data suggest that a visual or volatile signal alone—at least one that acts over distance—is not sufficient to promote antifungal activity in the nestmates. Non-volatile chemical signals, such as cuticular hydrocarbons [65] that are part of the ants' cuticle, may in theory still play an additional role. However, their perception would always require body contact, which promotes pathogen transfer at the same time. We conclude that low-level infections alone provide a sufficient explanation for an active social immunisation of nestmates. We then tested if it may be complemented by a passive transfer of antimicrobial substances among nestmates.

Passive Transfer of Antimicrobial Substances Is Unlikely We performed a “temporal-separation experiment” and allowed the exposed ant to interact with its nestmates for 48 h. In this period, the pathogen (a) lost its ability to be transferred (for confirmation see Materials and Methods) and (b) established an infection in the ants, likely triggering an immune response [53]–[55]. After this time, we separated the treated individual and its “early nestmates” and added five “new nestmates” to both (see Figure 5A,B). Three days later, we measured the antifungal activity of the new nestmates. We found no difference between new nestmates of control-treated versus fungus-exposed ants (Figure 5A; t test: t = −0.159, df = 18, p = 0.876) or between new nestmates of early nestmates to a control-treated versus exposed individual (Figure 5B; t test: t = −1.273, df = 18, p = 0.219). This reveals that nestmates do not show an increase in antifungal activity if pathogen transfer is excluded. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g005 Passive transfer of antimicrobials among the group members thus seems very unlikely as an explanation for social immunisation. However, such transferable substances might be upregulated in infected individuals and simply failed to elicit immunisation of nestmates in our experiment. We therefore also analysed both the fungus-exposed ant and its nestmates directly for the presence of potentially transferable antimicrobials 3 d after treatment. Although allogrooming rates among nestmates were low in both sham control and fungus-treated groups (Figure 2A), and trophallaxis rates were completely independent of treatment (Figure 2C), infected nestmates may be important in transferring antimicrobial substances, as their antifungal activity is higher than that of directly exposed ants, which suffer a much higher infection level (Figure 4). We tested whether transferable substances of fungus-exposed individuals or their nestmates had higher antifungal activity than those of control-treated individuals and their respective nestmates. For externally transferable substances via allogrooming, we measured the antifungal activity of (a) the cuticle and (b) the thorax containing the metapleural gland content, which is known to have antimicrobial function and to be secreted onto the cuticle [36]. We also measured the antifungal activity of (c) the trophallactic droplet that is produced in the ant's body and is transferred via social feeding. We found that neither the cuticles nor the thoraxes containing the metapleural gland nor the trophallactic droplets of fungus-exposed individuals showed a different antifungal activity than the respective body parts of control-treated individuals (Figure 5C; t tests; cuticle: t = 1.064, df = 10, p = 0.312; thorax: t = 0.224, df = 10, p = 0.828; trophallactic droplets: t = −0.594, df = 18, p = 0.560). The same was true for the nestmates (Figure 5D; t tests; cuticle: t = 0.107, df = 18, p = 0.916; thorax: t = 0.894, df = 18, p = 0.383; trophallactic droplets: t = −0.717, df = 18, p = 0.482). This result was not an artifact caused by a potential effect of the control treatment, as the antifungal activity in these individuals was not different from completely untreated ants (Materials and Methods). Taken together, we found no evidence for (a) a potential protective effect of nestmates in the absence of pathogen transfer and (b) potential upregulation of socially transferable antimicrobials in exposed colonies. This contrasts observations that trophallactic droplets obtained from bacteria-exposed ants had higher antibacterial activity than that of controls [16], making passive immunisation a likely mechanism involved in social immunisation of ant colonies after bacterial exposure [16], but not after fungal exposure. Instead, we documented that social interaction, most likely allogrooming, leads to pathogen transfer and sublethal low-level infections in the majority of nestmates of fungus-exposed individuals and that low-level infections are necessary and sufficient to induce an increased antifungal activity.

Nestmates Show Active Upregulation of Immune Genes Specific for Antifungal Defence To directly assess the effect of low-level infections on the immune response, we measured immune gene expression in nestmates using quantitative real-time PCR. We chose three immune genes known to be involved in the humoral and cellular defences of ants: (1) the antimicrobial peptide (AMP) defensin [66],[67], a soluble mediator that most closely resembles termicin, an antifungal peptide in termites [68],[69]; (2) prophenoloxidase (PPO), a key mediator of immune function in ants [70],[71] that is essential for the process of melanization upon infection by a variety of pathogens, including entomopathogenic fungi [72],[73]; and (3) cathepsin L, a lysosomal protease expressed in hemocytes [74], which has both antibacterial [75] and antiviral activity [76], but has not been implicated in antifungal responses. In Camponotus pennsylvanicus, another cathepsin (cathepsin D) was found to occur in higher amounts in the trophallactic droplets of ants after injection of heat-killed bacteria or LPS [16], suggesting the involvement of cathepsins in antibacterial responses in ants. We confirmed that our host ant, L. neglectus, also responds to bacterial infection with cathepsin upregulation. Septic injury with Bacillus thuringiensis led to upregulation of cathepsin L gene expression, but not PPO, or defensin expression, compared to pricked controls (Figure S4; defensin: t test; t = 0.186, df = 4, p = 0.862; PPO: t test; t = −1.448, df = 4, p = 0.221; cathepsin L: t test; t = −3.695, df = 4, p = 0.021; gene expression standardised to the housekeeping gene 18s rRNA). The choice of these three immune genes in this study therefore allowed us to examine the specific effects of social immunisation against the fungus M. anisopliae on immune pathways involved in insect defences. We compared mRNA levels of the three genes in nestmates of fungus-exposed individuals versus nestmates of control-treated individuals on day 3—that is, the first day that we observed an increase in their antifungal activity (Figure 1). After normalising to a housekeeping gene (18s rRNA), elevated expression was observed in nestmates of fungus-exposed individuals relative to nestmates of control-treated individuals for both defensin and PPO (Figure 6; defensin: Welch's t test; Welch t = −2.348, df = 26, p = 0.032; PPO: t test; t = −2.923, df = 26, p = 0.007), whereas cathepsin L showed no difference (t test; t = −0.094, df = 26, p = 0.926). This reveals an active upregulation of immune gene expression in nestmates of fungus-exposed ants and suggests the induction of a specific immune response distinct from immune responses to bacteria (Figure S4; [16]). Similar specific immune upregulation after fungal infection is known to occur in Drosophila [77]. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g006 To determine if the observed specificity in our candidate gene approach, which is limited to a small set of genes, reflects specificity at the functional level, we tested the nestmates' capacity to inhibit growth of the bacterium Arthrobacter globiformis in an “antibacterial activity assay.” We found that nestmates exhibited similar antibacterial activity for fungus and control treatment (Figure 7; t test: t = −0.644, df = 18, p = 0.528), revealing that social immunisation after fungal exposure of the colony is specific and does not lead to a protective effect against bacteria. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g007

Effects of Active Immunisation Via Low-Level Infections on Colony-Level Epidemiology We developed an epidemiological model to explore the adaptive value and colony-level long-term effects of social immunisation. We compared the effect of active versus passive immunisation in our ant-fungus system by extending classical SIS and SIR (Susceptible-Infectious-Recovered/Removed) models, which describe the progress of epidemics over time using the simplification that the diversity in the population can be reduced to a few states. Possible states in SIR models include individuals susceptible to the disease outbreak (S), infectious individuals (I), and recovered or dead individuals (R; [78],[79]). We included an active or passive immunisation mechanism by constructing a SIRM (Susceptible-Infectious-Removed-iMmune) model, in which ants can take five different states. Healthy nestmates are defined as susceptible (S) individuals, pathogen-exposed individuals as infectious (I) ones, and individuals dying from the disease are removed (R) from the model. Successful immunisation (by active or passive immunisation) leads to initially immune (M i ) individuals that may persist to create late-stage immune individuals (M l ; Figure 8). We describe the mean number of ants in each state by ordinary differential equations (ODEs; for details, see Text S2). We have thereby chosen a simple approach focusing on the comparison of active versus passive immunisation, but not taking into account spatial effects on epidemiology in societies that have been modelled elsewhere by cellular automata [27],[80],[81] or pair-wise approximations models [82]. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.1001300.g008 Ants can change their state by social interactions with each other and depending on their infection state (Figure 8A,B). Allogrooming reduces the fungus load of infectious (I), changing them to susceptible (S), but at the same time can increase the fungus load of the susceptible individuals (S), changing them to infectious (I). Active immunisation can occur when individuals receive a low-level infection and actively build up immunity, changing from infectious (I) to immune (M i ) with a given active immunisation rate. Under passive immunisation, susceptible (S) individuals change directly to the immune state (M i ) with a passive immunisation rate when receiving antimicrobial substances from infectious (I) individuals. Under the active immunisation scenario, initially immune ants (M i ) may then either die (R) if infection levels are too high and lead to the disease or enter into the later stage of immunity (M l ). Under passive immunisation, all initially immunised individuals become late-stage immune. Late-stage immune ants (M l ) can then lose their immunisation and become susceptible individuals (S; see Figure 8A,B and Text S2). Each transition is governed by a transition rate, which in total were fixed to similar ranges in order to allow easy model comparison. The following qualitative results did not depend on the precise rate values, so that we report only representative outcomes of our simulations in Figure 8C,D. We found that more individuals typically reach the immune state (M i , and turn into M l ) after passive immunisation (Figure 8C), as a single infectious individual may immunise multiple susceptible nestmates, whereas actively immunised ants need to first be in the infectious state themselves. Yet we found that infections die out (I becomes 0) more quickly under active immunisation (Figure 8D), leaving only a very small reservoir for individuals to become immunised. Moreover, active immunisation leads to a lower number of dead individuals (R). This is despite the fact that contraction of disease through pathogen transfer can only occur in the active route (with a risk of dying similar to our experimental outcome). Increasing this risk leads to higher death rates and lower immunisation in a linear relationship (simulations not shown). Taken together, active immunisation via pathogen transfer seems beneficial, as it allows more rapid disease elimination and produces lower death rates in colonies, except if the pathogen requires only a very low exposure dose to establish lethal infections in its host.