What does not kill you makes you peaceful: non-lethal fungal infection could reduce aggression towards strangers in ants

doi:10.21203/rs.3.rs-1305759/v1

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What does not kill you makes you peaceful: non-lethal fungal infection could reduce aggression towards strangers in ants

https://doi.org/10.21203/rs.3.rs-1305759/v1

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Many parasites interfere with the behaviour of their hosts. In social animals, such as ants, parasitic interference can cause changes on the level of the individual and also on the level of the society. The ant-parasitic fungus Rickia wasmannii influences the behaviour of Myrmica ants by expanding the host’s nestmate recognition template, thereby increasing the chance of the colony accepting infected non-nestmates. Infected ants consistently show an increase of the alkane tricosane (n-C23) in their cuticular hydrocarbon profiles. Although experimental application of single compounds often elicits aggression towards manipulated ants, we hypothesized that the increase of n-C23 might underlie the facilitated acceptance of infected non-nestmates. To test this, we mimicked fungal infection in M. scabrinodis by applying synthetic n-C23 to fresh ant corpses and observing the reaction of infected and uninfected workers to control and manipulated corpses. Infected ants appeared to be more peaceful towards infected but not uninfected non-nestmates. Adding n-C23 to uninfected corpses resulted in reduced aggression by uninfected ants. This supports the hypothesis that n-C23 acts as a “pacifying” signal. Parasitic interference with the nestmate discrimination of host ants might eventually change colony structure by increasing genetic heterogeneity and thus might significantly affect social evolution.

cuticular hydrocarbons

discrimination

fungal infection

manipulation

Myrmica scabrinodis

Rickia wasmannii

Parasites often affect the behaviour, morphology and/or physiology of their hosts in various degrees [1–6]. Parasite-induced modifications are generally quite well-defined in the case of individuals [2, 7–8]. In social organisms, though, substantial changes can also occur on the level of the society but are less obvious unless the social context is considered [9]. For example, such modifications might impair the capability of social animals to discriminate between related and unrelated individuals [10]. Sociality is typically founded on this capability to prevent non-relatives from benefiting from within-group altruistic actions without reciprocating [11].

In social insects, the main recognition cues and signals are chemical substances [12–14], although other signalling modes may also be of importance (vibroacoustic: e.g., [12, 15–16]; visual: [17–18]; tactile: [19]). The discrimination between strangers (both con- and allospecific) and nestmates is largely based on complex mixtures of long-chain cuticular hydrocarbons (CHC), which cover the surface of individuals. Cuticular profiles can consist of more than 100 different hydrocarbons, mostly linear alkanes, methyl-branched alkanes, and alkenes [20]. Both qualitative and quantitative differences in these profiles are used by social insects to recognize outsiders [21–24]. According to the ‘Gestalt’ model, the chemical profiles of the nestmates contribute to the formation of a colony-specific odour [25] and recognition signals are usually exchanged and homogenised among colony members via passive contact, allogrooming, or trophallaxis, but each individual retains its own chemical profile. Besides colony identity, this profile may encode additional information such as age, task, and sex [26–31]. It serves as a template during the recognition process and helps social insects to prevent potential freeloaders and parasites from entering their colonies [32].

Many parasites evolved mechanisms to infiltrate insect societies, including passive or active mimicry of host CHC profiles [33–34]. For example, larvae of Maculinea butterflies are known to synthesize CHCs specific to their host ants [35–36], while queens of the slave-making genus Polyergus almost instantly acquire CHCs through physical contact with their hosts [37]. Several fungi, viruses, mites, or tapeworms may elicit changes in the CHC profiles of their hosts within a few hours after infection [38–42]. Usually, these changes are restricted to a few infected individuals.

In contrast, the ectoparasitic, non-lethal laboulbenian fungus Rickia wasmannii can infect the entire colony without killing it [43–44] but changing the phenotype of infected individuals [43, 45–48]. Besides behavioural and morphological alterations, R. wasmannii interferes with the CHC profile of its Myrmica scabrinodis host ants [46]. It increases the variation of CHC profiles among infected colony members and opens the infected society towards strangers, such as infected non-nestmates, foreign queens, and parasitic Maculinea larvae. This causes substantial modifications on the social level, as revealed by our previous study [46]. In addition to the higher variability in their CHC-profiles, infected workers show a consistent increase in the relative abundance of two linear alkanes, n-C23 and n-C24. While n-C24 represents only 1–2% of the overall proportion of cuticular hydrocarbons, n-C23 contributes about 14% in uninfected workers and 19% in infected ones, suggesting its potential importance in shaping behaviour [46].

Previous studies in social Hymenoptera have shown that adding single components to the CHC profile elicits higher aggression [49–52]. In contrast, Myrmica ants reacted less aggressively to ants infected with R. wasmannii, despite the increased percentage of n-C23, making it a strong candidate for a pacifying compound. Here, we examine by experimental manipulation of the CHC profiles whether (a) n-C23 makes the infected hosts more amiable, toning down its aggression towards others, or (b) it pacifies the opponent towards the bearer, irrespective of the opponent’s health status.

Study species and site

Colonies of the ant Myrmica scabrinodis, both infected (I, N = 3) and uninfected (U, N = 3), were collected from the same population near Cluj-Napoca, Romania (46.92N, 23.73E, 410–460 m a.s.l.) as in the previous study [46]. Experimental colonies of M. scabrinodis were kept in plastic boxes (16cm × 10cm × 5cm) with wet foam bricks under controlled lab conditions (20°C, 12 L:12D cycles) with a food mixture of sugar and proteins [53] provided daily. Infected colonies were parasitized by the ectoparasitic fungus R. wasmannii, which obligatorily exploits Myrmica ants [54–56]. This fungus does not have a mycelium, and its thallus develops from a bicellular ascospore attached to the outer layer of the host cuticle [47, 57]. In our study population, the colony-level prevalence of the fungus was more than 50%, while the within-colony prevalence reached 100% in certain colonies [44]. The species identity of ants and their fungal infection status was assessed in the field and later confirmed in the lab using an Olympus SZ51 stereomicroscope.

Experiments

Dyadic behavioural tests were performed after four days of acclimatisation under laboratory conditions with worker ants from infected and uninfected colonies (figure 1). Since in dyadic tests with live workers it is hard to tell apart the reactions of the two individuals, we used one live worker and one freshly killed non-nestmate conspecific corpse (dummy) in each assay. This allowed us to precisely characterize the response of the live worker. Fresh dummies, obtained by freeze-killing workers (-20°C for 1 hr), are still perceived by ants as live ones due to the integrity of their CHC-profile and the low amounts of linoleic and oleic acid. These two acids are used by the ants to detect dead colony members, but they reach the threshold levels for corpse removal only a few hours after an individual’s death [58]. In each test, new workers and dummies were used.

To prove the role of CHC-profile in nestmate/non-nestmate discrimination in M. scabrinodis, we initially tested (1) the response of ants to hexane-washed dummies (see below for further details; N = 36). Then we performed unmanipulated assays (2) in which the reactions of live workers were tested to infected (I) and uninfected (U) dummies (N = 54) to assess the baseline aggression towards non-nestmates for both cases (figure 1).

In the next step (3), uninfected washed dummies were used for the application of extracts in order to exclude any tactile and visual cues that might arise from the presence of fungal thalli. We manipulated hexane-washed dummies obtained from workers from uninfected colonies by applying cuticular extracts from uninfected or infected workers (see below for further details) to them, and then we recorded the behavioural responses of live workers (N = 90; figure 1). Thus, we could reveal whether adding CHC-extracts to hexane-washed dummies elicits responses from workers matching responses to untreated dummies of similar infection status.

To elucidate the potential role of n-C23 (4), we recorded the reaction of workers towards uninfected dummies treated with synthetic n-C23 (N = 45; figure 1). Finally (5), we also tested the reactions of workers to hexane-washed dummies onto which we had applied cuticular extracts from uninfected workers supplemented with synthetic n-C23 (N = 45; figure 1).

a) Behavioural assays

Only workers older than 30 days were used since they represent the foraging workforce having an increased chance of meeting rivals and potential intruders and constitute the first line of colony defence. Therefore, their nestmate discrimination ability should be more pronounced than those of younger nurse workers. Old workers were selected based on their location in the nest arena and their darker cuticular pigmentation (brown), as traditionally used for age class estimation in Myrmica [69–60].

The assays were carried out using two connected transparent plastic tubes (3 cm long), which were initially separated by a small piece of red plastic foil. A single M. scabrinodis worker was placed in one tube, while the dummy was placed in the other tube. The worker was allowed to acclimatise for one minute before the red plastic foil was removed. After each assay, the plastic tubes were rinsed with ethanol (98%) and water, then wiped out and left to dry for at least 30 minutes. Assays were carried out with both infected and uninfected workers with three replicates per combination, and new individuals were used for each test. The observations started after the first contact of the live individual with the presented dummy and ended after three minutes. All interactions were recorded and categorised as: (1) grooming, (2) antennation, (3) carrying, (4) fleeing/retreating, (5) mandible gaping, (6) biting, (7) dragging, and (8) stinging. Grooming was considered a socio-positive behaviour, antennation and carrying were considered neutral, and the last five behaviours were considered aggressive. An aggression index (AI) for each encounter was calculated as follows: AI = the total number of aggressive responses divided by the total number of interactions [46, 61]. To eliminate or to reduce observational biases, the observer did not know the infection status of the alive worker.

b) Preparation of hexane-washed dummies

Only uninfected ants from uninfected colonies were used to prepare washed dummies used in behavioural assays. After thawing the freeze-killed corpses for 20 min at room temperature, they were placed in clean glass vials and submerged in 300 µl of hexane and manually shaken. After 5 minutes the ants were transferred to new clean glass vials and washed again with 300 µl of hexane. This operation was repeated 5 times. The hexane residues were left to evaporate for 10 minutes before the dummy was used for the behavioural assays.

c) Preparation of hexane-washed dummies supplemented with cuticular extracts

Both infected and uninfected workers were used as dummies. After freeze-killing them, two workers from the same colony were submerged in 200 µl of hexane and manually shaken. The extracts were transferred to clean glass inserts (0.2 ml) after 20 min. The hexane was evaporated under a stream of nitrogen and then the content of each glass insert was resuspended in 15 µl of hexane by rinsing the walls of the insert repeatedly using a glass syringe (25 µl, Hamilton, Bonaduz, Switzerland). The resuspended cuticular extract was then taken with another glass syringe (10 µl, Hamilton, Bonaduz, Switzerland). The tip of the syringe needle was then carefully attached to a hexane-washed dummy and extract was slowly applied to it, forming a small droplet uniformly covering the dummy. The hexane was allowed to evaporate for 10 minutes before the dummy was used for the behavioural assays.

d) Preparation of uninfected ant dummies supplemented with n-C23

Only uninfected workers were used as dummies. We prepared a hexane solution of n-C23 (8 ng/µl, Supelco, Bellefonte, Pennsylvania), and 10 µl (representing a dose of 80 ng) were then taken with a glass syringe (10 µl, Hamilton, Bonaduz, Switzerland). The tip of the syringe needle was then carefully attached to the uninfected corpse, and the solution was slowly applied to the dummy forming a small droplet uniformly covering it. The hexane was allowed to evaporate for 10 min before the behavioural assays. The dose of n-C23 applied to uninfected dummies (80 ng) was adjusted to shift its relative abundance to the levels found on the cuticle of ants infected by R. wasmannii (18.72 ± 5.25%; [46]).

e) Hexane-washed corpses treated with cuticular extracts from uninfected ants and n-C23

Hexane-washed uninfected corpses were treated with cuticular extracts from uninfected ants as described previously. After drying, they were treated additionally with 10 µl (representing 80 ng) of the n-C23 solution. The hexane was allowed to evaporate for 10 min under a stream of nitrogen before the behavioural assays.

f) Posterior verifications of CHC-profiles

Cuticular extracts from ant corpses that had been washed five times with hexane (dummies) were analysed to verify that the treatment had successfully removed most of the CHC profile. We also analysed the relative abundance of n-C23 in the CHC profile of uninfected workers treated with n-C23 to check whether it was similar to the levels found in infected ants. We individually extracted the CHC-profile of dummies (N = 4) and treated ants (N = 6) for 20 min in 200 µl of hexane. Subsequently, the extract was transferred to a clean glass insert (0.2 ml) and 8 µl of a hexane solution of n-eicosane (n-C20, Supelco, Bellefonte, Pennsylvania) (100 ng/µl, representing 800 ng) were added to each extract as an internal standard. Samples were then carefully dried under a stream of nitrogen before being re-suspended in a final volume of 20 µl of hexane. Two µl of each sample were analysed on a Shimadzu GC2010 gas chromatograph (GC) connected to a QP2010 plus mass spectrometer (MS; Shimadzu, Duisburg, Germany). The GC was equipped with a non-polar capillary column (BPX-5, 30 m length, 0.25 mm inner diameter, 0.25 µm film thickness; SGE Analytical Sciences, Milton Keynes, UK). Helium was used as carrier gas with a constant linear velocity of 50 cm s⁻¹. The initial oven temperature was 70°C and ramped at 30°C/min to 150°C. It was then increased to 320°C at a rate of 5°C/min and held for 10 min at 320°C. Splitless injection (2 mins) was performed at 280°C. Mass spectra were acquired in full scan mode every 0.3 s with a scan lapse of 0.1 s, the mass was m/z 40 to 600. Mass spectra were acquired in EI mode at 70 eV. The chromatograms were manually integrated to calculate the area of each peak of interest using the proportion of the sum over the area of all peaks.

Statistical analysis

Aggression indices were analysed using a generalised linear model approach (GLM, binomial error, maximum likelihood fit) with test combinations (I–I, I–U, U–I and U–U) as fixed factors for the behavioural assays. Relative abundances of n-C23 present on the cuticle of infected and uninfected old workers and experimental ants treated with n-C23 were compared using a linear model approach (LM, maximum likelihood fit). Chemical data of infected and uninfected old workers are derived from Csata et al. [46].

All statistics were performed using R 4.0.2 [62]. GLM was performed using glm functions in the lme4 package [63]. Tukey’s HSD test was used to calculate post-hoc comparisons on each factor in the model using the function glht from the R package multcomp [64]. The graphs were produced using the ggplot2 R package [65].

Behavioural assays

a) Baseline aggression

Neither uninfected nor infected M. scabrinodis workers showed any aggression towards dummies with removed CHC. This confirmed the role of CHC-profiles in the discrimination process and demonstrated the suitability of the applied CHC-removal procedure in producing ‘odourless’ dummies for further experimental manipulations.

The application of CHC-extracts originating from either infected or uninfected workers to CHC-removed dummies resulted in responses by alive individuals comparable to corresponding unmanipulated assays, confirming the accuracy of the CHC-transfer method (figure 2): U–I untreated vs U–I extract GLM z = 0.6, p = 0.54 (figure 2b,e); U–U untreated vs U–U extract GLM z = -0.9, p = 0.35 (figure 2a,d); I–I untreated vs I–I extract GLM z = -1.96, p = 0.051 (figure 2h,k); I–U untreated vs I–U extract GLM z = -1.1, p = 0.27 (figure 2g,j).

In unmanipulated assays, the level of aggression was significantly lower when both ants were infected compared to uninfected counterparts (I–I untreated vs U–U untreated GLM z = 3.7, p = 0.0002; figure 2a,h). Generally, infected ants were less aggressive towards infected corpses than healthy workers (GLM z = 2.66, p = 0.007; figure 2b,h), but such differences were not observed when it came to uninfected corpses (GLM z = 1.93, p = 0.06; figure 2a,g). Infected ants also treated infected corpses less aggressively compared to uninfected ones (GLM z = 2.34, p = 0.02; figure 2g,h), while healthy workers did not differentiate between cadavers of different health status (GLM z = 1.39, p = 0.16; figure 2a,b).

b) Manipulation of the CHC-profiles

The aggression of infected workers towards dummies was significantly lower towards dummies supplemented with cuticular extract from infected individuals in comparison to assays with uninfected counterparts (GLM U-U extract vs I-I extract GLM z = 2.94, p = 0.003; figure 2d,k). Contrary to the results of the unmanipulated assays, uninfected ants this time responded with a significantly reduced aggression towards hexane-washed dummies treated with cuticular extract from infected individuals than to uninfected dummies (GLM U–I extract vs U–U extract GLM z = 2.49, p = 0.01; figure 2d,e). No significant differences were detected in any other comparisons: ( I–I extract vs I–U extract; I–U extract vs U–U extract; I–I extract vs U–I extract; see Supplementary results, Table S1 for the exact statistical values).

The addition of n-C23 to uninfected dummies, which simulates the effect of the parasitic fungus on the CHC profile of infected ants (see figure 1.4), resulted in striking changes in the outcome of encounters involving uninfected workers. The level of aggression displayed by healthy ants decreased significantly (GLM U–U+n-C23 vs U–U z = 3.27, p = 0.001; figure 2a,c). In contrast, no differences were observed between the assays when the live counterpart was already infected (GLM I-U vs I–U+n-C23 z = 1.76, p = 0.07; figure 2g,i). Similar results were obtained when instead of untreated dummies, CHC-stripped dummies were used that were treated with CHC-extracts from untreated ants and additional n-C23 (see figure 2).

The supplementation with n-C23 of hexane washed dummies with CHC extract from healthy ants significantly changed the outcome of interactions both in uninfected and infected ants: GLM U–U extract vs U–U extract + n-C23 z = -3.87, p = 0.001 (figure 2d,f); I–U extract vs I–U extract + n-C23 GLM z = 2.13, p = 0.03 (figure 2j,l).

c) Chemical analyses of treatment efficiency

Control extracts from dummies that had been washed five times with hexane contained only traces of four to five CHCs out of 38 being present in non-washed workers (figure S1). These residues were from those compounds with the highest abundance in non-washed ants [46]. The abundance of these major compounds, however, was 10-15 times lower than in the unwashed ants. Therefore, we concluded that the applied method was suitable for obtaining dummies without relevant amounts of cuticular compounds.

The relative abundance of n-C23 in uninfected ants treated with the synthetic alkane was on average 22.4 ± 6.0% (mean ± SD) and did not differ from the relative abundance of n-C23 in untreated infected ants (LM t = 1.87, p = 0.11), thus confirming that the topical application of n-C23 on uninfected ant corpses was successful to achieve the relative abundance of this linear alkane present on infected ants. In untreated, uninfected ants, the relative abundance of n-C23 was only 13.8 ± 1.3% and differed significantly from the relative abundance present on uninfected n-C23-treated ants (LM t = 4.46, p < 0.001).

The current study reveals in social insects, for the first time the ‘pacifying’ role of a simple component of the cuticular hydrocarbon profile linked to a fungal pathogen. Appeasement effect of certain compounds (e.g., crematoenones) has already been reported in ants [66], however it involves two mutualistic ant species that regularly share a common nest. In our case it is the parasite that induces the behavioural changes through modification of the CHC profile.

Our first hypothesis according to which n-C23 supplemented (infected) individuals become peaceful towards everyone can be dismissed, since the almost complete lack of aggression occurred towards infected counterparts. However, the surplus of n-C23 does seem to elicit less aggression from the interacting partner, thus the second hypothesis is to be confirmed. The increased variation of CHC profile in infected colonies [46] makes infected colonies in general more tolerant towards strangers than uninfected ones. These all can contribute to a higher degree of heterogeneity of infected colonies as already addressed by Csata et al. [46] which could be advantageous for the fungal parasite. There is a fair chance that R. wasmannii tapped into its host nestmate recognition system tampering with a relatively simple security code. There is also a chance that n-C23 could have a wider effect in the genus Myrmica since R. wasmannii is present in many species belonging to this genus. Whether the effect of the compound goes beyond this parasite-host system needs to be addressed in further studies.

In insect societies, recognition of relatives and intruders is crucially important since altruistic actions need to be directed towards relatives. Some hydrocarbon classes seem to be more informative than others for nestmate recognition. In particular, methyl-branched alkanes and linear alkenes seem to provide more information than linear alkanes (e.g., 49,67-68], which are thought to be structurally more effective in preventing dehydration [69–70]. The role of alkanes is currently ambiguous and appears to differ between species. In honeybees (Apis mellifera), they do not seem to have any effect [71–72]. However, in the ants Formica japonica [73] and Linepithema humile [74], workers were unable to distinguish between non-nestmates and nestmates when only the respective alkane or alkene fraction of the cuticular profile was presented to them. When the chemical profiles of L. humile nestmates were supplemented with linear alkanes (from n-C23 to n-C30 and n-C33), the tested workers responded aggressively. A similar behaviour occurred in the workers of the European hornet, Vespa crabro [51].

In the light of the examples given above, the results of the present study are highly remarkable, as they show that n-C23 represents a ‘pacification’ cue that, when exceeding normal levels, attenuates aggressive responses in conspecifics. The mentioned changes and effect on the nestmate recognition system may also have an impact on the spread of pathogens through increased tolerance towards non-nestmates and young queens that seek to infiltrate in established colonies [46]. The reduced discrimination accuracy of infected ants might also compromise their ability to detect and remove other social parasites. For instance, a high degree of chemical mimicry has been shown recently in parasitic queens of the workerless inquiline species Myrmica karavajevi, which parasites M. scabrinodis colonies, a high degree of chemical mimicry of parasitic queens towards the host queens has been recently shown [75]. Interestingly, high percentages of n-C23 were detected in the CHC profiles of queens and newly eclosed males of M. karavajevi which are almost twice the level found in the CHC profiles of host queens. This high relative abundance of n-C23 might thus facilitate the parasite usurpation of host colonies and the safe eclosion of parasite males by reducing the aggressiveness of host workers [75].

We conclude that the pathogen-related relative increase of n-C23 plays a crucial role in the nestmate vs non-nestmate discrimination accuracy in M. scabrinodis. The reduced aggressiveness caused by n-C23 coming along with a hampered ability to discriminate against non-related conspecifics might facilitate the propagation of R. wasmannii and might be exploited by social parasites and other pathogens to sneak into M. scabrinodis colonies. Our work paves the way for future studies investigating whether this phenomenon is conserved across other eusocial insect species and exploring the physiological mechanisms underlying the increased tolerance towards pathogen-infected conspecifics.

Acknowledgements

We are grateful for the assistance of Kriszta Kincső Keresztes, Márta Osváth-Ferencz and Zsolt Fazakas during field collections. The current work was partially supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-1930. E.C. was supported by a research grant of the DAAD and by a grant of the Alexander von Humboldt Foundation.

Competing interests

We declare no competing interests.

Author contributions

E.C., L.P.C., J.R., J.H., B.M. contributed to the design of the experiments. E.C. and B.M. collected the materials. E.C., L.P.C. and B.M. conducted the behavioural experiments. L.P.C., E.C. and J.R. prepared the solutions and extracted the CHC compounds. L.P.C. analysed the chromatograms and carried out the statistical analysis of the CHC-compounds. E.C. and A.B. performed the data analyses of behavioural assays. The manuscript was written by E.C., L.P.C., B.M., J.R., A.B., and J.H. Further on, all authors contributed to revisions of the manuscript.

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There is NO Competing Interest.

Csataatal2022CHCDATA.csv
Dataset 2
CsataetalSupplementaryresults.docx
Supplementary results
Csataetal2022BehaviouralDATA.csv
Dataset 1
CsataetalSupplementaryresults.docx

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published 16 Feb, 2023

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What does not kill you makes you peaceful: non-lethal fungal infection could reduce aggression towards strangers in ants

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Abstract

Figures

Introduction

Materials And Methods

Study species and site

Experiments

a) Behavioural assays

b) Preparation of hexane-washed dummies

c) Preparation of hexane-washed dummies supplemented with cuticular extracts

d) Preparation of uninfected ant dummies supplemented with n-C23

e) Hexane-washed corpses treated with cuticular extracts from uninfected ants and n-C23

f) Posterior verifications of CHC-profiles

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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