Coming out from the shadows: facultative slave-making ants reveal their chemical identity during colony development

doi:10.21203/rs.3.rs-7846516/v1

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Coming out from the shadows: facultative slave-making ants reveal their chemical identity during colony development

https://doi.org/10.21203/rs.3.rs-7846516/v1

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One of the main challenges of the socially parasitic mode of life is bypassing the host's recognition ability, which ensures that altruistic behaviour is directed towards related individuals. Various chemical strategies have evolved to achieve this goal. The most widespread, used also by the obligate slave-making ants, is camouflage or mimicry of colony odour encoded in cuticular hydrocarbon (CHC) composition. However, recent studies have shown that facultative slave-makers employ a different strategy: they manipulate the slaves' recognition labels to make them resemble the parasite's CHC profile. We examined the limitations of this strategy by focusing on incipient F. sanguinea colonies, where slaves are the majority. Our study revealed that callow F. sanguinea ants initially suppress their species-specific odour profile, which develops gradually over time accompanied by an increase of CHC amount per surface area in slave-maker workers. This allows the slaves to familiarise themselves with the parasite's CHC. We found that callow ants produce lower amounts of CHC, and the relative abundance of certain compounds differs from what is observed in older ants. Additionally, preimaginal stages of F. sanguinea ants acquire CHC from the slaves, which are later incorporated into the imagines’ recognition labels. These findings support the proposition that the parasite's manipulation strategy is limited by the slaves' learning capacity, which is necessary to maintain colony cohesion. They also shed light on the selective pressures that might have led to the evolution of chemical mimicry in mature obligate slave-maker colonies.

slave-making

ants

chemical ecology

social parasitism

The evolution and maintenance of eusociality can be explained by kin selection theory, which claims that altruistic behaviour can be promoted by natural selection if it benefits related individuals (Hamilton, 1964). This is facilitated by spatially segregated family groups, often formed through brood care or philopatry. However, resources shared by the collaborating groups of individuals are potential targets for competitors and parasites. Thus, maintaining group cohesion and evolving a eusocial lifestyle requires a recognition system to identify and reject intruders. In social insects, these recognition cues are primarily hydrocarbons, found on the insect's body surface (Bonavita-Cougourdan, 1987; Lahav et al., 1999; Dani et al., 2001; Dani et al., 2005). While the composition of these compounds varies between colonies, it is relatively similar across members of a colony, providing a unique colony identity for distinguishing nestmates from intruders.

Many studies highlight the role of genetic factors in the formation of cuticular hydrocarbon (CHC) profile (Beye et al., 1997, Beye et al., 1998, van Zweden et al., 2010). Consequently, intracolonial genetic variation generates recognition label variability, which is counterbalanced by the continuous exchange of recognition cues among colony members, leading to the formation of a uniform colony-specific CHC profile (Boulay et al., 2004, Soroker et al., 2004, Soroker and Hefetz, 2000). Moreover, environmental factors, such as diet or nest material, have also been shown to affect the recognition label (Le Moli et al., 1992, Liang and Silverman, 2000). Taken together, changes in colony demography and composition, along with an unstable environment, can explain the reported temporal variation in a colony recognition label (Vander Meer et al., 1989, Provost et al., 1993, Nielsen et al., 1999). Consequently, the nestmate recognition system should rely on adjustable discrimination criteria to account for this variation.

The colony recognition signature is suggested to be learned by newly eclosed social insects through the perception of cues borne by the nestmates (Morel 1983, Errard 1984, Errard and Jaisson 1991; Errard 1994) or present in the nest material (Pfennig et al. 1983, Singer and Espelie 1996). Its neural representation is often referred to as a template. In some species, there is also evidence for pre-imaginal colony odour learning (Isingrini et al. 1985, Signorotti et al. 2014). The template plasticity in the newly eclosed ants allows them to be manipulated to form artificial mixed colonies composed of individuals belonging to different species or even subfamilies (Fielde 1903, Errard et al. 2006). This trait is exploited by social parasites (Jaisson 1971). For example, queens of the inquiline Polistes wasps contaminate the usurped paper nest with own recognition cues, leading to their acceptance by newly hatched host workers which use the nest material as a source of reference odour based on which the template is then developed (Gamboa i in. 1986, Kaib et al. 1993, Lorenzi et al. 1996, Turillazzi et al. 2000). Another example is provided by slave-making ant species which kidnap the host pupae. The emerged workers integrate into a parasite society that benefits from their labour. However, the genetic heterogeneity of such a mixed society might lead to the greater variability in recognition cues, which could compromise the accuracy of the odour neural template. Accordingly, aggression between heterospecific slaves, accompanied by the odour differences, has been found in colonies of Harpegnathos sublaevis (Heinze et al. 1994). The role of template plasticity in slave-making ants is underlined by the Temnothorax parvulus ants, which, in contrast to T. unifasciatus, show a reduced tendency to learn colony odour based on experiences from early adulthood. As a consequence, only the latter species is enslaved by Myrmoxenus ravouxi (Blatrix and Sermage 2005).

The host's reduced capacity to update the recognition template could exert selective pressure on the parasite to maintain a relatively stable and homogeneous recognition odour within the colony. This might be one of the reasons why social parasites express chemical camouflage well after successful host colony usurpation (Yamaoka 1990, Bonavita-Courgourdan et al. 1997, Kaib et al. 1993, Guillem et al. 2014, Kleeberg et al. 2016). On the other hand, the facultative slave-making ants of the Formica sanguinea group exhibit a distinct species-specific odour and promote it by transferring their own recognition cues to the slaves. As a result, the recognition label of the slaves is partly masked by the compounds characteristic of parasite workers (Włodarczyk 2016, Włodarczyk and Szczepaniak 2017, Scheckel 2021). Although this strategy seems effective in mature colonies where slaves are the minority of colony members, it might become less effective if the proportion of slaves increases. Moreover, host workers might respond aggressively to the slave-maker’s odour if they have not been sufficiently familiarized with it, as indicated by the observations of individuals from free-living F. fusca colonies, which aggressively defend their nest area against F. sanguinea but are permissive towards a dominant competitor (Włodarczyk 2023). This behaviour suggests the existence of an adaptive ability to recognise parasite based on the inherited template of its phenotype. Being detected might be especially risky for F. sanguinea workers during the initial phase of colony development when the parasite is outnumbered by the host ants, which have no prior experience living with heterospecific individuals other than the parasite queen. The ability of the of slave-maker for the successful promotion of own recognition cues might then be limited by the plasticity of the host workers' template as well as their ability to recognise parasitic ant. Integration into an incipient colony can be challenging for a slave-maker also because of the odour homogeneity among slaves, which are close relatives. This leads to more accurate intruder discrimination compared to colonies in which the slave workforce is made up of a mixture of ants from different colonies (Torres and Tsutsui 2016, Włodarczyk 2016). Thus, by using an exceptional chemical strategy in mature colonies, F. sanguinea provides a unique opportunity to test the extent to which host template plasticity can be efficiently exploited by a social parasite. More specifically, we hypothesise that F. sanguinea ants use chemical mimicry, camouflage or insignificance at the early stage of colony development due to the risk of being rejected by the host ants. Alternatively, if the host template is plastic enough, no change in the chemical strategy would be expected throughout the life cycle of the slave-maker colony. In our study, we paid special attention to callow ants since they might be at greater risk of being rejected by nestmates compared to older individuals, which have already been engaged in social interactions with other colony members.

Study material

We established 16 F. sanguinea incipient colonies in the laboratory, which were maintained for between one and four years. The colonies were initiated with dealate queens collected in the field. Queens were spotted after mating as they were penetrated the forest litter in search of colony founding opportunities. They were collected during four consecutive seasons starting from 2017 in Solniczki and Turczyn Forests near Białystok and, in one case, near Sejny (northeastern Poland). Each queen was provided with 80 to 230 Formica fusca pupae removed from one of 18 laboratory colonies or, in the case of one queen, from a field colony. Colonies were maintained in the plastic boxes (40 × 30 × 30 cm) with the floor covered with a thin layer of mineral soil and sawdust. Test tubes wrapped in aluminium foil and partially filled with water, and closed with a cotton plug, served nesting sites for ants. The inner surface of the nest box walls was coated with Fluon to prevent ants from escaping. Colonies were fed diluted honey, fresh apple pieces, as well as crickets (Acheta domesticus), greater wax moth (Galleria mellonella) caterpillars, and male honey bee larvae and pupae all killed by freezing. Each of the developing F. sanguinea colonies was subjected to at least one census followed by the removal of 3–6 slave-maker workers (if present) and a similar number of F. fusca slaves. The timing of censuses was chosen to cover various stages of colony development spanning a range of slave-to-slave-maker ratios. Some of the samples included callow slave-maker workers, which were identified by the brighter coloration due to the incomplete cuticle pigmentation. After removal, ants were killed by freezing and stored at − 22 ºC for subsequent chemical analyses. A total of 78 mature F. fusca ants, 65 mature and 32 callow F. sanguinea ants were used for CHC profiling. The F. sanguinea colonies also served as a source of pupae for the separation experiments described below. Moreover, workers from 12 out of 19 free-living F. fusca colonies used as sources for slaves were also sampled for chemical analyses (five to eight ants per colony).

Separation of the callow workers

Since cuticular hydrocarbon (CHC) profiles of F. sanguinea callow workers from the parent colonies could have been modified by interactions with other colony members (see Ichinose & Lenoir 2009), pupae were removed from thirteen experimental colonies and incubated until they reached the adult stage. If present, cocoons were removed manually once the ants became motile, which was visible through the silk envelope after gentle pressing with an entomological pin.

After emerging from the pupae, ants were placed in plastic Petri dishes (9 cm in diameter) equipped with wet cotton to maintain humidity, left in darkness at a temperature between 21.5 and 24 ºC, and provided with diluted honey water twice a week. Ants were kept in pairs since pilot trials had shown that isolated individuals suffer high moralities. Due to the variation of pupa emergence time, there was a delay until the second ant was placed on the Petri dish (mean = 16.97 hours, maximum = 39.92 hours).

Each pair of ants was assigned to one of four treatments which differed in the period before ants were collected and killed by freezing. One of the ants was selected at random from the pair and was subsequently used for CHC extraction. For each colony, assignment of experimental units to treatments was randomized. If the number of available pupae was enough, treatment replicates per colony were performed (yielding an average of 1.82 experimental units per colony per treatment). In case an ant died precociously, the respective experimental unit was discarded.

Effect of the environmental cues on CHC production

We were interested in whether callow F. sanguinea ants respond to the presence of slaves in a colony by actively adjusting their own recognition label (chemical mimicry via biosynthesis, see Lenoir et al. 2001). We obtained callow workers by removing F. sanguinea pupae from developing colonies, and separating them in pairs for 12–15 days as described above. However, instead of using ants, we placed 3 mm glass beads in a Petri dish and stabilised them by putting them on plastic rings. Each glass bead was coated with CHC profiles equivalent to that found on 2–4 ants. As the glass beads served as “dummy ants”, this allowed us to eliminate the influence of social interactions with slaves on the CHC profile of the tested ants. Twelve F. fusca free-living colonies and the twelve F. sanguinea colonies with less than 10% slaves served as a source of ants used as CHC donors. For each colony, 24–48 ants were killed by freezing, pooled and extracted together for 10 minutes in hexane. The extract was enriched with 15 µg of docosane used as an internal standard, evaporated, and re-dissolved in 48 µl of hexane. Each glass bead was coated with 8 µl of the final extract with the use of a gas chromatography syringe. This allowed to apply small droplets on the glass beads when performed under a microscope. For the control treatment, clean glass beads were used. Twelve F. sanguinea colonies served as a source of worker pupae. Each of the three treatments (slave-makers' CHC, slaves' CHC, and control) was performed in 0–2 replicates per colony. Several experimental trials were cancelled due to precocious ant death and replaced with new trials if pupae were still available. After 12–15 days, ants were killed by freezing and stored at − 22 ºC for subsequent CHC extraction according to the protocol described below. No internal standard was added during preparation of the extract.

CHC extraction and chemical analyses

Individual ants were placed in glass vials (2 ml) and extracted in 150 µl of hexane for 10 minutes. Subsequently, 15 µl of a hexane solution of docosane (12.5 µg/ml) was added to the extracts as an internal standard. Glass vials with the extracts were left open until the solvent evaporated, re-dissolved in 50 µl of hexane, transferred to 100 µl inserts, and stored at -22 ºC until analysis. Head width measurements were performed as a proxy for ant body size and used for CHC amount normalization.

We analysed the CHC extracts of all samples with an Agilent 6890 gas chromatograph coupled with an Agilent 5975 Mass Selective Detector (GC-MS, Agilent, Waldbronn, Germany): The GC (split/splitless injector in splitless mode for 1 min, injected volume 1 µl at 300°C) was equipped with a DB−5 Fused Silica capillary column (30 m x 0.25 mm ID, df = 0.25 µm; J&W Scientific, Folsom, USA). Helium served as carrier gas at a constant flow of F. The following temperature program was usedStart temperature 60°C, temperature increase by 5°C per min up to 300°C, isotherm at 300°C for 10 min.: The electron ionisation mass spectra (EI-MS) were acquired at an ionisation voltage of 70 eV (source temperature: 230°C). Chromatograms and mass spectra were recorded and quantified via integrated peak areas with the software HP Enhanced ChemStation G1701AA (version A.03.00; Hewlett Packard). CHC compounds were identified by the compound-specific retention indices and their detected diagnostic ions (Carlson et al. 1998).

Body surface area approximation

The chemical analyses yielded the amount of hydrocarbons spread over the body surface. However, to make a fair comparison across individuals, this measure needs to be normalised to account for body size differences.Therefore, for all individuals included in the CHC amount analyses, head width was measured. In addition, for a subset of samples we measured the planar projection areas of the three body parts: dorsal view of the head, the dorsal view of the thorax, and lateral view of the thorax. The body of ants was photographed under a stereomicroscope and the number of pixels within the body parts was quantified using OpenCV Python library tools. Appendages were removed either physically or digitally in the image editor. For each view, 9–12 individuals of each species were analyzed, covering a wide range of the size distribution (Online Resource Fig. S10). Body area was approximated from head as follows: second-order polynomial regression was applied to relate planar projection area to head width, separately for each species and each view. The model coefficients were then used to predict the area of individuals for which only head width was available (Online Resource Section 9). Then the estimated area (in pixels) was used as a divisor of the standard area to obtain a scaling factor for normalizing CHC amount. As the standard area, we used the sum of estimated planar projections of an F. sanguinea worker with a head width of 1.15 mm. Thus, CHC amounts were scaled as if they were extracted from the ants with similar body areas (exact normalization was not possible due to approximation error).

Statistical analyses

Marker peaks

To identify the peaks characteristic of either F. fusca or F. sanguinea ants, we used a sparse partial least squares discriminant analysis model (sPLS-DA ; Rohart et al. 2017) trained on the data collected in another study (Włodarczyk and Szczepaniak 2017), in which ants were sampled from eleven F. sanguinea dulotic colonies and 21 F. fusca free-living field colonies. The data representing peak relative abundances are compositional in nature and were therefore subjected to the centred log-ratio transformation (Aitchison 1986, Hervé et al. 2018) after adding a small constant (10^− 5) to avoid log(0) operations. Since we were interested in the importance of each of the original variables (peaks) in predicting sample species, the data was subjected to PCA. Otherwise, the model might have suffered from non-identifiability due to the correlation between the peaks. Moreover, with the lasso penalization of loading vectors, the discriminant model might have disregarded some variables, relying on a few that could be sufficient to discriminate between the two species. This would remove some biologically relevant data variation, compromising the model’s robustness when making predictions about species identity of samples representing a mixed phenotype. Noise in the data was reduced by retaining only the first principal components that accounted for at least 80% of the total variance. To identify marker peaks, we calculated the weights of the input variables that are typically used for inference of observation class, i.e., species identity in this case (for details, see Online Resource Section 2.1). Since these weights were assigned to principal components, we needed to reverse the data transformation by multiplying the weight matrix by the pseudoinverse of the rotation matrix, which is used to project the original variables onto principal components. The resulting weights represented the predictive power of each peak within the context of the discriminant analysis model. The sign of each weight indicated the species towards which a peak biased the classification. We used the bottom and top 0.2 quantiles of the final scores to select peaks characteristic of F. fusca and F. sanguinea, respectively.

Similarly, we used the sPLS-DA method to identify markers distinguishing callow and mature F. sanguinea individuals. In this case, a multilevel data structure was imposed on the model with colony-sample date combination as a grouping factor. Each sample was classified into one of the three groups: F. fusca slaves, callow F. sanguinea, and mature F. sanguinea. By incorporating F. fusca samples into the discriminant analysis we corrected for the potential effect of F. fusca slaves on the differences between CHC profiles of callow and mature F. sanguinea ants. The trained discriminant analysis model was used, as before, to retrieve weights representing the contribution of each variable to the classification score. Peaks within the top 0.2 quantile were classified as markers.

Since the discriminant analysis is designed to identify differences between pre-defined groups of observations, we needed to ensure that our model's performance exceeded that of randomly selected samples. Otherwise, the peak markers could merely be statistical artefacts. Consequently, we randomly shuffled the age status of F. sanguinea samples within each sample date/colony combination, and we ran the perf function from mixOmics package. This function performed a cross-validation using 75% of samples to train the model which was then used to make predictions on all F. sanguinea samples and to assess its accuracy we calculated AUROC sliding the score indicating prediction of callow ant. The same was done for the data set with true age status labels and the difference in AUROC was computed. The procedure was repeated 10³ times to produce the sampling distribution of the differences in AUROC. The p-value was calculated as a proportion of differences equal to or less than zero.

Species Identity Score

The model trained for the identification of species-characteristic peaks was also used to generate predictions for new samples in the form of a continuous numerical value (henceforth Species Identity Score). Higher values indicated a stronger match to F. sanguinea and a weaker resemblance to F. fusca. This approach provided insights into the development of chemical identity in slave-maker ants as their proportion in a colony increased. We also compared nestmates of different species and ages by calculating the difference in Species Identity Score and regressing it against the proportion of F. sanguinea ants in the colony. When multiple samples per individual category (species/age) were available, we computed the average CHC profile and used it to calculate the difference in Species Identity Score.

Quantitative changes in the CHC profile

We fitted linear mixed models to see how the total CHC amount changed as the proportion of slave-making workers in a colony was becoming greater. We used all compounds or subsets composed of the markers of F. fusca, callow F. sanguinea, or mature F. sanguinea ants. The full model comprised following random effects: intercept nested within colony, intercept nested within interaction of colony and sampling date, and slope nested within colony. When necessary, random terms were dropped one by one to ensure model convergence, avoid singularity issues, and pass diagnostic tests. This process was repeated until a model that met these criteria was obtained.

We also fitted linear mixed models to track changes in CHC amounts over time in separated callow ants. As before, we conducted separate analyses on the data subsets with marker peaks only. Since ants in Petri dishes were not individually marked and there was a time lag between the introduction of the first individual and its pairing, we could not determine the exact separation time of the individual that was selected for CHC extraction. Therefore, we used the mean separation time of both ants from the same experimental unit as an approximation. If necessary, predictor or response variable were transformed by power or log function.

We used the lme4 package in R to fit the models and the lmerTest package to obtain p-values for the coefficients. Model diagnostics were performed using the DHARMa R package (Hartig 2021), which evaluates scaled residuals obtained through simulations from the fitted model and tests their distribution using the Kolmogorov-Smirnov test (for quantile-quantile distribution), an outlier test, a dispersion test, and a uniformity test. Additionally, we assessed response residuals of the fitted models using the Shapiro-Wilk normality test.

Permutation tests

We investigated whether the newly eclosed slave-maker workers adopt the chemical camouflage strategy. To examine this, we compared their recognition labels to those of the free-living relatives of the slaves. This approach eliminated the confounding effect of F. sanguinea ants on their slaves, which can lead to similarity in recognition odours (Włodarczyk and Szczepaniak 2017). We used the Bray-Curtis method (implemented in the R package vegan; Oksanen et al. 2020) on sum-normalized data to compute chemical distances between CHC profiles. Accordingly, we calculated the chemical distance of separated F. sanguinea ants to free-living F. fusca ants from slaves' parent colonies (Fig. 1). Using Wilcoxon matched pairs test, we compared these distances against those to a randomly selected unrelated F. fusca colony. The distance for samples collected from the same colony were averaged, to account for non-independence of observations. Since the unrelated colony was assigned randomly we repeated the procedure 10³ times for each treatment and report the mean p-value along with its range across all tests.

Non-parametric tests of the difference of means

We used the Wilcoxon signed-rank test to check the effect of species and maturity on the total amount of CHC or proportions of marker compounds. The samples were paired according to the colony of origin, and samples from the same category and colony were averaged to avoid pseudoreplication. In all our analyses, when the absolute amounts of CHC were taken into consideration, the corresponding values were corrected by dividing by the square of head width, which was used as a proxy for the body surface area. A similar procedure was used to determine if the chemical distance to the CHC mixture applied to the glass dummy ants was different between treatment and control ants. The dissimilarity between profiles was calculated as explained in the section on permutation tests.

Marker chemical compounds

We identified 81 peaks in chromatograms from our samples representing hydrocarbons and their mixtures (Table 1; Online Resource Table S1). The marker identification procedure classified 15 peaks as being associated with F. sanguinea (Online Resource Table S1). On average, these accounted for 33.1% of the total CHC amount in this species and 5 % in the free-living F. fusca (based on field-collected samples, see Methods). In addition, the procedure identified 15 peaks indicative of free-living F. fusca, contributing 6.5% and 23.9% to the total CHC amount in F. sanguinea and free-living F. fusca, respectively. Comparison between ants sampled from the same laboratory colony did not reveal a significant difference between mature F. sanguinea and their slaves in the proportion of compounds that were identified as markers of either species, highlighting an efficient exchange of CHC among nestmates (Fig. 2A and B). The discriminant model trained on data with true age labels performed better in predicting the age of unseen samples compared to models run after age label shuffling (p-value < 10^-³, see Online Resource Section 3.2). Thus, callow ants are more distinct from the rest of F. sanguinea samples than a randomly taken subset.

Change of the CHC profile composition during colony development

In total, we analysed 68 samples from mature F. sanguinea workers and 78 samples from F. fusca workers. They were collected from the colonies with varying proportions of slave-maker workers, ranging from 0% (before the emergence of slave-maker workers, 17 samples) through 100% (no slaves, 2 samples; Fig. 4). Moreover, the CHC composition changed in a coordinated manner, as we found that normalized amount of hydrocarbons associated with F. sanguinea tended to increase on the cuticle of both mature slave-makers and slaves as the proportion of the slave-making workers in a colony increases (p-value for the model slope < 10^-⁹, <10^-¹¹ for F. sanguinea and F. fusca ants, respectively; Online Resource Section 4.3). Conversely, the mass of compounds associated with F. fusca decreased in mature individuals of both species (p-values: Fs: < 10^-3, Ff: <10^-2; Online Resource Section 4.4). In accordance with these results, the Species Identity Score increased with F. sanguinea proportion for both slave-makers (p-value < 10^-¹¹) and slaves (p-value < 10^-⁹; Online Resource Section 2.2.1).

Factors associated with the CHC amount

Callow ants were characterized by a relatively low amount of CHC (mean across samples normalized to standard body surface area = 2.01 ± 1.34 μg; Fig. 2C) compared to mature nestmates of the same species (standardized mean = 5.32 ± 2.82 μg; p-value in Wilcoxon signed-rank test < 10^-4). Moreover, the total amount of CHC on callow and mature F. sanguinea ants, but not on slaves, showed a positive trend as the proportion of slave-making ants in a colony increases (p-value for the model slope: <10^-2 and <10^-3 for mature and callow ants, respectively). The normalized CHC amount predicted by a linear model is three-fold larger on F. sanguinea compared to F. fusca assuming pure colonies, i.e., eliminating the influence of the other species (95% highest density interval: 2.1-4.3; Fig. 4, Online Resource Fig. S8). The difference between both species in the CHC concentration was also confirmed in paired comparisons of colony-averaged samples (Fig 2D).

Chemical identity of the callow F. sanguinea workers

Some of the peaks biased predictions of the discriminant model towards callow ants. This outcome was further verified by generating differential profiles derived by subtraction of CHC proportions of mature F. sanguinea ants from those of their callow nestmates, which were sampled at the same stage of colony development (Fig. 3). Among peaks with a substantial contribution to the overall profile, two (corresponding to the compounds 13-Me C27 and 13-; 11-; 9-MeC25) were consistently more abundant in the CHC blend of callow ant. In contrast, n-pentacosane (#20) and n-heptacosane (#41) were more abundant in the CHC profile of mature ants (Table 1, Fig. 3, Online Resource Table S2). Moreover, in callow F. sanguinea ants the proportion of n-alkanes in the CHC blend was lower as compared to the mature individuals (Fig. 2D, p-value < 10^-4). Two predominant n-alkanes, n-pentacosane and n-heptacosane, together constituting 21.5% and 8.5% of the total CHC amount in F. sanguinea and F. fusca, respectively (Włodarczyk and Szczepaniak 2017), received negative scores as callow markers. This indicates that they were underrepresented in the CHC profile of callow F. sanguinea ants (Online Resource Table S2). The specificity of the callow ant CHC profile is also highlighted by the fact that their Species Identity Score was lower than that of mature ants and not significantly different from that of slaves throughout the colony development (Online Resource Section 2.2). This indicates a bias toward the slave chemical signature in the callow slave-makers.

Formica sanguinea ants separated from their colonies before eclosion showed significantly smaller chemical distances to the free-living sisters of the slaves from their parent colonies than to unrelated free-living F. fusca ants for all but the longest separation periods (Fig. 1 and 5, Online Resource Section 7.1). Increasing chemical distances over time corroborate the tendency of F. sanguinea ants to diverge from the CHC profile of slaves. A similar pattern emerges when the analysis is restricted to the samples from ants that have formed silky envelopes (cocoons) during pupal stage, thereby excluding the effect of CHC absorption during that period, in contrast to what might have occurred in the case of naked pupae (Online Resource Section 7.2).

Effect of the presence of glass beads coated with recognition odours

We did not find any significant difference between control and treated ants in their chemical distance to colonies which served as a source of CHC applied to glass beads (Wilcoxon test, F. sanguinea CHC: p-value = 0.24, F. fusca CHC: p-value = 0.68; Online Resource Sections 8.1 and 8.2). This suggests that ants separated in pairs did not change their CHC profile to mimic that encountered on dummies. However, the tested ants came into the contact with the glass beads, as evidenced by a significantly greater abundance of docosane on the cuticle of the treated ants as compared to the control ones (Wilcoxon test, F. sanguinea: p-value = 0.007, F. fusca: p-value = 0.006; Online Resource Section 8.3). This compound was used to contaminate the CHC mixture applied to the glass bead surface (see Methods).

The results of this experiment suggest that F. sanguinea workers produce a small intrinsic amount of n-docosane (0.2% ± 0.4% of the total CHC amount, on average). However, this contribution is negligible for our calculations of CHC mass based on n-docosane as an internal standard, since after its addition it constituted a much larger fraction of the CHC profile (6.9% ± 4.2% on average).

Our study shows that the species-specific CHC profile of F. sanguinea ants develops gradually during colony development, starting from a point where it is more similar to that of the free-living host. This result reflects colony demography, where the parasite reproduces while the number of slaves declines due to mortality. A similar phenomenon was observed in colonies of the inquiline wasp Polistes atrimandibularis, which modifies the hydrocarbon composition of the usurped nest comb by introducing compounds specific to its own species. Following initial chemical mimicry by the parasite female, this process progresses until the fall of the colony (Bagnères et al. 1996). We also found that qualitative changes in the CHC profile are accompanied by an increase in body-size normalized CHC mass as the proportion of slave-makers in the colony rises. Furthermore, the normalized CHC mass is higher in F. sanguinea compared to their slaves. This trait suggests an adaptation to employ a strategy of chemical dominance, whereby the parasite influences the slaves to adopt a chemical identity similar to its own (Włodarczyk 2016, Włodarczyk and Szczepaniak 2017, Scheckel 2021). This strategy gradually replaces the chemical mimicry employed at the onset of the colony life cycle as the proportion of slaves decreases.

In the experiment in which separated callow F. sanguinea ants were exposed to dummy individuals in the form of glass beads covered with CHC blend, we found no effect of this artificial social environment on the pattern of CHC development in the tested ants. This result might indicate the insensitivity of ants’ internal program of CHC production to environmental cues. Alternatively, the glass beads might not have provided the relevant stimuli for a response in the test ants, as the response to semiochemicals might require the right context created by visual cues or motion (Orlova and Amsalem 2021). Nevertheless, callow ants did come into contact with the dummy ants, as indicated by the significantly higher proportion of the contaminant (n-docosane) on their cuticle compared to ants kept in the presence of clean glass beads.

The CHC profile of callow F. sanguinea ants and its formation support the idea that the chemical strategy used by F. sanguinea ants to deceive the host recognition system might be limited by the constraints of the slave template’s plasticity, coupled with the ability of slaves to recognise the parasite as a parasite. In particular, callow F. sanguinea workers are chemically closer to the F. fusca CHC signature than their mature counterparts. The finding is complemented by the result of our experiment that showed that F. sanguinea ants separated before hatching were similar in terms of their epicuticular chemistry to free-living relatives of their slaves, providing indirect evidence of the influence of slaves on the CHC profile of F. sanguinea ants (Fig. 1). The possible mechanism for this effect is the transmission of recognition cues from adult workers to larvae during feeding and/or their direct absorption by larvae from the adults' cuticle and nest (Boss et al. 2011). To our knowledge, this is the first evidence for the impact of the social environment of developing stages on the subsequent CHC odour of adult social insects. Since there has been no compelling evidence so far that social parasites use chemical mimicry (active biosynthesis) to adjust their CHC profile to match host odour at a colony level, we advocate interpreting our results in terms of chemical camouflage. Chemical similarity to host species has also been found in the callow P. rufescens workers separated from the rest of the colony (D'Ettorre et al. 2002). They possessed only trace amounts of hydrocarbons at emergence but expressed a CHC profile matching that of their host by the age of 5 days. Although D'Ettorre et al. (2002) postulate an inherited capacity to mimic the host recognition signature, based on our results, the post-emergence deposition of hydrocarbons sequestered during the larval stage is also a likely explanation for the outcome of their study.

We found that some compounds occur in higher proportions on the cuticle of newly hatched individuals as compared to mature slave-makers and slaves. Furthermore, the deposition of some other compounds on the cuticle is clearly reduced in callow individuals. Taken together, these findings indicate an age-dependent pattern of CHC changes in the studied species. As previously noted, the CHC profile characteristic of callow F. sanguinea is affected by the transfer of chemicals from the slaves. However, this phenomenon cannot fully explain why callow ants are chemically distinguishable from adults since the difference between both groups is maintained even in colonies with a negligible proportion of slaves. We therefore conclude that the maturation program in this species involves a change in the biosynthetic machinery underlying CHC production. Regardless of the proximate mechanism, we propose that the altered profile in the callow F. sanguinea is an adaptation leading to the confusion of host workers about slave-maker species identity. It is known that host workers often show inherited ability to recognise slave-makers, most likely based on the species-specific CHC profile (D'Ettorre et al. 2004, Brandt et al. 2005, Pammiger et al. 2011, Delattre et al. 2012). In particular, the presence of F. sanguinea workers near the nest of F. fusca excites the resident ants, provoking attacks on the parasite (Włodarczyk 2023). Thus, the appearance of slave-making workers in a colony might trigger antiparasitic behaviour in slave ants if the odour difference between species exceeds the tolerated deviations from their neural template (Reeve 1989). Therefore, for successful social integration of slave-maker workers in a society, odour familiarization must be gradual. Expressing an alternative variant of CHC profile by callow ants might deceive the host by pacifying its antiparasitic reaction. The full recognition label is developed later, when host workers are already partly familiarized with the scent of the parasite. It should be noted that important preadaptations could already be present in the free-living ancestors of F. sanguinea ants as compounds characteristic of callow workers are present in the free-living ant species Cataglyphis iberica (Dhabi et al. 1998). Moreover, age-related changes in the proportion of CHC blend components have been found in isolated newly hatched Polistes dominulus wasps (Panek et al. 2001). In contrast, no qualitative differences between mature and newly eclosed ants were reported in Aphaenogaster senilis (Ichinose and Lenoir 2009). Studies on related free-living species should determine whether the discussed traits of F. sanguinea chemical ecology have evolved as an adaptation to parasitic lifestyle.

Newly hatched F. sanguinea workers possess a reduced amount of hydrocarbons on their cuticle. This may hinder their discrimination by slaves, similar to how young Polyergus queens invading host colonies escape detection by resident workers by nearly lacking CHC on their cuticle (Johnson et al. 2001, Lenoir et al. 2001, Tsuneoka and Akino 2012). This strategy, known as chemical insignificance, is employed by many social parasites from distant phylogenetic groups (reviewed in Lorenzi and d'Ettorre 2020). Surprisingly, we did not observe an increased proportion of n-alkanes on the cuticle of callow F. sanguinea ants. This feature is also considered a variant of the chemical insignificance strategy (Lorenzi and d'Ettorre 2020), since n-alkanes play a smaller role in nestmate recognition than branched alkanes and alkenes (van Zweden and d'Ettorre 2010), and their overrepresentation in the chemical signature is widespread across social parasites (Nehring et al. 2015, Lorenzi and d'Ettorre 2020). Nevertheless, we found linear alkanes to be negatively associated with callow ants. We argue that they might have limited utility for achieving long-term acceptance of F. fusca ants. As a host to myriad social parasites (Martin et al. 2011), this species may have evolved more sophisticated mechanisms of nestmate recognition, integrating tactile, visual, and olfactory cues to make final acceptance or rejection decisions. In such cases, being chemically transparent might not prevent host aggression, as information from other modalities could indicate that the inspected object is a potential intruder, and the absence of the expected chemical label might itself signal potential threat.

Our results indicate that the time needed to attain a quantitatively complete CHC profile might be longer in F. sanguinea compared to that of free-living species (Lenoir et al., 2001; Ichinose and Lenoir, 2009). We found no significant change in CHC mass in separated workers over 40 days following eclosion. However, we cannot rule out the potential impact of stress associated with separation (even when paired with another individual) or a reduced diet composed mainly of sugars. Further studies are needed to elucidate this issue. Similarly, the positive correlation between CHC mass per body area unit in F. sanguinea and their proportion in the colony, which increased gradually during colony development, suggests an extended period before full CHC production capacity is reached in this species. This delay may give host ants time to adapt to the changing colony odour. Alternatively, recognition cues might be synthesised at full rate earlier but their transfer to the nestmates might not be balanced by the incoming flow due to lower CHC production in slaves. Thus, the lack of positive effect of the F. sanguinea proportion on CHC concentration in slaves could be explained by a reduced CHC biosynthesis rate in response to CHC acquisition from the social environment.

Age-dependent changes in the CHC chemistry of F. sanguinea ants may ultimately lead to the observed transition from chemical mimicry to chemical dominance as the colony grows, since the mean age of colony members is expected to increase until the emergence of new individuals is fully balanced by mortality of old ones. Overall, the observed changes during colony development suggest that the deception strategy of slave-makers should be adjusted to the actual proportion of slaves in the colony. This observation, coupled with the fact that in obligate slave-maker colonies, parasites typically comprise only 10–20% of colony members (Savolainen and Deslippe 1996 and references therein, Herbers and Foitzik 2002), may explain why these species use chemical mimicry, even though their colonies are separate from the host ones. In our opinion, chemical mimicry of obligate slave-makers cannot be solely explained as an adaptation to avoid detection by free-living host ants since, as empirical evidence shows, they are not particularly effective in achieving this (D'Ettorre et al. 2004, Brandt et al. 2005, Pammiger et al. 2011, Delattre et al. 2012). Instead, we advocate that the main driver of the evolution of chemical mimicry in slave-making ant species is the pressure to maintain colony integrity, which is challenged by potential slave rebellions or sabotage (Achenbach and Foitzik 2009, Czechowski and Godzińska 2014).

Author Contribution

T.W. conceived the project, designed the study, collected material, prepared chemical samples, designed and performed data analysis, and wrote the manuscript. T.S. processed chemical samples, identified compounds, and contributed to writing and editing the manuscript.

Acknowledgement

We are grateful to Joanna Gromek for her assistance in maintaining ant colonies. Part of the statistical analyses were carried out at the Computer Center of University of Białystok.

Data Availability

The mass spectra, in the form of .mzML, files are available through the Metabolomics Workbench ( [https://www.metabolomicsworkbench.org/](https:/www.metabolomicsworkbench.org) , ID 6516). The code used to process the raw data and generate the final results is available in the corresponding author’s GitHub repository as an *R* package: [https://github.com/TomVuod/sangchem](https:/github.com/TomVuod/sangchem) .

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Coming out from the shadows: facultative slave-making ants reveal their chemical identity during colony development

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