Innate and learned components of egg recognition in the ant Camponotus floridanus.

Insect societies discriminate against foreigners to avoid exploitation. In ants, helper workers only accept individuals with the familiar chemical cues of their colony. Similarly, unfamiliar eggs may get rejected at their first appearance in the nest. We investigated egg acceptance mechanisms by introducing different types of foreign eggs into worker groups of the ant Camponotus floridanus. Workers from established colonies familiar with queen-laid eggs always accepted eggs from highly fecund queens, but worker-laid eggs only after exposure for several weeks. Workers naive to eggs only rejected worker-laid eggs once they had prior exposure to eggs laid by highly fecund queens, suggesting that prior exposure to such eggs is necessary for discrimination. The general acceptance of eggs from highly fecund queens, irrespective of previous worker egg exposure, suggests an innate response to the queen pheromone these eggs carry. Workers learned to accept queen-laid eggs from different species, indicating high flexibility in learning egg-recognition cues. In incipient colonies with queen-laid eggs that carry a weak queen pheromone, worker-laid eggs were more likely to get accepted than queen-laid eggs from a different species, suggesting that the similarity of egg-recognition cues between the two types of C. floridanus eggs increases acceptance.

Low Intraspecific Aggression Level, Cuticular Hydrocarbons, and Polydomy in the Bullet Ant.

Ants use chemical cues known as cuticular hydrocarbons (CHCs) for both intraspecific and interspecific recognition. These compounds serve ants in distinguishing between nestmates and non-nestmates, enabling them to coexist in polydomous colonies characterized by socially connected yet spatially separated nests. Hence, the aim of this study was to investigate the intraspecific aggression level between nestmates and non-nestmates of the bullet ant Paraponera clavata (Fabricius, 1775), analyze and compare their CHCs, and evaluate the occurrence of polydomy in this species. We conducted aggression tests between foragers, both in laboratory and field settings. To identify the chemical profiles, we utilized gas chromatography coupled with mass spectrometry (GC-MS). We marked the foragers found at nest entrances and subsequently recaptured these marked ants to validate workers exchange among nests. Across all nests, a low intraspecific aggression level was observed within the same area. However, a significant difference in aggression correlated to distance between nests. Analysis of the cuticular chemical profile of P. clavata unveiled colony-specific CHCs, both qualitatively and quantitatively. Notably, we observed instances of ants from certain nests entering or exiting different nests. This behavior, in conjunction with the observed low intraspecific aggression despite differences in CHCs suggests polydomy for this species. Polydomy can offer several benefits, including risk spreading, efficient exploitation of resources, potential for colony size increasing and reduced costs associated with foraging and competition.

Highly diverse cuticular hydrocarbon profiles but no evidence for aggression towards non-kin in the ambrosia beetle Xyleborinus saxesenii.

Animal societies use nestmate recognition to protect against social cheaters and parasites. In most social insect societies, individuals recognize and exclude any non-nestmates and the roles of cuticular hydrocarbons as recognition cues are well documented. Some ambrosia beetles live in cooperatively breeding societies with farmed fungus cultures that are challenging to establish, but of very high value once established. Hence, social cheaters that sneak into a nest without paying the costs of nest foundation may be selected. Therefore, nestmate recognition is also expected to exist in ambrosia beetles, but so far nobody has investigated this behavior and its underlying mechanisms. Here we studied the ability for nestmate recognition in the cooperatively breeding ambrosia beetle Xyleborinus saxesenii, combining behavioural observations and cuticular hydrocarbon analyses. Laboratory nests of X. saxesenii were exposed to foreign adult females from the same population, another population and another species. Survival as well as the behaviours of the foreign female were observed. The behaviours of the receiving individuals were also observed. We expected that increasing genetic distance would cause increasing distance in chemical profiles and increasing levels of behavioural exclusion and possibly mortality. Chemical profiles differed between populations and appeared as variable as in other highly social insects. However, we found only very little evidence for the behavioural exclusion of foreign individuals. Interpopulation donors left nests at a higher rate than control donors, but neither their behaviours nor the behaviours of receiver individuals within the nest showed any response to the foreign individual in either of the treatments. These results suggest that cuticular hydrocarbon profiles might be used for communication and nestmate recognition, but that behavioural exclusion of non-nestmates is either absent in X. saxesenii or that agonistic encounters are so rare or subtle that they could not be detected by our method. Additional studies are needed to investigate this further.

A predatory social wasp does not avoid nestmates contaminated with a fungal biopesticide.

Fungus-based biopesticides have been used worldwide for crop pest control as a safer alternative to chemical pesticides such as neonicotinoids. Both agrochemicals can be lethal and may also trigger side effects on the behavioral traits of non-target social insects, which play a crucial role in providing essential biological pest control services in agroecosystems. Here, we evaluated whether a commercial formulation of the entomopathogenic fungus Beauveria bassiana or the neonicotinoid imidacloprid causes mortality in foragers of Mischocyttarus metathoracicus. These social wasps are natural enemies of caterpillars and other herbivorous insects and inhabit both urban and agricultural environments in Brazil. We also tested whether wasps discriminate between biopesticide-exposed and unexposed conspecifics. Through a combination of laboratory (survival assay) and field experiments (lure presentation), along with chemical analyses (cuticular hydrocarbon profiles), we showed that topic exposure to the label rate of each pesticide causes a lethal effect, with the biopesticide exhibiting a slower effect. Moreover, wasps do not discriminate biopesticide-exposed from unexposed conspecifics, likely because of the similarity of their cuticular chemical profiles 24 h after exposure. Overall, the delayed lethal time at the individual level, combined with the indistinctive chemical cues of exposure and the lack of discrimination by conspecifics suggests that the fungal biopesticide may ultimately pose a threat to the colony survival of this predatory wasp.

Cuticular hydrocarbon reception by sensory neurons in basiconic sensilla of the Japanese carpenter ant.

To maintain the eusociality of a colony, ants recognize subtle differences in colony-specific sets of cuticular hydrocarbons (CHCs). The CHCs are received by female-specific antennal basiconic sensilla and processed in specific brain regions. However, it is controversial whether a peripheral or central neural mechanism is mainly responsible for discrimination of CHC blends. In the Japanese carpenter ant, Camponotus japonicus, about 140 sensory neurons (SNs) are co-housed in a single basiconic sensillum and receive colony-specific blends of 18 CHCs. The complexity of this CHC sensory process makes the neural basis of peripheral nestmate recognition difficult to understand. Here, we electrophysiologically recorded responses of single basiconic sensilla to each of 18 synthesized CHCs, and identified CHC responses of each SN co-housed in a single sensillum. Each CHC activated different sets of SNs and each SN was broadly tuned to CHCs. Multiple SNs in a given sensillum fired in synchrony, and the synchronicity of spikes was impaired by treatment with a gap junction inhibitor. These results indicated that SNs in single basiconic sensilla were electrically coupled. Quantitative analysis indicated that the Japanese carpenter ants have the potential to discriminate chemical structures of CHCs based on the combinational patterns of activated SNs. SNs of ants from different colonies exhibited different CHC response spectra. In addition, ants collected from the same colony but bred in separate groups also exhibited different CHC response spectra. These results support the hypothesis that the peripheral sensory mechanism is important for discrimination between nestmate and non-nestmate ants.

Consistency of Cuticular Hydrocarbon Mixtures of Five Reticulitermes (Blattodea: Rhinotermitidae) Taxa From Northern California: Similarity Among Colonies and Seasonal Variation.

Cuticular hydrocarbon (CHC) mixtures from workers of five distinct CHC phenotypes of Reticulitermes Holmgren 1913 from two locations in northern California were examined from monthly collections taken over a 3-yr period. The objectives of this study were (1) to identify and quantify variations of the CHCs of multiple colonies of each of these phenotypes (= species or subspecies) to demonstrate consistency, (2) to assess the potential of CHC mixtures to separate or identify colonies within each phenotype, and (3) to detect any temporal changes in each of the hydrocarbons in the CHC mixtures. Nonmetric multidimensional scaling of all CHC mixtures of all samples collected at both locations separated the samples into five clearly visible, different groups of CHC phenotypes (taxa or species) of Reticulitermes. The degree of variability of the CHC mixtures among colonies of each phenotype was such that nonmetric multidimensional scaling did not separate or identify colonies. Strong seasonal fluctuations were evident in some of the CHCs of all five phenotypes and were significantly consistent with a sine curve. Maximum proportions of seasonal CHCs within a phenotype occurred in all seasons of the year but occurred mostly in the winter and summer. In general, the CHCs displaying maximum values in the winter were short-chained (C23-C27) methyl-branched alkanes, whereas the CHCs displaying maximum values in the summer were long-chained (C35-C43) methyl-branched alkanes, which likely influences water retention. These consistent chemical fingerprints are probably responsible for inter-phenotype recognition patterns and are thus useful for chemical taxonomy.

The Effect of Diet on Colony Recognition and Cuticular Hydrocarbon Profiles of the Invasive Argentine Ant, Linepithema humile.

Ants are some of the most abundant and ecologically successful terrestrial organisms, and invasive ants rank among the most damaging invasive species. The Argentine ant is a particularly well-studied invader, in part, because of the extreme social structure, known as unicoloniality, that occurs in introduced populations. Unicoloniality is characterized by the formation of geographically vast supercolonies, within which territorial behavior and intraspecific aggression are absent. Although there is considerable evidence supporting a genetic basis for the odor cues involved in colony recognition, some studies have suggested that diet may also influence colony recognition cues and, thus, colony structure. Here, we test the role for insect-derived recognition cues by performing a diet supplementation experiment in a natural field setting, and a more extreme dietary manipulation experiment in the lab. After one month, in both the field and the lab, we found that aggressive supercolonies remained aggressive toward each other and non-aggressive nests (from the same supercolony) remained non-aggressive, regardless of dietary treatment. In one lab treatment, we did observe a significant decrease in the level of aggression between different supercolonies that were fed the same diet, but aggression was still frequent. We did not see any evidence for cuticular hydrocarbon odor cues being transferred from prey to ants in any of the field treatments. In the more extreme lab treatment, however, several cuticular hydrocarbons were acquired from both roach and cricket insect prey (but not Drosophila). Based on these data, we conclude that dietary changes are unlikely to underlie changes in behavior or colony structure in Argentine ants in real-world settings. However, these results indicate that caution is warranted when interpreting the behaviors of animals that have been reared on diets that are substantially different from natural populations.

Side effects of a fungus-based biopesticide on stingless bee guarding behaviour.

Pathogenic fungi have been used worldwide to control crop pests and are assumed to pose negligible threats to the survival of pollinators. Although eusocial stingless bees provide essential pollination services and might be exposed to these biopesticides in tropical agroecosystems, there is a substantial knowledge gap regarding the side effects of fungal pathogens on behavioural traits that are crucial for colony functioning, such as guarding behaviour. Here, we evaluated the effect of Beauveria bassiana on the sophisticated kin recognition system of Tetragonisca angustula, a bee with morphologically specialized entrance guards. By combining behavioural assays and chemical analyses, we show that guards detect pathogen-exposed nestmates, preventing them from accessing nests. Furthermore, cuticular profiles of pathogen-exposed foragers contained significantly lower amounts of linear alkanes than the unexposed ones. Such chemical cues associated with fungal conidia may potentially trigger aggression towards pathogen-exposed bees, preventing pathogen spread into and among colonies. This is the first demonstration that this highly abundant native bee seems to respond in a much more adaptive way to a potentially infectious threat, outweighing the costs of losing foraging workforce when reducing the chances of fungal pathogen outbreaks within their colonies, than honeybees do.

Distinct colony boundaries and larval discrimination in polygyne red imported fire ants (Solenopsis invicta).

Evaluating the factors that promote invasive ant abundance is critical to assess their ecological impact and inform their management. Many invasive ant species show reduced nestmate recognition and an absence of boundaries between unrelated nests, which allow populations to achieve greater densities due to reduced intraspecific competition. We examined nestmate discrimination and colony boundaries in introduced populations of the red imported fire ant (Solenopsis invicta; hereafter, fire ant). Fire ants occur in two social forms: monogyne (colonies with a single egg-laying queen) and polygyne (colonies with multiple egg-laying queens). In contrast with monogyne nests, polygyne nests are thought to be interconnected due to the reduced antagonism between non-nestmate polygyne workers, perhaps because polygyne workers habituate the colony to an odour unique to Gp-9b -carrying adults. However, colony boundaries and nestmate discrimination are poorly documented, particularly for worker-brood interactions. To delimit boundaries between field colonies, we correlated the exchange of a 15 N-glycine tracer dissolved in a sucrose solution with social form. We also evaluated nestmate discrimination between polygyne workers and larvae in the laboratory. Counter to our expectations, polygyne colonies behaved identically to monogyne colonies, suggesting both social forms maintain strict colony boundaries. Polygyne workers also preferentially fed larval nestmates and may have selectively cannibalized non-nestmates. The levels of relatedness among workers in polygyne colonies was higher than those previously reported in North America (mean ± standard error: 0.269 ± 0.037). Our study highlights the importance of combining genetic analyses with direct quantification of resource exchange to better understand the factors influencing ant invasions.

Similarities in Recognition Cues Lead to the Infiltration of Non-Nestmates in an Ant Species.

Chemical cues are among the most important information-sharing mechanisms in insect societies, in which cuticular hydrocarbons play a central role, e.g., from nestmate recognition to queen signaling. The nestmate recognition mechanism usually prevents intruders from taking advantage of the resources stored in the nest. However, nestmate recognition is not unconditionally effective, and foreign individuals can sometimes infiltrate unrelated nests and take advantage of the colony resources. In this study, we investigated the role of overall colony odor profiles on the ability of conspecific workers to drift into unrelated colonies. We hypothesized that drifters would have higher chances of success by infiltrating colonies with the odor profiles most similar to their own nest, avoiding being detected as non-nestmates. By performing a drifting bioassay, we found that workers of the ant Formica fusca infiltrated unrelated conspecific colonies at a rate of 2.4%, significantly infiltrating colonies displaying CHC profiles most similar to their natal nests. Notably, methyl branched hydrocarbons seem to play a role as recognition cues in this species. In addition, we show that environmental rather than genetic factors are responsible for most contributions on the CHC phenotype, presenting ca. of 50% and 27.5% of explained variation respectively, and playing a major role in how worker ants detect and prevent the infiltration of non-nestmates in the colony. Hence, relying on cuticular hydrocarbons similarities could be a profitably evolutionary strategy by which workers can identify conspecific colonies, evade detection by guards, and avoid competition with genetic relatives.

Cuticular hydrocarbon profiles differ between ant body parts: implications for communication and our understanding of CHC diffusion.

Insect cuticular hydrocarbons (CHCs) serve as communication signals and protect against desiccation. They form complex blends of up to 150 different compounds. Due to differences in molecular packing, CHC classes differ in melting point. Communication is especially important in social insects like ants, which use CHCs to communicate within the colony and to recognize nestmates. Nestmate recognition models often assume a homogenous colony odor, where CHCs are collected, mixed, and redistributed in the postpharyngeal gland (PPG). Via diffusion, recognition cues should evenly spread over the body surface. Hence, CHC composition should be similar across body parts and in the PPG. To test this, we compared CHC composition among whole-body extracts, PPG, legs, thorax, and gaster, across 17 ant species from 3 genera. Quantitative CHC composition differed between body parts, with consistent patterns across species and CHC classes. Early-melting CHC classes were most abundant in the PPG. In contrast, whole body, gaster, thorax, and legs had increasing proportions of CHC classes with higher melting points. Intraindividual CHC variation was highest for rather solid, late-melting CHC classes, suggesting that CHCs differ in their diffusion rates across the body surface. Our results show that body parts strongly differ in CHC composition, either being rich in rather solid, late-melting, or rather liquid, early-melting CHCs. This implies that recognition cues are not homogenously present across the insect body. However, the unequal diffusion of different CHCs represents a biophysical mechanism that enables caste differences despite continuous CHC exchange among colony members.

Antenna Cleaning Is Essential for Precise Behavioral Response to Alarm Pheromone and Nestmate-Non-Nestmate Discrimination in Japanese Carpenter Ants (Camponotus japonicus).

Self-grooming of the antennae is frequently observed in ants. This antennal maintenance behavior is presumed to be essential for effective chemical communication but, to our knowledge, this has not yet been well studied. When we removed the antenna-cleaning apparatuses of the Japanese carpenter ant (C. japonicus) to limit the self-grooming of the antennae, the worker ants demonstrated the self-grooming gesture as usual, but the antennal surface could not be sufficiently cleaned. By using scanning electron microscopy with NanoSuit, we observed the ants' antennae for up to 48 h and found that the antennal surfaces gradually became covered with self-secreted surface material. Concurrently, the self-grooming-limited workers gradually lost their behavioral responsiveness to undecane-the alarm pheromone. Indeed, their locomotive response to the alarm pheromone diminished for up to 24 h after the antenna cleaner removal operation. In addition, the self-grooming-limited workers exhibited less frequent aggressive behavior toward non-nestmate workers, and 36 h after the operation, approximately half of the encountered non-nestmate workers were accepted as nestmates. These results suggest that the antennal sensing system is affected by excess surface material; hence, their proper function is prevented until they are cleaned.

Cuticular Hydrocarbon Profile of Parasitic Beetles, Aethina tumida (Coleoptera: Nitidulidae).

Cuticular hydrocarbons (CHCs) cover insects' bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs), Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.

Guarding Vibrations-Axestotrigona ferruginea Produces Vibrations When Encountering Non-Nestmates.

Flower visiting stingless bees store collected pollen and nectar for times of scarcity. This stored food is of high value for the colony and should be protected against con- and heterospecifics that might rob them. There should be high selective pressure on the evolution of mechanisms to discriminate nestmates from non-nestmates and to defend the nest, i.e., resources against intruders. Multimodal communication systems, i.e., a communication system that includes more than one sensory modality and provide redundant information, should be more reliable than unimodal systems. Besides olfactory signals, vibrational signals could be used to alert nestmates. This study tests the hypothesis that the vibrational communication mode plays a role in nest defense and nestmate recognition of Axestotrigona ferruginea. Substrate vibrations induced by bees were measured at different positions of the nest. The experiments show that guarding vibrations produced in the entrance differ in their temporal structure from foraging vibrations produced inside the nest. We show that guarding vibrations are produced during non-nestmate encounters rather than nestmate encounters. This further supports the idea that guarding vibrations are a component of nest defense and alarm communication. We discuss to whom the vibrations are addressed, and what their message and meaning are.

Limited dispersal and an unexpected aggression pattern in a native supercolonial ant.

Understanding how social groups function requires studies on how individuals move across the landscape and interact with each other. Ant supercolonies are extreme cooperative units that may consist of thousands of interconnected nests, and their individuals cooperate over large spatial scales. However, the inner structure of suggested supercolonial (or unicolonial) societies has rarely been extensively studied using both genetic and behavioral analyses. We describe a dense supercolony-like aggregation of more than 1,300 nests of the ant Formica (Coptoformica) pressilabris. We performed aggression assays and found that, while aggression levels were generally low, there was some aggression within the assumed supercolony. The occurrence of aggression increased with distance from the focal nest, in accordance with the genetically viscous population structure we observe by using 10 DNA microsatellite markers. However, the aggressive interactions do not follow any clear pattern that would allow specifying colony borders within the area. The genetic data indicate limited gene flow within and away from the supercolony. Our results show that a Formica supercolony is not necessarily a single unit but can be a more fluid mosaic of aggressive and amicable interactions instead, highlighting the need to study internest interactions in detail when describing supercolonies.

Desiccation Resistance and Micro-Climate Adaptation: Cuticular Hydrocarbon Signatures of Different Argentine Ant Supercolonies Across California.

Cuticular hydrocarbons (CHCs), the dominant fraction of the insects' epicuticle and the primary barrier to desiccation, form the basis for a wide range of chemical signaling systems. In eusocial insects, CHCs are key mediators of nestmate recognition, and colony identity appears to be maintained through a uniform CHC profile. In the unicolonial Argentine ant Linepithema humile, an unparalleled invasive expansion has led to vast supercolonies whose nestmates can still recognize each other across thousands of miles. CHC profiles are expected to display considerable variation as they adapt to fundamentally differing environmental conditions across the Argentine ant's expanded range, yet this variation would largely conflict with the vastly extended nestmate recognition based on CHC uniformity. To shed light on these seemingly contradictory selective pressures, we attempt to decipher which CHC classes enable adaptation to such a wide array of environmental conditions and contrast them with the overall CHC profile uniformity postulated to maintain nestmate recognition. n-Alkanes and n-alkenes showed the largest adaptability to environmental conditions most closely associated with desiccation, pointing at their function for water-proofing. Trimethyl alkanes, on the other hand, were reduced in environments associated with higher desiccation stress. However, CHC patterns correlated with environmental conditions were largely overriden when taking overall CHC variation across the expanded range of L. humile into account, resulting in conserved colony-specific CHC signatures. This delivers intriguing insights into the hierarchy of CHC functionality integrating both adaptation to a wide array of different climatic conditions and the maintenance of a universally accepted chemical profile.

Honey bee (Apis mellifera) sociability and nestmate affiliation are dependent on the social environment experienced post-eclosion.

Underpinning the formation of a social group is the motivation of individuals to aggregate and interact with conspecifics, termed sociability. Here, we developed an assay, inspired by vertebrate approaches to evaluate social behaviours, to simultaneously examine the development of honey bee (Apis mellifera) sociability and nestmate affiliation. Focal bees were placed in a testing chamber which was separated from groups of nestmates and conspecific non-nestmates by single-layer mesh screens. Assessing how much time bees spent contacting the two mesh screens allowed us to quantify simultaneously how much bees sought proximity and interaction with other bees and their preference for nestmates over non-nestmates. Both sociability and nestmate affiliation could be detected soon after emergence as an adult. Isolation early in adult life impaired honey bee sociability but there was no evidence for a critical period for the development of the trait, as isolated bees exposed to their hive for 24 h when as old as 6 days still recovered high levels of sociability. Our data show that, even for advanced social insects, sociability is a developmental phenomenon and experience dependent.

What are the Mechanisms Behind a Parasite-Induced Decline in Nestmate Recognition in Ants?

Social insects have developed sophisticated recognition skills to defend their nests against intruders. They do this by aggressively discriminating against non-nestmates with deviant cuticular hydrocarbon (CHC) signatures. Studying nestmate recognition can be challenging as individual insects do not only vary in their discriminatory abilities, but also in their motivation to behave aggressively. To disentangle the influence of signaling and behavioral motivation on nestmate recognition, we investigated the ant Temnothorax nylanderi, where the presence of tapeworm-infected nestmates leads to reduced nestmate recognition among uninfected workers. The parasite-induced decline in nestmate recognition could be caused by higher intra-colonial cue diversity as tapeworm-infected workers are known to exhibit a modified hydrocarbon signature. This in turn may broaden the neuronal template of their nestmates, leading to a higher tolerance towards alien conspecifics. To test this hypothesis, we exchanged infected ants between colonies and analyzed their impact on CHC profiles of uninfected workers. We demonstrate that despite frequent grooming, which should promote the transfer of recognition cues, CHC profiles of uninfected workers neither changed in the presence of tapeworm-infected ants, nor did it increase cue diversity among uninfected nestmates within or between colonies. However, CHC profiles were systematically affected by the removal of nestmates and addition of non-nestmates, independently from the ants' infection status. For example, when non-nestmates were present workers expressed more dimethyl alkanes and higher overall CHC quantities, possibly to achieve a better distinction from non-nestmates. Workers showed clear task-specific profiles with tapeworm-infected workers resembling more closely young nurses than older foragers. Our results show that the parasite-induced decline in nestmate recognition is not due to increased recognition cue diversity or altered CHC profiles of uninfected workers, but behavioral changes might explain tolerance towards intruders.

Facultative slave-making ants Formica sanguinea label their slaves with own recognition cues instead of employing the strategy of chemical mimicry.

Slave-making ant species use the host workforce to ensure normal colony functioning. Slaves are robbed as pupae from their natal nest and after eclosion, assume the parasite colony as their own. A possible factor promoting the successful integration of slaves into a foreign colony is congruence with the slave-makers in terms of cuticular hydrocarbons, which are known to play the role of recognition cues in social insects. Such an adaptation is observed in the obligate slave-making ant species, which are chemically adjusted to their slaves. To date, however, no reports have been available on facultative slave-making species, which represent an earlier stage of the evolution of slavery. Such an example is Formica sanguinea, which exploit F. fusca colonies as their main source of a slave workforce. Our results show that F. sanguinea ants have a distinct cuticular hydrocarbon profile, which contains compounds not present in free-living F. fusca ants from potential target nests. Moreover, enslaved F. fusca ants acquire hydrocarbons from their slave-making nestmates to such an extent that they become chemically differentiated from free-living, conspecific ants. Our study shows that F. sanguinea ants promote their own recognition cues in their slaves, rather than employing the strategy of chemical mimicry. Possible reasons why F. sanguinea is not chemically well adjusted to its main host species are discussed in this paper.

Discriminatory abilities of facultative slave-making ants and their slaves.

Intra-colony odor variability can disturb ants' ability to discriminate against intruders. The evolutionary relevance of this phenomenon can be revealed by studies on colonies of slave-making ants in which the parasite, and not the host, is subject to selection pressures associated with living in a mixed colony. We examined how the European facultative slave-making species Formica sanguinea and its F. fusca slaves perform in discriminating ants from alien colonies. Results of behavioral assays showed that slave-maker ants respond with hostility to conspecific individuals from alien colonies but are relatively tolerant to alien slaves. Furthermore, the behavior of slaves indicated a limited ability to discriminate ants from alien parasitic colonies. The subdivision of colony fragments into mixed and species-separated groups demonstrated that contact with the parasite is necessary for F. fusca slaves to be re-accepted by former nestmates after a period of separation from the stock colony. The results presented in this paper are consistent with the following hypotheses: (1) F. sanguinea ants, as opposed to their slaves, are adapted to discriminate alien individuals in the conditions of odor variability found in a mixed-species colony, (2) the recognition of slaves by F. sanguinea ants involves a dedicated adaptive mechanism that prevents aggression toward them, (3) the odor of slaves is strongly influenced by the parasite with beneficial effect on the colony integrity.