Pavement Ant Workers (<Emphasis Type="Italic">Tetramorium caespitum</Emphasis>) Assess Cues Coded in Cuticular Hydrocarbons to Recognize Conspecific and Heterospecific Non-Nestmate Ants

Most ants live in closed societies from which non-members are excluded through fighting or ritualized displays to protect colony resources. Nestmate recognition is the process by which ants discriminate nestmate from non-nestmate ants. Ants use cues coded in mixtures of long-chain hydrocarbon compounds on the cuticle as nestmate recognition cues. Pavement ants (Tetramorium caespitum) form conspicuous wars between neighboring colonies that are organized after workers meet and make the decision to fight after assessing nestmate recognition cues. These wars involve thousands of individuals. Fighting is ritualized and few ants die in the process. We identified 24 cuticular hydrocarbon compounds, above 1% in relative abundance, in the profile of pavement ants with chain lengths ranging from 15 to 31 carbon atoms. Cuticular lipids contained, in order of abundance: mono-methyl alkanes (45–56%), n-alkanes (range: 16–40% relative abundance), and alkenes (10–20%), with small or trace amounts of di-methyl, tri-methyl alkanes and fatty acids. Results from behavioral tests show that pavement ants assess information in cuticular hydrocarbon profiles to recognize both conspecific and heterospecfic (Pogonomyrmex occidentalis and Camponotus modoc) non-nestmate ants and that the relative abundance of methyl-branched alkanes and alkenes codes for nestmate status, at least for conspecific interactions. Our data add to a growing body of knowledge about how ants use cuticular hydrocarbon based nestmate recognition cues to prevent the intrusion of non-nestmates in to colony space.     

Blending of heritable recognition cues among ant nestmates creates distinct colony gestalt odours but prevents within‐colony nepotism

The evolution of sociality is facilitated by the recognition of close kin, but if kin recognition is too accurate, nepotistic behaviour within societies can dissolve social cohesion. In social insects, cuticular hydrocarbons act as nestmate recognition cues and are usually mixed among colony members to create a Gestalt odour. Although earlier studies have established that hydrocarbon profiles are influenced by heritable factors, transfer among nestmates and additional environmental factors, no studies have quantified these relative contributions for separate compounds. Here, we use the ant Formica rufibarbis in a cross‐fostering design to test the degree to which hydrocarbons are heritably synthesized by young workers and transferred by their foster workers. Bioassays show that nestmate recognition has a significant heritable component. Multivariate quantitative analyses based on 38 hydrocarbons reveal that a subset of branched alkanes are heritably synthesized, but that these are also extensively transferred among nestmates. In contrast, especially linear alkanes are less heritable and little transferred; these are therefore unlikely to act as cues that allow within‐colony nepotistic discrimination or as nestmate recognition cues. These results indicate that heritable compounds are suitable for establishing a genetic Gestalt for efficient nestmate recognition, but that recognition cues within colonies are insufficiently distinct to allow nepotistic kin discrimination.     

The role of cuticular hydrocarbons as chemical cues for nestmate recognition in the invasive Argentine ant (<Emphasis Type="Italic">Linepithema humile</Emphasis>)

Argentine ants (Linepithema humile) in their native South American range, like most other ant species, form spatially restricted colonies that display high levels of aggression toward other such colonies. In their introduced range, Argentine ants are unicolonial and form massive supercolonies composed of numerous nests among which territorial boundaries are absent. Here we examine the role of cuticular hydrocarbons (CHCs) in nestmate recognition of this highly damaging invasive ant using three supercolonies from its introduced range. We conducted behavioral assays to test the response of Argentine ants to workers treated with colonymate or non-colonymate CHCs. Additionally, we quantified the amount of hydrocarbons transferred to individual ants and performed gas chromatography-mass spectrometry (GC/MS) to qualitatively characterize our manipulation of CHC profiles. The GC/MS data revealed marked differences in the hydrocarbon profiles across supercolonies and indicated that our treatment effectively masked the original chemical profile of the treated ants with the profile belonging to the foreign individuals. We found that individual workers treated with foreign CHCs were aggressively rejected by their colonymates and this behavior appears to be concentration-dependent: larger quantities of foreign CHCs triggered higher levels of aggression. Moreover, this response was not simply due to an increase in the amount of CHCs applied to the cuticle since treatment with high concentrations of nestmate CHCs did not trigger aggression.The results of this study bolster the findings of previous studies on social insects that have implicated CHCs as nestmate recognition cues and provide insight into the mechanisms of nestmate recognition in the invasive Argentine ant. Received 6 February 2007; revised 31 May and 27 July 2007; accepted 16 August 2007.     

Nestmate recognition signals of the leaf-cutting ant Atta laevigata

Behavioral tests with field colonies of Atta laevigata were performed in order to identify the source of the odors used in nestmate recognition. We tested the postpharyngeal (PPG) and mandibular glands (MG) as putative organs producing chemical signals for nestmate recognition. Chemical analyses of PPG were also undertaken. With a series of bioassays, we confirmed that nestmate recognition is based on cephalic odors and that these odors come mainly from the mandibular gland secretion. We show chemical evidence that odors from MG are dispersed all over the cuticle. Although odors from PPG elicited colony-specific behavioral responses, the types of behaviors they elicited differed from those of nestmate recognition of whole ants or MG extracts. PPG secretion was characterized by long-chain alkanes and methyl branched alkanes of low volatility, whereas MG contained volatile ketones and alcohols.     

Wax on, wax off: nest soil facilitates indirect transfer of recognition cues between ant nestmates

Social animals use recognition cues to discriminate between group members and non-members. These recognition cues may be conceptualized as a label, which is compared to a neural representation of acceptable cue combinations termed the template. In ants and other social insects, the label consists of a waxy layer of colony-specific hydrocarbons on the body surface. Genetic and environmental differences between colony members may confound recognition and social cohesion, so many species perform behaviors that homogenize the odor label, such as mouth-to-mouth feeding and allogrooming. Here, we test for another mechanism of cue exchange: indirect transfer of cuticular hydrocarbons via the nest material. Using a combination of chemical analysis and behavioral experiments with Camponotus aethiops ants, we show that nest soil indirectly transfers hydrocarbons between ants and affects recognition behavior. We also found evidence that olfactory cues on the nest soil influence nestmate recognition, but this effect was not observed in all colonies. These results demonstrate that cuticular hydrocarbons deposited on the nest soil are important in creating uniformity in the odor label and may also contribute to the template.     

Disentangling environmental and heritable nestmate recognition cues in a carpenter ant

Discriminating between group members and strangers is a key feature of social life. Nestmate recognition is very effective in social insects and is manifested by aggression and rejection of alien individuals, which are prohibited to enter the nest. Nestmate recognition is based on the quantitative variation in cuticular hydrocarbons, which can include heritable cues from the workers, as well as acquired cues from the environment or queen-derived cues. We tracked the profile of six colonies of the ant Camponotus aethiops for a year under homogeneous laboratory conditions. We performed chemical and behavioral analyses. We show that nestmate recognition was not impaired by constant environment, even though cuticular hydrocarbon profiles changed over time and were slightly converging among colonies. Linear hydrocarbons increased over time, especially in queenless colonies, but appeared to have weak diagnostic power between colonies. The presence of a queen had little influence on nestmate discrimination abilities. Our results suggest that heritable cues of workers are the dominant factor influencing nestmate discrimination in these carpenter ants and highlight the importance of colony kin structure for the evolution of eusociality.     

Recognition in a Social Symbiosis: Chemical Phenotypes and Nestmate Recognition Behaviors of Neotropical Parabiotic Ants

Social organisms rank among the most abundant and ecologically dominant species on Earth, in part due to exclusive recognition systems that allow cooperators to be distinguished from exploiters. Exploiters, such as social parasites, manipulate their hosts’ recognition systems, whereas cooperators are expected to minimize interference with their partner’s recognition abilities. Despite our wealth of knowledge about recognition in single-species social nests, less is known of the recognition systems in multi-species nests, particularly involving cooperators. One uncommon type of nesting symbiosis, called parabiosis, involves two species of ants sharing a nest and foraging trails in ostensible cooperation. Here, we investigated recognition cues (cuticular hydrocarbons) and recognition behaviors in the parabiotic mixed-species ant nests of Camponotus femoratus and Crematogaster levior in North-Eastern Amazonia. We found two sympatric, cryptic Cr. levior chemotypes in the population, with one type in each parabiotic colony. Although they share a nest, very few hydrocarbons were shared between Ca. femoratus and either Cr. levior chemotype. The Ca. femoratus hydrocarbons were also unusually long–chained branched alkenes and dienes, compounds not commonly found amongst ants. Despite minimal overlap in hydrocarbon profile, there was evidence of potential interspecific nestmate recognition –Cr. levior ants were more aggressive toward Ca. femoratus non-nestmates than Ca. femoratus nestmates. In contrast to the prediction that sharing a nest could weaken conspecific recognition, each parabiotic species also maintains its own aggressive recognition behaviors to exclude conspecific non-nestmates. This suggests that, despite cohabitation, parabiotic ants maintain their own species-specific colony odors and recognition mechanisms. It is possible that such social symbioses are enabled by the two species each using their own separate recognition cues, and that interspecific nestmate recognition may enable this multi-species cooperative nesting.     

Nestmate recognition in the leaf-cutting antAtta laevigata

Summary We tested matureAtta laevigata colonies in the field to see if the ants used queen substances, environmental odours (in this case odours produced by the nest's fungi), an odour produced by each individual, or a gestalt odour (resulting from odours distributed between nestmates) as a discrimination signal for nestmate recognition. We found that nestmate recognition inA. laevigata appears to be largely based on an odour produced by each nestmate which appears to be concentrated in the head, although other odours may also be used. We found no evidence of genetic relatedness influencing the discrimination ability, nor did ants respond differently to neighbors in comparison to non-neighbors.     

Nest volatiles as modulators of nestmate recognition in the ant Camponotus fellah

When ants from alien colonies encounter each other the stereotypic reaction is usually one of aggressive behavior. It has been shown that factors such as queen-derived cues or nest-odors modulate this reaction. Here we examined whether nest volatiles affect nestmate recognition by observing the reaction of nestmates towards individual workers under one of four regimes: completely isolated; isolated but receiving a constant airflow from the mother colony; as previous but with the passage of nest volatiles towards the isolated ants blocked by adsorption on a SuperQ column; or reversed airflow direction-from the isolated ants to the nest interior. Ants that had been completely isolated for three weeks were subjected to aggressive behavior, but not those that had continued to receive airflow from the mother colony. Adsorbing the nest volatiles from the airflow by SuperQ abolished this difference, with these ants now also being subjected to aggression, indicating that nest volatiles can modulate nestmate recognition. Reverse airflow also reduced the level of aggression but to a lesser extent than airflow directed from the mother colony. In queenless colonies the overall aggression was reduced under all regimes, and there was no effect of flow, suggesting that the volatiles involved are queen-borne. The SuperQ adsorbed volatiles originated from Dufour's gland secretions of both workers and queen, implicating them in the process. Cuticular hydrocarbon profiles were not affected by exposure to nest volatiles, suggesting that the latter either constitute part of the recognition cues or affect worker behavior via a different, as yet elusive mechanism.     

Nest-area marking with faeces: a chemical signature that allows colony-level recognition in seed harvesting ants (Hymenoptera, Formicidae)

Summary. Nestmate recognition systems in ants are largely based on chemical signals. The hydrocarbon fraction of the lipid layer which covers the insect cuticle plays a determinant role in this context. Here we report a novel extension of nestmate and alien recognition – nest area marking with faeces containing the same hydrocarbons as the cuticle of workers – in a harvesting ant, Messor capitatus. Workers of M. capitatus deposit large quantities of brown-yellow material from the hindgut (termed spots) in the vicinity of the nest. Behavioural investigation showed that such spotting behaviour has a communicative value in the context of nest area identification. Anal fluids deposited in the nest surroundings contain colony-specific cues which the ants use to recognize their own nest areas, and distinguish them from foreign areas even in the absence of nestmate or alien ants. Chemical analyses by gas chromatography-mass spectrometry (GC-MS) of the contents of anal spots, rectal sacs, and cuticular extracts revealed that all contain the same long-chained linear and branched hydrocarbons in varying proportions. Importantly, multivariate analyses showed that the relative proportions of these compounds on the cuticle and in spots are colony-specific. This provides a mechanism by which spot marking could be used by workers to define and recognize their colony area, and would represent a simple extension of the existing nestmate recognition template based on colonial cuticular signatures. The ecological and sociobiological implications of these findings are discussed.Received 3 February 2004; revised 10 June 2004; accepted 14 June 2004.     

Postpharyngeal gland secretion as a modifier of aggressive behavior in the myrmicine antManica rubida

The role of postpharyngeal gland secretion in nestmate recognition was tested inManica rubida. Behavioral tests comprised two consecutive encounters between five ants and their color-marked nestmate. The first encounter utilized an untreated ant constituting a control, whereas in the second encounter the marked ant was treated with postpharyngeal gland exudate. Scoring was done using an aggression index obtained by direct observation and an agitation index deduced from a frame-by-frame analysis of videotapes of the various tests. When the glandular secretion originated from an alien ant, the ants became very agitated and were aggressive toward their nestmate. When the exudate originated from a nestmate, the ants generally remained calm, although their rates of self-grooming increased.     

Odour convergence and tolerance between nestmates through trophallaxis and grooming in the ant <Emphasis Type="Italic">Camponotus fellah</Emphasis> (Dalla Torre)

Summary Social isolation provides a useful tool to study nestmate recognition in ants. In Camponotus fellah, reintroduction of 10-day isolated (IS) workers to their colony resulted in intensive trophallaxis and grooming, while longer isolation periods generally provoked rejection of the IS ants. In the first experiment the behaviour of queenless (QL) and queenright (QR) workers towards 10-day IS workers was tested. Trophallaxis of QL or QR with IS workers was of similar magnitude, but was significantly higher than that among the QL or QR, or that between QL and QR workers. Allogrooming was mostly initiated by the resident non-isolated ants (QL or QR) possibly because they detected a slight mismatch between the IS ants odour and their own template, which represents the group odour. It appears that the presence/absence of the queen did not affect nestmate recognition cues of workers.The second experiment demonstrated that 20-day IS workers were strongly aggressed by colony guards, irrespective of whether they were QL or QR. However, if they were permitted to exchange trophallaxis and grooming with 5 young nestmates (companion ants) for 5 days before reintroduction to their colony, aggression was greatly reduced, irrespective of the origin of the companion ants (QR or QL). Chemical analysis showed a significant divergence between the hydrocarbon profiles of IS and both QL and QR groups, but a prior contact of the IS workers with companion ants resulted in re-convergence of their profile with that of the colony. These results demonstrate that nestmate recognition cues are exchanged between workers via trophallaxis and grooming and that they are not dominated by queen cues, two conditions that fulfil Gestalt nestmate recognition signals requirements.Received 26 February 2003; revised 24 July 2003; accepted 1 August 2003.     

Cryptic castes,social context and colony defence in a social bee,Tetragonula carbonaria

Division of labour in social insect colonies is facilitated in two ways: through temporal sharing of tasks or by morphologically specialised castes. In casteless species, colony defence is maintained by morphologically indistinct workers, who lack the obvious defensive specialisation of polymorphic species. Discrimination of intruders is carried out via antenna, which also detects defensive social cues such as alarm pheromones. Despite their functional importance however, antennal morphology is rarely considered in studies of nestmate recognition. We investigated antennal morphology and the necessity of social cues in mediating defensive behaviour across differentially tasked workers of a casteless social bee, Tetragonula carbonaria. Our results suggest that the current understanding of division of labour in casteless worker species remains poorly understood, with differences in antennal morphology and aggression creating morphologically and behaviourally distinct ‘cryptic castes’. Further, we found that defensive behaviour was only elicited near nest odours, highlighting the importance of mediating aggression among workers.     

Mating structure and nestmate relatedness in a communal bee, Andrena jacobi (Hymenoptera, Andrenidae), using microsatellites

Complex eusocial insect societies are generally matrifilial, suggesting kin selection has been of importance in their development. For simpler social systems, factors favouring their existence, in particular kin selection, have rarely been studied. Communal nesting is one of these simple social organizations, and is found in a diversity of insect species. To examine whether kin selection may play a role in the evolution and maintenance of communality, we estimated genetic relatedness of nestmate females of the facultatively communal bee, Andrena jacobi . Microsatellite loci were developed for this species and used to analyse individuals from two populations. Loci were variable, they were in heterozygote deficit and showed positive inbreeding coefficients. This may arise from nonrandom mating; previous observations (Paxton & Tengö 1996) indicate that a large proportion of females mate intranidally with nestmate males in their natal nests before first emerging. Nestmate relatedness was low, no different from zero for all loci in one population and for three of four loci in the other population. The large number of nestmates sharing a common nest (up to 594) may explain the low relatedness estimates, although relatedness was also independent of the number of females sharing a nest. Lack of inclusive fitness payoffs could constrain social evolution in this communal species.     

KIN RECOGNITION: FUNCTIONS AND MECHANISMS A REVIEW

1. The aim of this paper has been to review the theory behind kin recognition to examine the benefits individuals obtain by recognizing their kin and to review the mechanisms used by individuals in their recognition of kin. 2. The ability to discriminate between kin and non-kin, and between different classes of kin gives individuals advantages in fitness greater than individuals unable to recognize their kin. Four specific areas of benefit were considered: altruistic behaviour, co-operative behaviour, parental care and mate choice. Finally the possibility that kin recognition has arisen as a byproduct from some other ability was discussed. 3. Mechanisms of kin recognition were considered with respect to three essential components of kin recognition. The cue used to discriminate kin, how individuals classify conspecifics as kin, etc. and how the ability to recognize kin develops. 4. Individuals can use a number of cues to discriminate kin from non-kin. These were divided into cues presented by conspecifics (conspecific cues), of which three types were considered: individual, genetic and group/colony cues, and non-conspecific cues, environmental, state and no cues. Kin recognition could be achieved by use of all these cues. 5. How individuals classify their conspecifics as kin, etc. can be achieved in a number of ways; dishabituation or self-matching, which require no learning of kinship cues, or by phenotype matching or familiarity, both of which require the learning of kinship information. 6. It may be necessary for individuals to acquire information concerning kinship. This may be learned, and can be achieved in a number of ways; physiological imprinting, exposure learning or associative learning. Acquisition by these means is non-selective, in that the cues which are most salient in the individual's environment will be learned. Selectivity can be introduced into this process to increase the probability of acquiring kinship information by a number of means; learning from parents, sensitive periods for learning and prenatal learning. Finally, kinship information could be supplied by recognition genes. 7. A distinction is drawn between cues which are used by an individual in the discrimination of kin, discriminators, and cues which are used by individuals in the acquisition of information about kinship, acquisitors. 8. Experiments used to support previous categories of mechanisms of kin recognition were examined in the light of this discussion and it was found that the results were open to a number of different interpretations and yielded little specific information about the mechanisms of kin recognition. 9. It was concluded that there was much evidence, both theoretical and experimental to support the proposed benefits individuals gain from recognizing kin, but much more research is required before the mechanisms of kin recognition are fully understood.     

Interactional behaviors of the parasitic beetle Paussus favieri with its ant host Pheidole pallidula: the mimetic role of the acoustical signals

The social parasitic beetle Puussus favieri(Coleoptera,Carabidae,Paussini)performs different types of stridulations,which sclectively mimic those emitted by dif-ferent ant castes of its host Pheidole pallidula(Hymenoptera,Formicidae,Myrmicinae).However,the significance of this acoustical mimicry for the success of the parasitic strat-egy and the behaviors elicited in the host ants by stridulations was unknown.We reared Paussus favieri in Pheidole pallidula colonies and filmed their interacting behaviors.We analyzed in slow motion the behavior of ants near a stridulating beetle.We analyzed sep-arately trains of pulse(Pa+Pb,produced by repeated rubbings)and single pulse(Pc,produced by a single rubbing)of stridulations,clearly recognizable from the shaking up and down of the beetle hind legs.and associated them with differcent ant responscs.The full repertoire of sounds produced by P:favieri elicited benevolent responses both in workers and soldiers.We found that different signals elicit different(sometimes multiplc)bchaviors in ants,with different frequency in the two ant castes.However,Pc(alone or in conjunction with other types of pulses)appears to be the type of acoustic signal mostly responsible for all recorded behaviors.These results indicate that the acoustic channel plays a pivotal role in the host-parasite interaction.Finding that a parasite uses the acoustical channel so intensively,and in such a complicated way to trigger ant bchaviors,indicates that acoustic signals may be more important in ant societies than commonly recognized.     

Evolutionary transition from single to multiple mating in fungus-growing ants

Queens of leafcutter ants exhibit the highest known levels of multiple mating (up to 10 mates per queen) among ants. Multiple mating may have been selected to increase genetic diversity among nestmate workers, which is hypothesized to be critical in social systems with large, long-lived colonies under severe pressure of pathogens. Advanced fungus-growing (leafcutter) ants have large numbers (104-106 workers) and long-lived colonies, whereas basal genera in the attine tribe have small (< 200 workers) colonies with probably substantially shorter lifespans. Basal attines are therefore expected to have lower queen mating frequencies, similar to those found in most other ants. We tested this prediction by analysing queen mating frequency and colony kin structure in three basal attine species: Myrmicocrypta ednaella, Apterostigma collare and Cyphomyrmex longiscapus. Microsatellite marker analyses revealed that queens in all three species were single mated, and that worker-to-worker relatedness in these basal attine species is very close to 0.75, the value expected under exclusively single mating. Fungus growing per se has therefore not selected for multiple queen mating. Instead, the advanced and highly productive social structure of the higher attine ants, which is fully dependent on the rearing of an ancient clonal fungus, may have necessitated high genetic diversity among nestmate workers. This is not the case in the lower attines, which rear fungi that were more recently derived from free-living fungal populations.     

Insights into the microbial world associated with ants

Insects are among the most successful animals of the world in terms of species richness as well as abundance. Their biomass exceeds that of mammals by far. Among insects, ants are of particular interest not only because of their enormous ecological role in many terrestrial ecosystems, but also because they have developed an impressive behavioural repertoire. In fact, a key feature of the evolutionary success of ants is their ability to form complex societies with division of labour among individuals in a colony belonging to different castes such as workers and soldiers. In addition to these complex social interactions of ants, they have shown an extraordinary capacity to build up close associations with other organisms such as other insects, plants, fungi and bacteria. In the present review we attempt to provide an overview of the various symbiotic interactions that ants have developed with microorganisms.     

Nestmate recognition in sweat bees (Lasioglossum zephyrum): Does an individual recognize its own odour or only odours of its nestmates?

We investigated the olfactory mechanism by which guard bees of Lasioglossum zephyrum decide whether to admit conspecifics to their nests. First we set up colonies of young bees, consisting of sisters from a single family or a mixture of bees from two distinct families. These bees were then introduced into colonies other than their own. Our experimental evidence shows that guards learn the odours of their nestmates, then accept or reject other bees on the basis of the similarity of the latters' odours to those of the guards' nestmates. Guards act as though they do not use their own odour as a reference for nestmate recognition. This recognition mechanism enables individuals with different odours to live together; it may also enhance the operation of kin selection by providing a more complete basis for discriminating relatives from non-relatives. No evidence was found that nestmates acquire one another's odours. Such lack of odour transfer may be characteristic of early stages in the evolution of recognition mechanisms.     

The mechanisms of kin discrimination and the evolution of kin recognition in vertebrates: a critical re-evaluation

I re-examine the four most widely proposed mechanisms of kin discrimination among vertebrates and conclude that the current categorization of kin discrimination mechanisms has been counterproductive because it has a hindered a clear understanding of the basic mechanisms by which animals discriminate kin. I suggest that there likely is only one authentic mechanism of kin discrimination and that this mechanism is learning, particularly associative learning and habituation. Observed differences in the way animals discriminate between kin and non-kin are due only to the cues (e.g., individually-distinctive, family-distinctive, or self) that are used, and not to different mechanisms per se. I also consider whether kin discrimination is mediated by specially evolved kin recognition systems, defined as neural mechanisms that allow animals to directly classify conspecifics as either kin or non-kin. A preliminary analysis of vertebrate recognition systems suggests that specialized neural, endocrine, and developmental mechanisms specifically for recognizing kin have not evolved. Rather, kin discrimination results from an extension of other, non-specialized sensory and cognitive abilities of animals, and may be derived from other forms of social recognition, such as individual, group, or species recognition.