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.     

Acceptance by Honey Bee Guards of Non‐Nestmates is not Increased by Treatment with Nestmate Odours

Honey bee, Apis mellifera, entrance guards use chemical cues to discriminate nestmates from non‐nestmates. Previous research has shown that when wax combs are reciprocally swapped between two colonies, guards become more accepting of workers from the swap partner. However, when combs were transferred only one way, guards in the comb‐receiver colony became more accepting of bees from the comb‐donor colony, but not vice versa. Hence, the increased acceptance of non‐nestmates caused by reciprocal comb swapping was not because of introduced bees acquiring odours from the transferred combs, which was surprising because comb wax was known to affect the odour of bees. In the current experiment, we caused workers to acquire either nestmate or non‐nestmate odours by holding them for 15 min in a tube, which had previously held nestmates or non‐nestmates and then measured their acceptance by entrance guards of nestmate or non‐nestmate hives. When transferred workers had acquired odours of non‐nestmates, acceptance by their own colony’s guards significantly decreased to 66% from 91%. Conversely, the acceptance of non‐nestmates that had acquired odours of the guards’ own nestmates was unchanged, 25% vs. 25%. These results show that when equivalent changes in the odour of introduced bees are made, guards are more sensitive to changes that cause nestmates to acquire non‐nestmate odours than vice versa. These results are also a likely explanation for the earlier and surprising results from the unidirectional comb swap experiment ( Couvillon et al. 2007 ). We make a hypothesis for the underlying mechanism in terms of a multidimensional recognition cue space.     

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.     

Could learning of pollen odours by honey bees (Apis mellifera) play a role in their foraging behaviour?

Abstract. The role of pollen odour cues in the foraging behaviour of honey bees (Apis mellifera L.) is poorly understood. Using classical conditioning of the proboscis extension response, in which bees learn to associate an odour with a sucrose reward, the present study tests whether odours of bee-collected pollen from the hive environment or odours of fresh pollen on the anthers of flowers could be used in pollen foraging. Honey bees efficiently learn odours from field-bean (Vicia faba) bee-collected pollen and oilseed-rape (Brassica napus) bee-collected pollen, hand-collected pollen, anthers and whole flowers, demonstrating that honey bees can learn pollen odours associatively in biologically realistic concentrations. Honey bees learn pollen odours of oilseed rape better than field bean and, although they generalize these two odours, they easily distinguish between them in discrimination tests, suggesting that pollen odours may be used in species recognition/discrimination. There is little evidence that honey bees can recognize whole flowers based on previous experience of bee-collected pollen odour. However, they generalize the odours of oilseed-rape anthers and whole flowers, suggesting that anther pollen in situ may play a more prominent role than bee-collected pollen in foraging behaviour.     

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.     

An Easy Method to Test Odour Recognition in Songbird Hatchlings

We describe an easy method to test odour detection and recognition in 1‐d‐old zebra finch hatchlings (Taeniopygia guttata). Day‐old chicks beg in a stereotypical posture, which can be induced by directing gentle puffs of air from a plastic wash bottle near the face. We used this method to experimentally test whether begging duration of chicks was indicative of nest odour recognition. We manipulated the olfactory environment of 12 nests throughout incubation and hatching with either an artificial odour (orange oil) or with a neutral control (tap water). We then presented these two stimulus odours to 25 day‐old chicks and measured the duration of the first begging bout exhibited for each odour. Zebra finches hatched in a nest environment enriched with orange oil scent begged significantly longer when exposed to orange oil odour, compared to control hatchlings. Our simple testing procedure can be used to efficiently quantify odour recognition and/or preference in altricial songbirds at a very early developmental stage.     

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.     

Low intraspecific aggression level in the polydomous and facultative polygynous ant Ectatomma tuberculatum

Nestmate recognition is a key feature of social insects, as it preserves colony integrity. However, discrimination of non‐nestmates and nestmate recognition mechanisms are highly variable according to species and social systems. Here, we investigated the intraspecific level of aggression in the facultative polygynous and polydomous ant, Ectatomma tuberculatum Olivier (Hymenoptera: Formicidae: Ectatomminae), in a population with a strong genetic structure. We found that the intraspecific level of aggression was generally low in this population of E. tuberculatum. However, the level of aggression was significantly correlated with the geographical distance, suggesting that both genetic and environmental cues could be involved in nestmate recognition and discrimination mechanisms. Moreover, polydomy was confirmed by the absence of aggression between workers from nests at a distance of 3 m, while the level of aggression was significantly higher between workers from nests separated by a distance of 10 or 1300 m. Field experiments showed that the low level of aggression between neighbouring colonies was associated with shared foraging areas, which could suggest that familiarization processes may occur in this species. We propose that the particular social organization of this species, with secondary polygyny, polydomy, and budding, may have favoured a high acceptance threshold, because of the low probability of interactions with unrelated conspecifics, the high cost of erroneously rejecting nestmates, and the low cost of accepting non‐nestmate workers. The resulting open recognition system can thus allow privileged relationships between neighbouring colonies and promote the ecological dominance of E. tuberculatum in the mosaic of arboreal ants.     

Polistes dominulus (Hymenoptera: Vespidae) larvae possess their own chemical signatures

Social insects use cuticular hydrocarbons (CHCs) as recognition cues in a variety of social contexts, such as species and nestmate recognition. Discrimination of nestmates is an important requisite to avoid exploitation by unrelated individuals. In social wasps, use of CHCs in nestmate recognition has been demonstrated only among adults, whereas very little is known regarding brood recognition. We performed gas chromatography-mass spectrometry analyses of the CHCs of adults and larvae of the social wasp Polistes dominulus and found that larvae possess a characteristic chemical colony-specific pattern distinct from that of adults. Behavioural assays confirmed that these are recognized and discriminated by adults. Larval epicuticular substances are therefore sufficient for recognition of nestmate larvae by adults and demonstrate that wasps are able to discriminate between alien and nestmate larval odours.     

Affinity towards maternal odour and offspring dispersal in the common lizard

Kin competition is known to strongly influence the spacing behaviour of juveniles in the common lizard, a species which is only weakly social and has no parental care. We examined whether variation in offspring dispersal propensity was associated with variation in their ability to discriminate odours of their mothers versus unknown females. We caught pregnant females in two populations and reared them in the laboratory until parturition. Just after birth, we recorded the juvenile response to the odour of either their mother or an unrelated female. Mothers and offspring were then released at the mother's last capture point, and juvenile movement was monitored by recapture for 2 years. Philopatric individuals were more attracted to the odour of their mother and more repulsed by the odour of an unrelated female than dispersers. In this species, ability to discriminate maternal cues appears to be implied in the spacing behaviour of juveniles. Since juvenile males and females reacted in the same way, the main role of mother discrimination is likely to be the avoidance of kin competition by promoting juvenile dispersal. This does not necessary require mother discrimination to imply mother recognition, or not to be implied in other functions such as inbreeding avoidance. Further research is needed to clarify these points. We suggest that context-dependent kin discrimination may be involved in the spacing behaviour of a large number of species, even for those species where other social functions for kin discrimination have been proposed.     

Weak nestmate discrimination behavior in native and invasive populations of a yellowjacket wasp (<Emphasis Type="Italic">Vespula pensylvanica</Emphasis>)

In geographic regions with warm winters, invasive yellowjacket wasp colonies (genus Vespula) often exhibit polygyny (multiple queens) and persist for multiple years, despite these phenomena being rare in the native range. Here, we test the hypothesis that polygyny, caused by foreign queens being accepted into an existing colony, is the result of relaxed nestmate recognition in the invasive range, as has been observed in some supercolonial invasive ants. In bioassays with wild colonies in the field, we found that nestmate discrimination was weak in both invasive (Hawaii) and native (California) populations of Vespula pensylvanica, with significant nestmate discrimination in only ~?30% of trials. We also found that the diversity and variability of cuticular hydrocarbons, chemical compounds that mediate nestmate recognition, were not reduced in introduced populations, unlike several supercolonial invasive ant species. Our findings suggest that ancestral weak nestmate discrimination behavior of V. pensylvanica may make this species pre-adapted to transition to polygyny and extended colony lifespans when introduced into environments with benign winters that facilitate foreign queens joining existing colonies in late season.     

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.     

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.     

The smell of danger: chemical recognition of fish predators by the invasive crayfish Procambarus clarkii

1. Since avoiding predation can compromise animal fitness, prey are expected to respond to different predator species with an intensity appropriate to the level of risk. In fresh waters, the threat of predation is typically assessed by chemical cues, in particular by odours released by either injured/disturbed conspecifics (conspecific alarm odour) or predators (predator odours). Here, we used the most widely distributed crayfish in the world, the invasive North American Procambarus clarkii, to investigate the relative effectiveness of odours emitted by fish predators compared with conspecific alarm odour. We also tested whether P. clarkii is able to discriminate between fish predators of which it has ‘experience’ (either recent, via introduction to the same water body, or old, by sharing a native range), as well as between fish predators that pose low or high risk. 2. The study was carried out on introduced populations of P. clarkii from two sites, characterised by different fish assemblages: the Malewa River (a tributary of Lake Naivasha, Kenya) and Lake Trasimeno (Italy). Laboratory experiments consisted of three sequential phases (‘water’, ‘food’ and ‘smell’ phases) and five treatments. Treatments differed in the odour presented during the smell phase, i.e. no odour (plain water) and odours from either injured conspecifics or three fish species per site. Crayfish from the Malewa River population were confronted with the odours of largemouth bass (Micropterus salmoides), common carp (Cyprinus carpio) and tilapia (Tilapia zillii) (all introduced to Lake Naivasha but absent from the Malewa River), and those from the Lake Trasimeno population with the odours of the introduced largemouth bass and carp and the native chub (Squalius cephalus). Largemouth bass is the only predator that imposes a high risk to crayfish, and it also shares its native range with P. clarkii. We analysed the time spent by crayfish feeding, in locomotion and in adopting a raised or lowered posture. A reduction in the time spent feeding and in locomotion, and an increase in the time spent in the lowered posture were considered to indicate alarm. 3. Crayfish from both populations responded with a more pronounced reduction in feeding to conspecific alarm odour rather than to predator odours. Crayfish from the Malewa River reacted with the same intensity to the odours of the three fish species tested, whereas, in Lake Trasimeno, the odour of largemouth bass was significantly more threatening than the odours of the other two species. 4. Procambarus clarkii seems to perceive a general fish odour that alerts it to possible predation risk without the need of either a direct recent experience or via sharing a common native range. However, where they coexist with fish, crayfish become able to distinguish among species, adapting the intensity of their response to the effective risk. Our results confirm the relatively high learning capacity of P. clarkii reported in previous studies and suggest the existence of mechanisms that make predator recognition particularly efficient in this extraordinarily successful invader.     

A new ethological test to study nestmate recognition in adult ants

Summary An ethological test was designed to investigate nestmate recognition in adult ants. Contrary to classic dyadic tests, it consisted of a choice situation where a tested-worker was faced with nestmate and non-nestmate workers. These were kept alive, tied down, and thus immobilised in the neutral arena to record the tested-worker's reactions independently of the behaviour of the others. Such tests, applied to the ponerine ant,Ectatomma tuberculatum, enabled us to record various measures of discrimination and to limit aggressive behaviours between alien ants, which often leads to serious injury in dyadic tests. They also demonstrated the influence of age on discrimination behaviour in this species.     

Unexpected,well-developed nestmate recognition in laboratory colonies of polygyne imported fire ants (Hymenoptera: Formicidae)

Aggression bioassays were used to investigate nestmate recognition in polygyne laboratory colonies of the imported fire ant, Solenopsis invictaBuren. Unlike workers from polygyne field colonies, laboratory-maintained (>10 weeks) workers exhibited well-developed nestmate recognition. As in monogyne colonies of this species, both heritable and environmentally acquired (diet) odors provided recognition cues and were roughly additive in their effect. Within diet treatments, polygyne colonies responded in a graded fashion to polygyne conspecifics, monogyne conspecifics, and heterospecifics (S. richteri Forel),thus suggesting incipient genetic divergence between the two S. invictasocial forms. Hypotheses to account for the acute intraspecific discrimination observed in the laboratory are presented. Empirical testing of these hypotheses will illuminate ecological constraints and proximate mechanisms underlying the reduced intercolony discrimination associated with natural polygyne colonies of this and other ant species.     

Kin recognition in the ant Rhytidoponera confusa I. Environmental odour

The relative contribution of genetic and environmental components to the colony-specific recognition odour was assessed for the ant Rhytidoponera confusa, using an aggression assay. Colonies maintained in the laboratory for 6 months were significantly less aggressive to each other than they were to colonies that had been collected from the field within the previous 48 h. Colonies tested 12–24 h after collection from the field showed significantly more aggression against laboratory ants than did the same colonies after they had been kept in the laboratory for 4–5 weeks. Both results indicate that environmental odours can contribute to the colony odour. Nestmates supplied with different diets and nest materials for 4–5 weeks were attacked more than the controls. Though slight, the increase in aggression was significant. This suggests that environmental odours, though significant when odour differences are extreme enough, may nevertheless be less important than genetic odours in nature. This and other studies suggest this pattern may be common in social insects generally.     

Not one odour but two: A new model for nestmate recognition

Recognition systems play a key role in a range of biological processes, including mate choice, immune defence and altruistic behaviour. Social insects provide an excellent model for studying recognition systems because workers need to discriminate between nestmates and non-nestmates, enabling them to direct altruistic behaviour towards closer kin and to repel potential invaders. However, the level of aggression directed towards conspecific intruders can vary enormously, even among workers within the same colony. This is usually attributed to differences in the aggression thresholds of individuals or to workers having different roles within the colony. Recent evidence from the weaver ant Oecophylla smaragdina suggests that this does not tell the whole story. Here I propose a new model for nestmate recognition based on a vector template derived from both the individual’s innate odour and the shared colony odour. This model accounts for the recent findings concerning weaver ants, and also provides an alternative explanation for why the level of aggression expressed by a colony decreases as the diversity within the colony increases, even when odour is well-mixed. The model makes additional predictions that are easily tested, and represents a significant advance in our conceptualisation of recognition systems.     

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.