Nestmate Recognition Research Articles

Learning and perceptual similarity among cuticular hydrocarbons in ants

Nestmate recognition in ants is based on perceived differences in a multi-component blend of hydrocarbons that are present on the insect cuticle. Although supplementation experiments have shown that some classes of hydrocarbons, such as methyl branched alkanes and alkenes, have a salient role in nestmate recognition, there was basically no information available on how ants detect and perceive these molecules. We used a new conditioning procedure to investigate whether individual carpenter ants could associate a given hydrocarbon (linear or methyl-branched alkane) to sugar reward. We then studied perceptual similarity between a hydrocarbon previously associated with sugar and a novel hydrocarbon. Ants learnt all hydrocarbon-reward associations rapidly and with the same efficiency, regardless of the structure of the molecules. Ants could discriminate among a large number of pairs of hydrocarbons, but also generalised. Generalisation depended both on the structure of the molecule and the animal’s experience. For linear alkanes, generalisation was observed when the novel molecule was smaller than the conditioned one. Generalisation between pairs of methyl-alkanes was high, while generalisation between hydrocarbons that differed in the presence or absence of a methyl group was low, suggesting that chain length and functional group might be coded independently by the ant olfactory system. Understanding variations in perception of recognition cues in ants is necessary for the general understanding of the mechanisms involved in social recognition processes based on chemical cues.

Odor-Based Recognition of Familiar and Related Conspecifics: A First Test Conducted on Captive Humboldt Penguins (Spheniscus humboldti)

Studies of kin recognition in birds have largely focused on parent-offspring recognition using auditory or visual discrimination. Recent studies indicate that birds use odors during social and familial interactions and possibly for mate choice, suggesting olfactory cues may mediate kin recognition as well. Here, we show that Humboldt penguins (Spheniscus humboldti), a natally philopatric species with lifetime monogamy, discriminate between familiar and unfamiliar non-kin odors (using prior association) and between unfamiliar kin and non-kin odors (using phenotype matching). Penguins preferred familiar non-kin odors, which may be associated with the recognition of nest mates and colony mates and with locating burrows at night after foraging. In tests of kin recognition, penguins preferred unfamiliar non-kin odors. Penguins may have perceived non-kin odors as novel because they did not match the birds' recognition templates. Phenotype matching is likely the primary mechanism for kin recognition within the colony to avoid inbreeding. To our knowledge this is the first study to provide evidence of odor-based kin discrimination in a bird.

The role of wax and resin in the nestmate recognition system of a stingless bee, Tetragonisca angustula

Recent research has shown that entrance guards of the stingless bee Tetragonisca angustula make less errors in distinguishing nestmates from non-nestmates than all other bee species studied to date, but how they achieve this is unknown. We performed four experiments to investigate nestmate recognition by entrance guards in T. angustula. We first investigated the effect of colony odours on acceptance. Nestmates that acquired odour from non-nestmate workers were 63% more likely to be rejected while the acceptance rate of non-nestmates treated with nestmate odour increased by only 7%. We further hypothesised that guards standing on the wax entrance tube might use the tube as an odour referent. However, our findings showed that there was no difference in the acceptance of non-nestmates by guards standing on their own colony’s entrance tube versus the non-nestmate’s entrance tube. Moreover, treatment of bees with nestmate and non-nestmate resin or wax had a negative effect on acceptance rates of up to 65%, regardless of the origin of the wax or resin. The role of resin as a source of recognition cues was further investigated by unidirectionally transferring resin stores between colonies. Acceptance rates of nestmates declined by 37% for hives that donated resin, contrasting with resin donor hives where acceptance of non-nestmates increased by 21%. Overall, our results confirm the accuracy of nestmate recognition in T. angustula and reject the hypothesis that this high level of accuracy is due to the use of the wax entrance tubes as a referent for colony odour. Our findings also suggest that odours directly acquired from resin serve no primary function as nestmate recognition cues. The lack of consistency among colonies plus the complex results of the third and fourth experiments highlight the need for further research on the role of nest materials and cuticular profiles in understanding nestmate recognition in T. angustula.

Conspecific Ant Aggression is Correlated with Chemical Distance, but not with Genetic or Spatial Distance

Five possible mechanisms might underlie kin recognition in social groups: spatial location, familiarity through prior association, phenotype matching, recognition alleles, or rejecting unfamiliar cues. Kin recognition by phenotype matching relies on a strong correlation between genotype and phenotype. Aggression bioassays are the standard method for investigating recognition in animals, particularly social insect interactions among nestmates and non-nestmates. These bioassays typically pay little regard to how outcomes are determined by differences in chemical recognition cues of the test subjects, because the system of signal coding was unknown until recently. We exploited the known nestmate recognition system of the ant Formica exsecta to investigate aggression between 24 pairs of colonies across a range of chemical (Z9-alkene & n-alkanes), genetic, and spatial distances. The whole Z9-alkene chemical profile was the only significant (p<0.001) predictor of aggression levels. Aggression was a nonlinear step function of Z9-alkene chemical distance, where a small change in chemical profile resulted in a rapid behavioural transition from non-aggression to overt aggression. These findings raise questions surrounding our current understanding of recognition systems, because they support phenotype matching to a colony chemical profile without a significant genetic or spatial component.

Blochmannia endosymbionts and their host, the ant Camponotus fellah: Cuticular hydrocarbons and melanization

Carpenter ants (genus Camponotus) have mutualistic, endosymbiotic bacteria of the genus Blochmannia whose main contribution to their hosts is alimentary. It was also recently demonstrated that they play a role in improving immune function as well. In this study, we show that treatment with an antibiotic produces a physiological response inducing an increase in both the quantity of cuticular hydrocarbons and in the melanization of the cuticle probably due to a nutritive and immunological deficit. We suggest that this is because it enhances the protection the cuticle provides from desiccation and also from invasions by pathogens and parasites. Nevertheless, the cuticular hydrocarbon profile is not modified by the antibiotic treatment, which indicates that nestmate recognition is not modified.

Learning and discrimination of cuticular hydrocarbons in a social insect

Social insect cuticular hydrocarbon (CHC) mixtures are among the most complex chemical cues known and are important in nest-mate, caste and species recognition. Despite our growing knowledge of the nature of these cues, we have very little insight into how social insects actually perceive and discriminate among these chemicals. In this study, we use the newly developed technique of differential olfactory conditioning to pure, custom-designed synthetic colony odours to analyse signal discrimination in Argentine ants, Linepithema humile. Our results show that tri-methyl alkanes are more easily learned than single-methyl or straight-chain alkanes. In addition, we reveal that Argentine ants can discriminate between hydrocarbons with different branching patterns and the same chain length, but not always between hydrocarbons with the same branching patterns but different chain length. Our data thus show that biochemical characteristics influence those compounds that ants can discriminate between, and which thus potentially play a role in chemical signalling and nest-mate recognition.

Flagellar sensilla of the eusocial gall-inducing thrips Kladothrips intermedius and its kleptoparasite, Koptothrips dyskritus (Thysanoptera: Phlaeothripinae)

Insect antennal flagella host a multitude of sensory organs fulfilling different functions. Chemoreception, for example, is essential for insects in many contexts. Both olfaction and contact chemoreception are involved in host-plant selection, as well as in the integrity of insect societies, especially for nestmate recognition. Kladothrips intermedius is a eusocial gall-inducing thrips with two castes: dispersers and soldiers. Koptothrips dyskritus is a specialist in invading Kl. intermedius galls, killing the occupants, and thereby gaining the food and shelter offered by galls. In this study, we compared the morphology and ultrastructure of the flagellar sensilla of Kl. intermedius and Ko. dyskritus via scanning and transmission electron microscopy in order to facilitate future investigations of their sensory ecology, with an emphasis on chemical ecology. The two species show a very similar sensillar array. There are a few mechanosensory trichoid and a second type of mechanosensory sensilla, thermo-hygroreceptive sensilla, olfactory single-walled basiconic and double-walled coeloconic sensilla as well as contact chemoreceptive chaetic sensilla. The latter are sexually dimorphic in Kl. intermedius. Dispersers and soldiers of Kl. intermedius do not present noteworthy morphological differences, but the ultrastructural investigations revealed that soldiers have fewer ORNs, possibly an adaptation to their gall-cloistered lifestyle.

Incipient morphological castes in Polistes gallicus (Vespidae, Hymenoptera)

The occurrence of a pre-imaginal caste determination represents a sort of “point of no return” to eusociality. In some social insect taxa, including Polistes species, the occurrence of distinct queen and worker castes is still debated. Before this report, no clear morphological differences between reproductive and non-reproductive individuals were known. Here we show that several pre-imaginal morphological differences of the Van der Vecht organ occur between foundresses (queens) and workers. Geometric morphometrics revealed that queens are characterized by shape deformations of this organ, which is responsible for a typical allometric growth of the secretory area. This organ is predicted to be larger in foundresses compared to workers because its secretion is involved in defense against ants, in nestmate recognition and in preventing workers from challenging for direct reproduction. The results presented here indicate the existence of an incipient morphological caste determination Polistes gallicus and suggest that this species may have passed the “point of no return” for eusociality.

Socially peaceful: foragers of the eusocial bee Lasioglossum malachurum are not aggressive against non-nestmates in circle-tube arenas

Due to the universally found nestmate recognition in eusocial insects, it is predictable that non-nestmates interact aggressively. In sweat bees (Hymenoptera: Halictidae), this trend was largely shown for queen–queen interactions, but data on worker–worker interactions are still scarce and somehow controversial. We studied behavioural interactions between foragers of the eusocial and ground-nesting bee Lasioglossum malachurum within circle-tubes. Independently of colony membership, bees exhibited high frequencies of cooperative behaviours, together with lack of aggression and moderate avoidance of social interactions. The cooperative mutual passing was the most frequently recorded behaviour. Size difference between the opponents had no effects on cooperation or avoidance. In a heterospecific experiment, bee foragers were observed to react more aggressively and to pass very rarely towards cuckoo bees, suggesting that our results were not biased by the circle-tube methodology. Our results and comparisons with other bee species suggest that studying worker interactions may be not enough to predict the social organisation in bees. Whatever the evolutionary meaning of this generalised tolerance towards conspecifics, the present findings are somehow in agreement with recent studies showing that L. malachurum colonies may have imperfect nestmate recognition and often include a mixture of related and unrelated workers.

Cuticular Hydrocarbons Rather Than Peptides Are Responsible for Nestmate Recognition in Polistes dominulus

A colony of social insects is like a fortress where access is allowed only to colony members. The epicuticular mixture of hydrocarbons has been widely reported to be involved in nestmate recognition in insects. However, recent studies have shown that polar compounds (mainly peptides) are also present, mixed with hydrocarbons, on the cuticle of various insects, including the paper wasps of the genus Polistes. As these polar compounds are variable among Polistes species and are perceived by the wasps, this cuticular fraction could also be involved in nestmate recognition. Through MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time of Flight) mass spectrometry analysis, we assessed, for the first time, the intercolonial variability of the cuticular polar fraction of Polistes dominulus in order to evaluate its reliability as source of nestmate recognition cues. We then tested through behavioral assays the importance of the 2 isolated fractions (apolar and polar) in nestmate recognition by presenting them separately to colonies of P. dominulus. Our results showed that the cuticular polar compounds are not colony specific and they are not used by paper wasps to discriminate nestmates from non-colony members. On the contrary, we confirmed that the isolated cuticular hydrocarbons are the chemical mediators prompting nestmate recognition in paper wasps.

Nest membership determines the levels of aggression and cooperation between females of a supposedly communal digger wasp

Females of communal and eusocial Hymenoptera differ greatly in the treatment of conspecifics: communal species lack the nest membership-bias treatment via nestmate (NM) recognition, which is typical of eusocial Hymenoptera. Therefore, an analysis of social interactions can suggest whether a given bee or wasp is communal or eusocial. For this reason, we observed females of the digger wasp Cerceris rubida in experimental dyadic encounters. Because this species had previously been labeled "communal," we expected to find little evidence of a NM bias with respect to cooperation or aggression when paired with NMs and non-nestmates (NNM). Contrary to expectation, females were highly cooperative toward NMs and highly aggressive toward NNMs. Variation in the intensity of aggression toward NNMs partially stems from the degree to which the paired females differed in size. From these results and quantitative comparisons with studies on solitary, communal, and eusocial bee species, it appears that C. rubida has a more complex social organization than previously believed and, in particular, that females of this species exhibit NM recognition, a trait associated with eusociality.

Taste Perception in Honey Bees

Taste is crucial for honeybees for choosing profitable food sources, resins, water sources, and for nestmate recognition. Peripheral taste detection occurs within cuticular hairs, the chaetic and basiconic sensilla, which host gustatory receptor cells and, usually a mechanoreceptor cell. Gustatory sensilla are mostly located on the distal segment of the antennae, on the mouthparts, and on the tarsi of the forelegs. These sensilla respond with varying sensitivity to sugars, salts, and possibly amino acids, proteins, and water. So far, no responses of receptor cells to bitter substances were found although inhibitory effects of these substances on sucrose receptor cells could be recorded. When bees are free to express avoidance behaviors, they reject highly concentrated bitter and saline solutions. However, such avoidance disappears when bees are immobilized in the laboratory. In this case, they ingest these solutions, even if they suffer afterward a malaise-like state or even die from such ingestion. Central processing of taste occurs mainly in the subesophageal ganglion, but the nature of this processing remains unknown. We suggest that coding tastants in terms of their hedonic value, thus classifying them in terms of their palatability, is a basic strategy that a central processing of taste should achieve for survival.

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.

Hydrocarbons on Harvester Ant (Pogonomyrmex barbatus) Middens Guide Foragers to the Nest

Colony-specific cuticular hydrocarbons are used by social insects in nestmate recognition. Here, we showed that hydrocarbons found on the mound of Pogonomyrmex barbatus nests facilitate the return of foragers to the nest. Colony-specific hydrocarbons, which ants use to distinguish nestmates from non-nestmates, are found on the midden pebbles placed on the nest mound. Midden hydrocarbons occur in a concentration gradient, growing stronger near the nest entrance, which is in the center of a 1-2 m diameter nest mound. Foraging behavior was disrupted when the gradient of hydrocarbons was altered experimentally. When midden material was diluted with artificial pebbles lacking the colony-specific hydrocarbons, the speed of returning foragers decreased significantly. The chemical environment of the nest mound contributes to the regulation of foraging behavior in harvester ants.

Workers select mates for queens: a possible mechanism of gene flow restriction between supercolonies of the invasive Argentine ant

Some invasive ants form large networks of mutually non-aggressive nests, i.e., supercolonies. The Argentine ant Linepithema humile forms much larger supercolonies in introduced ranges than in its native range. In both cases, it has been shown that little gene flow occurs between supercolonies of this species, though the mechanism of gene flow restriction is unknown. In this species, queens do not undertake nuptial flight, and males have to travel to foreign nests and cope with workers before gaining access to alien queens. In this study, we hypothesized that male Argentine ants receive interference from workers of alien supercolonies. To test this hypothesis, we conducted behavioral and chemical experiments using ants from two supercolonies in Japan. Workers attacked males from alien supercolonies but not those from their own supercolonies. The level of aggression against alien males was similar to that against alien workers. The frequency of severe aggression against alien males increased as the number of recipient workers increased. Cuticular hydrocarbon profiles, which serve as cues for nestmate recognition, of workers and males from the same supercolony were very similar. Workers are likely to distinguish alien males from males of their own supercolony using the profiles. It is predicted that males are subject to considerable aggression from workers when they intrude into the nests of alien supercolonies. This may be a mechanism underlying the restricted gene flow between supercolonies of Argentine ants. The Argentine ant may possess a distinctive reproductive system, where workers participate in selecting mates for their queens. We argue that the aggression of workers against alien males is a novel form of reproductive interference.

Survival and growth of parasitic Maculinea alcon caterpillars (Lepidoptera, Lycaenidae) in laboratory nests of three Myrmica ant species

The Alcon blue butterfly (Maculinea alcon) parasitizes the nests of several Myrmica ant species. In Denmark, it uses M. rubra and M. ruginodis, but never M. scabrinodis. To further examine the basis of this specificity and local co-adaptation between host and parasite, the pattern of growth and survival of newly-adopted caterpillars of M. alcon in Myrmica subcolonies was examined in the laboratory. M. alcon caterpillars were collected from three populations differing in their host use, and reared in laboratory nests of all three ant species collected from each M. alcon population. While there were differences in the pattern of growth of caterpillars from different populations during the first few months after adoption, which depended on host ant species and the site from which the ants were collected, there was no evidence of major differences in final size achieved. Survival was, however, much higher in nests of M. rubra than in nests of M. ruginodis and M. scabrinodis, even for caterpillars from a population that is never known to use M. rubra as a host in the field. The caterpillars of M. alcon thus do not show local adaptation in their pattern of growth and survival, but instead show a pattern that may reflect different nestmate recognition abilities of the host ants, related to their sociogenetic organisation. The pattern of observed host ant use in the field seems to result from a combination of differences in local host availability and locally adapted infectivity, modulated by smaller differences in survivorship in the nests of the different host ants.

Evolution of cuticular hydrocarbon diversity in ants

The cuticular hydrocarbons (CHCs) of ants provide important cues for nest-mate and caste recognition. There is enormous diversity in the composition of these CHCs, but the manner in which this diversity has evolved is poorly understood. We gathered data on CHC profiles for 56 ant species, relating this information to their phylogeny. We deduced the mode of evolution of CHC profiles by reconstructing character evolution and then relating the number of changes in CHC components along each branch of the phylogeny to the length of the branch. There was a strong correlation between branch length and number of component changes, with fewer changes occurring on short branches. Our analysis thereby indicated a gradual mode of evolution. Different ant species tend to use specific CHC structural types that are exclusive of other structural types, indicating that species differences may be generated in part by switching particular biosynthetic pathways on or off in different lineages. We found limited, and contradictory, evidence for abiotic factors (temperature and rainfall) driving change in CHC profiles.

Colony and Caste Specific Cuticular Hydrocarbon Profiles in the Common Japanese Hornet, Vespa analis (Hymenoptera, Vespidae)

In this study, the influence of individual cuticular hydrocarbons on nestmate recognition in Japanese hornets was investigated. Cuticular hydrocarbons were extracted from the individuals of several colonies of Japanese hornets, Vespa analis Fablicius (Hymenoptera: Vespidae), and analyzed by combined gas chromatography and mass spectrometry (GC-MS). In the case of V. analis, the results of the GC-MS analyses showed 12 major components and 20 minor components. The results showed some intraspecific variation in the quantities of each component of the profiles even within the same colony and same cast. The number of cuticular hydrocarbon compounds of V. analis was relatively high compared with that of V. crabro. Discriminant analysis of the cuticular hydrocarbon profiles of the workers and queens showed that the wasps could be grouped by colony and by caste even if the colonies were located at a close distance. The cuticular hydrocarbon profiles of workers and males were different. Discipline: Insect pest Additional key words: Asian vespines, GC-MS analysis, nestmates recognition *Corresponding author: e-mail tokoro@affrc.go.jp Received 12 May 2010; accepted 15 November 2010.

Nestmate recognition in the stingless bee Frieseomelitta varia (Hymenoptera, Apidae, Meliponini): sources of chemical signals

Social insects use cuticular lipids for nestmate recognition. These lipids are chiefly hydrocarbons that can be endogenously produced or acquired from the environment. Although these compounds are already described as coming from different sources for different groups of social insects, nothing is known about the source of cuticular hydrocarbons in stingless bees. We used behavioural recognition tests and cuticle chemical investigation to elucidate the role of endogenous and environmentally based cues for nestmate recognition in the stingless bee Frieseomelitta varia. We found that although newly emerged workers present specific cuticle patterns according to their nest origin, these compounds are not used for nestmate recognition, since newly emerged workers are broadly accepted in different colonies. The cerumen used in nest construction played an important role in recognition behaviour. Twenty minutes of contact with foreign cerumen was sufficient to increase the rejection rates of nestmates and separate the groups of workers according to their chemical profile. On the other hand, tests of feeding on a common diet showed no effect on chemical cuticle pattern or recognition behaviour.

Analysis of chemical signals in red fire ants by gas chromatography and pattern recognition techniques

Gas chromatographic (GC) profiles of cuticular hydrocarbon extracts obtained from individual and pooled ant samples were analyzed using pattern recognition techniques. Clustering according to the biological variables of social caste and colony were observed. Pooling individual extracts enhanced the recognition of patterns in the GC profile data characteristic of colony. Evidently, the contribution of the ant's individual pattern to the overall hydrocarbon profile pattern can obscure information about colony in the GC traces of cuticular hydrocarbon extracts obtained from red fire ants. Re-analysis of temporal caste and time period data on the cuticular hydrocarbon patterns demonstrates that sampling time and social caste must be taken into account to avoid unnecessary variability and possible confounding. This and the fact that foragers could not be separated from reserves and brood-tenders in all five laboratory colonies studied suggests that cuticular hydrocarbons as a class of sociochemicals cannot model every facet of nestmate recognition in Solenopsis invicta which in turn suggests a potential role for other compounds in the discrimination of alien conspecifics from nestmates.