Chemical mimicry of the ant Oecophylla smaragdina by the myrmecophilous spider Cosmophasis bitaeniata: Is it colony-specific?

Workers of most social insects can distinguish between nestmates and non-nestmates, and actively attack the latter if they attempt to intrude into the nest or surrounding territory. Nevertheless, there are many records of heterospecific organisms living within the nests of social insects, and they are thought to gain access through chemical mimicry. The salticid spider Cosmophasis bitaeniata lives within the leaf nests of the ant Oecophylla smaragdina, where it preys on the ant larvae. We investigated, using behavioural bioassays and chemical analyses, whether the previously reported resemblance of the cuticular hydrocarbons of ant and spider was colony-specific. Behavioural experiments revealed that the spiders can distinguish between nestmate and non-nestmate major workers and are less inclined to escape when confined with ants that are nestmates. More significantly, C. bitaeniata were more likely to capture ant larvae from nestmate minor workers than non-nestmate minor workers. The chemical analyses revealed that the cuticular hydrocarbon profiles of the spiders and the major workers of the ant colonies were colony-specific. However, the hydrocarbon profiles of C. bitaeniata do not match those of the major workers of O. smaragdina from the same colony. Perhaps the colony-specific cuticular hydrocarbon profiles of C. bitaeniata function to obtain prey from the minor workers rather than avoid eliciting aggression from the major workers.     

Food resources, chemical signaling, and nest mate recognition in the ant Formica aquilonia

Animals such as social insects that live in colonies can recognizeintruders from other colonies of the same or different speciesusing colony-specific odors. Such colony odors usually haveboth a genetic and an environmental origin. When within-colonyrelatedness is high (i.e., one or very few reproductive queens),colonies comprise genetically distinct entities, and recognitionbased on genetic cues is reliable. However, when nests containmultiple queens and colonies comprise multiple nests (polydomy),the use of purely genetically determined recognition labelsmay become impractical. This is due to high within-colony geneticheterogeneity and low between-colony genetic heterogeneity.This may favor the use of environmentally determined recognitionlabels. However, because nests within polydomous colonies maydiffer in their microenvironment, the use of environmental labelsmay also be impractical unless they are actively mixed amongthe nests. Using a laboratory experiment, we found that bothisolation per se and diet composition influenced the cuticularchemical profiles in workers of Formica aquilonia. In addition,the level of aggression increased when both the proportionsof dietary ingredients and the availability of food were altered.This suggests that increased aggression was mediated by changesin the chemical profile and that environmental cues can mediaterecognition between colonies. These results also suggest thatthe underlying recognition cues are mutable in response to extrinsicfactors such as the amount and the composition of food.     

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.     

No Experimental Evidence for Host Ant Related Oviposition in a Parasitic Butterfly

The ability of adult butterflies of the genus Maculinea to locate their host ants prior to oviposition has been the subject of much discussion. We studied the egg laying behavior of the dusky large blue Maculinea nausithous whose larvae parasitize colonies of the ant Myrmica rubra. Flowerheads of the initial food plant were sprinkled with soil from ant nests, which contain chemicals involved in the nest recognition behavior of ants. The experiment was conducted to determine whether ant-released chemicals may act as oviposition cues and whether intraspecific competition for suitable plants may force female butterflies to alternative decisions. Host plant choice was not influenced by the presence of nest-derived host-ant cues. Density dependent shifts to less suitable host plants could not be ascertained nor changes in egg laying behavior across the flight period. The observed egg distribution could be primarily explained by host plant characteristics and environmental variability among sites. The result confirms the theory that host ant dependent oviposition appears to be a disadvantageous strategy in the face of resource limitation within ant colonies and the immobility of caterpillars.     

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.     

Intraspecific nestmate recognition in two parabiotic ant species: acquired recognition cues and low inter-colony discrimination

Parabiotic ants—ants that share their nest with another ant species—need to tolerate not only conspecific nestmates, but also nestmates of a foreign species. The parabiotic ants Camponotus rufifemur and Crematogaster modiglianii display high interspecific tolerance, which exceeds their respective partner colony and extends to alien colonies of the partner species. The tolerance appears to be related to unusual cuticular substances in both species. Both species possess hydrocarbons of unusually high chain lengths. In addition, Cr. modiglianii carries high quantities of hereto unknown compounds on its cuticle. These unusual features of the cuticular profiles may affect nestmate recognition within both respective species as well. In the present study, we therefore examined inter-colony discrimination within the two parabiotic species in relation to chemical differentiation. Cr. modiglianii was highly aggressive against workers from alien conspecific colonies in experimental confrontations. In spite of high inter-colony variation in the unknown compounds, however, Cr. modiglianii failed to differentiate between intracolonial and allocolonial unknown compounds. Instead, the cuticular hydrocarbons functioned as recognition cues despite low variation across colonies. Moreover, inter-colony aggression within Cr. modiglianii was significantly influenced by the presence of two methylbranched alkenes acquired from its Ca. rufifemur partner. Ca. rufifemur occurs in two varieties (‘red’ and ‘black’) with almost no overlap in their cuticular hydrocarbons. Workers of this species showed low aggression against conspecifics from foreign colonies of the same variety, but attacked workers from the respective other variety. The low inter-colony discrimination within a variety may be related to low chemical differentiation between the colonies. Ca. rufifemur majors elicited significantly more inter-colony aggression than medium-sized workers. This may be explained by the density of recognition cues: majors carried significantly higher quantities of cuticular hydrocarbons per body surface.     

Parametric and non-parametric masking of randomness in sequence alignments can be improved and leads to better resolved trees

Background

Ants typically distinguish nestmates from non-nestmates based on the perception of colony-specific chemicals, particularly cuticular hydrocarbons present on the surface of the ants' exoskeleton. These recognition cues are believed to play an important role in the formation of vast so-called supercolonies that have been described for some invasive ant species, but general conclusions about the role of these cues are hampered by only few species being studied. Here we use data on cuticular hydrocarbons, aggression and microsatellite genetic markers to investigate the interdependence of chemical recognition cues, genetic distance and nestmate discrimination in the pharaoh ant (Monomorium pharaonis), a widespread pest species, and ask whether introduced populations of this species are genetically differentiated and exhibit intraspecific aggression.

Results

Microsatellite analyses of a total of 35 colonies from four continents revealed extremely high levels of genetic differentiation between almost all colonies (F _ST = 0.751 ± 0.006 SE) and very low within-colony diversity. This implies that at least 34 and likely hundreds more independent lineages of this ant have spread worldwide. Aggression tests involving workers from 14 different colonies showed only low levels of aggression, even between colonies that were geographically and/or genetically very distant. Chemical analyses of groups of worker ants showed that all colonies had the same cuticular compounds, which varied only quantitatively among colonies. There was a positive correlation between geographical and genetic distance, but no other significant relationships were detected between aggression, chemical profile, genetic distance and geographical distance.

Conclusions

The pharaoh ant has a global invasion history of numerous independent introductions resulting in genetically highly differentiated colonies typically displaying surprisingly low levels of intraspecific aggression, a behaviour that may have evolved in the native range or by lineage selection in the introduced range.     

Variation between and within colonies in the termite: morphology, genomic DNA, and behaviour

We investigate the structure between and within colonies of Schedorhinotermes lamanianus (Isoptera: Rhinotermitidae) at a cluster of foraging galleries in Shimba Hills National Reserve, Kenya. Three independent methods (morphometrics of minor soldiers, multilocus fingerprinting from genomic DNA of workers, and aggression tests between workers) yielded concordant results concerning number and spatial extent of colonies as well as variation between and within colonies. At least three colonies exist in our study area. Genetic data reveal that the largest colony is genetically and spatially substructured in three subsidiary nests, which may form reproductive units. These subsidiary nests were not completely isolated as we were able to document exchange of workers. Subsidiary nests may facilitate foundation of colonies by budding which may generate isolation by distance (population viscosity).     

Does cooperation mean kinship between spatially discrete ant nests?

Eusociality is one of the most complex forms of social organization, characterized by cooperative and reproductive units termed colonies. Altruistic behavior of workers within colonies is explained by inclusive fitness, with indirect fitness benefits accrued by helping kin. Members of a social insect colony are expected to be more closely related to one another than they are to other conspecifics. In many social insects, the colony can extend to multiple socially connected but spatially separate nests (polydomy). Social connections, such as trails between nests, promote cooperation and resource exchange, and we predict that workers from socially connected nests will have higher internest relatedness than those from socially unconnected, and noncooperating, nests. We measure social connections, resource exchange, and internest genetic relatedness in the polydomous wood ant Formica lugubris to test whether (1) socially connected but spatially separate nests cooperate, and (2) high internest relatedness is the underlying driver of this cooperation. Our results show that socially connected nests exhibit movement of workers and resources, which suggests they do cooperate, whereas unconnected nests do not. However, we find no difference in internest genetic relatedness between socially connected and unconnected nest pairs, both show high kinship. Our results suggest that neighboring pairs of connected nests show a social and cooperative distinction, but no genetic distinction. We hypothesize that the loss of a social connection may initiate ecological divergence within colonies. Genetic divergence between neighboring nests may build up only later, as a consequence rather than a cause of colony separation.     

Polygyny reduces rather than increases nestmate discrimination cue diversity in Formica exsecta ants

Although the majority of social insect colonies are headed by a single queen, some species possess nests that contain numerous reproductive queens (polygyny), a trait that is particularly widespread amongst the ants. Polygyny is often associated with a lack of conspecific inter-nest aggression between workers. This is hypothesised to result from increased nestmate cue diversity within nests, since polygynous nests are more genetically diverse than monogynous nests. Alternatively, it may reflect the common origin of polygynous nests that form polydomous networks. We exploit the recent discovery that the nestmate discrimination system in the ant Formica exsecta is based on cuticular hydrocarbons to investigate cue (Z9-alkenes) diversity in several monogynous and polygynous populations. Contrary to previous predictions, in all polygynous populations, the variation between nests in the Z9-alkene profiles was reduced relative to that found in monogynous populations. However, nest-specific Z9-alkene profiles with little variation amongst nestmate workers were still maintained irrespective of nest type or population. This suggests a very effective gestalt mechanism that homogenises the chemical discrimination cues, despite genetic diversity within colonies. Although the reduction in variation between nests was associated with reduced worker aggression on the population level, it cannot totally explain the weak aggression associated with polygynous populations.     

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.     

Chemical basis of nest-mate discrimination in the ant Formica exsecta.

Distinguishing nest-mates from non-nest-mates underlies key animal behaviours, such as territoriality, altruism and the evolution of sociality. Despite its importance, there is very little empirical support for such a mechanism in nature. Here we provide data that the nest-mate recognition mechanism in an ant is based on a colony-specific Z9-alkene signature, proving that surface chemicals are indeed used in ant nest-mate recognition as was suggested 100 years ago. We investigated the cuticular hydrocarbon profiles of 10 Formica exsecta colonies that are composed almost entirely of a Z9-alkene and alkane component. Then we showed that worker aggression is only elicited by the Z9-alkene part. This was confirmed using synthetic Z9-alkene and alkane blends matched to the individual colony profiles of the two most different chemical colonies. In both colonies, only glass beads with 'nest-mate' alkene profiles received reduced aggression. Finally, changing the abundance of a single Z9-alkene on live ants was shown to significantly increase the aggression they received from nest-mates in all five colonies tested. Our data suggest that nest-mate discrimination in the social insects has evolved to rely upon highly sensitive responses to relatively few compounds.     

Societies Drifting Apart? Behavioural,Genetic and Chemical Differentiation between Supercolonies in the Yellow Crazy Ant Anoplolepis gracilipes

Background

In populations of most social insects, gene flow is maintained through mating between reproductive individuals from different colonies in periodic nuptial flights followed by dispersal of the fertilized foundresses. Some ant species, however, form large polygynous supercolonies, in which mating takes place within the maternal nest (intranidal mating) and fertilized queens disperse within or along the boundary of the supercolony, leading to supercolony growth (colony budding). As a consequence, gene flow is largely confined within supercolonies. Over time, such supercolonies may diverge genetically and, thus, also in recognition cues (cuticular hydrocarbons, CHC''s) by a combination of genetic drift and accumulation of colony-specific, neutral mutations.

Methodology/Principal Findings

We tested this hypothesis for six supercolonies of the invasive ant Anoplolepis gracilipes in north-east Borneo. Within supercolonies, workers from different nests tolerated each other, were closely related and showed highly similar CHC profiles. Between supercolonies, aggression ranged from tolerance to mortal encounters and was negatively correlated with relatedness and CHC profile similarity. Supercolonies were genetically and chemically distinct, with mutually aggressive supercolony pairs sharing only 33.1%±17.5% (mean ± SD) of their alleles across six microsatellite loci and 73.8%±11.6% of the compounds in their CHC profile. Moreover, the proportion of alleles that differed between supercolony pairs was positively correlated to the proportion of qualitatively different CHC compounds. These qualitatively differing CHC compounds were found across various substance classes including alkanes, alkenes and mono-, di- and trimethyl-branched alkanes.

Conclusions

We conclude that positive feedback between genetic, chemical and behavioural traits may further enhance supercolony differentiation through genetic drift and neutral evolution, and may drive colonies towards different evolutionary pathways, possibly including speciation.     

Nest-moving by the polydomous ant <Emphasis Type="Italic">Cataglyphis iberica</Emphasis>

In this paper we analyze emigration from nests by the polydomous ant Cataglyphis iberica. Social carrying of workers of this species between different nests of the colony is frequent. In Bellaterra (Barcelona, NE Spain), we monitored field emigration of C. iberica by noting for each nest the migratory behavior of C. iberica workers and, when the nests were attacked by another ant species, Camponotus foreli, we noted the number of C. foreli workers involved in the attacks. Emigration of C. iberica from nests was highly variable. We suggest the main factor determining emigration by this species was attack by workers of C. foreli, so emigration from C. iberica nests was much faster when harassment by C. foreli increased. The system of multiple nests of C. iberica enables this species to abandon attacked nests and to reinstall their population in other nests of the same colony. This reduces risk to the colonies because the route between the different nests is well known by transporter workers.     

Colony insularity through queen control on worker social motivation in ants

We investigated the relative contribution of the queen and workers to colony nestmate recognition cues and on colony insularity in the Carpenter ant Camponotus fellah. Workers were either individually isolated, preventing contact with both queen and workers (colonial deprived, CD), kept in queenless groups, allowing only worker-worker interactions (queen deprived, QD) or in queenright (QR) groups. Two weeks post-separation QD and QR workers were amicable towards each other but both rejected their CD nestmates, which suggests that the queen does not measurably influence the colony recognition cues. By contrast, aggression between QD and QR workers from the same original colony was apparent only after six months of separation. This clearly demonstrates the power of the Gestalt and indicates that the queen is not a dominant contributor to the nestmate recognition cues in this species. Aggression between nestmates was correlated with a greater hydrocarbon (HC) profile divergence for CD than for QD and QR workers, supporting the importance of worker-worker interactions in maintaining the colony Gestalt odour. While the queen does not significantly influence nestmate recognition cues, she does influence colony insularity since within 3 days QD (queenless for six months) workers from different colony origins merged to form a single queenless colony. By contrast, the corresponding QR colonies maintained their territoriality and did not merge. The originally divergent cuticular and postpharyngeal gland HC profiles became congruent following the merger. Therefore, while workers supply and blend the recognition signal, the queen affects worker-worker interaction by reducing social motivation and tolerance of alien conspecifics.     

Testing the adjustable threshold model for intruder recognition on Myrmica ants in the context of a social parasite

Social insect colonies are like fortresses, well protected and rich in shared stored resources. This makes them ideal targets for exploitation by predators, parasites and competitors. Colonies of Myrmica rubra ants are sometimes exploited by the parasitic butterfly Maculinea alcon. Maculinea alcon gains access to the ants' nests by mimicking their cuticular hydrocarbon recognition cues, which allows the parasites to blend in with their host ants. Myrmica rubra may be particularly susceptible to exploitation in this fashion as it has large, polydomous colonies with many queens and a very viscous population structure. We studied the mutual aggressive behaviour of My. rubra colonies based on predictions for recognition effectiveness. Three hypotheses were tested: first, that aggression increases with distance (geographical, genetic and chemical); second, that the more queens present in a colony and therefore the less-related workers within a colony, the less aggressively they will behave; and that colonies facing parasitism will be more aggressive than colonies experiencing less parasite pressure. Our results confirm all these predictions, supporting flexible aggression behaviour in Myrmica ants depending on context.     

The Rules of Aggression: How Genetic,Chemical and Spatial Factors Affect Intercolony Fights in a Dominant Species,the Mediterranean Acrobat Ant Crematogaster scutellaris

Nest-mate recognition plays a key role in the biology of ants. Although individuals coming from a foreign nest are, in most cases, promptly rejected, the degree of aggressiveness towards non nest-mates may be highly variable among species and relies on genetic, chemical and environmental factors. We analyzed intraspecific relationships among neighboring colonies of the dominant Mediterranean acrobat ant Crematogaster scutellaris integrating genetic, chemical and behavioral analyses. Colony structure, parental relationships between nests, cuticular hydrocarbons profiles (CHCs) and aggressive behavior against non nest-mates were studied in 34 nests located in olive tree trunks. Bayesian clustering analysis of allelic variation at nine species-specific microsatellite DNA markers pooled nests into 14 distinct clusters, each representing a single colony, confirming a polydomous arrangement of nests in this species. A marked genetic separation among colonies was also detected, probably due to long distance dispersion of queens and males during nuptial flights. CHCs profiles varied significantly among colonies and between nests of the same colony. No relationship between CHCs profiles and genetic distances was detected. The level of aggressiveness between colonies was inversely related to chemical and spatial distance, suggesting a ‘nasty neighbor’ effect. Our findings also suggest that CHCs profiles in C. scutellaris may be linked to external environmental factors rather than genetic relationships.     

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.     

The relationship between the distribution of worker sizes and new worker production in the ant Formica neorufibarbis

While it is commonly assumed that variation in worker sizes within a single ant colony increases colony efficiency, there is little causal evidence of a link between worker size variation and colony performance. I tested whether the range of worker sizes within colonies of the ant species Formica neorufibarbis affected new worker production. Removing large workers from colonies lowered the rate of new worker production. A study of unmanipulated colonies indicated that colonies did not maintain a full range of worker sizes; mean worker head widths varied from 0.89-1.24 mm. Colonies naturally missing large workers did not have lower rates of worker production, suggesting that the relative size, not the absolute size, of workers within colonies was important. These are the first results to directly link the range of worker sizes to a component of colony fitness in a natural setting.     

Nest demographics and foraging behavior of <Emphasis Type="Italic">Apterostigma collare</Emphasis> Emery (Hymenoptera,Formicidae) provide evidence of colony independence

Apterostigma collare Emery is a highly derived fungus-growing ant within the Tribe Attini whose small, fungal nests are found in tropical rain forests. This study focuses on determining the colony structure of A. collare, specifically searching for evidence of polydomy or independence. We surveyed and observed nests in the field, and performed foraging bioassays and dissected nests in the laboratory. We determined the size and contents of nests in field populations. Nests found near other nests were not statistically different in size compared to nests found alone. There was also no statistical difference between near and lone nests regarding the presence of a queen in the nest. Most nests contained one queen with brood and workers, regardless of their proximity to other nests. Observations also were made of foraging and trail-marking behaviors. Foraging activity observed in the field revealed that workers left the nest area and followed trails upwards into the canopy, but they did not interact with foragers from other nearby nests. In a laboratory foraging arena, foragers marked a trail to a food source by dragging the gaster. Bioassays showed that A. collare workers preferred their own foraging trails, but not those of other conspecific colonies. All results suggest that each nest represents an independent colony, supporting a previous report that nests found in close proximity do not constitute a polydomous colony. Received 19 July 2006; revised 23 March 2007; accepted 6 June 2007.