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 of nestmate eggs in the ant Formicafusca is based on queen derived cues

Inclusive fitness benefits depend on recognizing the right individuals to interact with. Social insect nests protect themselves from non-kin intruders through nestmate recognition based on chemical cues. The recognition cues on adult individu- als are from a mixture of genetic and environmental sources, but the ontogeny and use of recognition cues on eggs has not been previously assessed. We studied recognition by workers of eggs that were either nestmates or non-nestmates, and the ontogeny of recognition cues on eggs in the ant Formica fusca, a species with precise egg recognition abilities. Workers were able to dis- criminate among freshly laid eggs with no nest derived cues on them, and the egg surface chemicals varied among nests in these eggs, suggesting that queen derived cues are used in nestmate recognition. The results are discussed in the light of their implica- tions on deceptive social parasite strategies and within colony conflicts     

Cuticular hydrocarbon dynamics in young adult Polistes dominulus (Hymenoptera: Vespidae) and the role of linear hydrocarbons in nestmate recognition systems

In social insects, cuticular hydrocarbons (CHCs) play an important role in nestmate discrimination processes, but young individuals are usually not discriminated. We studied CHC changes in young workers of the social wasp Polistes dominulus. A quantitative estimation demonstrated that total quantities of CHCs increased after emergence, with branched alkanes increasing drastically when compared with other classes of hydrocarbons. The relative quantity of longer-chain compounds increased with respect to shorter ones; unsaturated compounds decreased. These changes might reduce the capacity of the cuticle to acquire compounds of environmental origin. We then tested whether individuals acquire hydrocarbons from the environment, and whether this capability equally characterises newly emerged and mature wasps. We exposed wasps of two age classes (adults younger or older than 24 h) to four linear hydrocarbons in turn, and observed how nestmates reacted to their re-introduction into the natal colony. Exposed young wasps elicited significantly more aggressive responses than control sisters; but treated wasps older than 24 h were generally accepted by nestmates. Chemical assays showed that exposed young wasps readily absorbed hydrocarbons; older ones did not incorporate hydrocarbons, suggesting that the chemical profiles of mature wasps are less prone to chemical shifts than those of newly emerged wasps.     

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.     

Interspecific recognition in Chilean parabiotic ant species

Summary In Chile, Camponotus morosus and Solenopsis gayi sometimes co-inhabit a single nest, seemingly in a parabiotic association. To elucidate the nature of this association we conducted behavioural tests that measured aggression between homo- and allospecific ants. These tests revealed that C. morosus was aggressive towards alien conspecific and allospecific ants, but tolerated allospecific individuals from the same parabiotic society as well as allospecific individuals from a different parabiotic colony. In contrast S. gayi was much more tolerant towards alien ants whether homo- or allospecific and irrespective of their colony of origin, parabiotic or non-parabiotic.Chemical analyses showed that each species possess a distinct cuticular hydrocarbons profile. Moreover, each species tended to keep its specific profile even when living in association with the other species, in spite of very little acquired allospecific chemicals in C. morosus, dismissing chemical mimicry as the basis of the peaceful co-existence. We hypothesise that the switch from aggression to tolerance as a consequence of parabiotic association exhibited by C. morosus is due to a familiarisation as well as memorisation of the allospecific colonial odour. The finding that the parabiotic C. morosus was tolerant to S. gayi, even if they originated from a non-parabiotic nest points to an odour generalisation ability in this species.Received 12 July 2002; revised 15 January and 11 April 2003; accepted 23 April 2003.     

Absence of aggression but not nestmate recognition in an Australian population of the Argentine ant Linepithema humile

Intraspecific aggression is rare within introduced populations of the Argentine ant Linepithema humile, and colonies exhibit a structure known as unicoloniality, in which aggression among nests is atypical. We document a similar form of colony structure in an introduced population of Argentine ants in Victoria, Australia, in which aggression is extremely rare among nests ranging over hundreds of kilometres. However, using a highly sensitive behavioural bioassay we found that workers display subtle differences in their behaviour towards non-nestmates and nestmates. In particular, non-nestmates consistently engage in antennating behaviour with greater frequency than nestmates, perhaps providing a mechanism for homogenization of nest odour. Further, we found that non-nestmates at seaport sites (where populations may derive from multiple introductions) antennate each other with greater frequency than their counterparts from non-seaport sites. These data suggest that the Victorian population of L. humile may comprise multiple independent introductions. Received 4 July 2007; revised 15 January and 4 March 2008; accepted 4 March 2008.     

Role of cuticular hydrocarbons in the chemical recognition between ant species in the Pachycondyla villosa species complex

Cuticular hydrocarbons (HCs) play important roles in insect communication but few studies clearly demonstrate the direct link between HCs and nestmate recognition. Therefore, cuticular lipids were extracted from ants, their HC and non-HC fractions as well as the three principal classes of HCs (n-alkanes, branched alkanes and alkenes) were purified and tested using an immobilizing "joust" device which allowed quantification of early pairwise behavioural responses, mandibular opening and antennal retraction, without occurrence of subsequent damages as in classic dyadic encounters. Chemical recognition of ants was studied at three levels of interactions (intra-colonial, intra-specific and inter-specific). Three closely related species already chemically characterized were used: Pachycondyla villosa (Pv), P. inversa (Pi) and P. subversa (Ps). Each species had its own behavioural responses. Moreover, responses of Pi and Ps towards Pv were significantly longer, than they were between themselves whereas Pv ants were equally aggressive towards Pi and Ps. These differences are in agreement with the results of the cluster analysis of the cuticular HCs profiles that place Pi closer to Ps. In support of the idea that components of cuticular lipids profiles are important for recognition, we found that only the HC fraction and its branched subfraction elicited a behavioural response of Ps workers. It is suggested that internally branched methyl- and dimethylalkanes are involved in recognition behaviour.     

Early learning of volatile chemical cues leads to interspecific recognition between two ant species

Nestmate recognition in social insects generally involves matching a label to the template that is acquired through the early learning of non-volatile cuticular hydrocarbon cues. However, a possible role of the volatile chemical cues that exist in the nest, and which may also affect template formation, has not been studied. We investigated this possibility using experimental mixedspecies groups composed of the two ant species Manica rubida and Formica selysi. The experimental set-up either allowed full contact between workers of the two species or interspecific contact was hindered or prohibited by a single or a double mesh. After three months, workers of M. rubida ants were selected as focal ants for aggression tests including the following target ants: F. selysi workers from the same mixed-species group (for each of the three rearing conditions) or from a single-species group (control). Workers of M. rubida were always amicable towards their group-mates, irrespective of the experimental group (contact, single or double mesh). However, M. rubida that were not imprinted on F. selysi, expressed high levels of aggression towards the non-familiar F. selysi workers. The finding that F. selysi workers in the mixed-species groups appeared familiar to their M. rubida group-mates even without physical contact between them, suggests that the volatile cues produced by F. selysi affected nestmate recognition in M. rubida. In an attempt to identify these volatile cues we performed SPME analysis of the head space over groups of F. selysi workers. The findings revealed that F. selysi Dufour’s gland constituents, with undecane as the major product, are released into the head space, rendering them likely candidates to affect template formation in M. rubida. Analysis of Dufour’s gland secretion of F. selysi revealed a series of volatile alkanes, including undecane as a major product. These alkanes were not present in the glandular secretion of M. rubida, whose secretion was mainly composed of isomers of farnesene. We therefore hypothesize that callow M. rubida workers in the mixed-species groups had become imprinted by the above alkanes (in particular undecane, being the major heterospecific volatile in the head space) and incorporated them into their own template. Received 18 October 2007; revised 2 January 2008; accepted 7 January 2008.     

Discrimination of nestmate workers and drones in honeybees

Summary Discrimination of nestmates from non-nestmates has mainly been investigated in female social insects. Little is known about discrimination of males. Here we show that under natural conditions at the nest entrance, honeybee workers can discriminate nestmate drones from non-nestmate drones as effectively as they can discriminate nestmate workers from non-nestmate workers.     

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.     

Hydrocarbon-released nestmate aggression in the Argentine ant, Linepithema humile, following encounters with insect prey

Argentine ants, Linepithema humile, were attacked by their nestmates following contact with a particular prey item, the brown-banded cockroach, Supella longipalpa. Contact with prey, as brief as 2 min, provoked nestmate aggression. Argentine ants contaminated with hydrocarbons extracted from S. longipalpa also released nestmate aggression behavior similar to that released by the whole prey item, confirming the involvement of hydrocarbons. In contrast to S. longipalpa, little or no nestmate aggression was induced by other ant prey from diverse taxa. A comparison of prey hydrocarbon profiles revealed that all hydrocarbons of S. longipalpa were very long chain components with 33 or more carbons, while other prey had either less, or none, of the very long chain hydrocarbons of 33 carbons or greater. We identified the hydrocarbons of S. longipalpa and some new groups of long chain hydrocarbons of L. humile. The majority of S. longipalpa hydrocarbons were 35 and 37 carbons in length with one to three methyl branches, and closely resembled two previously unidentified groups of compounds from L. humile of similar chain length. The hydrocarbons of S. longipalpa and L. humile were compared and their role in the Argentine ant nestmate recognition is discussed.     

Nestmate recognition and identification of cuticular hydrocarbons composition in the swarm founding paper wasp Ropalidia opifex

Eusocial polistine wasps comprise species in which new colonies are founded by single wasps or groups of foundresses (independent founding species) or by swarms of queens and workers (swarm founding species). The first ones have relatively small societies generally comprising only one egg-laying queen, while the second ones have larger colonies where several egg-laying queens are usually present. These differences in social organisation are expected to influence the acceptance threshold of conspecifics in the colony and the intra-colony communication modalities. In this paper, we showed by field experiments that Ropalidia opifex (a swarm founding species) presents a good discrimination between alien and nestmate conspecifics. Moreover by GC/MS, we identified 19 cuticular hydrocarbons in this species, and we demonstrated that cuticular signature in this species depends mainly on colony membership, not showing any correlation with fertility.     

Host recognition cues of the granary weevil parasitoid Lariophagus distinguendus

Host recognition was examined in Lariophagus distinguendus (Förster), a parasitoid of larvae of the granary weevil Sitophilus granarius (L.) that live endophytically in wheat grains. On encountering a grain infested with S. granarius, females of L. distinguendus behave in a set sequence. First they show antennal drumming on the grain, second they tap with the tip of the abdomen on the grain surface, third they drill into the grain and then insert their ovipositor. Bioassays revealed that drumming and drilling was stimulated by non-volatile chemicals present on the grain-host complex. Host faeces and herbivore damaged grain material stimulated the most activity, followed by artificially damaged grain, and healthy grain. This is the first report on non-volatile chemicals released from herbivore-damaged seeds as signals for foraging parasitoids. Volatile chemicals from the faeces alone were not active. Experiments on the use of physical cues revealed that the presence of a three-dimensional structure increased the response towards chemicals from the faeces. The shape (ovoid or rectangular solid) and colour (brown or white) of the structure had no impact. Thus, physical cues alone were insufficient to stimulate host recognition behaviour, but acted by increasing the response towards the chemical stimuli.     

Intra- and interspecific nestmate recognition inMelipona workers (Hymenoptera: Apidae)

The possible significance of nestmate recognition in prevention of robbing and parasitism in three species of stingless bees was assessed. Nestmate discrimination abilities vary among them; Melipona quadrifasciataworkers attacked 74% of nonnestmate conspecifics that were encountered, while M. scutellarisand M. rufiventriswere less discriminating, attacking only 14 and 60% of non-nestmates, respectively. In tests of interspecific interactions, M. quadrifasciataand M. scutellariswere the least mutually tolerant of all species pairs tested. Tests with Apis melliferashowed a high degree of intolerance by two of the three Meliponaspecies.     

Effects of nest paper hydrocarbons on nest and nestmate recognition in colonies ofPolistes metricus say

The role of nest paper hydrocarbons in nest and nestmate recognition for the social waspPolistes metricus was examined. Newly emergedP. metricus workers maintained in the laboratory spent four days alone on a fragment of nest paper that was subjected to one of the following tretments: untreated, extracted with hexane to remove surface hydrocarbons, or extracted with extract reapplied. Test wasps were returned to their natal nest with nestmates and observed for 1 h. Time spent on nest by test wasp and its behaviors were recorded. Wasps exposed to untreated and reapplied nest fragments spent an average of 34.13 and 31.75 min on their nests, respectively, while wasps from extracted fragments averaged 17.19 min. Behavior of wasps exposed to extracted paper differed significantly from wasps exposed to paper with hydrocarbons. These results suggest that exposure to nest paper hydrocarbons is important for both nest and nestmate recognition.     

Divide and conquer: Multicolonial structure,nestmate recognition,and antagonistic behaviors in dense populations of the invasive ant Brachymyrmex patagonicus

The ecological success of ants has made them abundant in most environments, yet inter‐ and intraspecific competition usually limit nest density for a given population. Most invasive ant populations circumvent this limitation through a supercolonial structure, eliminating intraspecific competition through a loss of nestmate recognition and lack of aggression toward non‐nestmates. Native to South America, Brachymyrmex patagonicus has recently invaded many locations worldwide, with invasive populations described as extremely large and dense. Yet, in contrast with most invasive ants, this species exhibits a multicolonial structure, whereby each colony occupies a single nest. Here, we investigated the interplay between genetic diversity, chemical recognition, and aggressive behaviors in an invasive population of B. patagonicus. We found that, in its invasive range, this species reaches a high nest density with individual colonies located every 2.5 m and that colony boundaries are maintained through aggression toward non‐nestmates. This recognition and antagonism toward non‐nestmates is mediated by chemical differentiation between colonies, as different colonies exhibit distinct chemical profiles. We highlighted that the level of aggression between colonies is correlated with their degree of genetic difference, but not their overall chemical differentiation. This may suggest that only a few chemical compounds influence nestmate recognition in this species or that weak chemical differences are sufficient to elicit aggression. Overall, this study demonstrates that invasive ant populations can reach high densities despite a multicolonial structure with strong aggression between colonies, raising questions about the factors underlying their ecological success and mitigating negative consequences of competitive interactions.     

Nest surface hydrocarbons facilitate nestmate recognition for the social wasp,Polistes metricus Say (Hymenoptera: Vespidae)

Nestmate recognition was observed inPolistes metricus Say workers only if the workers were exposed to their nest surface hydrocarbons after eclosion. If the newly emerged workers were never exposed to the nest hydrocarbons as adults, they showed no discrimination between nestmates and nonnestmates. Furthermore, the newly emerged workers were accepted more readily by their experienced nestmates than by experienced nonnestmates only if the newly emerged wasps had been exposed to the nest surface hydrocarbons. This reciprocal recognition implies that the nest recognition cues are nest surface hydrocarbons that are learned and that may be acquired byP. metricus workers as adults on the natal nest.     

Nestmate recognition in a neotropical polygynous wasp

In social insects, nestmate recognition systems can be dynamic and modulated in response to various kinds of genetic and environmental cues. For example, multiple-queen colonies can possess weak recognition abilities relative to single-queen colonies, due to broader exposure to heritable and environmentally derived nestmate recognition cues.We conducted field experiments to examine nestmate recognition ability in a neotropical polygynous wasp, Polybia paulista. Despite the fact that the effective queen number in P. paulista is the highest ever recorded in polygynous wasps, this species exhibits a well functioning nestmate recognition system, which allows colony entry only to nestmate individuals. Similar to other social Hymenoptera, young wasps express colony specific chemical signatures within several days after emergence. This is the first study to show that the polygynous epiponine wasp is able to distinguish nestmates from non-nestmates. Received 23 May 2006; revised 6 October 2006; accepted 23 October 2006.     

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.