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-informative recognition cues in ants

Although social groups are characterized by cooperation, they are also often the scene of conflict. In non-clonal systems, the reproductive interests of group members will differ and individuals may benefit by exploiting the cooperative efforts of other group members. However, such selfish behaviour is thought to be rare in one of the classic examples of cooperation--social insect colonies--because the colony-level costs of individual selfishness select against cues that would allow workers to recognize their closest relatives. In accord with this, previous studies of wasps and ants have found little or no kin information in recognition cues. Here, we test the hypothesis that social insects do not have kin-informative recognition cues by investigating the recognition cues and relatedness of workers from four colonies of the ant Acromyrmex octospinosus. Contrary to the theoretical prediction, we show that the cuticular hydrocarbons of ant workers in all four colonies are informative enough to allow full-sisters to be distinguished from half-sisters with a high accuracy. These results contradict the hypothesis of non-heritable recognition cues and suggest that there is more potential for within-colony conflicts in genetically diverse societies than previously thought.     

Behavioral and Electroantennogram Responses of Phorid fly <Emphasis Type="Italic">Pseudacteon tricuspis</Emphasis> (Diptera: Phoridae) to Red Imported Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis> Odor and Trail Pheromone

The phorid fly, Pseudacteon tricuspis is an introduced parasitoid of the red imported fire ant, Solenopsis invicta in the United States. Previous studies show that phorid flies are attracted to host ant workers at disturbed colonies, to colonies exhibiting aggressive interspecific interactions, and to fire ant mating flights. In a series of behavioral and electroantennogram (EAG) experiments, we confirm the possible use of fire ant odor as cues for host location by P. tricuspis. We tested the response of P. tricuspis of different sex and mating status to several host-related odor stimuli including live fire ant workers, extracts of worker whole body, head, thorax, and abdomen, and (E,E)-α-farnesene, a trail pheromone component of Solenopsis fire ants. Results from Y-tube olfactometer bioassays demonstrated the attraction of mated female P. tricuspis to live S. invicta workers. In addition, extracts of S. invicta worker whole body and thorax elicited strong olfactometer response in female flies (mated and unmated) and mated males, but not in unmated males. Pseudacteon tricuspis did not show significant attraction to extracts of S. invicta worker head and abdomen, or to (E,E)-α-farnesene, irrespective of sex and mating status. In EAG experiments, female and male P. tricuspis showed significant EAG response to extracts of worker whole body, head, and abdomen, and to a less extent, thorax extract, but not to (E,E)-α-farnesene. Females showed slightly greater EAG response than males, but EAG response was not affected by mating status. These results suggest that fire ant thorax is likely the source of kairomones used as host location cues by P. tricuspis, and support the hypothesis that fire ant worker trail pheromones are not likely used by P. tricuspis for host location.     

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.     

Nest and colony-specific spectra in the weaver ant Oecophylla smaragdina

Animals in social groups need to differentiate between group members and others. In very large groups, such as those formed by many ant species, it is not possible to rely on individually specific cues to identify colonymates. Instead, recognition must be based on the colony-specific cues. Individual ant colonies tend to have a specific chemical gestalt that is maintained by the continual exchange of chemicals between workers. In very large polydomous colonies, the exchange of chemicals may be limited between nests within the colony, resulting in inter-nest variation in colony odour that might hinder identification of colonymates or conspecific intruders. We used near-infrared spectroscopy to explore variation in the chemical profile between and within colonies of the weaver ant Oecophylla smaragdina. We found that differences between colonies were reflected in the position, amplitude and width of spectral peaks, while differences between nests within colonies were reflected mainly in amplitude. Furthermore, in the context of colonymate recognition, the behaviour of the ants themselves was positively correlated with colony-specific spectral characteristics, rather than with nest-specific characteristics. Thus, colony spectra have features that are not obscured by intra-colonial variation and may potentially encode the chemical characteristics used by workers to identify colonymates.     

红火蚁入侵对棉花粉蚧近距离扩散的促进作用

[背景]红火蚁与棉花粉蚧入侵到同一地区,因侵入生境重叠而相遇,进而产生互惠关系。这种互惠关系对红火蚁、棉花粉蚧的生存、扩散传播、入侵的意义和作用规律、机制等是需要解释的生态学问题。[方法]采用田问试验生态学的方法,通过迁移红火蚁蚁巢、向扶桑上接粉蚧等观察研究了红火蚁入侵对棉花粉蚧近距离扩散的影响。[结果]随着离蚁巢距离的增大,扶桑上工蚁数量逐渐减少,距离为1．0、2．0m时数量较多;发生该蚁区域距蚁巢2．0、3．0m扶桑感染粉蚧比率（75％、45％）显著高于无红火蚁区（25％、10％）,其感染比率与工蚁数量呈显著正相关,符合方程Y=0．0042X＋0．1992。[结论与意义]红火蚁入侵促进了棉花粉蚧的近距离扩散,扩散范围在2—3m。研究结果可为深入了解红火蚁与棉花粉蚧协同入侵规律等提供支持。     

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.     

Repellency of callicarpenal and intermedeol against workers of imported fire ants (Hymenoptera: Formicidae)

Callicarpenal and intermedeol are two insect-repellent terpenoids isolated from leaves of American beautyberry (Callicarpa americana L.; Verbenaceae) and Japanese beautyberry (Callicarpa japonica Thunb.). The repellency of these two terpenoids against workers of red imported fire ants, Solenopsis invicta Buren, black imported fire ants, Solenopsis richteri Forel, and a hybrid of these two species was evaluated using digging bioassays. In a multiple choice digging bioassay using two colonies from each species and their hybrid, callicarpenal showed significant repellency at concentration as low as 50 ppm against both red imported fire ant colonies and 6.25 ppm against all black imported fire ant and hybrid colonies. Intermedeol showed significant repellency at concentration as low as 1.50 ppm against both red imported fire ant colonies and 6.25 ppm against all black imported fire ant and hybrid colonies. In total, 15 colonies, five colonies from each species and the hybrid, were tested on callicarpenal and intermedeol at 50 ppm in a two-choice digging bioassay. Both callicarpenal and intermedeol showed repellency against all colonies, and intermedeol showed significantly greater repellency than callicarpenal against both species and their hybrid.     

Aggressive interactions between Solenopsis invicta and Linepithema humile (Hymenoptera: Formicidae) under laboratory conditions

The Argentine ant, Linepithema humile (Mayr), and the red imported fire ant, Solenopsis invicta Buren, are natural agonists in their country of origin. Since the first report of L. humile in California in 1907 its range expanded statewide, displacing native ant species wherever it spread. Since the discovery of established populations of S. invicta in southern California in 1998, it has been restricted to discrete areas of southern California. However, as these discrete populations expand, they are encountering large populations of L. humile, which are effective competitors for available resources and are particularly aggressive in their encounters with other ant species such as S. invicta. Most Dolichoderine ants such as L. humile do not prefer to forage on baits made with defatted corn grit and soybean oil typically used in red imported fire ant control programs. Applications of these baits in areas where distributions of these species overlap might selectively affect populations of S. invicta and give L. humile a competitive advantage. Three laboratory experiments were conducted to determine the competitive outcomes between S. invicta pitted against L. humile: 1) agonistic behavior of workers in small arenas, 2) colony interactions with different population ratios, and 3) the effects of pyriproxyfen on the competitiveness of S. invicta against L. humile. Populations of S. invicta consisting of major workers killed more L. humile than did minors or a mixture of majors and minors. When paired against L. humile colonies consisting of 1,100 workers, colonies consisting of 38 S. invicta workers were easily defeated by L. humile. Colonies consisting of 450 S. invicta workers plugged their nest entrances, but they were ultimately defeated by L. humile after 13 d. The S. invicta colonies consisting of 1,100 workers took control of the bridge connecting the colonies, invaded the L. humile colony, killed the Argentine ant queens, and removed their brood. Pyriproxyfen-treated fire ant workers took significantly longer to chase the Argentine ants from a connecting bridge than did untreated fire ants. Thus, fire ant baits may have long-term effects on intercolonial aggression between S. invicta and L. humile, especially when Argentine ant populations are high in the summer.     

Chemical communication of queen supergene status in an ant

Traits of interest to evolutionary biologists often have complex genetic architectures, the nature of which can confound traditional experimental study at single levels of analysis. In the fire ant Solenopsis invicta, the presence of a Mendelian ‘supergene’ is both necessary and sufficient to induce a shift in a fundamental property of social organization, from single‐queen (monogyne) to multiple‐queen (polygyne) colonies. This selfish genetic element, termed the Social b (Sb) supergene, contains > 600 genes that collectively promote its fitness by inducing the characteristic polygyne syndrome, in part by causing polygyne workers to accept only queens bearing the Sb element (a behaviour termed ‘worker Sb discrimination’). Here, we employ a newly developed behavioural assay to reveal that polygyne workers, many of which bear the Sb element, employ chemical cues on the cuticle of queens to achieve worker Sb discrimination, but we found no evidence for such pheromonally mediated worker Sb discrimination in monogyne workers, which universally lack the Sb element. This polygyne worker Sb discrimination was then verified through a ‘green beard’ effect previously described in this system. We thus have demonstrated that the Sb element is required both for production of relevant chemical cues of queens and for expression of the behaviours of workers that collectively result in worker Sb discrimination. This information fills a critical gap in the map between genotype and complex phenotype in S. invicta by restricting the search for candidate genes and molecules involved in producing this complex social trait to factors associated with the Sb element itself.     

Comparing electroantennogram and behavioral responses of two Pseudacteon phorid fly species to body extracts of Black, Red and Hybrid imported fire ants, Solenopsis spp

Several phorid fly species were introduced to the southern United States for biological control of the invasive imported fire ants, Solenopsis richteri (Black), Solenopsis invicta (Red), and their Hybrid S. richteri×S. invicta (Hybrid). It has been previously reported that the Jaguariuna biotype of Pseudacteon tricuspis and the Formosan biotype of Pseudacteon curvatus could distinguish among the three fire ant species with greater preference for Hybrid and Red fire ants. We hypothesized that phorid flies might use host derived chemical cues to differentiate ant species. To determine possible differential olfactory sensitivity of phorid fly species to different fire ant species, we compared electroantennogram (EAG) and behavioral responses of both sexes of P. tricuspis and P. curvatus to body extracts of Black, Red and Hybrid fire ants. As worker sizes of Black and Hybrid fire ants used in this study were much larger than that of Red fire ant (the average weight for Black, Red and Hybrid workers was 1.707, 0.863, 1.223mg per ants, respectively), at doses of 0.01, 0.1, 1 worker equivalent, body extracts of Black and Hybrid fire ant elicited significantly greater EAG response in both sexes of P. tricuspis than that of Red fire ant. Similarly, the EAG response in female P. curvatus to body extract of Black fire ant was significantly greater than to body extract of Red fire ant. To eliminate worker size influence on EAG response in phorid flies, we conducted a second EAG study using a dose of 1mg ant equivalent (body extract from 1mg of worker). No difference in EAG responses was recorded to body extract obtained from the same amount of workers among the three fire ant species (we consider viable Hybrid fire ant as a species in this paper), suggesting that worker size differences contributed to difference in EAG response in the first EAG study. In both EAG studies, male P. tricuspis showed significantly greater EAG responses than male P. curvatus to all three fire ant species. In four-way olfactometer bioassay, worker body extracts of all three fire ant species were equally attractive to P. tricuspis and P. curvatus (i.e. both phorid fly species did not show any preferences among the three fire ant species). Together, the results of the EAG and behavior studies suggest that parasitic phorid flies utilize host derived non-polar compounds from worker ants extracted out by hexane for host location but not for host preference, since both fly species are not able to distinguish among the body extracts of the three fire ant species. Future study will investigate possible involvement of polar compounds and/or non-chemical cues in mediating host preference by phorid flies.     

Polyandry and colony genetic structure in the primitive ant Nothomyrmecia macrops

The Australian endemic ant Nothomyrmecia macrops is considered one of the most ‘primitive’ among living ants. We investigated the genetic structure of colonies to determine queen mating frequencies and nestmate relatedness. An average of 18.8 individuals from each of 32 colonies, and sperm extracted from 34 foraging queens, were genotyped using five highly variable microsatellite markers. Queens were typically singly (65%) or doubly mated (30%), but triple mating (5%) also occurred. The mean effective number of male mates for queens was 1.37. No relationship between colony size and queen mate number was found. Nestmate workers were related by b=0.61 ± 0.03, significantly above the threshold under Hamilton’s rule over which, all else being equal, altruistic behaviour persists, but queens and their mates were unrelated. In 25% of the colonies we detected a few workers that could not have been produced by the resident queen, although there was no evidence for worker reproduction. Polyandry is for the first time recorded in a species with very small mature colonies, which is inconsistent with the sperm‐limitation hypothesis for the mediation of polyandry levels. Facultative polyandry is therefore not confined to the highly advanced ant genera, but may have arisen at an early stage in ant social evolution.     

To b or not to b: a pheromone-binding protein regulates colony social organization in fire ants

A major distinction in the social organization of ant societies is the number of reproductive queens that reside in a single colony. The fire ant Solenopsis invicta exists in two distinct social forms, one with colonies headed by a single reproductive queen and the other containing several to hundreds of egg-laying queens. This variation in social organization has been shown to be associated with genotypes at the gene Gp-9. Specifically, single-queen colonies have only the B allelic variant of this gene, whereas multiple-queen colonies always have the b variant as well. Subsequent studies revealed that Gp-9 shares the highest sequence similarity with genes encoding pheromone-binding proteins (PBPs). In other insects, PBPs serve as central molecular components in the process of chemical recognition of conspecifics. Fire ant workers regulate the number of egg-laying queens in a colony by accepting queens that produce appropriate chemical signals and destroying those that do not. The likely role of GP-9 in chemoreception suggests that the essential distinction in colony queen number between the single and multiple-queen form originates from differences in workers' abilities to recognize queens. Other, closely related fire ant species seem to regulate colony social organization in a similar fashion.     

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

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

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.     

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.     

Kin recognition in the ant Rhytidoponera confusa II. Gestalt odour

Two models have been proposed for the transfer of genetically determined colony odour pheromones in social insects. The ‘Gestalt’ model suggests a complete transfer of pheromones amongst all nestmates whereas the ‘Individualistic’ model suggests no significant odour transfer with nestmates bearing individually distinct odours. In experiments with the ant Rhytidoponera confusa, colonies showed significantly more aggression (12% aggression) towards nestmates that had been kept in cages with non-nestmates than they did to nestmate controls (0% aggression). Colonies also showed significantly less aggression against non-nestmates that had been kept with nestmates in a separate cage (84% aggression) than they did to non-nestmate controls (100% aggression). A second experiment indicated that workers can absorb colony-specific odours from the nest materials of other colonies. However, whilst both experiments indicated a partial Gestalt component to the colony odour (ca. 28%), the Individualistic component seemed to be more important (ca. 72%).     

Getting carbohydrates in ants: damage of the young cereal sprouts by ants of the genus Myrmica Latreille, 1804 (Hymenoptera: Formicidae), to obtain plant sap

Summary

Carbohydrate food is of high importance for survival of ant colonies. Ants are known to use sugary excretions (honeydew) of various insects, nectar of floral and extrafloral nectaries, and even sap of some trees. However, the ability of ants to use sap of herbaceous plants has not been mentioned. This is the first evidence that ants of the genus Myrmica can intentionally ‘cut off’ young cereal sprouts to obtain plant sap. The investigation was carried out in a laboratory in 2018 and 2019 and involved three ant species of the genus Myrmica [12 colonies of M. rubra (Linnaeus, 1758); eight colonies of M. ruginodis Nylander, 1846; and five colonies of M. scabrinodis Nylander, 1846]. First observations were made occasionally in 10 ant colonies during the study of ant–aphid interactions. After three days of carbohydrate starvation, ants were supplied with the plants of wheat infested with aphids of Schizaphis graminum (Rondani, 1852). Within the first day in addition to ordinary trophobiotic relations with aphids, the workers of all the studied colonies demonstrated unexpected behaviour: they ‘cut off’ some sprouts and collected sap of these plants. The experimental investigation in 15 ant colonies of various sizes (about 150, 300 and 500 workers) supplied with the plants infested or non-infested with aphids has shown that getting sap of herbs depends greatly on ant colony needs and available resources. The number of damaged plants was much higher both in the larger colonies of ants and in the absence of aphids. This way of getting carbohydrates allows ants to quickly obtain some extra food needed to maintain colony viability and seems to be one of the mechanisms promoting survival of ants in conditions of acute carbohydrate deficiency. At the same time, ants avoid using plant sap when there are more available alternative carbohydrate resources.     

Task-Related Variation of Cuticular Hydrocarbon Profiles Affect Nestmate Recognition in the Giant ant Dinoponera quadriceps

Recognition seems to be one of the more remarkable characteristics of social groups. In social insects, cuticular hydrocarbons are important for colonial recognition by providing a chemical signature for colony members. The acceptance threshold model predicts that colony members will accept conspecifics when the levels of nest mate cues dissimilarities are below the acceptance threshold. We tested the hypothesis that the encounter of a guard ant worker with a nurse may cause a delay in the process of recognition, because nurses from distinct colonies may share greater amount of chemical compounds. Dinoponera quadriceps guard workers decreased their effectiveness to recognize nurses rather than did to foragers. Alien foragers received significantly more bites and other stronger acts than non-nestmate nurses when they were experimentally introduced. In addition, guards took significantly more time to react against non-nestmate nurses than against alien foragers. Analysis of the cuticular hydrocarbon profiles corroborated our behavioural analysis that nurses from distinct colonies overlap greater amount of cuticular hydrocarbons.     

Differences in nestmate recognition for drones and workers in the honeybee, Apis mellifera (L.)

Nestmate recognition is the basic mechanism for rejecting foreign individuals and is essential for maintaining colony integrity in insect societies. However, in honeybees, Apis mellifera, both workers and males occasionally gain access to foreign colonies in spite of nest guards (=drifting). Instead of conducting direct behavioural observations, we inferred nestmate recognition for males and workers from the genotypes of naturally drifting individuals in honeybee colonies. We evaluated the degree of polyandry of the resident queens, because nestmate recognition theory predicts that the genotypic composition of insect colonies may affect the recognition precision of guards. Workers (N=1346) and drones (N=407) from 38 colonies were genotyped using four DNA microsatellite loci. Foreign bees were identified by maternity testing. The proportion of foreign individuals in a host colony was defined as immigration. Putative mother queens were identified if a queen's genotype corresponded with the genotype of a drifted individual. The proportion of a colony's individuals in the total number of drifted individuals was defined as emigration. Drones immigrated significantly more frequently than workers. The impact of polyandry was significantly different between drones and workers. Whereas drones immigrated more readily into less polyandrous colonies, worker immigration was not correlated with the degree of polyandry of the host colony. Furthermore, colonies with high levels of emigrated drones did not show high levels of emigration for workers, and colonies that adopted many workers did not adopt many foreign drones. Our data indicate that genetically derived odour cues are important for honeybee nestmate recognition in drones and show that different nestmate recognition mechanisms are used to identify drones and workers.