Aggression and worker control of caste fate in a multiple-queen wasp, Parachartergus colobopterus

Although famously cooperative, social insect colonies harbour considerable potential for genetic conflict among colonymates. This conflict may be expressed behaviourally as aggression by workers. We investigated aggression in 34 colonies of the wasp Parachartergus colobopterus, by evaluating the characteristics of both instigators and victims of aggressive interactions. We estimated genetic relatedness and queen number using DNA microsatellites and found that workers and emerging females should be most in conflict over the caste of the latter when there are many queens on the nest. We found that aggressive interactions are more likely to involve older workers attacking either males or younger workers, and that victim and aggressor females have more ovarian development than randomly sampled colonymates. Moreover, mated females with low levels of ovarian development relative to active queens were also more likely to be aggressors and victims than were randomly sampled females. Aggression among females supports the hypothesis that older workers use aggression towards younger females as a means of policing the development of emerging females into queens. Workers also may use aggression to suppress the reproduction of some mated females. Our findings thus support the hypothesis that genetic conflicts of interest motivate worker aggression in swarm-founding wasp colonies.     

Queen phenotype and behaviour during cooperative colony founding in Polyrhachis moesta

Summary. Founding queens of the arboreal ant Polyrhachis moesta aggregate independently of kinship and cooperate in caring for their brood. In field studies, the number of queens in a founding nest varied from 1 to 8. The number of queens in the nests with multiple queens decreased significantly with time after the nuptial flight, resulting in monogynous or oligogynous nests. Single- and multiple-founding queens did not differ in characteristics representing nutritional states or body size immediately after the nuptial flight. Wet body weight decreased as days passed, whereas head width of founding queens who overwintered successfully were relatively larger. In laboratory studies, founding queens performed liquid food exchanges more frequently with queens from other founding nests or immature colonies than with those from the same nests. Queens in founding nests and immature colonies were observed to show no aggression against non-nestmate queens, whereas queens in established colonies showed aggressive behaviours against non-nestmates. This indicates that founding queens change drastically in their aggression levels before and after colony establishment. Multiple-founding queens started laying eggs earlier than single-founding queens under laboratory conditions. Higher brood productivity and lower brood mortality were observed in multiple-queen nests. These potential advantages in multiple-queen founding may support the cooperative association among unrelated founding queens.Received 1 December 2003; revised 25 March and 20 May 2004; accepted 3 June 2004.     

Sociogenomics of Cooperation and Conflict during Colony Founding in the Fire Ant Solenopsis invicta

One of the fundamental questions in biology is how cooperative and altruistic behaviors evolved. The majority of studies seeking to identify the genes regulating these behaviors have been performed in systems where behavioral and physiological differences are relatively fixed, such as in the honey bee. During colony founding in the monogyne (one queen per colony) social form of the fire ant Solenopsis invicta, newly-mated queens may start new colonies either individually (haplometrosis) or in groups (pleometrosis). However, only one queen (the “winner”) in pleometrotic associations survives and takes the lead of the young colony while the others (the “losers”) are executed. Thus, colony founding in fire ants provides an excellent system in which to examine the genes underpinning cooperative behavior and how the social environment shapes the expression of these genes. We developed a new whole genome microarray platform for S. invicta to characterize the gene expression patterns associated with colony founding behavior. First, we compared haplometrotic queens, pleometrotic winners and pleometrotic losers. Second, we manipulated pleometrotic couples in order to switch or maintain the social ranks of the two cofoundresses. Haplometrotic and pleometrotic queens differed in the expression of genes involved in stress response, aging, immunity, reproduction and lipid biosynthesis. Smaller sets of genes were differentially expressed between winners and losers. In the second experiment, switching social rank had a much greater impact on gene expression patterns than the initial/final rank. Expression differences for several candidate genes involved in key biological processes were confirmed using qRT-PCR. Our findings indicate that, in S. invicta, social environment plays a major role in the determination of the patterns of gene expression, while the queen''s physiological state is secondary. These results highlight the powerful influence of social environment on regulation of the genomic state, physiology and ultimately, social behavior of animals.     

Variation in social structure alters queen body mass and productivity in the desert seed-harvester ant Messor pergandei

Parent–offspring conflict theory predicts conflict between parents and their offspring over per-offspring resource investment. Across the range of the desert seed-harvester ant, Messor pergandei, daughter queens use three different social strategies during colony founding that are expected to alter the optimal level of parental investment. To test whether social strategy variation is associated with shifts in body mass, we surveyed queen live mass over 3 years at 25 sites that spanned the range of behavioral strategies and founding group sizes. To test whether reduction in parental investment into individual offspring negatively impacts their productivity, queens were individually isolated and allowed to produce a single worker cohort under common garden conditions. Queen live mass was highly variable, from 24 mg on average at the site with the lightest queens to 1.5 times that size, 37 mg, at the site with the heaviest queens. As predicted by parent–offspring conflict over investment, solitary colony founding sites contained the heaviest queens, followed by secondary monogyny. Polygynous queens were lightest, with a strong negative relationship between group size and live mass. Reductions in body mass had a negative effect on queen productivity across all queen social types; however, queens from sites where queen–queen aggression is typical were significantly more efficient at brood rearing, resulting in lower mass loss during founding per unit offspring biomass. This may represent an adaptation to queen competition to gain a strength advantage over potential rivals.     

Octopamine and cooperation: octopamine regulates the disappearance of cooperative behaviours between genetically unrelated founding queens in the ant

We investigated whether octopamine (OA) is associated with the disappearance of cooperation in Polyrhachis moesta ant queens. Queens of P. moesta facultatively found the colony with genetically unrelated queens. The founding queens perform frequent food exchange with these non-related queens and partake in cooperative brood rearing, whereas single colony queens exclude non-related queens via aggressive behaviour. Thus, aggression is a factor that reduces cooperation. Given that aggression is generally associated with brain OA in insects, we hypothesized that OA controls the behavioural change in cooperation in the ant queen, via an increase in aggression. To test this hypothesis, we compared the amounts of OA and related substances in the brain between founding and colony queens, and observed the interaction of founding queens following oral OA administration. The brain OA levels in colony queens were significantly higher than those in founding queens. Oral administration of OA to founding queens caused significantly less trophallaxis and allogrooming behaviour than in the control founding queens, but with no significant increase in aggression. These results suggest that OA promotes the disappearance of cooperation in founding queens of P. moesta. This is the first study to reveal the neuroendocrine mechanism of cooperation in ant queens.     

Restricted gene flow between two social forms in the ant Formica truncorum

We studied genetic differentiation between two social forms (M-type: single queen, independent nest founding; P-type: multiple queens, dependent nest founding, building of colonial networks) of the ant Formica truncorum in a locality where the social types characterize two sympatric populations. The genetic results indicate restricted gene flow between the social forms. Female gene flow between the forms appears to be absent as they did not share mitochondrial haplotypes. Significant nuclear differentiation and the distribution of private alleles suggest that male gene flow between the forms is weak. However, the assignment analysis indicates some gene flow with P males mating with M females. The results have potentially important implications concerning social evolution within the forms but they need to be confirmed in other localities before they can be generalized. The colonies in the M-type population have earlier been shown to produce split sex ratios, depending on the mating frequency of the queens. The inferred gene flow from the P to the M type means that the split sex ratio is partly suboptimal, possibly because the P populations are not long-lived enough to influence the behavioural decisions in the M colonies.     

Evidence that queens do not influence nestmate recognition in Argentine ants

Summary Introduced populations of many invasive ants exhibit low levels of intraspecific aggression. Argentine ants (Linepithema humile), for example, maintain expansive supercolonies in many parts of their introduced range. Recent studies demonstrate that variation in nestmate recognition in L. humile can derive from both environmental and genetic sources. In some ants, pheromones emitted by queens also influence nestmate-recognition behavior. To test if such a phenomenon occurs in Argentine ants, we examined whether levels of intraspecific aggression vary as a function of queen presence or absence in experimental lab colonies. For each of four known supercolonies from southwestern California, we set up a pair of experimental colonies and randomly assigned replicates within each pair to treatment (queen removal) and control (no queen removal) groups. Using two different behavioral assays, we then measured aggressive behavior for ten days, removed queens from colonies in the treatment group, and continued to monitor aggression in both experimental groups for an additional 65 days. Both assays yielded qualitatively similar results: intraspecific aggression remained high throughout the experiment in both experimental groups. These results suggest that L. humile queens fail to influence levels of intraspecific aggression in introduced populations.Received 2 June 2003; revised 1 September 2003; accepted 18 September 2003.     

Long-term memory of individual identity in ant queens

Remembering individual identities is part of our own everyday social life. Surprisingly, this ability has recently been shown in two social insects. While paper wasps recognize each other individually through their facial markings, the ant, Pachycondyla villosa, uses chemical cues. In both species, individual recognition is adaptive since it facilitates the maintenance of stable dominance hierarchies among individuals, and thus reduces the cost of conflict within these small societies. Here, we investigated individual recognition in Pachycondyla ants by quantifying the level of aggression between pairs of familiar or unfamiliar queens over time. We show that unrelated founding queens of P. villosa and Pachycondyla inversa store information on the individual identity of other queens and can retrieve it from memory after 24h of separation. Thus, we have documented for the first time that long-term memory of individual identity is present and functional in ants. This novel finding represents an advance in our understanding of the mechanism determining the evolution of cooperation among unrelated individuals.     

Evolution of gene expression in fire ants: the effects of developmental stage, caste, and species

Ants provide remarkable examples of equivalent genotypes developing into divergent and discrete phenotypes. Diploid eggs can develop either into queens, which specialize in reproduction, or workers, which participate in cooperative tasks such as building the nest, collecting food, and rearing the young. In contrast, the differentiation between males and females generally depends upon whether eggs are fertilized, with fertilized (diploid) eggs giving rise to females and unfertilized (haploid) eggs giving rise to males. To obtain a comprehensive picture of the relative contributions of gender (sex), caste, developmental stage, and species divergence to gene expression evolution, we investigated gene expression patterns in pupal and adult queens, workers, and males of two species of fire ants, Solenopsis invicta and S. richteri. Microarray hybridizations revealed that variation in gene expression profiles is influenced more by developmental stage than by caste membership, sex, or species identity. The second major contributor to variation in gene expression was the combination of sex and caste. Although workers and queens share equivalent diploid nuclear genomes, they have highly distinctive patterns of gene expression in both the pupal and the adult stages, as might be expected given their extraordinary level of phenotypic differentiation. Overall, the difference in the proportion of differentially expressed genes was greater between workers and males than between workers and queens or queens and males, consistent with the fact that workers and males share neither gender nor reproductive capability. Moreover, between-species comparisons revealed that the greatest difference in gene expression patterns occurred in adult workers, a finding consistent with the fact that adult workers most directly experience the distinct external environments characterizing the different habitats occupied by the two species. Thus, much of the evolution of gene expression in ants may occur in the worker caste, despite the fact that these individuals are largely or completely sterile. Analyses of gene expression evolution revealed a combination of positive selection and relaxation of stabilizing selection as important factors driving the evolution of such genes.     

Genetic caste determination in Pogonomyrmex harvester ants imposes costs during colony founding

Some populations of Pogonomyrmex harvester ants comprise genetically differentiated pairs of interbreeding lineages. Queens mate with males of their own and of the alternate lineage and produce pure-lineage offspring which develop into queens and inter-lineage offspring which develop into workers. Here we tested whether such genetic caste determination is associated with costs in terms of the ability to optimally allocate resources to the production of queens and workers. During the stage of colony founding, when only workers are produced, queens laid a high proportion of pure-lineage eggs but the large majority of these eggs failed to develop. As a consequence, the number of offspring produced by incipient colonies decreased linearly with the proportion of pure-lineage eggs laid by queens. Moreover, queens of the lineage most commonly represented in a given mating flight produced more pure-lineage eggs, in line with the view that they mate randomly with the two types of males and indiscriminately use their sperm. Altogether these results predict frequency-dependent selection on pairs of lineages because queens of the more common lineage will produce more pure-lineage eggs and their colonies be less successful during the stage of colony founding, which may be an important force maintaining the coexistence of pairs of lineages within populations.     

Raiders from the sky: slavemaker founding queens select for aggressive host colonies

Reciprocal selection pressures in host-parasite systems drive coevolutionary arms races that lead to advanced adaptations in both opponents. In the interactions between social parasites and their hosts, aggression is one of the major behavioural traits under selection. In a field manipulation, we aimed to disentangle the impact of slavemaking ants and nest density on aggression of Temnothorax longispinosus ants. An early slavemaker mating flight provided us with the unique opportunity to study the influence of host aggression and demography on founding decisions and success. We discovered that parasite queens avoided colony foundation in parasitized areas and were able to capture more brood from less aggressive host colonies. Host colony aggression remained consistent over the two-month experiment, but did not respond to our manipulation. However, as one-fifth of all host colonies were successfully invaded by parasite queens, slavemaker nest foundation acts as a strong selection event selecting for high aggression in host colonies.     

Variation in queen size across a behavioral transition zone in the ant Messor pergandei

Summary. Ant colonies should be selected to optimally allocate resources to individual reproductive offspring so as to balance production costs with offspring fitness gains. Different modes of colony founding have different size-dependent fitness functions, and should thus lead to different optimal queen sizes. We tested whether a behavioral transition from solitary colony founding (haplometrosis) to group colony founding (pleometrosis) across the range of the ant Messor pergandei was associated with a difference in queen size or condition. Both winged gynes and founding queens were significantly smaller and lighter at pleometrotic than at haplometrotic sites, with an abrupt shift in these characters across the 8.5 km-wide behavioral transition zone. Both the mutualistic advantages of grouping and among-queen competition within associations are likely to be important in selecting for smaller queen size in pleometrotic populations.Received 16 January 2004; revised 13 August 2004; accepted 16 August 2004.     

Female aggression predicts mode of paternity acquisition in a social lizard

Individual differences in behaviour are ubiquitous in nature. Despite the likely role of selection in maintaining these differences, there are few demonstrations of their fitness consequences in wild populations and, consequently, the mechanisms that link behavioural variation to variation in fitness are poorly understood. Specifically, the consequences of consistent individual differences in behaviour for the evolution of social and mating strategies have rarely been considered. We examined the functional links between variation in female aggression and her social and mating strategies in a wild population of the social lizard Egernia whitii. We show that female Egernia exhibit temporally consistent aggressive phenotypes, which are unrelated to body size, territory size or social density. A female''s aggressive phenotype, however, has strong links to her mode of paternity acquisition (within- versus extra-pair paternity), with more aggressive females having more offspring sired by extra-pair males than less aggressive females. We discuss the potential mechanisms by which female aggression could underpin mating strategies, such as the pursuit/acceptance of extra-pair copulations. We propose that a deeper understanding of the evolution and maintenance of social and mating systems may result from an explicit focus on individual-level female behavioural phenotypes and their relationship with key reproductive strategies.     

Caste totipotency and conflict in a large-colony social insect

In most social insects with large, complex colonies workers and queens are morphologically quite distinct. This means that caste determination must occur prior to adulthood. However workers and queens in the swarm-founding epiponine wasps are often morphologically indistinguishable, or nearly so, suggesting that caste determination in these wasps could be quite different. To determine the extent of caste lability in the epiponine, Parachartergus colobopterus, we removed all the queens from one colony and all but one from another colony. Worker aggression diminished after queen removal. A week later the colony with no queens had a new, young cohort of mated queens. These must have been either adults or pupae at the time of queen removal, and so could not have been fed any differently from workers. Relatedness patterns confirmed that these new queens would normally have been workers and not queens. A model of inclusive fitness interests shows that workers ought to suppress new queen production, except at low queen numbers, a prediction supported by our empirical results. The patterns of social conflict over queenship resulting from swarm founding in a many-queen society may help to explain the unusually weak caste differentiation in the epiponines.     

Changes in mating system and social structure of the ant Petalomyrmex phylax are associated with range expansion in Cameroon

Past climate shifts have led to major oscillations in species distributions. Hence historical contingencies and selective processes occurring during such phases may be determinants for understanding the forces that have shaped extant phenotypes. In the plant-ant Petalomyrmex phylax (Formicinae), we observed spatial variation in number of queens in mature colonies, from several queens (high polygyny) in the median part of its distribution to a moderate number of queens (weak polygyny) or even only a single queen (monogyny) in the southwesternmost populations. This variation did not correlate with indicators of variation in current nest site availability and colony turnover, the supposedly determinant selective forces acting on gyny in ants. We show here that the variation in social structure correlates with a historical process corresponding to a progressive colonization of coastal southern Cameroon by the ant. Using microsatellite markers, we observed a clear pattern of isolation by distance except for the southernmost populations. Measures of genetic variability that do not take into account allele size were at equilibrium in all except the southernmost populations, suggesting recent foundation of the latter. Measures of genetic diversity taking into account allele size showed a clinal north-south decrease in variance of allele size. We propose that southern populations have yet to regain allele size variance after bottlenecks associated with the foundation of new populations, and that this variance is regained over time. Hence variation in social structure mirrors an old but still active southward colonization process or metapopulation dynamics, possibly in association with an expansion of the rain forest habitat during the late Holocene. A low number of queens in ant colonies is typically associated with strong dispersal capacity. We therefore suggest that the initial founders of new populations belong to the monogynous to weakly polygynous phenotype, and that queen number progressively increases in older populations.     

Simple inheritance,complex regulation: Supergene‐mediated fire ant queen polymorphism

The fire ant Solenopsis invicta exists in two alternate social forms: monogyne nests contain a single reproductive queen and polygyne nests contain multiple reproductive queens. This colony‐level social polymorphism corresponds with individual differences in queen physiology, queen dispersal patterns and worker discrimination behaviours, all evidently regulated by an inversion‐based supergene that spans more than 13 Mb of a “social chromosome,” contains over 400 protein‐coding genes and rarely undergoes recombination. The specific mechanisms by which this supergene influences expression of the many distinctive features that characterize the alternate forms remain almost wholly unknown. To advance our understanding of these mechanisms, we explore the effects of social chromosome genotype and natal colony social form on gene expression in queens sampled as they embarked on nuptial flights, using RNA‐sequencing of brains and ovaries. We observe a large effect of natal social form, that is, of the social/developmental environment, on gene expression profiles, with similarly substantial effects of genotype, including: (a) supergene‐associated gene upregulation, (b) allele‐specific expression and (c) pronounced extra‐supergene trans‐regulatory effects. These findings, along with observed spatial variation in differential and allele‐specific expression within the supergene region, highlight the complex gene regulatory landscape that emerged following divergence of the inversion‐mediated Sb haplotype from its homologue, which presumably largely retained the ancestral gene order. The distinctive supergene‐associated gene expression trajectories we document at the onset of a queen’s reproductive life expand the known record of relevant molecular correlates of a complex social polymorphism and point to putative genetic factors underpinning the alternate social syndromes.     

Colony social structure in native and invasive populations of the social wasp Vespula pensylvanica

Social insects rank among the most invasive of terrestrial species. The success of invasive social insects stems, in part, from the flexibility derived from their social behaviors. We used genetic markers to investigate if the social system of the invasive wasp, Vespula pensylvanica, differed in its introduced and native habitats in order to better understand variation in social phenotype in invasive social species. We found that (1) nestmate workers showed lower levels of relatedness in introduced populations than native populations, (2) introduced colonies contained workers produced by multiple queens whereas native colonies contained workers produced by only a single queen, (3) queen mate number did not differ significantly between introduced and native colonies, and (4) workers from introduced colonies were frequently produced by queens that originated from foreign nests. Thus, overall, native and introduced colonies differed substantially in social phenotype because introduced colonies more frequently contained workers produced by multiple, foreign queens. In addition, the similarity in levels of genetic variation in introduced and native habitats, as well as observed variation in colony social phenotype in native populations, suggest that colony structure in invasive populations may be partially associated with social plasticity. Overall, the differences in social structure observed in invasive V. pensylvanica parallel those in other, distantly related invasive social insects, suggesting that insect societies often develop similar social phenotypes upon introduction into new habitats.     

Workers influence royal reproduction

Understanding which parties regulate reproduction is fundamental to understanding conflict resolution in animal societies. In social insects, workers can influence male production and sex ratio. Surprisingly, few studies have investigated worker influence over which queen(s) reproduce(s) in multiple queen (MQ) colonies (skew), despite skew determining worker-brood relatedness and so worker fitness. We provide evidence for worker influence over skew in a functionally monogynous population of the ant Leptothorax acervorum. Observations of MQ colonies leading up to egg laying showed worker aggressive and non-aggressive behaviour towards queens and predicted which queen monopolized reproduction. In contrast, among-queen interactions were rare and did not predict queen reproduction. Furthermore, parentage analysis showed workers favoured their mother when present, ensuring closely related fullsibs (average r = 0.5) were reared instead of less related offspring of other resident queens (r ≤ 0.375). Discrimination among queens using relatedness-based cues, however, seems unlikely as workers also biased their behaviour in colonies without a mother queen. In other polygynous populations of this species, workers are not aggressive towards queens and MQs reproduce, showing the outcome of social conflicts varies within species. In conclusion, this study supports non-reproductive parties having the power and information to influence skew within cooperative breeding groups.     

Caste‐biases in gene expression are specific to developmental stage in the ant Formica exsecta

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co‐opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of ‘toolkit’ genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA‐Seq to compare caste‐biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste‐biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste‐biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste‐biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste‐biased at least in some life stages in F. exsecta, and the caste‐biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.     

Multilocus Evolution in Fire Ants: Effects of Selection, Gene Flow and Recombination

The reproductive success of individual fire ant queens (Solenopsis invicta) previously has been shown to be strongly influenced by their genotype at a single enzyme-encoding gene, designated Pgm-3. This paper presents evidence that a second, tightly linked gene, designated Gp-9, is under similarly strong selection in these ants. Selection appears to act independently on the two genes and is detectable in only one of the two social forms of this species (the ``polygyne' social form, in which nests contain multiple fertile queens). Strong directional selection on Pgm-3 in this form involves worker destruction of all queens with genotype Pgm-3(AA) before they reproduce. Selection on Gp-9 is more complex, involving both lethality of all Gp-9(bb) females and a strong or even complete survival advantage to reproductive queens with the heterozygous genotype Gp-9(Bb). Pgm-3 and Gp-9 are tightly linked (r(f) = 0.0016) and exhibit strong gametic phase disequilibrium in introduced populations in the U.S. This disequilibrium seems not to have stemmed from the founder event associated with the introduction, because the same associations of alleles found in the U.S. apparently occur also in two native populations in Argentina. Rather, selection acting independently on Pgm-3 and Gp-9, in conjunction with gene flow from the alternate, ``monogyne' social form (in which nests contain a single fertile queen), may explain the origin of disequilibrium between the two loci in polygyne fire ants.