Honeybees possess a structurally diverse and functionally redundant set of queen pheromones.

Queen pheromones, which signal the presence of a fertile queen and induce workers to remain sterile, play a key role in regulating reproductive division of labour in insect societies. In the honeybee, volatiles produced by the queen's mandibular glands have been argued to act as the primary sterility-inducing pheromones. This contrasts with evidence from other groups of social insects, where specific queen-characteristic hydrocarbons present on the cuticle act as conserved queen signals. This led us to hypothesize that honeybee queens might also employ cuticular pheromones to stop workers from reproducing. Here, we support this hypothesis with the results of bioassays with synthetic blends of queen-characteristic alkenes, esters and carboxylic acids. We show that all these compound classes suppress worker ovary development, and that one of the blends of esters that we used was as effective as the queen mandibular pheromone (QMP) mix. Furthermore, we demonstrate that the two main QMP compounds 9-ODA and 9-HDA tested individually were as effective as the blend of all four major QMP compounds, suggesting considerable signal redundancy. Possible adaptive reasons for the observed complexity of the honeybee queen signal mix are discussed.

The origin and evolution of social insect queen pheromones: Novel hypotheses and outstanding problems.

Queen pheromones, which signal the presence of a fertile queen and induce daughter workers to remain sterile, are considered to play a key role in regulating the reproductive division of labor of insect societies. Although queen pheromones were long thought to be highly taxon-specific, recent studies have shown that structurally related long-chain hydrocarbons act as conserved queen signals across several independently evolved lineages of social insects. These results imply that social insect queen pheromones are very ancient and likely derived from an ancestral signalling system that was already present in their common solitary ancestors. Based on these new insights, we here review the literature and speculate on what signal precursors social insect queen pheromones may have evolved from. Furthermore, we provide compelling evidence that these pheromones should best be seen as honest signals of fertility as opposed to suppressive agents that chemically sterilize the workers against their own best interests.

Biological activity of the enantiomers of 3-methylhentriacontane, a queen pheromone of the ant Lasius niger.

Queen pheromones are essential for regulation of the reproductive division of labor in eusocial insect species. Although only the queen is able to lay fertilized eggs and produce females, in some cases workers may develop their ovaries and lay male-destined eggs, thus reducing the overall colony efficiency. As long as the queen is healthy, it is usually in the workers' collective interest to work for the colony and remain sterile. Queens signal their fertility via pheromones, which may have a primer effect, affecting the physiology of workers, or a releaser effect, influencing worker behavior. The queen pheromone of the ant Lasius niger was among the first queen pheromones of social insects to be identified. Its major component is 3-methylhentriacontane (3-MeC31), which is present in relatively large amounts on the queen's cuticle and on her eggs. 3-MeC31 regulates worker reproduction by inhibiting ovarian development. Most monomethyl-branched hydrocarbons can exist in two stereoisomeric forms. The correct stereochemistry is fundamental to the activity of most bioactive molecules, but this has rarely been investigated for methyl-branched hydrocarbons. Here, we tested the bioactivity of the (S)- and (R)-enantiomers of 3-MeC31, and found that whereas both enantiomers were effective in suppressing worker ovarian development, (S)-3-MeC31 appeared to be more effective at suppressing aggressive behavior by workers. This suggests that the natural pheromone may be a mixture of the two enantiomers. The enantiomeric ratio produced by queens remains unknown because of the small amounts of the compound available from each queen.

Conservation of Queen Pheromones Across Two Species of Vespine Wasps.

Social insects are known for their reproductive division of labor between queens and workers, whereby queens lay the majority of the colony's eggs, and workers engage mostly in non-reproductive tasks. Queens produce pheromones that signal their presence and fertility to workers, which in turn generally remain sterile. Recently, it has been discovered that specific queen-characteristic cuticular hydrocarbons (CHCs) function as queen pheromones across multiple lineages of social insects. In the common wasp, Vespula vulgaris, several long-chain linear alkanes and 3-methylalkanes were shown to act as queen signals. Here, we describe similar bioassays with a related species of highly eusocial vespine wasp, the Saxon wasp, Dolichovespula saxonica. We show that a blend of queen-characteristic hydrocarbons that are structurally related to those of the common wasp inhibit worker reproduction, suggesting conservation of queen pheromones across social wasps. Overall, our results highlight the central importance of CHCs in chemical communication among social insects in general, and as conserved queen pheromones in these social wasps in particular.

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

Chemical Strategies of the Beetle Metoecus Paradoxus, Social Parasite of the Wasp Vespula Vulgaris.

The parasitoid beetle Metoecus paradoxus frequently parasitizes colonies of the common wasp, Vespula vulgaris. It penetrates a host colony as a larva that attaches itself onto a foraging wasp's body and, once inside the nest, it feeds on a wasp larva inside a brood cell and then pupates. Avoiding detection by the wasp host is crucial when the beetle emerges. Here, we tested whether adult M. paradoxus beetles avoid detection by mimicking the cuticular hydrocarbon profile of their host. The beetles appear to be chemically adapted to their main host species, the common wasp, because they share more hydrocarbon compounds with it than they do with the related German wasp, V. germanica. In addition, aggression tests showed that adult beetles were attacked less by common wasp workers than by German wasp workers. Our results further indicated that the host-specific compounds were, at least partially, produced through recycling of the prey's hydrocarbons, and were not acquired through contact with the adult host. Moreover, the chemical profile of the beetles shows overproduction of the wasp queen pheromone, nonacosane (n-C29), suggesting that beetles might mimic the queen's pheromonal bouquet.

Crozier's paradox revisited: maintenance of genetic recognition systems by disassortative mating.

BACKGROUND: Organisms are predicted to behave more favourably towards relatives, and kin-biased cooperation has been found in all domains of life from bacteria to vertebrates. Cooperation based on genetic recognition cues is paradoxical because it disproportionately benefits individuals with common phenotypes, which should erode the required cue polymorphism. Theoretical models suggest that many recognition loci likely have some secondary function that is subject to diversifying selection, keeping them variable. RESULTS: Here, we use individual-based simulations to investigate the hypothesis that the dual use of recognition cues to facilitate social behaviour and disassortative mating (e.g. for inbreeding avoidance) can maintain cue diversity over evolutionary time. Our model shows that when organisms mate disassortatively with respect to their recognition cues, cooperation and recognition locus diversity can persist at high values, especially when outcrossed matings produce more surviving offspring. Mating system affects cue diversity via at least four distinct mechanisms, and its effects interact with other parameters such as population structure. Also, the attrition of cue diversity is less rapid when cooperation does not require an exact cue match. Using a literature review, we show that there is abundant empirical evidence that heritable recognition cues are simultaneously used in social and sexual behaviour. CONCLUSIONS: Our models show that mate choice is one possible resolution of the paradox of genetic kin recognition, and the literature review suggests that genetic recognition cues simultaneously inform assortative cooperation and disassortative mating in a large range of taxa. However, direct evidence is scant and there is substantial scope for future work.

Do cuticular hydrocarbons provide sufficient information for optimal sex allocation in the ant Formica exsecta?

Split sex ratio theory predicts that when kin structure varies among colonies of social insects, in order to maximize the inclusive fitness, colonies with relatively high sister-sister relatedness should specialize in producing reproductive females, whereas in those with relatively low sister-sister relatedness workers should bias their sex ratio towards males. However, in order to achieve this, workers need to be able to reliably assess the type of colony in which they live. The information on colony kin structure may be encoded in cuticular hydrocarbons (CHCs), assuming that genetic variability translates accurately into chemical variability. However, in genetically heterogeneous colonies, too accurate information may encourage the pursuit of individual interests through nepotistic behavior and reduce colony efficiency or cause social disruption. In this study, we estimated how well variability of CHC recognition cues reflects colony kin structure in the ant Formica exsecta. Our results show that CHC variability does not covary with kin structure or the overall genetic diversity of the colony, and that patrilines and matrilines can have distinct CHC profiles in some but not all colonies. However, within-colony relatedness remains the key determinant of colony sex ratios. Based on our results, CHC variability cannot serve as accurate information on within-colony relatedness, kin structure, or full-sib affiliation, nor do workers seem to use colony CHC variability as a proxy for sex-ratio adjustment. The use of this type of information thus could lead workers to make mistakes, and it remains unclear how colonies of Formica exsecta adjust offspring sex ratio to their optimal value.

Parent of origin gene expression in the bumblebee, Bombus terrestris, supports Haig's kinship theory for the evolution of genomic imprinting.

Genomic imprinting is the differential expression alleles in diploid individuals, with the expression being dependent on the sex of the parent from which it was inherited. Haig's kinship theory hypothesizes that genomic imprinting is due to an evolutionary conflict of interest between alleles from the mother and father. In social insects, it has been suggested that genomic imprinting should be widespread. One recent study identified parent-of-origin expression in honey bees and found evidence supporting the kinship theory. However, little is known about genomic imprinting in insects and multiple theoretical predictions must be tested to avoid single-study confirmation bias. We, therefore, tested for parent-of-origin expression in a primitively eusocial bee. We found equal numbers of maternally and paternally biased expressed genes. The most highly biased genes were maternally expressed, offering support for the kinship theory. We also found low conservation of potentially imprinted genes with the honey bee, suggesting rapid evolution of genomic imprinting in Hymenoptera.

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.

Specialization in policing behaviour among workers in the ant Pachycondyla inversa.

Most animal societies are non-clonal and thus subject to conflicts. In social insects, conflict over male production can be resolved by worker policing, i.e. eating of worker-laid eggs (WLE) or aggression towards reproductive workers. All workers in a colony have an interest in policing behaviour being expressed, but there can be asymmetries among workers in performing the actual behaviour. Here, we show that workers of the ant Pachycondyla inversa specialize in policing behaviour. In two types of behavioural assays, workers developed their ovaries and laid eggs. In the first experiment, reproductive workers were introduced into queenright colonies. In the second experiment, WLE were introduced. By observing which individuals policed, we found that aggressive policing was highly skewed among workers that had opportunity to police, and that a similar tendency occurred in egg policing. None of the policing workers had active ovaries, so that policing did not incur a direct selfish benefit to the policer. This suggests that policing is subject to polyethism, just like other tasks in the colony. We discuss several hypotheses on the possible causes of this skew in policing tasks. This is the first non-primate example of specialization in policing tasks without direct selfish interests.

Conserved queen pheromones in bumblebees: a reply to Amsalem et al.

In a recent study, Amsalem, Orlova & Grozinger (2015) performed experiments with Bombus impatiens bumblebees to test the hypothesis that saturated cuticular hydrocarbons are evolutionarily conserved signals used to regulate reproductive division of labor in many Hymenopteran social insects. They concluded that the cuticular hydrocarbon pentacosane (C25), previously identified as a queen pheromone in a congeneric bumblebee, does not affect worker reproduction in B. impatiens. Here we discuss some shortcomings of Amsalem et al.'s study that make its conclusions unreliable. In particular, several confounding effects may have affected the results of both experimental manipulations in the study. Additionally, the study's low sample sizes (mean n per treatment = 13.6, range: 4-23) give it low power, not 96-99% power as claimed, such that its conclusions may be false negatives. Inappropriate statistical tests were also used, and our reanalysis found that C25 substantially reduced and delayed worker egg laying in B. impatiens. We review the evidence that cuticular hydrocarbons act as queen pheromones, and offer some recommendations for future queen pheromone experiments.

Hormonal pleiotropy helps maintain queen signal honesty in a highly eusocial wasp.

In insect societies, both queens and workers produce chemicals that reliably signal caste membership and reproductive status. The mechanisms that help to maintain the honesty of such queen and fertility signals, however, remain poorly studied. Here we test if queen signal honesty could be based on the shared endocrine control of queen fertility and the production of specific signals. In support of this "hormonal pleiotropy" hypothesis, we find that in the common wasp, application of methoprene (a juveline hormone analogue) caused workers to acquire a queen-like cuticular hydrocarbon profile, resulting in the overproduction of known queen pheromones as well as some compounds typically linked to worker fertility. By contrast, administration of precocene-I (a JH inhibitor) had a tendency to have the opposite effect. Furthermore, a clear gonadotropic effect of JH in queens was suggested by the fact that circulating levels of JH were ca. 2 orders of magnitude higher in queens than those in workers and virgin, non-egg-laying queens, even if methoprene or precocene treatment did not affect the ovary development of workers. Overall, these results suggest that queen signal honesty in this system is maintained by queen fertility and queen signal production being under shared endocrine control.

Dual effect of wasp queen pheromone in regulating insect sociality.

Eusocial insects exhibit a remarkable reproductive division of labor between queens and largely sterile workers [1, 2]. Recently, it was shown that queens of diverse groups of social insects employ specific, evolutionarily conserved cuticular hydrocarbons to signal their presence and inhibit worker reproduction [3]. Workers also recognize and discriminate between eggs laid by the queen and those laid by workers, with the latter being destroyed by workers in a process known as "policing" [4, 5]. Worker policing represents a classic example of a conflict-reducing mechanism, in which the reproductive monopoly of the queen is maintained through the selective destruction of worker-laid eggs [5, 6]. However, the exact signals used in worker policing have thus far remained elusive [5, 7]. Here, we show that in the common wasp, Vespula vulgaris, the pheromone that signals egg maternity and enables the workers to selectively destroy worker-laid eggs is in fact the same as one of the sterility-inducing queen signals that we identified earlier [3]. These results imply that queen pheromones regulate insect sociality in two distinct and complementary ways, i.e., by signaling the queen's presence and inhibiting worker reproduction, and by facilitating the recognition and policing of worker-laid eggs.

Queen signaling in social wasps.

Social Hymenoptera are characterized by a reproductive division of labor, whereby queens perform most of the reproduction and workers help to raise her offspring. A long-lasting debate is whether queens maintain this reproductive dominance by manipulating their daughter workers into remaining sterile (queen control), or if instead queens honestly signal their fertility and workers reproduce according to their own evolutionary incentives (queen signaling). Here, we test these competing hypotheses using data from Vespine wasps. We show that in natural colonies of the Saxon wasp, Dolichovespula saxonica, queens emit reliable chemical cues of their true fertility and that these putative queen signals decrease as the colony develops and worker reproduction increases. Moreover, these putative pheromones of D. saxonica show significant conservation with those of Vespula vulgaris and other Vespinae, thereby arguing against fast evolution of signals as a result of a queen-worker arms race ensuing from queen control. Lastly, levels of worker reproduction in these species correspond well with their average colony kin structures, as predicted by the queen signaling hypothesis but not the queen control hypothesis. Altogether, this correlative yet comprehensive analysis provides compelling evidence that honest signaling explains levels of reproductive division of labor in social wasps.

Conserved class of queen pheromones stops social insect workers from reproducing.

A major evolutionary transition to eusociality with reproductive division of labor between queens and workers has arisen independently at least 10 times in the ants, bees, and wasps. Pheromones produced by queens are thought to play a key role in regulating this complex social system, but their evolutionary history remains unknown. Here, we identify the first sterility-inducing queen pheromones in a wasp, bumblebee, and desert ant and synthesize existing data on compounds that characterize female fecundity in 64 species of social insects. Our results show that queen pheromones are strikingly conserved across at least three independent origins of eusociality, with wasps, ants, and some bees all appearing to use nonvolatile, saturated hydrocarbons to advertise fecundity and/or suppress worker reproduction. These results suggest that queen pheromones evolved from conserved signals of solitary ancestors.

Social evolution: when promiscuity breeds cooperation.

In social evolution theory, it has become common wisdom that close family ties should promote cooperative behaviour. Yet, in social insects, evidence is accumulating that queen promiscuity and low relatedness sometimes work better.

The evolution of honest queen pheromones in insect societies.

Social insect workers are often capable of reproduction, but will not do so in the presence of a fertile queen. In large societies, queens are expected to produce a pheromone that honestly signals her dominance and/or fertility, to which workers respond by suppressing the development of their ovaries and by preventing other workers from reproducing (worker policing). However, what maintains the honesty of such queen pheromones is still under discussion. The explanation that an honest queen signal evolves simply because it serves the interest of all colony members does not seem to hold, since it is undermined by the fitness benefits of direct reproduction of workers at the individual level. A better explanation may be found in the idea that queen pheromones are difficult to produce for subordinate individuals, either because policing workers attack them, or because queen pheromones are intrinsically costly chemicals. Here, I discuss some of the arguments for and against these hypotheses and the evolutionary scenarios that each would lead to.

Rapid decision-making with side-specific perceptual discrimination in ants.

BACKGROUND: Timely decision making is crucial for survival and reproduction. Organisms often face a speed-accuracy trade-off, as fully informed, accurate decisions require time-consuming gathering and treatment of information. Optimal strategies for decision-making should therefore vary depending on the context. In mammals, there is mounting evidence that multiple systems of perceptual discrimination based on different neural circuits emphasize either fast responses or accurate treatment of stimuli depending on the context. METHODOLOGY/PRINCIPAL FINDINGS: We used the ant Camponotus aethiops to test the prediction that fast information processing achieved through direct neural pathways should be favored in situations where quick reactions are adaptive. Social insects discriminate readily between harmless group-members and dangerous strangers using easily accessible cuticular hydrocarbons as nestmate recognition cues. We show that i) tethered ants display rapid aggressive reactions upon presentation of non-nestmate odor (120 to 160 ms); ii) ants' aggressiveness towards non-nestmates can be specifically reduced by exposure to non-nestmate odor only, showing that social interactions are not required to alter responses towards non-nestmates; iii) decision-making by ants does not require information transfer between brain hemispheres, but relies on side-specific decision rules. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that first-order olfactory processing centers (up to the antennal lobes) are likely to play a key role in ant nestmate recognition. We hypothesize that the coarse level of discrimination achieved in the antennal lobes early in odor processing provides enough information to determine appropriate behavioral responses towards non-nestmates. This asks for a reappraisal of the mechanisms underlying social recognition in insects.