Mandibular Gland Pheromone Research Articles

A Hitchhiker’s Ride: The Honey Bee Louse Braula Coeca (Diptera: Braulidae) Selects its Host by Eavesdropping

The bee louse Braula spp. had until recently a distribution coincident with its host the honey bee. The adult fly usually attaches to a worker honey bee and steals food from its mouth. However, not all worker bees carry Braula spp. and the mechanism used by Braula spp. to select hosts is not well understood. Using choice remounting bioassays and chemical analyses, we determined host selection and the cues used by B. coeca, a species associated with the African honey bee Apis mellifera scutellata. Braula coeca successfully remounted bees from which they were initially removed and preferred their mandibular gland pheromones (MDG) over those of bees not carrying them. The bee lice did not show any preference for the cuticular hydrocarbons of both types of workers. Chemical analyses of the MDG extracts, revealed quantitative differences between the two categories of workers, with workers carrying B. coeca having more of the queen substance (9-oxo-2(E)-decenoic acid) and worker substance (10-hydroxy-2(E)-decenoic). Braula coeca showed a dose response to the queen substance, indicating its ability to use host derived kairomones as cues that allowed it to benefit from trophallactic dominance by individuals that have a higher probability of being fed by other workers.

Fighting ability and the toxicity of raiding pheromone in an obligate kleptoparasite, the stingless bee Lestrimelitta niitkib

The evolution of obligate kleptoparasitism, the theft of food, has led to remarkable innovations, including physical weapons and chemical signals that can evolve into chemical weapons. Stingless bees in the genus Lestrimelitta are excellent examples of this phenomenon because they are obligate kleptoparasites that no longer collect floral resources and instead steal brood resources from other bees. Their ability to raid successfully is thus essential to their fitness even when they fight species that are physically bigger, have larger defense forces, or both. We conducted morphometric analyses, quantified Lestrimelitta niitkib mandibular gland pheromone (MGP) components, and carried out individual fighting trials between L. niitkib and the stingless bee Scaptotrigona mexicana, a common victim species, to shed light on the detailed reasons for their success at robbing. Measurements showed that L. niitkib mandibles have thicker exoskeleton cuticles and overall greater width, particularly in the medial and proximal sections, than S. mexicana, which is quite similar in body size. In all fights, L. niitkib bit victims and released MGP, as it does during raids. Scaptotrigona mexicana victims exhibited significantly increased uncoordinated behaviors and showed partial or complete paralysis. We analyzed and quantified the major components of MGP, which consisted of large quantities of geranial (mean of 253 μg) and neral (48 μg) per bee. Microinjections of 1 bee equivalent (BE) of natural or synthetic MGP and ≥ 0.1 BE of geranial significantly increased deleterious behaviors and paralysis as compared to control injections. We suggest that the large quantities of MGP used during raiding have led to an unexpected outcome, a semiochemical evolving the additional function of a toxin, and contribute to the ability of Lestrimelitta to rob its victims.Significance statementKleptoparasites, organisms that steal food resources, employ multiple physical and chemical tools to survive. The success of kleptoparasitism requires a balance between honesty and coercion in interspecific communication. The genus Lestrimellita consists of a group of kleptoparasitic stingless bee species that raid other bee colonies for food and therefore depend upon winning these raids. However, why they succeed remains not fully understood. We studied differences in morphology between L. niitkib and its victims, the pheromones they release during raids, and ran individual fight trials between L. niitkib and a common victim to identify why they are successful. We suggest that the release of pheromones at the beginning of raids, in concert with the pheromone’s toxicity, has been combined to improve L. niitkib’s ability to successfully rob.

Queen honey bee (Apis mellifera) pheromone and reproductive behavior are affected by pesticide exposure during development

Pollinator diversity and abundance in North America have been at a steep decline over the last two decades due to the combinatorial effects of several environmental and anthropogenic stressors. In particular, managed honey bees (Apis mellifera) face multiple health risks including nutritional stress, exposure to pests and pathogens, poor queen quality, and pesticide contamination, which cause problems at the individual and colony levels. One of the gravest problems faced by honey bees is parasitization by the mite Varroa destructor, which is typically controlled through the application of miticides such as tau-fluvalinate, coumaphos, and amitraz. In addition to miticides, colonies are also exposed to pesticides brought by foragers from agricultural settings, including the fungicide chlorothalonil and the insecticide chlorpyrifos. Here, we explored whether exposure of wax to combinations of these pesticides during development affects honey bee queen physiology and worker behavior. To do this, we reared queens in plastic cups coated with molten beeswax that was either pesticide-free or containing field-relevant concentrations of tau-fluvalinate and coumaphos, amitraz, or chlorothalonil and chlorpyrifos. Once queens mated naturally, we placed them in observation hives to measure egg-laying rate and worker retinue size. We then dissected the queens and used the contents of their mandibular glands to measure worker attractiveness in caged bioassays and to analyze their chemical components using GC-MS. Exposure of wax to field-relevant concentrations of the tested pesticides during queen development significantly lowered the adult queens’ egg-laying rate and worker retinue size. Miticide exposure during development also lowered the attractiveness of queen mandibular gland contents to workers and affected the relative amounts of the glands’ chemical components. Our results support the ideas that mandibular gland pheromones act as honest indicators of queen reproductive fitness and that pesticide exposure of wax during bee development is an important and concerning factor impairing honey bee health. Pollinator health is currently being threatened by multiple environmental factors, including poor nutrition, habitat fragmentation, and pesticide contamination. In particular, honey bee health is facing challenges caused by combinations of these factors, leading to an annual mortality of over 30% of all managed colonies in the USA. The conditions in which honey bee queens are reared can cause severe physiological and behavioral effects in adults, which in turn can affect the behavior of workers that tend to those queens, thus affecting colony productivity, growth, and survival. In this study, we show that queens reared in beeswax contaminated with miticides or agrochemicals lay fewer eggs over time and exhibit mandibular gland pheromone profiles that differ from those of queens reared in pesticide-free wax. Workers discern these differences, preferentially investigating the gland contents and forming larger retinues (thus performing more tending behaviors) for queens reared in pesticide-free wax. These results indicate that pesticide contamination of the queen-rearing wax directly affects queen physiology and behavior and further affirm that queen mandibular pheromones seem to act as honest indicators to workers of a queen’s reproductive capacity and overall health.

Larval Pheromone Disrupts Pre-Excavation Aggregation of Cactoblastis cactorum (Lepidoptera: Pyralidae) Neonates Precipitating Colony Collapse

Newly eclosed larvae of Cactoblastis cactorum Berg (Lepidoptera: Pyralidae) contain their activity to an arena formed at the base of their eggstick, marked with a mandibular gland pheromone. Laboratory and field studies were undertaken to determine if these pre-excavation aggregations, essential to their successful penetration of the host plant, could be disrupted with mandibular gland extract causing the incipient colonies to perish. Cladodes or whole plants were sprayed with the pheromone, obtained by extracting macerated caterpillars in hexane, hexane only, or left unsprayed, and the survivorship of caterpillars that eclosed from eggsticks attached to the cladodes recorded at a later date. In 4 separate experiments, the average survivorship of C. cactorum larvae from cohorts on cladodes sprayed with the extract (15%) differed markedly from survivorship of caterpillar cohorts on cladodes treated with the solvent only (84%) or left untreated (80%). This differential mortality was attributed to the elicitation of the independent dispersal of the caterpillars by the mandibular gland pheromone and their failure to reaggregate in numbers sufficient to mount a successful attack on the host plant. The potential for managing pest populations of caterpillars employing this target-specific alternative to conventional pesticides is discussed.

Hydroxylation patterns associated with pheromone synthesis and composition in two honey bee subspecies Apis mellifera scutellata and A. m. capensis laying workers

Colony losses due to social parasitism in the form of reproductive workers of the Apis mellifera capensis clones results from the production of queen-like pheromonal signals coupled with ovarian activation in these socially parasitic honey bees. While the behavioral attributes of these social parasites have been described, their genetic attributes require more detailed exploration. Here, we investigate the production of mandibular gland pheromones in queenless workers of two sub-species of African honey bees; A. m. scutellata (low reproductive potential) and A. m. capensis clones (high reproductive potential). We used standard techniques in gas chromatography to assess the amounts of various pheromone components present, and qPCR to assess the expression of cytochrome P450 genes cyp6bd1 and cyp6as8, thought to be involved in the caste-dependent hydroxylation of acylated stearic acid in queens and workers, respectively. We found that, for both subspecies, the quality and quantity of the individual pheromone components vary with age, and that from the onset, A. m. capensis parasites make use of gene pathways typically upregulated in queens in achieving reproductive dominance. Due to the high production of 9-hydroxy-decenoic acid (9-HDA) the precursor to the queen substance 9-oxo-decenoic acid (9-ODA) in newly emerged capensis clones, we argue that clones are primed for parasitism upon emergence and develop into fully fledged parasites depending on the colony's social environment.

Increased response to sequential infections of honeybee, Apis mellifera scutellata, colonies by socially parasitic Cape honeybee, A. m. capensis, workers

Cape honeybee, Apis mellifera capensis, workers can be social parasites and host colonies can defend themselves by rejection of such workers. Using the pseudo-clonal obligate parasitic lineage of A. m. capensis and wild-type A. m. capensis workers, which are facultative parasites, we show that host colonies significantly increase their defence behaviour towards social parasites upon secondary exposure. Most obligate and facultative social parasites were rejected before they could even produce significant amounts of the queen-like mandibular gland pheromone secretion or activate their ovaries. This suggests that other signals, like cuticular hydrocarbons, could be used by host colonies to identify potential invaders. Honeybee colonies seem to be able to utilise these potential cues, learn from their initial exposure to hive intruders and enable them to improve their defensive behaviour during subsequent infestations, resulting in increased removal rates of parasites.

Reproductive parasitism by worker honey bees suppressed by queens through regulation of worker mandibular secretions

Social cohesion in social insect colonies can be achieved through the use of chemical signals whose production is caste-specific and regulated by social contexts. In honey bees, queen mandibular gland pheromones (QMP) maintain reproductive dominance by inhibiting ovary activation and production of queen-like mandibular gland signals in workers. We investigated whether honey bee queens can control reproductively active workers of the intraspecific social parasite Apis mellifera capensis, parasitising A. m. scutellata host colonies. Our results show that the queen’s QMP suppresses ovarian activation and inhibits the production of QMP pheromone signals by the parasitic workers, achieved through differential expression of enzymes involved in the biosynthesis of these pheromones at two points in the biosynthetic pathway. This is the first report showing that honey bee queens can regulate reproduction in intraspecific social parasites and deepens our understanding of the molecular mechanisms involved in the regulation of worker reproduction in social insects.

Control of mandibular gland pheromone synthesis by alternative splicing of the CP-2 transcription factor gemini in honeybees (Apis mellifera carnica)

The honeybee queen’s mandibular gland pheromones (QMP) are essential for the suppression of worker reproduction. Worker ovary activation is regulated by alternative splicing of a CP2-transcription factor named gemini. Since workers with activated ovaries also produce QMP in their mandibular glands, we tested whether alternative splicing of gemini also controls mandibular gland pheromone biosynthesis in workers using RNA interference. Altering the splice pattern of gemini resulted in enhanced levels of the queen-specific components of the mandibular gland pheromone in queenless honeybee workers, suggesting that gemini functions as a pleiotropic regulatory switch influencing both ovary activation and resulting in QMP synthesis in workers. Because the QMP produced by these workers suppresses ovary activation in other workers, gemini seems to be a key regulatory gene affecting reproductive hierarchies among workers in queenless colonies.

Turning workers into false queens: the role of exogenous pheromones in regulating reproduction in worker honey bees.

One of the responses that honey bee workers can make in the event of queen loss is to develop into false queens. False queens are workers that exhibit both behavioural and physiological traits similar to those of a true queen. However, the presence of more than one false queen in a colony distorts the established hierarchies. As transformation into a false queen occurs after emergence as an adult, we tested the effect of worker mobile pheromone carriers (PCs) treated with exogenously supplied pheromones on their nestmates. The PCs carried either synthetic mandibular gland pheromones or pheromones extracted from Apis melliferacapensis parasitic workers. Only the PCs attracted retinues of workers, increased pheromone production and activated their ovaries, becoming false queens. Pheromones from A. m.capensis workers were more effective than extracts of commercially available synthetic queen pheromones in eliciting these effects. Using this simple mobile pheromone delivery system, we have shown that carrying amounts of exogenous pheromone can induce pheromone production in the carrier, resulting in the production of false queens within experimental groups. Possible implications of using this technique to modify and regulate worker reproduction in colonies are discussed.

Computational genome-wide survey of odorant receptors from two solitary bees Dufourea novaeangliae (Hymenoptera: Halictidae) and Habropoda laboriosa (Hymenoptera: Apidae)

Olfactory/odorant receptors (ORs) probably govern eusocial behaviour in honey bees through detection of cuticular hydrocarbons (CHCs) and queen mandibular gland pheromones (QMP). CHCs are involved in nest-mate recognition whereas QMP acts as sex pheromone for drones and as retinue pheromone for female workers. Further studies on the effect of eusociality on the evolution of ORs are hindered by the non-availability of comprehensive OR sets of solitary species. We report complete OR repertoires from two solitary bees Dufourea novaeangliae (112 ORs) and Habropoda laboriosa (151 ORs). We classify these ORs into 34 phylogenetic clades/subfamilies. Differences in the OR sets of solitary and eusocial bees are observed in individual subfamilies like subfamily 9-exon (putative CHC receptors) and L (contains putative QMP receptor group). A subfamily (H) including putative floral scent receptors is expanded in the generalist honey bees only, but not in the specialists. On the contrary, subfamily J is expanded in all bees irrespective of their degree of social complexity or food preferences. Finally, we show species-lineage specific and OR-subfamily specific differences in the putative cis-regulatory DNA motifs of the ORs from six hymenopteran species. Out of these, [A/G]CGCAAGCG[C/T] is a candidate master transcription factor binding site for multiple olfactory genes.

Reproductive traits and mandibular gland pheromone of anarchistic honey bee workers Apis mellifera occurring in China

In honey bee colonies, workers, in particular of “anarchistic” lineages, can activate their ovaries and lay eggs, even in the presence of the queen. We identified three queenright colonies showing typical signs of worker reproduction. To characterize this new lineage, we extracted the mandibular gland and analyzed it using gas chromatography. The total amounts of the five main components of the mandibular gland, namely methyl p-hydroxyben-zoate (HOB), 9-oxo-2(E)-decenoic acid (9-ODA), (S)-9-hydroxy-(E)-2-decenoic acid (9-HDA), 10-HDA, and 10-hydroxyde-canoic acid (10-HDAA) were significantly higher in the mandibular gland profiles of workers with activated ovaries (AWs, 8.88 ± 1.71 μg) compared to workers with inactivated ovaries (IAWs, 4.00 ± 2.09 μg). Furthermore, the chemical profiles of IAWs were dominated by the “worker substances” 10-HDA (34.64 ± 8.19 %) and its precursor 10-HDAA (22.88 ± 4.95 %), while the chemical profiles in AWs were dominated by the precursor of the queen substance 9-HDA (40.04 ± 7.55 %). The ratios of two precursor substances 10-HDAA/9-HDA of IAWs were more worker like (>1.0) whereas AWs were more queen like (≤1.0). These results suggest that the mandibular pheromones of anarchistic workers resemble a more queen-like reproductive active profile and that these workers may represent a reversion to a more basal reproductive phenotype.

Mandibular gland pheromone contents in workers and queens of Apis mellifera adansonii

Secretions from the mandibular glands of honeybees have been studied extensively, with those of queens dominated by ώ-9 fatty acids and ώ-10 fatty acids dominating those of non-laying workers. Apis mellifera adansonii (Latreille) is one of the widely distributed subspecies of African honeybees. However, its mandibular gland pheromones have not been analysed previously. Using gas chromatography, we analysed the composition of mandibular gland pheromones in workers and queens of A. mellifera adansonii from Nigeria. Qualitatively, workers and queens have similar pheromone profiles to those previously reported in other African subspecies of honeybees. We found 9-ODA and high amounts of its precursor 9-hydroxy-2 (E)-decenoic acid (9-HDA) in workers, thus showing that they produce queen-like signals under queen-right conditions. We also found geographic variation in the pheromone profiles and morphometric characters of these workers, suggesting different pheromone and morphoclusters from the different ecological and climatological regions inhabited by A. m. adansonii in Nigeria.

Genomic analysis of the interactions between social environment and social communication systems in honey bees (Apis mellifera)

Social context is often a primary regulator of social behavior, but genes that affect or are affected by social context have rarely been investigated. In social insects, caste specific pheromones are key modulators of social behavior, e.g., in honey bees the queen mandibular gland (MG) pheromone mediates reproductive dominance, its absence prompting ovary activation and queen pheromone production in workers. Here, we investigate the effect of social environment on genome-wide expression patterns in the MG, to determine how social context modulates expression of genes that, in turn alter social environment. We used microarrays to examine the MGs of virgin and mated queens, and queenright (QR) and queenless (QL) workers with or without activated ovaries. Approximately 2554 transcripts were significantly differentially expressed among these groups, with caste and social context being the main regulators of gene expression patterns, while physiological state (ovary activation) only minimally affecting gene expression. Thus, social context strongly regulates expression of genes, which, in turn, shape social environment. Among these, 25 genes that are putatively involved in caste selective production of the fatty-acid derived MG pheromone were differentially expressed in queens and workers. These genes whose functions correspond with enzymatic or transport processes emphasize the occurrence of disparate pheromone biosynthetic pathways for queens and workers, adding another dimension regarding the regulation of these important pheromones. Gene ontology analysis also revealed genes of different functional categories whose expression was impacted by caste or by the social environment, suggesting that the MG has broader functions than pheromone biosynthesis.

Bioassay of the mandibular gland pheromones of Apis florea on the foraging activity of dwarf honey bees

SummaryIn this study, we tested the responses (attractiveness or repellence) of two species of dwarf honey bee (Apis andreniformis and Apis florea) foragers to the synthetic mandibular gland pheromones 1-eicosanol, eicosane, heneicosane and 2-heptanol. The pheromone concentrations were varied from 0.0, 0.1, 0.5, 1.0, 5.0 and 10.0% (v/v) in n-hexane. A. andreniformis foragers were repelled by 5.0% eicosane and 5.0 and 10.0% heptanol, while 0.5 and 1.0% 1-eicosanol, 0.1, 0.5, 1.0, 5.0 and 10.0% eicosane, 0.5 and 1.0% heneicosane and 0.1, 0.5, 1.0, 5.0 and 10.0% 2-heptanol repelled A. florea foragers. A. andreniformis foragers were attracted to 0.1% 1-eicosanol, 10.0% eicosane, 1.0, 5.0 and 10.0% heneicosane and 0.1% 2-heptanol. None of the pheromones were attractive to A. florea foragers.

Using sensillum potential analysis to quantify pheromone sensing of the antennal sensilla of Apis florea Fabricius (1787), foragers and guards

Odor perception via the antennal sensilla in most honeybee species is poorly studied. We measured the antennal sensillum potential in response to Apis florea mandibular gland pheromone and showed that it is robust and reliable in forager and guard bees. Mandibular gland pheromone may be involved in signaling alarm or foraging resource depletion. Changes of antennal sensilla placodea potential of A. florea foragers and guards were measured after exposure to three concentrations of the synthetic pheromones, 2-heptanone and ( Z)-11-eicosen-1-ol, using a potentiostat connected to an e-corder (ED401) with microelectrodes. The resting sensillum potential of A. florea foragers and guards were −55.37 ± 3.44 and −52.85 ± 5.34 mV, respectively. The sensillum potential of bees exposed to 1.0%, 5.0% and 10.0% ( Z)-11-eicosen-1-ol were −23.35 ± 0.98, −16.78 ± 1.94 and −24.24 ± 8.20 mV, respectively, in foragers, and −21.95 ± 3.21, −21.42 ± 4.73 and −13.54 ± 4.16 mV, respectively, for guards. Exposure of bees to 1.0%, 5.0% and 10.0% 2-heptanone induced sensillum potentials of −10.64 ± 2.44, −44.88 ± 2.41 and −48.84 ± 4.40 mV, respectively, in foragers and 15.85 ± 9.38, −25.48 ± 1.43 and −15.52 ± 6.61 mV, respectively, in guards. The highest sensillum potential was recorded in foragers exposed to 1.0% 2-heptanone. In general, except for the response to 1.0% 2-heptanone, the sensillum potentials of all bees to ( Z)-11-eicosen-1-ol were higher than that of 2-heptanone. These results show that A. florea antennal sensilla in foragers and guard bees exhibit a stronger response to ( Z)-11-eicosen-1-ol as compared to 2-heptanone. Our results also provide useful comparative data to explore olfactory perception in non-model honey bee species.

Responses of Queenright and Queenless Workers of Apis Cerana to 9-keto-2(E)-decenoic Acid, a Pheromonal Constituent of the Mandibular Gland

In dequeened honeybee colonies ovarian activation occurs in some workers, and the pheromonal bouquets of these laying workers become more queen-like. In the Asiatic honeybee, Apis cerana, we compared the amount of 9-keto-2(E)-decenoic acid (9-ODA), a mandibular gland pheromone component, between non-laying workers from queenright colonies and laying workers from queenless colonies, and further, applied synthetic 9-ODA to workers to determine whether they discriminate workers with activated ovaries based on the level of this compound. Levels of 9-ODA were higher in laying workers from dequeened colonies than in non-laying workers from queenright colonies. In both queenright and queenless colonies, workers attacked more workers treated with 9-ODA than control-treated workers. These results suggest that detection of pseudoqueens in A. cerana is mediated by changes in 9-ODA.

Worker reproduction in mixed-species colonies of honey bees

To explore reasons for the unusually high rates of worker ovary activation in Apis cerana, we investigated the reproductive physiology of workers in conspecific and mixed-species colonies of A. cerana and its sibling species Apis mellifera under queenright and queenless conditions. In both queenless and queenright conspecific colonies, the proportion of A. cerana workers with activated ovaries was significantly greater than the proportion of A. mellifera workers with activated ovaries. Apis cerana workers showed significantly greater ovary activation in queenright mixed-species colonies than in conspecific queenright colonies. Moreover, there was significantly greater ovary activation in A. cerana workers in mixed-species colonies headed by A. mellifera queens than A. mellifera workers in mixed-species colonies headed by A. cerana queens. Apis mellifera workers in conspecific queenless colonies showed significantly greater ovary activation than those in mixed-species queenless colonies. Quantification of the chemical components of mandibular gland pheromones of queens of the 2 species showed that they are similar. Combined, our results show that queen signals have been preserved between the 2 species and that workers respond to the heterospecific signals from queens. However, despite the similarity in the queen mandibular gland fatty acid secretions between the 2 species, workers respond differentially toward hetero- and conspecific queens. Key words: Apis cerana, Apis mellifera, honest signals, ovary activation, pheromones, queen mandibular pheromone, worker. [Behav Ecol]

The role of the queen mandibular gland pheromone in honeybees (Apis mellifera): honest signal or suppressive agent?

Queen pheromones interfere with worker reproduction in social insects. However, there is still an unresolved question as to whether this pheromone acts as an “honest” signal for workers, giving a reliable indication of the queen’s reproductive value, or as a suppressive agent, inhibiting worker reproduction independent of the queen’s reproductive capacity. In honeybees (Apis mellifera), the queen’s mandibular gland secretion, a mix of fatty acids and some aromatic compounds, is crucial for regulating the reproductive division of labor in the colony inhibiting ovary development in workers. We quantified the mandibular gland secretions of virgin, drone-laying, and naturally mated queens using gas chromatography to test whether the queens’ mating, ovary activation, or the reproductive value for workers correlated with the composition of the secretion. Although the absolute amounts of the “queen substance” 9-oxo-2(E)-decenoic acid (9-ODA) were similar among the three groups, the proportions of 9-ODA decreased with increasing reproductive quality. Furthermore, the ratios of queen to worker compounds were similar in all three treatment groups, irrespective of the reproductive capacity. A multivariate analysis including all six compounds could not separate drone-laying queens from naturally mated ones, both with active ovaries but only the latter ensuring colony survival. We suggest that the mandibular gland pheromones are unlikely to function as reliable indicators of queen reproductive value and rather operate as an agent to suppress worker reproduction. This does not exclude the possibility that other “honest” pheromone signals exist in the honeybee colony, but these would have to arise from other semiochemicals, which could be produced by both the queen and the brood.

Pheromonal dominance and the selection of a socially parasitic honeybee worker lineage (Apis mellifera capensis Esch.)

The recent invasion by self-replicating socially parasitic Cape honeybee workers, Apis mellifera capensis, of colonies of the neighbouring African subspecies Apis mellifera scutellata represents an opportunity to study evolution of intraspecific parasitism in real time. As honeybee workers compete pheromonally for reproductive dominance, and as A. m. capensis workers readily produce queen-like pheromones, we hypothesized that these semiochemicals promoted the evolution of intraspecific social parasitism. Remarkably, the offspring of a single worker became established as a parasite in A. m. scutellata's range. This could have resulted from extreme selection among different clonal parasitic worker lineages. Using pheromonal contest experiments, we show that the selected parasitic lineage dominates in the production of mandibular gland pheromones over all other competitors to which it is exposed. Our results suggest that mandibular gland pheromones played a key role in the evolution of intraspecific social parasitism in the honeybee and in the selection of a single genotype of parasitic workers.

Reversible royalty in worker honeybees (Apis mellifera) under the queen influence

In most social insects, worker sterility is reversible, and in the absence of the queen, at least some workers develop ovaries and lay male-destined eggs. In the honeybee, reproductive workers also produce queen-characteristic mandibular and Dufour’s pheromones. The evolution of worker sterility is still under debate as to whether it is caused by queen manipulation (queen-control hypothesis) or represents worker fitness maximization (worker-control hypothesis). In this study, we investigated whether worker fertility and royal pheromone production are reversible under the queen influence. To that effect, we induced ovary activation and queen pheromone production in workers by rearing them as queenless (QL) groups. These workers were subsequently reintroduced into queenright (QR) microcolonies for 1 week, and their ovary status and queen pheromone levels were monitored. Workers reintroduced into QR, but not QL colonies, showed a clear regression in ovary development and levels of the queen pheromones. This is the first demonstration that worker sterility and/or fertility is reversible and is influenced by the queen. These results also emphasize the robustness of the coupling between ovary activation and royal pheromone production, as well as lending credence to the queen-control hypothesis. The dynamics of queen pheromone production in QL workers supports the role of Dufour’s gland pheromone as a fertility signal and that of the mandibular gland pheromone in dominance hierarchies.