Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.)

Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour''s glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour''s gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour''s gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.     

Effect of honey bee queen mating condition on worker ovary activation

The presence of the honey bee queen reduces worker ovary activation. When the queen is healthy and fecund, this is interpreted as an adaptive response as workers can gain fitness from helping the queen raise additional offspring, their sisters. However, when the queen is absent, workers activate their ovaries and lay unfertilized eggs that become males. Queen pheromones are recognised as a factor affecting worker ovary activation. Recent work has shown that queen mandibular pheromone composition changes with queen mating condition and workers show different behavioural responses to pheromone extracts from these queens. Here, we tested whether workers reared in colonies with queens of different mating condition varied in level of ovary activation. We also examined the changes in the chemical composition of the queen mandibular glands to determine if the pheromone blend varied among the queens. We found that the workers activated their ovaries when queens were unmated and had lower ovary activation when raised with mated queens, suggesting that workers detect and respond adaptively to queens of differing mating status. Moreover, variation in queen mandibular gland’s chemical composition correlated with the levels of worker ovary activation. Although correlative, this evidence suggests that queen pheromone may act as a signal of queen mating condition for workers, in response to which they alter their level of ovary activation.     

Multiple functions of fire ant Solenopsis invicta mandibular gland products

Alarm pheromones of social insects are best known for their role in the defence and maintenance of colony integrity. Previous studies with the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) demonstrate that the mandibular glands of workers (sterile females) and male and female sexuals produce an alarm pheromone, 2‐ethyl‐3,6‐dimethylpyrazine. The function of alarm pheromones in worker ants is well understood and divergent from the function of these compounds in the winged sexual forms. The present study quantifies the amount of pyrazine in the mandibular glands from male and female alate sexuals, as well as queens. Pyrazine production in female alates starts in the late pupal stage and increases until they reach mating flight‐ready maturity; however, after mating flight participation, the pyrazine level declines by >50%. Interestingly, mature male alates lose >85% of their mandibular gland pyrazine during mating flight activity. The results of the present study indicate that male and female sexuals use mandibular gland secretions for mating flight initiation and during mating flights. Furthermore, the ontogeny of mandibular gland products (pyrazine as the marker) from newly‐mated queens to mature colony queens shows a more than two‐fold increase in the amount of pyrazine by 6 months after mating. However, this is followed by a decline to trace amounts in mature colony queens (>2 years old), suggesting a function for mandibular gland products during colony development. Multifunctional use of social insect pheromones is well documented and data are reported in the present study suggesting new roles for mandibular gland products in fire ants.     

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.     

Comparative morphology of cephalic exocrine glands among castes of the black ant Lasius niger

The glandular system is crucially involved in main aspects of ant social life. The function of glands has been primarily studied in the workers (the non-reproductive individuals in a colony). In contrast, little information is available on queens (the reproductive females in a colony) or males in spite of the obvious functional differences between these castes. Here we report a comparison of the general morphology of the mandibular, propharyngeal and postpharyngeal glands between the three castes of the black ant Lasius niger. The analysis clearly shows that all these cephalic glands differ in relative size between castes and suggests a link between gland structure and its behavioral role in queens, workers and males. In particular, males present a hypertrophied mandibular gland. This is consistent with the fact that these glands might be the source of the sex pheromone in this caste. By contrast, queens exhibited the most developed postpharyngeal glands. This is consistent with the production of particular cues by queens for workers to help them to distinguish between reproductive and non-reproductive females. Finally, the propharyngeal glands were most developed in the worker caste and of similar relative size in males and queens. Their function is still enigmatic.     

Effects of insemination quantity on honey bee queen physiology

Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.     

Glandular Epithelium as a Possible Source of a Fertility Signal in Ectatomma tuberculatum (Hymenoptera: Formicidae) Queens

The wax layer covering the insect''s cuticle plays an important protective role, as for example, uncontrolled water loss. In social insects, wax production is well-known in some bees that use it for nest building. Curiously, mated-fertile queens of the ant Ectatomma tuberculatum produce an uncommon extra-wax coat and, consequently queens (mated-fertile females) are matte due to such extra cuticular hydrocarbon (CHC) coat that covers the cuticle and masks the brightness of the queens'' cuticle while gynes (virgin-infertile queens) are shiny. In this study, histological analysis showed differences in the epidermis between fertile (i.e., queens or gynes with highly ovarian activity) and infertile females (gynes or workers with non developed ovaries). In fertile females the epidermis is a single layer of cubic cells found in all body segments whereas in infertile females it is a thin layer of flattened cells. Ultrastructural features showed active secretory tissue from fertile females similar to the glandular epithelium of wax-producing bees (type I gland). Different hypotheses related to the functions of the glandular epithelium exclusive to the E. tuberculatum fertile queens are discussed.     

Queen substances from the abdomen of the honey bee queen

Summary The secretion of the mandibular glands of a honey bee queen enables the worker bees to react to the presence of their queen. Extirpating the mandibular glands of the queen does not prevent that she is accepted by her colony. Hitherto this was attributed to contamination of the queen's body by mandibular gland substances during or preceding the extirpation. When, however, these glands are extirpated before they have secreted any material and the queens are inseminated artificially, the colonies still accept these queens. A normal-sized retinue, the absence of emergency cell building and the absence of activation of the worker's ovaries indicate that such a queen is still able to maintain her social position. This supports Verheijen-Voogd's (1959) conclusion that the queen's influence on her workers has a behavioural basis (chemoreception) rather than a biochemical one.Laboratory experiments reveal that apart from the mandibular gland substances other queen pheromones are produced in glands on the abdomen, most probably in the glands described by Renner and Baumann (1964).     

Chemical signals of queens in kin recognition of honeybees,Apis mellifera L.

Summary The volatile signals of live queens as well as head and tergite extracts were analyzed by capillary gas chromatography. Multivariate analysis revealed a significant decrease in the variability of the volatile signals of queens the more closely related they were. Though extracts of the tergal glands showed the same phenomenon, head extracts of closely related queens had no significantly reduced variabilities compared to extracts of unrelated queens. Workers, tested in a metabolic bioassay, could not discriminate among head extracts of related and unrelated queens. Therefore the classical queen substance and the semiochemicals of the mandibular glands are unlikely to be used as kin recognition labels by the workers. Workers could, however, discriminate among the odours of tergite extracts and the volatile signals of queens, indicating that the signal of the tergal gland secretions is a possible source for kin recognition labels which the mandibular gland secretions are not. Furthermore the results show that learning is strongly involved in the kin recognition system of honeybees.     

Functional morphology of the postpharyngeal gland of queens and workers of the ant Monomorium pharaonis (L.)

Eelen D., Børgesen L.W. and Billen J. 2006. Functional morphology of the postpharyngeal gland of queens and workers of the ant Monomorium pharaonis (L.). —Acta Zoologica (Stockholm) 87 : 101–111 The postpharyngeal gland (PPG) is unique to ants and is the largest exocrine gland in their head. In queens of the pharaoh's ant, Monomorium pharaonis, the gland contains approximately 15 finger‐like epithelial extensions on each side and opens dorsolaterally in the posterior pharynx. In these ants the PPG morphology varies considerably according to age and mating status. The epithelial thickness increases with age and reaches a maximum at 3 weeks in both virgin and mated queens. A considerable expansion of the lumen diameter occurs in both groups between 4 and 7 days. Virgin queens release their secretion into the gland lumen from an age of 7 days, whereas mated queens accumulate large amounts of secretion in their epithelium. The increasing epithelial thickness, together with the increasing lumen diameter, the presence of numerous inclusions in the epithelium and the release of secretion, are indicative for increasing gland activity. The gland ultrastructure indicates involvement in lipid metabolism and de novo synthesis of lipids. The PPG of workers consists of 12 finger‐like tubes at each side. There is a significant difference in epithelial thickness between nurses and repletes and between nurses and foragers. We suggest the PPG serves different purposes in pharaoh's ants: it is likely that the PPG of workers and virgin queens is used to feed larvae. In mated queens the gland probably plays a role in providing the queen with nutritious oils for egg production. The PPG may also function in signalling species nestmate and caste identity, as well as in the reproductive capacity of the queens.     

The queen in relation to wax secretion and comb building in honeybees

Summary The role of the queen in relation to wax secretion and comb building in honeybees was analyzed with respect to queen status (mated, virgin and dead queens and queenlessness), and pheromones of the head and abdominal tergite of queens. Worker variables considered were colony size, percentage of bees bearing wax scales, wax scale weight, and weight of constructed combs.The amount of wax recovered from festoon bees and the percentage of festoon bees bearing wax were independent of queen status, the pheromones of queens and access to the queen. Colonies with full access to freely moving mated queens always constructed significantly more comb than those headed by virgin or dead queens as well as all permutations of caged and division board queens whose mandibular glands and/or abdominal tergite glands were operative or not.Despite pheromonal similarity of virgin queens to mated ones, colonies headed by virgin queens constructed as little comb as did queenless colonies. The bouquets of the mandibular glands did not differ significantly among queens nor was the amount of comb constructed correlated with pheromonal bouquet. Comb building is greatest among colonies having full access to freely moving queens but the stimulus for such building is not attributable to the 90DA, 9HDA and 10HDA components of the queen's mandibular gland secretions.     

Mating Triggers Queen Elimination by Workers of Japanese Harvester Ant (<Emphasis Type="Italic">Messor aciculatus</Emphasis>)

During reproduction, ant colonies produce winged queens. These new queens usually leave the nest to mate and can then establish a new nest. If the new nest is close to an existing colony, it will be in competition with the existing colony. Therefore, workers will kill any mated queens they find outside the colony during the reproductive season. In this study, factors that might determine whether workers eliminate queens were investigated. Mating status (mated or unmated), colony origin (same or different to tested workers) and mating partners (inbred or outbred) of the queens of Japanese harvester ants (Messor aciculatus) were manipulated and the workers’ behavior towards the queens was observed. Mated queens were always attacked by workers, though this was not affected by either colony origin or mating partners. These results suggest that mating status triggers elimination of queens by workers, and that the colony origin and mating partner are unlikely to be important roles in elimination of queens.     

中华蜜蜂上颚腺的组织结构及其胚后发育

【目的】上颚腺（mandibular gland）是昆虫重要的外分泌腺,它产生的化学物质在昆虫的种内信息交流中起重要的作用。本研究目的在于了解中华蜜蜂Apis cerana cerana上颚腺的组织结构以及胚后发育特点。【方法】本研究通过组织形态学、BrdU免疫组织化学等技术,对中华蜜蜂上颚腺的结构和发育过程进行了比较研究。【结果】中华蜜蜂的上颚腺在不同级型间差异显著,蜂王的面积最大,工蜂较小,而雄蜂退化。上颚腺出现在末龄幼虫到预蛹阶段,细胞分裂活动的高峰期发生在蛹发育的第1天,随后分裂细胞数减少,并一直持续到蛹发育的第6天结束。在上颚腺发育早期,由分泌细胞分化的内膜就已经出现,并一直保持到成虫。【结论】本研究为昆虫上颚腺的发育和功能研究提供了理论基础。     

Exocrine glands in the legs of the social wasp Vespula vulgaris

This study brings a survey of the exocrine glands in the legs of Vespula vulgaris wasps. We studied workers, males, virgin queens as well as mated queens. A variety of 17 glands is found in the different leg segments. Among these, five glands are novel exocrine structures for social insects (trochanter-femur gland, ventrodistal tibial gland, distal tibial sac gland, ventral tibial gland, and ventral tarsomere gland). Most leg glands are present in the three leg pairs of all castes. This may indicate a mechanical function. This is likely for the numerous glands that occur near the articulation between the various leg segments, where lubricant production may be expected. Other possible functions include antenna cleaning, acting as a hydraulic system, or pheromonal. Further research including leg-related behavioural observations and chemical analyses may help to clarify the functions of these glandular structures in the legs.     

Source of alate excitant pheromones in the red imported fire antSolenopsis invicta (Hymenoptera: Formicidae)

At the onset of mating flights inSolenopsis invicta, workers swarm excitedly over the mound as alates prepare to fly. Previous studies demonstrated that this excitement is stimulated by the male and female alates. We investigated the glandular source(s) of pheromones produced by the alates that cause excitement. The only common female and male alate body part that elicited excitement when crushed was the head. Within the head, excised mandibular glands were found to be responsible for worker excitement. Fire ant workers are very sensitive to external stimuli and some excitement was elicited by crushed female gasters and male thoraces, but the response was never as significant as with crushed heads. Tests with summer and winter alates revealed similar results, except that gasters of winter female alates had a greater excitant effect than did gasters of summer female alates. This may be due to the production of attractant pheromones by the poison glands of overwintering female alates. We conclude that the mandibular gland is the source of alate excitant pheromones.     

The function of the post-pharyngeal glands of the red imported fire ant, Solenopsis invicta buren

Using radiolabelled triglycerides and fatty acids we have shown that these 2 lipids are absorbed into the haemolymph of the imported fire ant, Solenopsis invicta Buren from the post-pharyngeal gland. The post-pharyngeal glands attain their greatest weight and contain the highest hexane extractable lipid in establishing queens which have just reared the first minum brood. The lipid content of the digestive system is greatest in queens initiating a mating flight with the majority of the lipid contained in the crop. During colony establishment by the queen the lipid content of the crop moves forward, some is fed to the developing larvae and some moves into the post-pharyngeal gland. The hexane extractable lipid from the postpharyngeal gland of newly mated queens consists of hydrocarbons, sterols. tri-, di-, and monoglycerides and free fatty acids with a trace of wax esters.Excision of the post-pharyngeal glands from mated established queens causes no noticeable change in their behaviour or the behaviour they elicit from the remainder of the colony. However, post-pharyngeal glandectomized females lost weight and died in ca. 2 months although their crop and midgut contained food. The post-pharyngeal glands therefore appear to function in much the same way as a gastric caecum.     

Abdominal microbial communities in ants depend on colony membership rather than caste and are linked to colony productivity

Gut bacteria aid their host in digestion and pathogen defense, and bacterial communities that differ in diversity or composition may vary in their ability to do so. Typically, the gut microbiomes of animals living in social groups converge as members share a nest environment and frequently interact. Social insect colonies, however, consist of individuals that differ in age, physiology, and behavior, traits that could affect gut communities or that expose the host to different bacteria, potentially leading to variation in the gut microbiome within colonies. Here we asked whether bacterial communities in the abdomen of Temnothorax nylanderi ants, composed largely of the gut microbiome, differ between different reproductive and behavioral castes. We compared microbiomes of queens, newly eclosed workers, brood carers, and foragers by high‐throughput 16S rRNA sequencing. Additionally, we sampled individuals from the same colonies twice, in the field and after 2 months of laboratory housing. To disentangle the effects of laboratory environment and season on microbial communities, additional colonies were collected at the same location after 2 months. There were no large differences between ant castes, although queens harbored more diverse microbial communities than workers. Instead, we found effects of colony, environment, and season on the abdominal microbiome. Interestingly, colonies with more diverse communities had produced more brood. Moreover, the queens' microbiome composition was linked to egg production. Although long‐term coevolution between social insects and gut bacteria has been repeatedly evidenced, our study is the first to find associations between abdominal microbiome characteristics and colony productivity in social insects.     

The evolution of food-producing glands in eusocial bees (Apoidea,Hymenoptera)1

A food-producing role for cephalic exocrine glands has arisen independently in both taxa of highly eusocial bees, Apis and Meliponini. With several exceptions, there is little evidence that food is produced by glands of solitary bees or by most bees at lower levels of sociality. We suggest that this association with sociality is due to four adaptive features of these glands: (1) food from the glands allows feces from queens and larvae to have a small volume, (2) the queen's fecundity can be increased, (3) nutrient recovery via cannibalism can be facilitated, and (4) rearing of emergency replacement queens is accelerated. Acceleration of the rearing of other castes and of queens in the normal process of colony fission is not clearly an advantage ascribed to these glands. Trophic eggs produced by meliponine colony workers are analogous to the secretions from food-producing glands in Meliponini and Apis workers.     

The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes

Neither the overall differences in ovariole number nor the caste-specifically modulated expression of vitellogenin can fully explain the striking caste differences in honey bee reproduction, in particular the mechanisms that block oogenesis in virgin queens and in workers kept in the presence of a queen. For this reason we investigated the initial stages of oogenesis in queens in relation to mating status and in workers exposed to different social conditions. A striking feature in ovarioles of both castes was a considerably elongated terminal filament which consisted not only of normal terminal filament cells but also contained apparently undifferentiated cells that were tentatively considered as stem cells. BrdU incorporation was detected in the upper germarium, as well as in the terminal filament. Cytoskeleton analysis by TRITC-phalloidin labeling for F-actin, and immunofluorescence detection for β-tubulin did not reveal structural differences in the early oogenesis steps between queens and queenless workers. In contrast, queenright workers showed signs of a disorganized microtubule and microfilament system that could explain the histological evidence for progressive cell death observed in the germaria. In addition to cytoplasmic tubulin we also detected marked intranuclear foci indicating the presence of nuclear β_II-tubulin.     

Queen recognition by brood-rearing workers of the ant Myrmica rubra L

The means by which queens communicate their presence to workers and so influence broodrearing, in particular caste determination, have been analysed; both chemical and topographical stimuli are used. This follows from the fact that though dead inseminated queens are effective, the following are not: dead virgins (same shape but different smell), lipid extracted mature queens (same shape but no smell), parts of inseminated queens alone or together (same smell but different shape). This also rules out a drug-like influence on workers or larvae. The chemicals are not produced in the two main head glands, the mandibular and the pharyngeal, as no loss of potency follows their excision. The abdomen is the most likely source of a substance, the emission of which must vary seasonally.