Effects of Instrumental Insemination and Insemination Quantity on Dufour’s Gland Chemical Profiles and <Emphasis Type="Italic">Vitellogenin</Emphasis> Expression in Honey Bee Queens (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Honey bee queens (Apis mellifera) mate in their early adult lives with a variable number of males (drones). Mating stimulates dramatic changes in queen behavior, physiology, gene expression, and pheromone production. Here, we used virgin, single drone- (SDI), and multi-drone- (MDI) inseminated queens to study the effects of instrumental insemination and insemination quantity on the pheromone profiles of the Dufour’s gland, and the expression of the egg-yolk protein, vitellogenin, in the fat body. Age, environmental conditions, and genetic background of the queens were standardized to specifically characterize the effects of these treatments. Our data demonstrate that insemination and insemination quantity significantly affect the chemical profiles of the Dufour’s gland secretion. Moreover, workers were more attracted to Dufour’s gland extract from inseminated queens compared to virgins, and to the extract of MDI queens compared to extract of SDI queens. However, while there were differences in the amounts of some esters between MDI queens and the other groups, it appears that the differences in behavioral responses were elicited by subtle changes in the overall chemical profiles rather than dramatic changes in specific individual chemicals. We also found a decrease in vitellogenin gene expression in the fat body of the MDI queens, which is negatively correlated with the quantities of Dufour’s gland content. The possible explanations of this reduction are discussed.     

Regulation of Reproduction in the Primitively Eusocial Wasp <Emphasis Type="Italic">Ropalidia marginata</Emphasis>: on the Trail of the Queen Pheromone

Queens and workers are not morphologically differentiated in the primitively eusocial wasp, Ropalidia marginata. Upon removal of the queen, one of the workers becomes extremely aggressive, but immediately drops her aggression if the queen is returned. If the queen is not returned, this hyper-aggressive individual, the potential queen (PQ), will develop her ovaries, lose her hyper-aggression, and become the next colony queen. Because of the non-aggressive nature of the queen, and because the PQ loses her aggression by the time she starts laying eggs, we hypothesized that regulation of worker reproduction in R. marginata is mediated by pheromones rather than by physical aggression. Based on the immediate loss of aggression by the PQ upon return of the queen, we developed a bioassay to test whether the queen’s Dufour’s gland is, at least, one of the sources of the queen pheromone. Macerates of the queen’s Dufour’s gland, but not that of the worker’s Dufour’s gland, mimic the queen in making the PQ decrease her aggression. We also correctly distinguished queens and workers of R. marginata nests by a discriminant function analysis based on the chemical composition of their respective Dufour’s glands.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

Hexyl Decanoate, the First Trail Pheromone Compound Identified in a Stingless Bee, Trigona recursa

Foragers of many species of stingless bees guide their nestmates to food sources by means of scent trails deposited on solid substrates between the food and the nest. The corresponding trail pheromones are generally believed to be produced in the mandibular glands, although definitive experimental proof has never been provided. We tested the trail following behavior of recruits of Trigona recursa in field experiments with artificial scent trails branching off from natural scent trails of this stingless bee. First-time recruits (newcomers) did not follow these trails when they were laid with pure solvent or mandibular gland extract. However, they did follow trails made with labial gland extract. Chemical analyses of labial gland secretions revealed that hexyl decanoate was the dominant component (72.4 ± 1.9% of all volatiles). Newcomers were significantly attracted to artificial trails made with synthetic hexyl decanoate, demonstrating its key function in eliciting scent-following behavior. According to our experiments with T. recursa, the trail pheromone is produced in the labial glands and not in the mandibular glands. Hexyl decanoate is the first component of a trail pheromone identified and proved to be behaviorally active in stingless bees.     

Constituents of mandibular and Dufour's glands of an australianPolyrhachis weaver ant

Worker ants ofPolyrhachis (Cyrtomyrma) ?doddi collectively discharge the secretions of their large mandibular glands when their nest is disturbed. The major glandular compounds of workers are 6-methylhept-5-en-2-one and phenylacetaldehyde oxime. Other components identified are mellein, 6-methylhept-5-en-2-one oxime, phenylacetonitrile, phenyiacetaldehyde, benzaldehyde, and several alkanes and alkenes. The mandibular gland secretions of queens differ from those of workers only quantitatively. Large queens have considerably more of most components, the small queens have much less of volatile components. There is a pronounced sexual difference: the major components of the male's secretions are octanoic acid and mellein, with geranic acid, 8-heptadecene, 2-methylbutanoic acid, and 9-nonadecene present in lesser amounts. Workers ofP. ?doddi also have unusually large Dufour's glands containing a large array of hydrocarbons, of which tridecane is the major component, but α-farnesene, pentadecane, and heptadecene are also present in large quantities.     

Virgin Queen Mandibular Gland Signals of Apis mellifera capensis Change with Age and Affect Honeybee Worker Responses

The mandibular gland secretions of Apis mellifera capensis virgin queens were analyzed by gas chromatography–mass spectroscopy. Changes in the patterns of the mandibular gland volatiles of A. m. capensis virgin queens were followed from emergence until 14-d old. Ontogenetic changes in the mandibular gland secretions were largely quantitative in nature, delineating the age categories (global R = 0.612, P = 0.001), except for 7- and 14-d-old queens, which cannot be separated on their mandibular gland profiles (P = 0.2). (E)-9-Oxodec-2-enoic acid (9ODA) contributes most and most consistently to the dissimilarity between groups as well as the similarity within groups. Worker reactions to introduced virgin queens of various ages were recorded. Workers showed a significant increase in hostile reactions as queens aged (r = 0.615, N = 20, P < 0.05). Consequently, worker reactions and relative 9ODA production exhibit a positive queen age-dependent response.     

Semiochemicals of the honeybee queen mandibular glands

The ontogeny of the five queen mandibular gland semiochemicals that initiate and maintain the retinue behavior of worker honeybees was investigated by quantitative splitless capillary gas chromatography. No detectable pheromone is present at the time of eclosion, but decenoic acid levels build up rapidly during the first week of the queen's life. Two aromatic components attain detectable levels later, with the more plentiful methylp-hydroxybenzoate preceding the 4-hydroxy-3-methoxyphenylethanol. Pheromone levels are maximal in mature, mated, laying queens. The ratio of (R,E)-(–)-9-hydroxy-2-decenoic acid to the (S,E)-(+) enantiomer increases with the age of the queen. Pheromone levels in queen mandibular glands are largely unaffected by queen banking, restraint with workers in mailing cages, and limited storage on dry ice. All major body parts of typical queens, especially the head and legs, have sufficient mandibular exudate to be highly attractive to worker bees.     

Queen Volatiles as a Modulator of <Emphasis Type="BoldItalic">Tetragonisca angustula</Emphasis> Drone Behavior

Tetragonisca angustula mating occurs during the virgin queen nuptial flight, usually in the presence of a drone congregation area (DCA). The presence of virgin queen pheromone is considered the trigger for DCA establishment, although this has not been demonstrated experimentally. We established meliponaries, in different habitats, with T. angustula virgin queens during the main drone reproduction period. Eight DCAs were observed in urban areas, and all established outside or near colonies containing at least one virgin queen. The accumulation of drones in the DCAs occurred from 08:00 to 18:00 h and over 3–35 days. The number of drones in DCAs ranged from 60 to 2,000. In field trials, drones were attracted to virgin queens and also, unexpectedly, to physogastric queens. Volatiles collected from both virgin and physogastric queens elicited strong electoantennogram (EAG) responses from drones. Virgin and physogastric queen volatiles were qualitatively similar, but quantitatively different, in chemical composition. The queen’s abdomen was the principal source of these compounds. Isopropyl hexanoate (IPH), the most abundant compound in virgin queen volatiles and one of the most abundant in physogastric queen volatiles, was identified as one of the compounds that elicited EAG responses and was demonstrated to attract drones in a field test.     

Species- and Caste-Determined Mandibular Gland Signals in Honeybees (Apis)

Queens and workers of five honeybee species (Apis mellifera A. cerana A. dorsata A. floreaand A. andreniformis) were analyzed for their mandibular gland components. In A. melliferathe queen mandibular pheromone consists of 9-hydroxy- and 9-keto-2(E)-decenoic acids. (9-HDA and ODA), methyl p-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenyl-ethanol (HVA), and is responsible for retinue attraction, among other functions. In retinue bioassays with workers of A. cerana (whose queens lack HVA), ODA, 9-HDA, and HOB were sufficient to elicit maximal retinue behavior. This suggests that the known queen mandibular pheromone components detected in mandibular glands of A. cerana queens constitute the functional queen mandibular pheromone in this species. Both castes of A. mellifera produce 10- and 8-carbon acids that are functionalized at the last position in the chain, and these are the predominant compounds found in worker mandibular glands. Workers of the other species also had these compounds, along with 9-HDA and ODA that are normally not present in A. mellifera worker glands. Queens and workers of each species had a unique combination of mandibular compounds. The aromatic compounds were characteristic of queens from the cavity-nesting speciesA. mellifera (HOB and HVA) and A. cerana (HOB). These two species also had more pronounced differences in the mandibular blends of queens and workers than the open-nesting speciesA. dorsata A. floreaand A. andreniformis. Our results indicate that the more derived cavity-nesting species of Apis have evolved greater caste-specific differences between queens and workers and a higher number of queen pheromone components, compared to the open-nesting species.     

Sex Pheromone of Queens of the Slave-Making Ant, Polyergus breviceps

The sex attractant pheromone produced in mandibular glands of queens of the slave-making ant Polyergus breviceps has been identified as a blend of methyl 6-methylsalicylate and 3-ethyl-4-methylpentanol. In field trials, each compound alone was completely unattractive to males, whereas blends of the two compounds attracted hundreds of males within a couple of hours.     

Why not be a queen? Regioselectivity in mandibular secretions of honeybee castes

Both female castes of the honeybee (Apis mellifera L.) synthesize hydroxylated 2(E)-decenoic acids in their mandibular glands. Queens produce 9-hydroxy-2(E)-decenoic acid as part of their primer pheromone, while workers produce the regioisomeric 10-hydroxy acid, probably as a larval food source and an antiseptic secretion. Both workers and queens are biosynthetically competent to produce the other caste's dominant hydroxylated compound, as both isomers can be detected in queens and workers. We investigated the source of the caste-determined regioselectivity of hydroxy acid biosynthesis by investigating the production and interconversion of these compounds in isolated worker honeybee mandibular glands with specifically deuterated precursors. Gas chromatographic-mass spectroscopic identification of the labeled product indicates that octadecanoic acid is converted into 10-hydroxy-2(E)-decenoic acid with higher efficiency than either hexadecanoic or decanoic acids. 10-Hydroxydecanoic acid is readily converted into 10-hydroxy-2(E)-decenoic acid as expected in the -oxidation process. The saturated and unsaturated 10-hydroxy acids are oxidized to the corresponding ten carbon diacids.     

<Emphasis Type="Italic">E</Emphasis>-2-Ethylhexenal, <Emphasis Type="Italic">E</Emphasis>-2-Ethyl-2-Hexenol,Mellein, and 4-Hydroxymellein in <Emphasis Type="Italic">Camponotus</Emphasis> Species from Brunei

E-2-ethyl-2-hexen-1-ol (1), mellein (4), and 4-hydroxymellein (5) were identified as the major volatile compounds in the head and/or thorax of Camponotus quadrisectus. Neither 1 nor 5 have been previously detected in insects. Also identified were small amounts of m-cresol (2) and 6-methyl salicylic acid (3). E-2-ethylhexenal (6) and small amounts of 3 were identified in heads of Camponotus irritibilis from Kuala Belalong, Brunei. Compounds 2–4 occur in other Bornean camponotines with hypertrophied mandibular glands, and 4 is widespread in the tribe. The possibility of semiochemical parsimony (multiple functions) for these mandibular gland compounds is reviewed in the context of existing data on mandibular gland products of other camponotines, reported biological activities of the compounds, and secondary loss of metapleural glands in this ant group.     

Reevaluation of the Role of Mandibular Glands in Regulation of Reproduction in Bumblebee Colonies

Possible pheromonal control of worker reproduction was tested in Bombus terrestris. The mode of assay included exposure of callow workers to extracts originating from different queen parts and measuring the effect on the in vitro biosynthesis of juvenile hormone (JH), the apparent gonadotropin in this species. Both queen total body extracts applied to dummies consisting of oven-dried or Soxhlet-washed virgin queen bodies and cuticular washes applied to living virgin queens effectively inhibited the biosynthesis of JH in callow workers. None of the five exocrine glands (mandibular, hypopharyngeal, salivary, Dufour's, and tarsal) demonstrated inhibitory activity. Likewise, the use of synthetic 3-hydroxy acids, found in queen mandibular glands, were ineffective in blocking JH biosynthesis in queenless workers. The results suggest that the queen may use a primer pheromone spread on the epicuticle as a means to inhibit worker reproduction. However, our results are not consistent with the prevailing hypothesis that in B. terrestris the main source of the pheromone that inhibits worker reproduction is in the queen's mandibular glands.     

Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.)

The composition of the five-component honey bee queen mandibular gland pheromone (QMP) of mated European honey bee queens was compared to those of virgin and drone-laying (i.e., laying only haploid unfertilized eggs that develop into males), European queens and Africanized mated queens. QMP of mated European queens showed significantly greater quantities of individual components than all queen types compared, except for a significantly greater quantity of 9-hydroxy-(E)-2-decenoic acid (9-HDA) found in Africanized queens. Glands of European drone-laying queens contained quantities intermediate between virgin and mated queens, reflecting their intermediate reproductive state and age. QMP ontogeny shifts from a high proportion of 9-keto-(E)-2-decenoic acid (ODA) in young unmated queens to roughly equal proportions of ODA and 9-HDA in mated queens. A biosynthetic shift occurs after mating that results in a greater proportion of 9-HDA, methylp-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) production, accompanied by a decreased proportion of ODA. Africanized QMP proportions of ODA and 9-HDA were significantly different from European queens. A quantitative definition of a queen equivalent of QMP is proposed for the various queen types, and a standard queen equivalent for mated European honeybee queen mandibular gland pheromone is adopted as 200µg ODA, 80µg 9-HDA, 20µg HOB, and 2 µg HVA.     

Synthesis of 18-octadecanolide,a major constituent of dufour’s gland secretion ofColletes bees

18-Octadecanolide, a major constituent of Dufour’s gland secretion ofColletes bees, was synthesized by polymerization followed by depolymerization of 18-hydroxyoctadecanoic acid which in turn was obtained by hydrogenation of kamlolenic acid (18-hydroxy, 9-cis, 11-trans, 13-trans-octadecatrienoic acid) isolated fromMallotus philippinensis seed oil. The structures of the intermediates as well as the final lactone were established by infrared, proton nuclear magnetic resonance and mass spectrometric methods.     

Queen Sex Pheromone of the Slave-making Ant, Polyergus breviceps

Workers of the slave-making ant, Polyergus breviceps, raid nests of Formica ants and return with Formica pupae that mature into worker ants in the slave-makers’ colony. These Formica workers then tend the Polyergus brood, workers, and reproductives. During raids in the mating season, winged virgin Polyergus queens accompany the workers in the raiding columns. During the raid, the virgin queens release a pheromone that attracts males that quickly mate with the queens. We report the identification, synthesis, and bioassay of the sex attractant pheromone of the queens as an approximately 1:6 ratio of (R)-3-ethyl-4-methylpentan-1-ol and methyl 6-methylsalicylate. The ants produce exclusively the (R)-enantiomer of the alcohol, and the (S)-enantiomer has no biological activity, neither inhibiting nor increasing attraction to blends of methyl 6-methylsalicylate with the (R)-enantiomer.     

The Mandibular Gland Secretions of the Leaf-Cutting Ants Atta sexdens sexdens and Atta opaciceps Exhibit Intercaste and Intercolony Variations

The mandibular gland secretions of worker castes from wild colonies of the leaf-cutting ants Atta sexdens sexdens and Atta opaciceps were analyzed quantitatively by gas chromatography–mass spectrometry. The secretions comprised a complex mixture of volatile, mainly oxygenated compounds, and their profiles exhibited considerable qualitative and quantitative variations among species and castes. The known alarm pheromone 4-methyl-3-heptanone was common to both species. The elevated relative proportions of this ketone found in the secretions of gardeners and generalists suggest that such castes are primarily responsible for the production and release of the alarm pheromone. Quantitative variations (but no qualitative differences) in the profiles of secretions of soldiers from different colonies of A. sexdens sexdens were detected, supporting the view that intraspecific colony recognition is mediated through mandibular gland secretions. Subsequent laboratory assays showed that, among the compounds identified by GC–MS, 4-methyl-3-heptanone elicited a strong alarm response in workers of A. sexdens sexdens and A. opaciceps.     

Venom Alkaloid and Cuticular Hydrocarbon Profiles Are Associated with Social Organization,Queen Fertility Status,and Queen Genotype in the Fire Ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Queens in social insect colonies advertise their presence in the colony to: a) attract workers’ attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype.     

Mandibular Gland Secretion in Different Castes of the Leaf-Cutter Ant Atta laevigata

Gas chromatography analyses and behavioral assays showed that Atta laevigata, a highly polymorphic ant species, has a mandibular gland secretion that varies with castes. All castes contain 4-methyl-3-heptanone as the main component and its concentration is proportional to head size. Small workers and soldiers, but not medium size workers, also contain 4-methyl-3-heptanol. Queens show variations in their chemical composition after mating, as virgin males contain a secretion dominated by 4-methyl-3-heptanol, and, in a lesser proportion, 4-methyl-3-heptanone. In mated males these proportions are inverted. The compounds 4-methyl-6-hepten-3-one, 4-methyl-4-hepten-3-one, 6-methyl-tetradecene, and 2,6-dimethyl-2-dodecene are found only in queens. The behavioral response elicited by the secretion is mainly alarm, which is elicited more strongly by glands of larger workers. The results suggest that chemical castes, behavioral castes, and morphological castes overlap in this species.     

Chemistry of the Cephalic Labial Gland Secretions of Male Bombus morrisoni and B. rufocinctus, Two North American Bumblebee Males with Perching Behavior

The labial gland secretions from males of the North American bumblebees Bombus morrisoni Cresson and B. rufocinctus Cresson were analyzed by gas chromatography/mass spectrometry. In both species, 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl acetate was found as the major compound of a complex mixture of alkenols, acetates, hydrocarbons, and wax-type esters. In addition to a mixture of saturated and mono-unsaturated straight chain hydrocarbons, the labial gland of both species contained the isoprenoid hydrocarbons (6E, 10E)-3,7,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene [β-springene], three isomers of 3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene [α-springene], and two further unidentified cyclic diterpenes. In B. morrisoni, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and 3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-ol were detected as characteristic alcohols, as well as small amounts of 9-hexadecenol and hexadecanol. Furthermore, a large peak of hexadecyl dodecanoate and minor amounts of 9-hexadecenyl, 9-octadecenyl, and eicosenyl 9-tetradecenoate were found as typical esters in this species. In B. rufocinctus, 9-hexadecenol, hexadecanol, and 9-octadecenol were present in considerable amounts, with their acetates and 9-tetradecenoic, tetradecanoic, hexadecanoic, and 11-octadecenoic acids. The chemical composition of cephalic labial glands in male bumblebees with perching behavior is discussed.