Dufour's Gland Research Articles

Queen-signal modulation of worker pheromonal composition in honeybees.

Worker sterility in honeybees is neither absolute nor irreversible. Whether under queen or worker control, it is likely to be mediated by pheromones. Queen-specific pheromones are not exclusive to queens; workers with activated ovaries also produce them. The association between ovarian activation and queen-like pheromone occurrence suggests the latter as providing a reliable signal of reproductive ability. In this study we investigated the effect of queen pheromones on ovary development and occurrence of queen-like esters in workers' Dufour's gland. Workers separated from the queenright compartment by a double mesh behaved like queenless workers, activating their ovaries and expressing a queen-like Dufour's gland secretion, confirming that the pheromones regulating both systems are non-volatile. Workers with developed ovaries produced significantly more secretion than sterile workers, which we attribute primarily to increased ester production. Workers separated from the queenright compartment by a single mesh displayed a delayed ovarian development, which we attribute to interrupted transfer of the non-volatile pheromone between compartments. We suggest that worker expression of queen-like characters reflects a queen-worker arms race; and that Dufour's gland secretion may provide a reliable signal for ovarian activation. The associative nature between ovary development and Dufour's gland ester production remains elusive.

Chemical composition of the dufour gland secretion in queens of Melipona bicolor (Hymenoptera, Meliponini)

We describe differences in the chemical composition of the Dufour gland secretion of virgin and physogastric queens of Melipona bicolor through gas chromatography and mass spectrometry. The Dufour gland secretion of virgin queens consists only of hydrocarbons, while that of physogastric queens contains, besides these, a variety of other compounds, such as isobutyrate and acetate esters. Such differences may indicate the queen fecundity condition and the oxygenated compounds of the physogastrics secretion may help to increase their attractiveness.

Egg marking pheromones of anarchistic worker honeybees (Apis mellifera)

In honeybees, worker policing via egg eating enforces functional worker sterility in colonies with a queen and brood. It is thought that queens mark their eggs with a chemical signal, indicating that their eggs are queen-laid. Worker-laid eggs lack this signal and are, therefore, eaten by policing workers. Anarchistic worker honeybees have been hypothesized to circumvent worker policing by mimicking the queen egg-marking signal. We investigated this phenomenon by relating chemical profiles of workers and their eggs to egg acceptability. We found that the ability of some workers (anarchistic workers in queenright colonies and deviant workers from a queenless colony) to lay more acceptable eggs is due to them producing significant amounts of queen-like esters from their Dufour's gland. These esters appear to be transferred to eggs during laying and increase egg survival. However, these esters cannot be the normal queen egg-marking signal, as they are generally absent from queen-laid eggs and only increase the short-term persistence of worker-laid eggs, because only 7?30% of anarchistic worker-laid eggs persisted to hatching versus 91?92% of queen-laid eggs. All workers can produce some esters, but only workers that greatly increase their ester production lay more acceptable eggs. The production of esters appears to be a flexible response, as anarchistic workers reared in queenless colonies did not increase their ester production, while some deviant workers in queenless colonies did increase their ester production.

Dufour gland secretion in the harvester ant genus Pogonomyrmex

Dufour gland secretions of the harvester ants Pogonomyrmex barbatus, P. rugosus and P. maricopa were investigated. The glandular blends exhibit species specific patterns, but more importantly, based on indiviual profiles of Dufour gland contents of colony members it is possible to separate the colonies from each other in each species. It is possible that these collective colony specific cues serve as longer lasting trunk route markers. Behavioral tests demonstrate that workers of P. rugosus prefer homing routes marked by members of their own colony to routes marked by a foreign conspecific colony.

Conservation of Bio synthetic pheromone pathways in honeybees Apis.

Social insects use complex chemical communication systems to govern many aspects of their life. We studied chemical changes in Dufour's gland secretions associated with ovary development in several genotypes of honeybees. We found that C28-C38 esters were associated only with cavity nesting honeybee queens, while the alcohol eicosenol was associated only with their non-laying workers. In contrast, both egg-laying anarchistic workers and all parasitic Cape workers from queenright colonies showed the typical queen pattern (i.e. esters present and eicosenol absent), while egg-laying wild-type and anarchistic workers in queenless colonies showed an intermediate pattern, producing both esters and eicosenol but at intermediate levels. Furthermore, neither esters nor eicosenol were found in aerial nesting honeybee species. Both esters and eicosenol are biosynthetically similar compounds since both are recognizable products of fatty acid biosynthesis. Therefore, we propose that in honeybees the biosynthesis of esters and eicosenol in the Dufour's gland is caste-regulated and this pathway has been conserved over evolutionary time.

Occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier (Hymenoptera, Meliponini)

The occurrence, morphology and ultrastructure of the Dufour gland in Melipona bicolor Lepeletier, 1836 are presented. The Dufour gland is not present in workers. In virgin queens the gland cells show characteristics of low activity, which are described in the text. In physogastric queens the gland epithelium is higher and the cells more active than in virgin queens, showing numerous basal plasmic membrane invaginations impregnated by an electrondense material, increased apical invaginations and accumulation of substances that will be released to the gland lumen in the subcuticular space. Therefore, the data show that the Dufour gland is more developed in physogastric than in virgin queens, indicating a possible involvement of the Dufour gland in the reproduction of this species.

Erratum

AbstractIn the paper “Novel diterpenoids and hydrocarbons in the Dufour gland of the ectoparsitoid Habrobracon hebetor (Say&) (Hymenoptera: Braconidae)” by: Howard et al. (Arch. Insect Biochem. Physiol. 54:95–109, 2003), sentences were mistakenly transposed on pages 98 and 103. The corrected paragraphs in which they should have appeared are herein reprinted. The misplaced sentences (now correctly placed) appear in boldface type.

A Comparative Cytochemical Study of the Dufour Gland in the Eusocial Bee Apis mellifera Linne, 1758 and Melipona bicolor Lepeletier, 1836

Dufour glands of Apis mellifera and Melipona bicolor were studied under light and transmission electron microscopy, using the cytochemical techniques of mercury bromophenol blue for protein detection, imidazole-buffered osmium tetroxide selective staining of unsaturated lipids, lanthanum nitrate for intercellular junction identification and zinc-iodide-osmium tetroxide for cytoplasmic endomembrane visualization. The results in both species corroborated the lipid nature of the gland secretion and showed in A. mellifera the poverty of the synthetic machinery in the worker gland cells in comparison with the queen, as expected by previous biochemical analyses. The pathway of the exogenous compounds of the secretion is intracellular, since substances can penetrate the cell folds and intercellular junctions, but their access to the gland lumen is barred by the apical intercellular junctions.

Dufour gland of the digger wasp Liris niger: structure and developmental and biochemical aspects.

The tubiform Dufour gland in the digger wasp species Liris niger is about 1.0 mm long ( 0.15 mm). An alternating arrangement of longitudinal and circumferential bundles of striated muscle fibers surrounds the gland. The Dufour gland, together with the venom gland, enters the sting base and terminates in the sting. The glandular epithelium is monolayered. Glands about 3 day after imaginal ecdysis have an empty lumen but a thick lining epithelium. The gland cells are characterized by a well-developed vesicular smooth endoplasmic reticulum, sparse rough ER and numerous free ribosomes. They also exhibit several electron-lucent vesicles and autophagic vacuoles. Secretion of electron-dense material via the gland cuticle into the gland lumen is apparent. Glands more than 20 days after imaginal ecdysis display a large lumen and a thin epithelium. The cells show signs of degeneration with numerous cytolytic inclusions. Dufour gland liquid contains numerous polypeptides of molecular weights ranging from 14 to about 200 kDa. In addition the secretion consists predominantly of straight-chain hydrocarbons, accompanied by small amounts of esters. The major hydrocarbons are pentadecane and ( Z)-8-heptadecene. Dufour gland secretion may have several functions: (1). the polypeptides might be involved in the gluing process of the eggs, while (2). the hydrocarbon oils may function as lubricants for the lancets and (3). might soften the secretion, thus allowing easier application of the glue. The lipophilic volatile material (4). might also be involved in pheromonal signaling.

Novel diterpenoids and hydrocarbons in the Dufour gland of the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae).

Chemical constituents contained in the Dufour gland of the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were characterized. Three terpenes, beta-springene, a homo-beta-springene, and a homo-geranyllinalool constitute approximately 37% of the gland components, with the remaining 63% all being hydrocarbons. The hydrocarbons consist of a homologous series of n-alkanes (n-C21 to n-C31), a trace amount of 3-methyl C23, a homologous series of internally methyl-branched alkanes (11-methyl C23 to 13-methyl C35), one dimethylalkane (13,17-dimethyl C33), a homologous series of monoenes (C(25:1) to C(37:1)) with the double bonds located at Delta9, Delta13 and Delta15 for alkenes of carbon number 25 to 31 and at Delta13 and Delta15 for carbon numbers 33 to 37 and three homologous dienes in very low amounts with carbon numbers of 31, 32, and 33. The terpenoid and hydrocarbon composition of the Dufour gland was similar in virgin and mated females. However, in contrast to the hydrocarbons, the amount of beta-springene and homo-geranyllinalool increased significantly with time after adult emergence from the cocoon. Although many hydrocarbons in the Dufour gland are the same as those on the cuticle of this species [Howard and Baker, Arch. Insect Biochem. Physiol. 53:1-18 (2003)], substantial differences also occur. Of particular note is the chain length of alkenes and location of the double bonds: cuticular alkenes have a chain length of C23 to C29 and double bond locations at Delta5, Delta7, and Delta9, whereas the Dufour gland alkenes contains a greater range of carbon numbers and have no Delta5 or Delta7 alkenes. The Dufour gland contains only one of the long-chain dimethylalkanes found on the cuticle. Also, no terpenoids are found on the cuticle, and the Dufour gland contains none of the secondary wax esters that are major components on the cuticle. GC-MS analysis of lipids carried in the hemolymph of H. hebetor indicated that all hydrocarbons found on both the cuticle and in the Dufour gland are present, as are some of the wax esters. However, none of the terpenoids were detected in the hemolymph. This suggests that the hydrocarbons are synthesized in other tissues or cells, probably by oenocytes, and differentially partitioned between the cuticle and the Dufour gland. The terpenoids are most likely synthesized within the Dufour gland. Analysis of surface lipids from eggs laid within 18 h indicated that no diterpenoids were present. Rather, the lipids present on the eggs were n-alkanes, monomethylalkanes, alkenes, and secondary alcohol wax esters. This composition did not reflect that of the Dufour gland, hence eggs are not being coated with Dufour gland components during oviposition.

Trail pheromones and Dufour gland contents in three Camponotus species ( C. castaneus, C. balzani, C. sericeiventris : Formicidae, Hymenoptera)

By means of gas chromatography, gas chromatographic coupled mass spectrometry and behavioral analysis the major trail pheromone components from the hindgut of the formicine species Camponotus castaneus, C. balzani and C. sericeiventris were identified. The trail pheromone of C. castaneus is 3,5-dimethyl-6-(1-methylpropyl)- tetrahydro-2H-pyran- 2-one, and that of the other two species is 3,4-dihydro-8-hydroxy-3,5,7-trimethylisocoumarin. Although both compounds release precise trail following behavior in the respective species, the major recruitment signal in the three Camponotus species appears to be formic acid discharged from the poison gland. The composition of the Dufour gland secretions of C. castaneus and C. sericeiventris is similar, but that of the hypertrophied Dufour gland of C. balzani is very different from any other Camponotus Dufour gland content described up to date: it contains large amounts of esters, the major compound of which is octyl hexanoate, which makes up 97% a/a of the total volatiles.

Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant Myrmecia gulosa.

In many species of social insects, the cuticular hydrocarbons of adults vary with both colony identity and individual physiology (oogenesis). Such variations have been shown in some ants and social wasps to function in nestmate recognition, but as yet there is no demonstration of their use by workers to recognize egg layers. We report that in the ant Myrmecia gulosa, workers can discriminate queens and fertile workers from infertile individuals based on distinctive blends of long-chained hydrocarbons present both on the cuticle and in the postpharyngeal gland. The purified hydrocarbon fraction of cuticular extracts from queens elicited high interest in workers, unlike the nonhydrocarbon fraction. However, both fractions were necessary to trigger a response of maximal intensity. In contrast, extracts of mandibular and Dufour glands from queens or infertile workers were not treated differentially by workers. We suggest that cuticular hydrocarbons function as pheromones allowing for recognition of the queen as well as egg-laying workers.

Investigation by solid-phase microextraction and gas chromatography/mass spectrometry of trail pheromones in ants.

The Dufour's gland content of workers of two ant species of the genus Messor has been analyzed by solid-phase microextraction and gas chromatography with mass spectrometry (GC/MS). The structures of the compounds in the pheromonal mixtures have been determined. In both cases only one intact gland, inserted in a properly dimensioned capillary vial, is sufficient to produce a clean and fully interpretable GC/MS profile. It is worth noting that, for the first time in Messor ants, farnesol has been detected as a minor component of glandular secretion in Messor capitatus.

Occurrence, Morphology and Chemical Composition of the Dufour Gland in Melipona bicolor Lepeletier, 1836 (Hymenoptera, Meliponini)

The Dufour gland is an organ associated to the bee’s female reproductive apparatus [1]. The gland produces mainly hydrocarbons and other organic oxygenated substances that play diverse pheromonal roles according to the bee ecology [2], but often related with reproduction. In eussocial bees its function is unknown, although probably is also involved in reproduction, marking queen’s eggs [3] or indicating the queen reproductive status [4]. In order to throw some more light on the Dufour gland function in eussocial bees, it was done a study on the occurrence, morphology and chemical composition of this gland in workers and queens of Melipona bicolor, a polygynic neotropical stingless bee. The results showed that the Dufour gland was not present in workers. In virgin queens the gland cells showed characteristics of low activity, presenting few basal and apical plasmic membrane invaginations and few organelles in the cytoplasm (Fig. 1). In physogastric queens the gland epithelium was higher and the cells more active than in virgin queens, showing numerous basal plasmic membrane invaginations that formed a labyrinth, which was filled of an electrondense material (Fig. 2) and is involved with taking up substances from the haemolymph. The apical plasmic membrane invaginations were increased and the subcuticular space filled with material produced by the gland cells, which after will make part of the gland lumen secretion. Therefore, by the morphology view point the Dufour gland is more developed in physogastric than in virgin queens, what is in agreement with chemical analysis of the gland secretion, which showed that virgin and physogastric queen differ reasonably. The virgin queen secretion presents only hydrocarbons and the physogastric hydrocarbons and esters (Table I). The present results showed that this gland in M. bicolor is possibly involved in reproduction. According to preliminary bioassays the secretion allows the workers to distinguish the queens reproductive status and help the physogastric queens to recruit workers for the provisioning and oviposition process, known as P.O.P.

Secretions of stingless bees: the Dufour glands of some Frieseomelitta species (Apidae, Meliponinae)

The first recorded analyses of meliponine bee Dufour gland secretions by gas chromatographic- mass spectrometry on three species of Frieseomelitta showed these glands contain a mixture of oxygenated compounds and terpenoids with some hydrocarbons. In F. varia the major substances are eicosenal, 1-eicosenol and 2-pentadecanone. In F. sylvestrii they are pentacosene, nonadecanal and heptacosene. F. silvestrii languida, with the largest glands and the most complex mixture, has geranylfarnesol, followed by 1-tetradecanol and tetradecanal, making it quite different from F. silvestrii. Frieseomelitta sylvestrii / Frieseomelitta varia / Frieseomelitta sylvestrii languida / geranylfarnesol

Morphology and chemistry of Dufour glands in four ectoparasitoids: Cephalonomia tarsalis, C. waterstoni (Hymenoptera: Bethylidae), Anisopteromalus calandrae, and Pteromalus cerealellae (Hymenoptera: Pteromalidae)

The venom apparatus of four hymenopterous parasitoids, including two bethylids, C. tarsalis (Ashmead) and C. waterstoni (Gahan), and two pteromalids, A. calandrae (Howard) and P. cerealellae (Ashmead), were removed and the associated Dufour glands characterized with respect to their external morphology and chemistry. Dufour glands in all four species have a characteristic translucent appearance that apparently results from their lipid content. The stalked Dufour glands of C. tarsalis and C. waterstoni are pear-shaped and have overall lengths of approximately 0.2 and 0.15 mm, respectively. The thin venom glands are bifurcate and insert through a fine duct into the transparent ovoid- to pear-shaped venom reservoir in these bethylids. In A. calandrae and P. cerealellae the Dufour glands are elongated, tubular structures of ca. 0.35 and 0.8 mm in length, respectively, that constrict to a short stalk that empties into the common oviduct. The venom glands in these pteromalids are simple elongated structures that insert into the sac-like venom reservoir through a fine duct. The chemistry of the volatile contents of the Dufour gland in these four species differs considerably. C. tarsalis Dufour glands contain the same hydrocarbon components as found on the cuticle of this species (Ann. Entomol. Soc. Am. 91:101–112 (1998)), and no other chemicals. The Dufour glands of C. waterstoni also contain only hydrocarbons, most of which are the same as the cuticular hydrocarbons (Ann. Entomol. Soc. Am. 85:317–325 (1992)), but in addition the Dufour gland contains ca. 3% of a mixture of 2,17- and 2,19-dimethyl C 23. A. calandrae Dufour gland chemistry is somewhat more complex than that of either of the two bethylids, but like the bethylids, only hydrocarbons are present. The carbon number range is from C 30 to C 39 and consists of a mixture of n-alkanes (C 30-C 38); 3-, 5-, 7-, 9-, 11-, 12-, 13-, 14-, 15- and 17-methyl alkanes; 3,7- and 3,11-dimethyl alkanes; 5,9- and 5,17-dimethyl alkanes; 7,11-, 9,13-, 13,17-, 14,18- and 15,19-dimethyl alkanes; 3,7,11- and 3, 9,15-trimethyl alkanes; and 3,7,11,15-tetramethyl alkanes. The cuticular hydrocarbons of this species have not been previously reported, but they are the same as the Dufour gland hydrocarbons. The Dufour glands of P. cerealellae contain both hydrocarbons and two long-chain aldehydes. Most of the hydrocarbons are identical to those found on the cuticle of this species (Ann. Entomol. Soc. Am. 94:152–158 (2001)), but in addition, 5,9-dimethyl C 27, 5,13-, 5,17- and 5,19-dimethyl C 35, 12- and 14-methyl C 36, 12,16- and 13,17-dimethyl C 36, 13-methyl C 37 and 13,17-dimethyl C 37 are present. The two aldehydes detected in glands from P. cerealellae are n-tetracosanal (C 23CHO) and n-hexacosanal (C 25CHO).

Honeybee egg-laying workers mimic a queen signal

In the honeybee, Dufour's gland secretion is caste specific and constitutes a component of the multi-sourced queen signal. As predicted, it is attractive to workers, which form a retinue around the scented source. Bioassays reveal the ester fraction and not the hydrocarbons to be the active constituents. This function of the esters was corroborated by assays with the synthetic queen-esters mixture, which successfully mimicked the queen's secretion. As predicted from the queen-like secretion exhibited by egg-laying workers, their glandular secretion was also attractive to nestmates, albeit to a lesser degree than that of the queen; while that of non-egg-laying workers was totally inactive. The evolution of the multiple queen signals in honeybees can be regarded as a component in an arms race between queen and workers. We hypothesize that in response to a reduced sensitivity to a certain queen signal, queen honeybees were selected to develop an alternative signaling-source. Dufour's gland seems to be one of these sources.

New 1-alk(en)yl-1,3,5-trihydroxycyclohexanes from the Dufour gland of the African ant Crematogaster nigriceps

The Dufour gland of the African ant Crematogaster nigriceps contains a mixture of at least eight 1-heptadec(en)yl- and 1-nonadec(en)yl-1,3,5-trihydroxycyclohexane derivatives, the mono-unsaturated components being the major ones (about 65%). They are accompanied by small amounts of ( Z, Z)-dienic derivatives (<10%). The structures, the relative and absolute configurations, and the preferred conformation of these new compounds have been established by spectroscopic and chemical methods, whereas the positions of the double bond in the alkenyl chains were determined by DMDS derivatisation followed by linked scan EIMS.

Comparative survey of abdominal gland secretions of the ant subfamily Ponerinae.

The chemical contents of three abdominal glands were investigated in representative species of the ponerine ants. The Dufour glands of 14 species show a wide variety of contents. In Mystrium camillae and Proceratium itoi, no volatile substances were found in either the Dufour or venom glands. In Ectatomma sp., Diacamma ceylonense, Diacamma indicum, Pachycondyla obscuricornis, and Pachycondyla striata, volatile chemicals were found in the venom glands as well as in the Dufour glands. Platythyrea punctata was examined, but unusually it does not have a Dufour gland and its venom gland contained no volatile substances. Epoxides were found in ants for the first time in the Dufour glands of Amblyopone reclinata. Venom glands of Pachycondyla tarsata were also found to contain volatile material, including bitter-tasting cyclic dipeptides. In all, 16 species have been added to the list of those examined. All of the 27 known analyses of Dufour glands, 21 analyses of venom glands, and 4 of pygidial glands of workers of ponerine ant species have been brought together in order to seek some pattern in the type of glandular contents. Although the great majority of species produce hydrocarbons in their worker Dufour glands, and some have terpenes, there is no observable pattern for this gland on a tribe or genus level. Volatile compounds have been found in the venom glands of some species of the tribe Ponerini only. The information on pygidial glands is still too fragmentary for any conclusions.

The mandibular glands as a source of sexual pheromones in virgin queens of Polyergus rufescens (Hymenoptera, Formicidae)

> ‘Female‐calling syndrome’ is the reproductive strategy adopted by the European slave‐making ant Polyergus rufescens, where a winged female disperses a short distance from the nest, ascends to an elevated position (at the top of a blade of grass) and is joined shortly thereafter by conspecific males. This suggests the probable use of very effective sexual calling pheromones by females. Here, the role of the mandibular glands of winged females is demonstrated to be a source of sexual pheromones. The secretion of these glands (among the structures tested: the mandibular, Dufour, poison and pygidial glands and the head without mandibular glands) appears to have a strong attractive effect on males and also elicits male copulatory behaviour.