Dufour's Gland Research Articles

Blacklegged ticks, Ixodes scapularis, reduce predation risk by eavesdropping on communication signals of Formica oreas thatching ants.

Ticks spend most of their life inhabiting leaf litter and detritus where they are protected from sun but preyed upon by ants. Ants secrete chemical communication signals to coordinate group tasks such as nest defence. Ticks that avoid ant semiochemicals-as indicators of ant presence-would reduce predation risk by ants. We tested the hypotheses that: (i) chemical deposits from the thatching ant Formica oreas deter blacklegged ticks, Ixodes scapularis, (ii) deterrent semiochemicals originate from the ants' poison and/or Dufour's gland(s), and (iii) tick-deterrent semiochemicals serve as alarm-recruitment pheromone components in F. oreas. In two-choice olfactometer bioassays, filter paper soiled with ant chemical deposits significantly deterred female and male ticks. Poison and Dufour's gland extracts deterred ticks in combination but not alone. Gas chromatographic-mass spectrometric analyses of gland extracts revealed formic acid as the major constituent in the poison gland and eight hydrocarbons as constituents in the Dufour's gland. Synthetic formic acid and hydrocarbons deterred ticks only when combined. F. oreas workers sprayed both formic acid and hydrocarbons when distressed. A synthetic blend of these compounds elicited alarm-recruitment responses by F. oreas in behavioural bioassays. All results combined indicate that ticks eavesdrop on the ants' communication system.

Ontogenetic morphological changes of the venom apparatus in 4 eupelmid egg parasitoids.

Parasitoid wasps, notably egg parasitoids of the family Eupelmidae (Hymenoptera: Chalcidoidea), a key natural enemy of insect pests, offer a sustainable approach to pest management in agriculture. This study investigated the venom apparatus's developmental dynamics across 4 species of eupelmid egg parasitoids: Anastatus. japonicus, Anastatus fulloi, Mesocomys trabalae and Mesocomys albitarsis. A comprehensive anatomical investigation revealed differences in the dimensions of the venom apparatus across different developmental stages in adult females. We found that the venom apparatus of these 4 studied species consists of a venom gland and a reservoir with an associated Dufour's gland. As the length of post-emergence increases, a significant enlargement in the venom apparatus is evident across all the studied parasitoid species. Notably, M. albitarsis consistently exhibites the shortest venom gland length, whereas that of A. fulloi is the longest among the observed species. At the high day age, the width of venom glands of the 2 Mesocomys species surpasses those of the Anastatus species; for the volume of the venom reservoir, there is a steady increase in all 4 species before the age of 6–7 days, with a decline on 8th day, especially for A. japonicus. This research provided new insights into the developmental trajectories of venom apparatus in eupelmid egg parasitoids and the potential impact of venom potency on their success.

Chemical Resemblance of Egg Surface Compounds and Dufour's Gland in Two Neotropical Polistinae Wasps Polistes versicolor (Olivier) and Mischocyttarus metathoracicus (de Saussure, 1854).

Chemical communication plays a major role in regulating social dynamics in social insect colonies. The most studied class of chemical compounds are the cuticular hydrocarbons (CHCs), compounds with high molecular weight that cover the insect body. CHCs are used in nestmate recognition and to signal reproductive status. Brood, in the form of larvae and eggs, is known to participate in chemical communication and social dynamics by performing hunger behaviour and inducing interaction with adults and conferring nest and maternity identity. CHCs of adults and egg surface compounds are similar in composition in social insect species. The main source of egg compounds is proposed to be Dufour's gland, an accessory reproductive gland found in several Hymenoptera females. There is still a lack of information about the level of similarity among CHCs, compounds of egg surface and Dufour's gland for several wasp species, which could provide correlational evidence about the origins of egg-marking compounds. Thus, we investigated whether egg surface compounds were more similar to CHCs or Dufour's gland secretions in two Neotropical primitively eusocial wasp species, Polistes versicolor (Olivier) and Mischocyttarus metathoracicus (de Saussure, 1854). As expected, there was a higher chemical similarity between eggs and Dufour's gland secretions in both studied species, supporting the hypothesis that this gland is the source of chemical compounds found over the eggs in these two primitively eusocial species.

Chemical Components of Dufour's and Venom Glands in Camponotus japonicus (Hymenoptera, Formicidae).

The Dufour's and venom glands are the most developed glands connected to the female reproductive organs, playing important roles in defense, foraging, information exchange, and reproduction in ants. The main chemical secretions of these glands vary among species and even among castes of the same species. In this study, we analyzed the chemical components of the Dufour's and venom glands in different castes of Camponotus japonicus (original worker, minor worker, major worker, gyne, and queen) using gas chromatography-mass spectrometry (GC-MS) with two sample processing methods (hexane solution and solid-phase microextraction). The secretion of the Dufour's gland is characterized by a high ratio of alkanes, with n-undecane being the dominant secretion in all castes except the original workers. The venom gland's secretion mainly includes alkanes, acids, ketones, and alcohols, with formic acid and n-undecane being the dominant components. Additionally, the chemical composition and proportion of the main components vary significantly among castes, which may be closely related to the division of labor in their social life. This study provides basic information to further understand the function of these two glands in the social life of ants.

Multi-Omic Identification of Venom Proteins Collected from Artificial Hosts of a Parasitoid Wasp.

Habrobracon hebetor is a parasitoid wasp capable of infesting many lepidopteran larvae. It uses venom proteins to immobilize host larvae and prevent host larval development, thus playing an important role in the biocontrol of lepidopteran pests. To identify and characterize its venom proteins, we developed a novel venom collection method using an artificial host (ACV), i.e., encapsulated amino acid solution in paraffin membrane, allowing parasitoid wasps to inject venom. We performed protein full mass spectrometry analysis of putative venom proteins collected from ACV and venom reservoirs (VRs) (control). To verify the accuracy of proteomic data, we also collected venom glands (VGs), Dufour's glands (DGs) and ovaries (OVs), and performed transcriptome analysis. In this paper, we identified 204 proteins in ACV via proteomic analysis; compared ACV putative venom proteins with those identified in VG, VR, and DG via proteome and transcriptome approaches; and verified a set of them using quantitative real-time polymerase chain reaction. Finally, 201 ACV proteins were identified as potential venom proteins. In addition, we screened 152 and 148 putative venom proteins identified in the VG transcriptome and the VR proteome against those in ACV, and found only 26 and 25 putative venom proteins, respectively, were overlapped with those in ACV. Altogether, our data suggest proteome analysis of ACV in combination with proteome-transcriptome analysis of other organs/tissues will provide the most comprehensive identification of true venom proteins in parasitoid wasps.

Chemical signatures of egg maternity and Dufour’s gland in Vespine wasps

Cuticular hydrocarbons (CHCs) are often used in the chemical communication among social insects. CHCs can be used in nestmate recognition and as queen pheromones, the latter allows the regulation of the reproductive division of labor. In the common wasp Vespula vulgaris, CHCs and egg-marking hydrocarbons are caste-specific, being hydrocarbon queen pheromones and egg maternity signals. Whether these compounds are conserved among other Vespinae wasps remains unknown. Queens, virgin queens, reproductive workers, and workers belonging to four different wasp species, Dolichovespula media, Dolichovespula saxonica, Vespa crabro, and Vespula germanica, were collected and studied. The cuticular hydrocarbons, egg surface, and Dufour's gland composition were characterized and it was found that chemical compounds are caste-specific in the four species. Quantitative and qualitative differences were detected in the cuticle, eggs, and Dufour's gland. Some specific hydrocarbons that were shown to be overproduced in the cuticle of queens were also present in higher quantities in queen-laid eggs and in their Dufour's gland. These hydrocarbons can be indicated as putative fertility signals that regulate the division of reproductive labor in these Vespine societies. Our results are in line with the literature for V. vulgaris and D. saxonica, in which hydrocarbons were shown to be conserved queen signals. This work presents correlative evidence that queen chemical compounds are found not only over the body surface of females but also in other sources, such as the Dufour's gland and eggs.

Morphology and Ultrastructure of the Female Reproductive Apparatus of an Asexual Strain of the Endoparasitoid Meteorus pulchricornis (Wesmael) (Hymenoptera, Braconidae).

Meteorus pulchricornis (Wesmael) is a solitary endoparasitoid of lepidopteran pests and a good candidate for the control of Spodoptera frugiperda. To elucidate the structure of the female reproductive apparatus, which may play a role in facilitating successful parasitism, we presented the description of the morphology and ultrastructure of the whole female reproductive system in a thelytokous strain of M. pulchricornis. Its reproductive system includes a pair of ovaries without specialized ovarian tissues, a branched venom gland, a venom reservoir, and a single Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer, possibly an egg surface protector, coats the surface of mature eggs. The venom gland consists of secretory units (including secretory cells and ducts) with abundant mitochondria, vesicles and end apparatuses in the cytoplasm, and a lumen. The venom reservoir is comprised of a muscular sheath, epidermal cells with few end apparatuses and mitochondria, and a large lumen. Furthermore, venosomes are produced by secretory cells and delivered into the lumen via the ducts. As a result, myriad venosomes are observed in the venom gland filaments and the venom reservoir, suggesting that they may function as a parasitic factor and have important roles in effective parasitism.

Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens.

Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic basis are known in only a handful of species. Here we examined the gene expression associated with pheromone biosynthesis of two main chemical classes: esters and terpenes, using the social bee Bombus impatiens. We conducted chemical and RNA-seq analyses of the Dufour's gland, an exocrine gland producing a plethora of pheromones regulating social behavior in hymenopteran species. The Dufour's gland contains mostly long-chained hydrocarbons, terpenes and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufour's gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic basis is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies candidate genes and pathways regulating caste-specific chemical signals in social insects.

Revisiting the trail pheromone components of the red imported fire ant, Solenopsis invicta Buren.

Ants use species-specific trail pheromones to coordinate their sophisticated foraging behavior. During the past decades, many trail pheromone components with various structures have been identified in ants, including the red imported fire ant, Solenopsis invicta, a notorious invasive species worldwide. Four compounds, Z,E- (ZEF) and E,E-α-farnesene (EEF), Z,E- (ZEHF) and E,E-α-homofarnesene (EEHF), have been reported as components of S. invicta trail pheromone. However, another study reported an analog of α-farnesene, Z,Z,Z-allofarnesene, as a key trail pheromone component. These contrasting results caused some uncertainty about the trail pheromone composition in S. invicta. In this study, we synthesized ZEF and EEF, ZEHF and EEHF, and reanalyzed the chemicals in the Dufour gland extract and in the trail pheromone fraction of S. invicta worker body extract. The reported isomers of farnesene and homofarnesene were detected and showed trail-following activity, with ZEF as the major compound, while no allofarnesene was found, neither in the Dufour gland extract nor in the whole-body extract. Our results confirm ZEF and EEF, ZEHF and EEHF as trail pheromone components of S. invicta.

Histology and ultrastructure of glands associated with the sting apparatus of the swarm-founding wasp Protopolybia exigua (Hymenoptera: Vespidae: Polistinae)

Social insects present a great diversity of exocrine glands, which are linked to fundamental roles of social life, and their morphological characterization represents the first step toward the knowledge of their function. We here describe the exocrine glands present in the sting apparatus of queens and workers of the wasp Protopolybia exigua. Histological analysis of serial sections under light microscopy revealed a glandular epithelium in the spiracular plate, the quadrate plate, and the gonostyli, while the last two in addition also contain clusters of class-3 cells. A big cluster of class-3 cells was also found in the oblong plate of some workers and queens. These findings reveal that a variety of glands, in addition to the common venom gland and Dufour gland, are present in association with the sting apparatus, of which three glands are novel reports for the exocrine repertoire of social insects. The position of the glands suggests a role in lubrication to provide mobility of the sting and venom ejection and/or participation in alarm behavior, while a function related to reproduction is also possible. The peculiar characteristics displayed by swarm-founding wasps are linked with exocrine gland secretions, making their investigation in Epiponini wasps a vast and interesting field.

The ant abdomen: The skeletomuscular and soft tissue anatomy of Amblyopone australis workers (Hymenoptera: Formicidae).

Recent studies of insect anatomy evince a trend towards a comprehensive and integrative investigation of individual traits and their evolutionary relationships. The abdomen of ants, however, remains critically understudied. To address this shortcoming, we describe the abdominal anatomy of Amblyopone australis Erichson, using a multimodal approach combining manual dissection, histology, and microcomputed tomography. We focus on skeletomusculature, but additionally describe the metapleural and metasomal exocrine glands, and the morphology of the circulatory, digestive, reproductive, and nervous systems. We describe the muscles of the dorsal vessel and the ducts of the venom and Dufour's gland, and characterize the visceral anal musculature. Through comparison with other major ant lineages, apoid wasps, and other hymenopteran outgroups, we provide a first approximation of the complete abdominal skeletomuscular groundplan in Formicidae, with a nomenclatural schema generally applicable to the hexapod abdomen. All skeletal muscles were identifiable with their homologs, while we observe potential apomorphies in the pregenital skeleton and the sting musculature. Specifically, we propose the eighth coxocoxal muscle as an ant synapomorphy; we consider possible transformation series contributing to the distribution of states of the sternal apodemes in ants, Hymenoptera, and Hexapoda; and we address the possibly synapomorphic loss of the seventh sternal-eighth gonapophyseal muscles in the vespiform Aculeata. We homologize the ovipositor muscles across Hymenoptera, and summarize demonstrated and hypothetical muscle functions across the abdomen. We also give a new interpretation of the proximal processes of gonapophyses VIII and the ventromedial processes of gonocoxites IX, and make nomenclatural suggestions in the context of evolutionary anatomy and ontology. Finally, we discuss the utility of techniques applied and emphasize the value of primary anatomical research.

Cuticular and Dufour's Gland Chemistry Reflect Reproductive and Social State in the Facultatively Eusocial Sweat Bee Megalopta genalis (Hymenoptera: Halictidae).

Queen pheromones evolved independently in multiple eusocial insect lineages, in which they mediate reproductive conflict by inhibiting worker ovarian development. Although fundamentally important for reproductive division of labor - the hallmark of eusociality - their evolutionary origins are enigmatic. Here, we analyze cuticular and Dufour's gland chemistries across alternative social and reproductive phenotypes in Megalopta genalis bees (tribe Augochlorini, family Halictidae) that facultatively express simple eusociality. Reproductive bees have distinct overall glandular and cuticular chemical phenotypes compared with non-reproductive workers. On the cuticle, a likely site of signal transmission, reproductives are enriched for certain alkenes, most linear alkanes, and are heavily enriched for all methyl-branched alkanes. Chemicals belonging to these compound classes are known to function as fertility signals in other eusocial insect taxa. Some macrocyclic lactones, compounds that serve as queen pheromones in the other eusocial halictid tribe (Halictini), are also enriched among reproductives relative to workers. The intra-population facultative eusociality of M. genalis permits direct comparisons between individuals expressing alternative reproductive phenotypes - females that reproduce alone (solitary reproductives) and social queens - to highlight traits in the latter that may be important mediators of eusociality. Compared with solitary reproductives, the cuticular chemistries of queens are more strongly differentiated from those of workers, and furthermore are especially enriched for methyl-branched alkanes. Determining the pheromonal function(s) and information content of the candidate signaling compounds we identify will help illuminate the early evolutionary history of queen pheromones, chemical signals central to the organization of insect eusocial behavior.

An uneasy alliance: a nesting association between aggressive ants and equally fierce social wasps.

Although the Neotropical territorially dominant arboreal ant Azteca chartifex Forel is very aggressive towards any intruder, its populous colonies tolerate the close presence of the fierce polistine wasp Polybia rejecta (F.). In French Guiana, 83.33% of the 48 P. rejecta nests recorded were found side by side with those of A. chartifex. This nesting association results in mutual protection from predators (i.e., the wasps protected from army ants; the ants protected from birds). We conducted field studies, laboratory-based behavioral experiments and chemical analyses to elucidate the mechanisms allowing the persistence of this association. Due to differences in the cuticular profiles of the two species, we eliminated the possibility of chemical mimicry. Also, analyses of the carton nests did not reveal traces of marking on the envelopes. Because ant forager flows were not perturbed by extracts from the wasps' Dufour's and venom glands, we rejected any hypothetical action of repulsive chemicals. Nevertheless, we noted that the wasps "scraped" the surface of the upper part of their nest envelope using their mandibles, likely removing the ants' scent trails, and an experiment showed that ant foragers were perturbed by the removal of their scent trails. This leads us to use the term "erasure hypothesis." Thus, this nesting association persists thanks to a relative tolerance by the ants towards wasp presence and the behavior of the wasps that allows them to "contain" their associated ants through the elimination of their scent trails, direct attacks, "wing-buzzing" behavior and ejecting the ants.

Chemical Characterization of Young Virgin Queens and Mated Egg-Laying Queens in the Ant Cataglyphis cursor: Random Forest Classification Analysis for Multivariate Datasets.

Social insects are well known for their extremely rich chemical communication, yet their sex pheromones remain poorly studied. In the thermophilic and thelytokous ant, Cataglyphis cursor, we analyzed the cuticular hydrocarbon profiles and Dufour's gland contents of queens of different age and reproductive status (sexually immature gynes, sexually mature gynes, mated and egg-laying queens) and of workers. Random forest classification analyses showed that the four groups of individuals were well separated for both chemical sources, except mature gynes that clustered with queens for cuticular hydrocarbons and with immature gynes for Dufour's gland secretions. Analyses carried out with two groups of females only allowed identification of candidate chemicals for queen signal and for sexual attractant. In particular, gynes produced more undecane in the Dufour's gland. This chemical is both the sex pheromone and the alarm pheromone of the ant Formica lugubris. It may therefore act as sex pheromone in C. cursor, and/or be involved in the restoration of monogyny that occurs rapidly following colony fission. Indeed, new colonies often start with several gynes and all but one are rapidly culled by workers, and this process likely involves chemical signals between gynes and workers. These findings open novel opportunities for experimental studies of inclusive mate choice and queen choice in C. cursor.

Egg-laying behavior and morphological and chemical characterization of egg surface and egg attachment glue of the digger wasp Ampulex compressa (Hymenoptera, Ampulicidae)

For providing their offspring females of the digger wasp species Ampulex compressa hunt cockroaches, paralyze them and attach as a rule one egg to the coxa of one of the mid legs of their prey. We observed the egg-laying behavior and examined with light- and scanning microscopy (i) nearly mature eggs from ovaries of freshly dissected females and (ii) eggs immediately after their deposition on the coxae of their prey. The length of the white bean-shaped eggs varied between 2.2 and 3.0 mm, their diameter between 0.66 and 0.72 mm, and their weight between 345 and 832 μg. The surface of fresh, untreated eggs shows even at higher magnifications (>20.000×) a smooth appearance. However, after conventional fixation, dehydration with ethyl-alcohol and critical-point drying the egg-surface exhibited a little bit texture. The eggs are at two-third of their underside glued to the coxa of the prey. With the naked eye the glue appears as a compact mass. The eggs may be mechanically removed from the substrate (their attachment site); however, in doing so the viscous attachment glue appears in a more fibrous consistence. The polypeptide composition washed off the egg surface and the glue revealed no similarities, whereas the molecular mass of two polypeptides were similar between glue and the Dufour's gland contents.

Morphology and ultrastructure of Dufour’s and venom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae)

The Dufour’s and venom glands are two important exocrine glands in ants. In this study, the morphology and fine structure of these two glands are described in the ant Camponotus japonicus Mayr. The Dufour glands have a characteristic bilobed shape and show a difference in size and color between the female castes (minor and major workers, alate and dealate queens). The external surface of Dufour’s gland shows different features among the female castes. It appears more hypertrophied in major workers than in the other castes, indicating for a more pronounced function. The cells of the glandular epithelium in Dufour’s gland are characterized by abundant mitochondria, basal invaginations and developed muscle fibres. The venom gland is morphologically similar in the different female castes, and consists of a venom reservoir, a convoluted gland and a bifurcated free secretory filament, with the convoluted gland appearing as a large cap lying on top of the reservoir. Cells of the convoluted gland and the free secretory filaments contain numerous mitochondria and a prominent end apparatus. The results will contribute to understanding the functional morphology of these glands among the different castes in Camponotus ants.

Proteomic analysis in the Dufour's gland of Africanized Apis mellifera workers (Hymenoptera: Apidae).

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour’s glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour’s gland contributing to a more complete understanding of the biological functions of this gland in honeybees.

Morphology and ultrastructure of the Dufour gland of Myzinum sp. (Tiphiidae)

Morphology and ultrastructure of the Dufour gland of Myzinum sp. (Tiphiidae)

Support for the reproductive ground plan hypothesis in a solitary bee: links between sucrose response and reproductive status.

In social bees, foraging behaviour is correlated with reproductive status and sucrose sensitivity via endocrine pathways. This association led to the hypothesis that division of labour in social insect societies is derived from an ancestral ground plan that functions to synchronize dietary preferences with reproductive needs in solitary insects. However, the relationship between these traits is unknown for solitary bees, which represent the ancestral state of social bees. We used the proboscis extension response assay to measure sucrose response among reproductive females of the solitary alkali bee (Nomia melanderi) as a function of acute juvenile hormone (JH) treatments and reproductive physiology. We also tested long-term effects of JH on reproductive development in newly emerged females. JH did not have short-term effects on reproductive physiology or sucrose response, but did have significant long-term effects on ovary and Dufour's gland development. Dufour's gland size, not ovary development, was a significant predictor of sucrose response. This provides support for the reproductive ground plan hypothesis, because the Dufour's gland has conserved reproductive functions in bees. Differing results from this study and honeybees suggest independent origins of division of labour may have evolved via co-option of different components of a conserved ground plan.

Juvenile hormone, but not nutrition or social cues, affects reproductive maturation in solitary alkali bees (Nomia melanderi).

Eusocial insect colonies are defined by extreme variation in reproductive activity among castes, but the ancestral conditions from which this variation arose are unknown. Investigating the factors that contribute to variation in reproductive physiology among solitary insects that are closely related to social species can help to fill this gap. We experimentally tested the role of nutrition, juvenile hormone (JH) and social cues on reproductive maturation in solitary alkali bees (Halictidae: Nomia melanderi). We found that alkali bee females emerge from overwintering with small Dufour's glands and small ovaries, containing oocytes in the early stages of development. Oocyte maturation occurs rapidly, and is staggered between the two ovaries. Lab-reared females reached reproductive maturity without access to mates or nesting opportunities, and many had resorbed oocytes. Initial activation of these reproductive structures does not depend on pollen consumption, though dietary protein or lipids may be necessary for long-term reproductive activity. JH is likely to be a limiting factor in alkali bee reproductive activation, as females treated with JH were more likely to develop mature oocytes and Dufour's glands. Unlike for related social bees, the effects of JH were not suppressed by the presence of older, reproductive females. These results provide valuable insight into the factors that influence reproductive activity in an important native pollinator, and those that may have been particularly influential in the evolution of reproductive castes.