Jewel Wasp Research Articles

A new lineage of Cretaceous jewel wasps (Chalcidoidea: Diversinitidae).

Jewel wasps (Hymenoptera: Chalcidoidea) are extremely species-rich today, but have a sparse fossil record from the Cretaceous, the period of their early diversification. Three genera and three species, Diversinitus attenboroughi gen. & sp. n., Burminata caputaeria gen. & sp. n. and Glabiala barbata gen. & sp. n. are described in the family Diversinitidae fam. n., from Lower Cretaceous Burmese amber. Placement in Chalcidoidea is supported by the presence of multiporous plate sensilla on the antennal flagellum and a laterally exposed prepectus. The new taxa can be excluded from all extant family level chalcidoid lineages by the presence of multiporous plate sensilla on the first flagellomere in both sexes and lack of any synapomorphies. Accordingly, a new family is proposed for the fossils and its probable phylogenetic position within Chalcidoidea is discussed. Morphological cladistic analyses of the new fossils within the Heraty et al. (2013) dataset did not resolve the phylogenetic placement of Diversinitidae, but indicated its monophyly. Phylogenetically relevant morphological characters of the new fossils are discussed with reference to Cretaceous and extant chalcidoid taxa. Along with mymarid fossils and a few species of uncertain phylogenetic placement, the newly described members of Diversinitidae are among the earliest known chalcidoids and advance our knowledge of their Cretaceous diversity.

Ampulexins: A New Family of Peptides in Venom of the Emerald Jewel Wasp, Ampulex compressa

The parasitoid wasp Ampulex compressa injects venom directly into the brain and subesophageal ganglion of the cockroach Periplaneta americana, inducing a 7 to 10 day lethargy termed hypokinesia. Hypokinesia presents as a significant reduction in both escape response and spontaneous walking. We examined aminergic and peptidergic components of milked venom with HPLC and MALDI-TOF mass spectrometry. HPLC coupled with electrochemical detection confirmed the presence of dopamine in milked venom, while mass spectrometry revealed that the venom gland and venom sac have distinct peptide profiles, with milked venom predominantly composed of venom sac peptides. We isolated and characterized novel α-helical, amphipathic venom sac peptides that constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin 1, into the subesophageal ganglion of cockroaches resulted in a short-term increase in escape threshold. Neither milked venom nor venom peptides interfered with growth of Escherichia coli or Bacillus thuringiensis on agar plates, and exposure to ampulexins or milked venom did not induce cell death in Chinese hamster ovary cells (CHO-K1) or Hi5 cells ( Trichoplusia ni).

HsTRPA of the Red Imported Fire Ant,Solenopsis invicta, Functions as a Nocisensor and Uncovers the Evolutionary Plasticity of HsTRPA Channels

Solenopsis invicta, the red imported fire ant, represents one of the most devastating invasive species. To understand their sensory physiology, we identified and characterized their Hymenoptera-specific (Hs) TRPA channel, SiHsTRPA. Consistent with the sensory functions of SiHsTRPA, it is activated by heat, an electrophile, and an insect repellent. Nevertheless, SiHsTRPA does not respond to most of the honey bee ortholog (AmHsTRPA)-activating compounds. The jewel wasp ortholog (NvHsTRPA) is activated by these compounds even though it outgroups both AmHsTRPA and SiHsTRPA. Characterization of AmHsTRPA/SiHsTRPA chimeric channels revealed that the amino acids in the N terminus, as well as ankyrin repeat 2 (AR2) of AmHsTRPA, are essential for the response to camphor. Furthermore, amino acids in ARs 3 and 5-7 were specifically required for the response to diallyl disulfide. Thus, amino acid substitutions in the corresponding domains of SiHsTRPA during evolution would be responsible for the loss of chemical sensitivity. SiHsTRPA-activating compounds repel red imported fire ants, suggesting that SiHsTRPA functions as a sensor for noxious compounds. SiHsTRPA represents an example of the species-specific modulation of orthologous TRPA channel properties by amino acid substitutions in multiple domains, and SiHsTRPA-activating compounds could be used to develop a method for controlling red imported fire ants.

Embryo Microinjection and Transplantation Technique for <em>Nasonia vitripennis</em> Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

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.

Unique sequence organization and small RNA expression of a "selfish" B chromosome.

B chromosomes are found in numerous plants and animals. These nonessential, supernumerary chromosomes are often composed primarily of noncoding DNA repeats similar to those found within transcriptionally "silenced" heterochromatin. In order to persist within their resident genomes, many B chromosomes exhibit exceptional cellular behaviors, including asymmetric segregation into gametes and induction of genome elimination during early development. An important goal in understanding these behaviors is to identify unique B chromosome sequences and characterize their transcriptional contributions. We investigated these properties by examining a paternally transmitted B chromosome known as paternal sex ratio (PSR), which is present in natural populations of the jewel wasp Nasonia vitripennis. To facilitate its own transmission, PSR severely biases the sex ratio by disrupting early chromatin remodeling processes. Through cytological mapping and other approaches, we identified multiple DNA repeats unique to PSR, as well as those found on the A chromosomes, suggesting that PSR arose through a merger of sequences from both within and outside the N. vitripennis genome. The majority of PSR-specific repeats are interspersed among each other across PSR's long arm, in contrast with the distinct "blocks" observed in other organisms' heterochromatin. Through transcriptional profiling, we identified a subset of repeat-associated, small RNAs expressed by PSR, most of which map to a single PSR-specific repeat. These RNAs are expressed at much higher levels than those arising from A chromosome-linked repeats, suggesting that in addition to its sequence organization, PSR's transcriptional properties differ substantially from the pericentromeric regions of the normal chromosomes.

Functional characterization of the transcriptional regulatory elements of three highly expressed constitutive genes in the jewel wasp, Nasonia vitripennis.

The jewel wasp, Nasonia vitripennis Ashmead (Hymenoptera: Pteromalidae), is an easily reared parasitoid that is providing an ever increasingly malleable model for examining the biology and genetics of Hymenoptera. Utilizing genomic and transcriptome resources, 5' upstream transcriptional regulatory sequences (TREs) from three highly expressed genes were identified and cloned. Criteria for TRE selection included the presence of an adjacent gene 5' of the translation initiation site. One gene was methylated whereas the other two were nonmethylated. Each TRE, heat-shock protein 70 (hsp70), activator of 90 kDa hsp ATPase protein 1 (hsp90A), and lipid storage droplet surface-binding protein 1 (lsdp) was linked with enhanced green fluorescent protein (EGFP) coding sequence and cloned into both pDP9e somatic and piggyBac germline transformation vectors. EGFP expression patterns under control of each TRE were compared with patterns of DsRed fluorescence produced from the transformation vector cassette. Functional activity of each TRE was observed in cultured Spodoptera frugiperda 9 (Sf9) cells and Drosophila melanogaster as well as in N.vitripennis embryos demonstrating that all three sequences had functional transcriptional regulatory activity in three different insect orders. Identification and functional characterization of these three TREs will provide critical and necessary resources for further genetic analyses of N. vitripennis, Hymenoptera and other insects.

Genome Silencing and Elimination: Insights from a "Selfish" B Chromosome.

B chromosomes are non-essential components of numerous plant and animal genomes. Because many of these “extra” chromosomes enhance their own transmission in ways that are detrimental to the rest of the genome, they can be thought of as genome parasites. An extreme example is a paternally inherited B chromosome known as paternal sex ratio (PSR), which is found in natural populations of the jewel wasp Nasonia vitripennis. In order to ensure its own propagation, PSR severely biases the wasp sex ratio by converting diploid female-destined embryos into transmitting haploid males. This action occurs at the expense of the other paternally inherited chromosomes, which fail to resolve during the first round of division and are thus eliminated. Recent work has revealed that paternal genome elimination by PSR occurs through the disruption of a number of specific histone post-translational modifications, suggesting a central role for chromatin regulation in this phenomenon. In this review, we describe these recent advances in the light of older ones and in the context of what is currently understood about the molecular mechanisms of targeted genome silencing and elimination in other systems.

Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9

The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.

Morphology and ultrastructure of the male reproductive system of the jewel wasp, Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The genital systems of insects are undoubtedly of great key for the success of mating and population reproduction. In the meantime, morphological and ultrastructural characteristics of the reproductive systems also provide valuable evidences for the studies of taxonomy, reproductive biology and evolutionary biology. More previously, the reproductive system of female Nasonia vitripennis was already described. To complement the information of reproductive systems from this species, the male reproductive system was here dissected and characterized by light microscopy and transmission electron microscopy. Results showed that the system follows the pattern of most Hymenoptera species: paired testes with one follicle, deferent ducts, seminal vesicles, accessory glands and a fused ejaculatory duct ultimately connected to the external aedeagus. Histologically, the testes, seminal vesicles and accessory glands are all composed of three portions, all of which include epithelial and luminal regions. In addition, both lipidic inclusions and nuclei are presented in the three organs with variable sizes and shapes. Spermatozoa are observed in the testes and seminal vesicles, and also vary considerably in size and shape, suggesting different phases of spermatogenesis. Mitochondria are abundant in seminal vesicles and accessory glands. Notably, membranous inclusions and Golgi complexes are found only in seminal vesicles, whereas secretory granules are presented only in accessory glands, being indicative of organ or age specificity. Together, this study complements the information of the reproductive systems from N. vitripennis, and provides an extensive resource for taxonomy, reproductive biology and evolutionary biology.

A \u2018selfish\u2019 B chromosome induces genome elimination by disrupting the histone code in the jewel wasp Nasonia vitripennis

Intragenomic conflict describes a phenomenon in which genetic elements act ‘selfishly’ to gain a transmission advantage at the expense of the whole genome. A non-essential, selfish B chromosome known as Paternal Sex Ratio (PSR) induces complete elimination of the sperm-derived hereditary material in the jewel wasp Nasonia vitripennis. PSR prevents the paternal chromatin from forming chromosomes during the first embryonic mitosis, leading to its loss. Although paternally transmitted, PSR evades self-elimination in order to be inherited. We examined important post-translational modifications to the DNA packaging histones on the normal genome and the PSR chromosome in the fertilized embryo. Three histone marks – H3K9me2,3, H3K27me1, and H4K20me1 – became abnormally enriched and spread to ectopic positions on the sperm’s chromatin before entry into mitosis. In contrast, other histone marks and DNA methylation were not affected by PSR, suggesting that its effect on the paternal genome is specific to a subset of histone marks. Contrary to the paternally derived genome, the PSR chromosome was visibly devoid of the H3K27me1 and H4K20me1 marks. These findings strongly suggest that PSR causes paternal genome elimination by disrupting at least three histone marks following fertilization, while PSR avoids self-elimination by evading two of these marks.

Do Quiescence and Wasp Venom-Induced Lethargy Share Common Neuronal Mechanisms in Cockroaches?

The escape behavior of a cockroach may not occur when it is either in a quiescent state or after being stung by the jewel wasp (Ampulex compressa). In the present paper, we show that quiescence is an innate lethargic state during which the cockroach is less responsive to external stimuli. The neuronal mechanism of such a state is poorly understood. In contrast to quiescence, the venom-induced lethargic state is not an innate state in cockroaches. The Jewel Wasp disables the escape behavior of cockroaches by injecting its venom directly in the head ganglia, inside a neuropile called the central complex a ‘higher center’ known to regulate motor behaviors. In this paper we show that the coxal slow motoneuron ongoing activity, known to be involved in posture, is reduced in quiescent animals, as compared to awake animals, and it is further reduced in stung animals. Moreover, the regular tonic firing of the slow motoneuron present in both awake and quiescent cockroaches is lost in stung cockroaches. Injection of procaine to prevent neuronal activity into the central complex to mimic the wasp venom injection produces a similar effect on the activity of the slow motoneuron. In conclusion, we speculate that the neuronal modulation during the quiescence and venom-induced lethargic states may occur in the central complex and that both states could share a common neuronal mechanism.

Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest.

An optimized approach to germ-free rearing in the jewel wasp Nasonia.

Development of a Nasonia in vitrogerm-free rearing system in 2012 enabled investigation of Nasonia-microbiota interactions and real-time visualization of parasitoid metamorphosis. However, the use of antibiotics, bleach, and fetal bovine serum introduced artifacts relative to conventional rearing of Nasonia. Here, we optimize the germ-free rearing procedure by using filter sterilization in lieu of antibiotics and by removing residual bleach and fetal bovine serum. Comparison of these methods reveals no influence on larval survival or growth, and a 52% improvement in adult production. Additionally, adult males produced in the new germ-free system are similar in size to conventionally reared males. Experimental implications of these changes are discussed.

An insect with a delta-12 desaturase, the jewel wasp Nasonia vitripennis, benefits from nutritional supply with linoleic acid.

The availability of linoleic acid (LA; C18:2(∆9,12)) is pivotal for animals. While vertebrates depend on a nutritional supply, some invertebrates, including the parasitic wasp Nasonia vitripennis, are able to synthesize LA from oleic acid (OA; C18:1(∆9)). This raises the question as to whether these animals nevertheless benefit from the additional uptake of LA with the diet. LA plays an important role in the sexual communication of N. vitripennis because males use it as a precursor for the synthesis of an abdominal sex pheromone attracting virgin females. We reared hosts of N. vitripennis that were fed diets enriched in the availability of stearic acid (SA: C18:0), OA or LA. N. vitripennis males developing on the different host types clearly differed in both the fatty acid composition of their body fat and sex pheromone titres. Males from LA-enriched hosts had an almost fourfold higher proportion of LA and produced significantly more sex pheromone than males from SA (2.2-fold) and OA (1.4-fold) enriched hosts, respectively. Our study demonstrates that animals being able to synthesize important nutrients de novo may still benefit from an additional supply with their diet.

Bioinformatic and Functional Analysis of Venom from the Jewel Wasp Ampulex compressa

The parasitoid jewel wasp Ampulex compressa exploits its host, the American cockroach Periplaneta americana, as a food source for its young. The host is subdued through direct envenomation into the central nervous system, inducing a days‐long “zombie‐like state” referred to as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions intact. This condition is reversible, provided that egg deposition is prevented. To facilitate elucidation of biochemical mechanisms underlying venom‐induced hypokinesia, we developed a comprehensive inventory of the venom by combining: 1) next‐generation RNA sequencing, transcriptomic analysis of venom gland components, and 2) proteomics of venom apparatus extracts and whole milked venom via mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology. These analyses yielded a total of 201 proteins and peptides in the venom, 105 with Pfam domains, 146 similar to other proteins in the NCBI‐nr or Uniprot databases, and 45 novel proteins absent in global databases. A. compressa venom proteins include members of the M13 family of proteases (11), phospholipase A2 isoforms (10), along with hyaluronidase, chitinase, icarapin, venom acid phosphatase, serpins, and signaling peptides including tachykinin, corazonin, and myosuppressin. Differential expression analysis and proteomics of the two major tissues of the venom apparatus ‐ venom sac and venom gland ‐identified tissue‐specific expression of key venom components.We show that A. compressa venom tachykinins (AcVTK) are able to activate the cockroach brain tachykinin receptor with high affinity (EC50 ~1 nM) in vitro. Injection of synthetic AcVTK into the subesophageal ganglion, an established target of the wasp venom, increases escape response threshold for one hour post‐treatment. These results indicate that tachykinin may participate in the behavioral changes resulting from envenomation of cockroach cerebral ganglia.We isolated and characterized two novel, paralogous venom sac peptides found in milked venom. These peptides are alpha‐helical, amphipathic, and constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin‐1, into the subesophageal ganglion of cockroaches resulted in a short‐term increase in escape threshold.Our findings show that the venom of A. compressa is a rich collection of both well‐known and novel, pharmacologically diverse compounds. Our study provides new and comprehensive information on A. compressa venom contents that inform hypotheses regarding its mode of action.

The clock gene period is essential for the photoperiodic response in the jewel wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

The role of the clock gene period (per) in photoperiodism was investigated in the jewel wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Typically, females reared under long days lay eggs that develop to adulthood without developmental interruption, whereas those reared under short days lay eggs that enter larval diapause. We found that RNA interference (RNAi) of per disrupts the photoperiodic response; RNAi wasps failed to produce diapause-destined eggs in response to short days. This indicates that per is essential for the photoperiodic response in this species. Exposure to low temperature swiftly affects maternal physiology and prompts N. vitripennis females to lay diapause-destined eggs. Interestingly, per RNAi females also laid diapause-destined eggs in response to the low temperature treatment in the current study. This finding indicates that per RNAi did not disrupt the maternal physiology that was directly involved in production of diapause-destined eggs, but it did affect photoperiodic perception. We also investigated the expression patterns of several clock genes, per, mammalian-type cryptochrome (cry-m), cycle (cyc), PAR-domain protein 1 (Pdp1), and clockwork orange (cwo), to determine whether their expression patterns were affected by different photoperiods.

Identification of Genes Uniquely Expressed in the Germ-Line Tissues of the Jewel Wasp Nasonia vitripennis.

The jewel wasp Nasonia vitripennis is a rising model organism for the study of haplo-diploid reproduction characteristic of hymenopteran insects, which include all wasps, bees, and ants. We performed transcriptional profiling of the ovary, the female soma, and the male soma of N. vitripennis to complement a previously existing transcriptome of the wasp testis. These data were deposited into an open-access genome browser for visualization of transcripts relative to their gene models. We used these data to identify the assemblies of genes uniquely expressed in the germ-line tissues. We found that 156 protein-coding genes are expressed exclusively in the wasp testis compared with only 22 in the ovary. Of the testis-specific genes, eight are candidates for male-specific DNA packaging proteins known as protamines. We found very similar expression patterns of centrosome associated genes in the testis and ovary, arguing that de novo centrosome formation, a key process for development of unfertilized eggs into males, likely does not rely on large-scale transcriptional differences between these tissues. In contrast, a number of meiosis-related genes show a bias toward testis-specific expression, despite the lack of true meiosis in N. vitripennis males. These patterns may reflect an unexpected complexity of male gamete production in the haploid males of this organism. Broadly, these data add to the growing number of genomic and genetic tools available in N. vitripennis for addressing important biological questions in this rising insect model organism.

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta, Hymenoptera, Sphecidae): Structural and biochemical aspects

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 μm (∅ 125 μm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.