Proteomic analysis of the venom of the social wasp Apoica pallens (Hymenoptera: Vespidae)

Wasps are a diverse group of insects that possess a sting apparatus associated with a venom gland, which is used for predation and colony defense. The biochemistry of Hymenoptera venom has been evaluated in relation to allergy and immunology, and proteomics has been shown to be a powerful tool for the identification of compounds with pharmacological potential. Data on wasps venom the of genus Apoica are scarce, so the objective of the present work was to identify the venom proteins of the eusocial wasp Apoica pallens, as a first step towards further investigation of applied uses of the venom and its protein constituents. The venom proteins were separated by two-dimensional gel electrophoresis, followed by MALDI-TOF/TOF mass spectrometry. A total of 259 spots were detected, with molecular weights from 4.9 to 141 kDa. Thirty of these proteins were identified and classified into eight functional categories: allergen, enzyme, metabolism, structural, environmental response, proteoglycan, active in DNA and RNA, and unknown function. Due to the few available proteomic data for wasp venom, many proteins could not be identified, which makes studies with proteomic analysis of Hymenoptera venom even more important.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

Identification of proteins from venom of the paralytic spider wasp, Cyphononyx dorsalis

The solitary spider wasp Cyphononyx dorsalis is well known to hunt spiders: it uses its stinger to paralyze its prey to feed its larva. This wasp venom was fractionated by bioassay-guided chromatography. Cation-exchange chromatography indicated that the pI value of the active principle was >6.5. 2D-PAGE analysis of the active fraction obtained by gel permeation chromatography showed three major spots of proteins. Two that appeared at pI of >6.5 were analyzed by in-gel digestion and protein sequencing. Three proteins were identified: an arginine kinase-like protein that was highly homologous to that of honeybee, an elastase like-protein that was homologous to that of fire ant, and an unknown protein that was not homologous to any protein in the database. Recombinant proteins expressed in E. coli were purified and used for bioassay. The results showed that the arginine kinase-like protein exhibited paralytic activity against spiders with the same characteristic symptoms as the crude venom.     

Regulatory mediators in the venom of Chelonus sp.: their biosynthesis and subsequent processing in homologous and heterologous systems

Following titration of the contents of the venom gland reservoir, the rate of biosynthesis of venom proteins was sufficiently rapid over the next 6-24 hrs to restore their titer to the level initially synthesized during early adulthood. There was no evidence of processing of smaller molecular weight components from much larger forms. Although most proteins were stable in young host embryos, two specific processing products of a 32.5 kDa venom protein were found in such hosts. The natural injection of venom proteins into either very old embryos or young embryos subsequently held at 4 degrees C for six days resulted in rapid degradation to biologically inactive forms. These data are the first report of direct examination of the biosynthesis of wasp venom proteins and the first analysis of the processing of specific hymenopteran venom proteins in target tissues.     

Alteration of the protein composition of bothrops jararaca venom and venom gland by isoproterenol treatment

• 1.1. The protein composition of Bothrops jararaca venom and venom gland was analyzed through SDS-PAGE, after isoproterenol (IPR) treatment.
• 2.2. Some proteins (47, 48, 57 and 72 kDa) were detected in the gland homogenate from the control but not from the IPR-treated samples.
• 3.3. Three proteins (26.5, 44.5 and 53 kDa) were detected in the venom gland from IPR-treated snakes but not from the venom gland from the control.
• 4.4. In the venom samples proteins of 41 and 74 kDa were detected only in the IPR treated samples, while proteins of 17 and 28 kDa were detected only in the control.
• 5.5. The biological activity of the venom did not change with IPR treatment.

     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects

Females of the parasitoid digger wasp species Liris niger hunt crickets as food for their future brood. The wasps paralyse the prey by injecting their venom directly into the CNS. The venom is produced in a gland consisting of two ramified glandular tubules terminating in a common reservoir. The reservoir contents enter the sting bulb via a ductus venatus. Secretory units of dermal gland type III line the two free gland tubules, the afferent ducts to the reservoir and the cap region within the reservoir. Secretion products of tubules reach the reservoir through the cuticle-lined central funnel. Secretory cells in the distal and middle parts of the tubules contain extensive rough endoplasmic reticulum and numerous electron-dense vesicles, whereas secretory cells of the afferent ducts and the cap region of the reservoir lack electron-dense vesicles and the endoplasmic reticulum is poorly developed. The secretory apparatus undergoes age-related changes. The secretory units in the venom gland tubules and inside the reservoir complete differentiation 1 day after imaginal ecdysis. After 30 days, massive autolytic processes occur in the secretory cells and in the epithelial cells of the reservoir. Analysis of the polypeptide composition demonstrates that the venom reservoir contains numerous proteins ranging from 3.4 to 200 kDa. A dominant component is a glycoprotein of about 90 kDa. In contrast the polypeptide composition of Dufour's gland is completely different and contains no glycoproteins. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets reveals that all of the major proteinaceous constituents become secreted. Thus the secreted venom contains exclusively proteins present in the soluble contents of the venom gland.     

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.     

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris

Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.     

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.     

Molecular and biochemical analysis of an aspartylglucosaminidase from the venom of the parasitoid wasp Asobara tabida (Hymenoptera: Braconidae)

The most abundant venom protein of the parasitoid wasp Asobara tabida was identified to be an aspartylglucosaminidase (hereafter named AtAGA). The aim of the present work is the identification of: 1) its cDNA and deduced amino acid sequences, 2) its subunits organization and 3) its activity. The cDNA of AtAGA coded for a proαβ precursor molecule preceded by a signal peptide of 19 amino acids. The gene products were detected specifically in the wasp venom gland (in which it could be found) under two forms: an (active) heterotetramer composed of two α and two β subunits of 30 and 18 kDa respectively and a homodimer of 44 kDa precursor. The activity of AtAGA enzyme showed a limited tolerance toward variations of pH and temperatures. Since the enzyme failed to exhibit any glycopeptide N-glycosidase activity toward entire glycoproteins, its activity seemed to be restricted to the deglycosylation of free glycosylasparagines like human AGA, indicating AtAGA did not evolve a broader function in the course of evolution. The study of this enzyme may allow a better understanding of the functional evolution of venom enzymes in hymenopteran parasitoids.     

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Differential gene expression profiles in the venom gland/sac of Orancistrocerus drewseni (Hymenoptera: Eumenidae)

To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Orancistrocerus drewseni Saussure (1857), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 498 expressed sequence tags (EST) were clustered and assembled into 205 contigs (94 multiple sequences and 111 singletons). About 65% (134) of the contigs had matched BLASTx hits (E≤10^?4). Among these, 115 contigs had similarity to proteins with assigned molecular function in the Gene Ontology database, and most of them (112 contigs, 83%) were homologous to genes from Hymenoptera, particularly to Apis mellifera (98 contigs). The contigs encoding hyaluronidase and phospholipase A2, known to be main components of wasp venoms, were found in high frequencies (27 and 4%, respectively, as judged by the number of ESTs) in the gene ontology category of catalytic activity. Full‐length open reading frames of hyaluronidase and phospholipase A2 were characterized and their abundance in the venom gland/sac was confirmed by quantitative real‐time PCR. Several contigs encoding enzymes, including zinc‐metallopeptidases that are likely involved in the processing and activation of venomous proteins or peptides, were also identified from the library. Discovery of venom gland/sac‐specific genes should promote further studies on biologically active components in the venom of O. drewseni. © 2009 Wiley Periodicals, Inc.     

Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein

Venom from Pimpla hypochondriaca, an endoparasitoid of pupae, was size-fractionated using gel filtration chromatography and analysed by SDS-PAGE in the presence and absence of reducing agent. A complex mixture of more than 20 venom constituents was identified which ranged in M(r) between approximately 5 and 100 kDa. Venom from a wide range of size fractions inhibited the motility of larval haemocytes and prevented the formation of cell aggregates when analysed in vitro, indicating that anti-haemocytic activity is mediated by multiple venom components. Sephadex A25 beads injected into the haemocoel of pupae were encapsulated within 24h. This reaction was abolished when the pupae were injected with 30 microg of venom protein, equivalent to one-fifth of a venom sac, 1h prior to implantation of the beads, confirming that venom suppresses encapsulation in pupae. Using random 5' end sequencing of a P. hypochondriaca venom gland cDNA library, we have isolated a cDNA encoding a 25.3 kDa protein containing a signal peptide and having sequence similarity to serine proteases. The N-terminal sequence of six residues from two venom proteins of 28 and 30 kDa was the same and identical to amino acids encoded by the cDNA, confirming that two mass forms of the protein are secreted into the venom sac. The N-terminal sequence of both venom proteins began nine residues towards the C terminus following the predicted signal sequence cleavage site, suggesting that the proteins are proteolytically processed before or during storage in the venom sac. The general applicability of using random 5' sequencing to identify cDNAs encoding secretory products is discussed.     

Proteins synthesized and secreted by larvae of the ectoparasitic wasp, Eulophus pennicornis

A microscopic examination of Eulophus pennicornis larvae on their host Lacanobia oleracea, revealed that peristaltic waves travelled from the anterior to posterior end of the feeding wasp larvae, and vice versa. In addition, when wasp larvae were immersed in PBS in vitro, they released a variety of proteins, with molecular weights ranging from (at least) 14 to 200 kDa. Amongst these was a protein with an estimated molecular weight similar to that of the 27 kDa parasitism-specific protein (PSP) detected in plasma from parasitized L. oleracea [Richards and Edwards, Insect Biochem Mol Biol 29:557-569 (1999)]. Similar results were obtained when the wasp larvae were incubated on balls of cotton wool soaked in tissue culture medium or sucrose, i.e., conditions that resemble their natural feeding behaviour. These results (and others) indicate that the wasp larvae release proteins, putatively through their mouth. Protein synthesis studies using (35)S-methionine indicated that the wasp larvae synthesize and secrete a variety of proteins in vitro, including one with a molecular weight corresponding to that of the L. oleracea 27 kDa PSP. As expected, only a portion of the total proteins synthesized by the parasitoid larvae were subsequently secreted. In addition, the autoradiogram of secreted proteins contained significantly fewer bands than silver-stained SDS gels of proteins released into PBS or onto cotton wool. Thus, some of the additional bands detected on the latter gels are thought to represent proteins that were not of wasp origin. Instead, these proteins released by the wasp larvae are speculated to be derived from their gut and, as such, probably represent proteins derived from host haemolymph and ingested during feeding. This possibility was supported by an electrophoretic analysis of homogenate supernatants prepared from wasp larvae with or without their gut contents. These studies indicated that the gut contents of the larval parasitoid contributes several distinct bands to the total protein profile. The ability of E. pennicornis larvae to synthesize, secrete, and release proteins is discussed with reference to those produced by endoparasitoid larvae. Published 2001 Wiley-Liss, Inc.     

Changes in the haemolymph proteome of Spodoptera littoralis induced by the parasitoid Chelonus inanitus or its polydnavirus and physiological implications

The egg-larval parasitoid Chelonus inanitus induces in its host Spodoptera littoralis two major developmental effects, namely a precocious onset of metamorphosis followed by a developmental arrest in the prepupal stage. Along with each egg, the wasp injects polydnavirus and venom into the host egg. The polydnavirus has been shown to play a major role in inducing the developmental arrest while the parasitoid larva is instrumental in inducing the precocious onset of metamorphosis. Here we report that experimental dilution of haemolymph of polydnavirus-containing larvae can partially prevent the developmental arrest while injection of native, but not of heat-treated, haemolymph or plasma from polydnavirus-containing larvae into nonparasitized larvae could induce developmental arrest in 14-15% of the larvae. This illustrates that heat-labile factors present in haemolymph play a role in causing developmental arrest. Injection of parasitoid medium increased the proportion of larvae entering metamorphosis precociously while injection of antibodies against a parasitoid-released protein had the opposite effect; this indicates that this protein and possibly other parasitoid-released substances are involved in inducing the precocious onset of metamorphosis. Analysis of the plasma proteome of nonparasitized, parasitized and polydnavirus-containing larvae revealed that the developmental effects are associated with only minor differences: eleven low abundant viral or virus-induced proteins and five parasitoid-released proteins were seen at specific stages of the host.     

Venom induces alarm behaviour in the social wasp Polybioides raphigastra (Hymenoptera: Vespidae): an investigation of alarm behaviour, venom volatiles and sting autotomy

Chemicals from the venom gland elicited alarm behaviour and attack in the Asian polistine wasp Polybioides raphigastra. When presented with crushed venom glands workers of this wasp respond with a mass stinging attack. Gas chromatography–mass spectrometry analyses show that the major volatiles in the venom gland are alkanes, monounsaturated alkenes and 2-alcohols. Several pyrazines, a spiroacetal and aromatics were also identified as trace compounds. The anatomy and morphology of the sting apparatus are reported, and we describe sting autotomy in this wasp. This is the first such report for the Ropalidiinae. The structures responsible for autotomy are likely to be large barbs present on the sting lancets, and a conspicuous tooth present on the medial side of the left lancet. Sting autotomy in P. raphigastra probably plays an important role in the localization of sites of attack by wasps defending the nest.