The N-glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.     

Basophil Activation Test Using Recombinant Allergens: Highly Specific Diagnostic Method Complementing Routine Tests in Wasp Venom Allergy

Background

Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test.

Methods

sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom.

Results

The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum.

Conclusions

BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.     

Prey Diets and Population-Densities of the Wasps

Prey collected by Vespula vulgaris and V. germanica were sampled by intercepting foragers returning to nests at two sites in scrubland-pasture near Hamilton. About 12% of returning foragers carried animal prey and 5% carried wood pulp. The remaining 83% carried no external load. The most common prey item for both species was Diptera, followed by Lepidoptera and Araneae (spiders). Even in similar habitats the two species collected different prey, with V. germanica collecting more Diptera and V. vulgaris more Lepidoptera. The prey changed slightly over time at both sites, V. germanica and V. vulgaris collected an estimated 1800 g and 600 g of prey nest-1 season-1 respectively. The wasps collected an estimated 75 000 prey loads ha-1 season-1 at Mystery Creek and 12 000 at Ruakura. This represents biomasses of 50 and 470 g ha-1, respectively, which are at least an order of magnitude lower than estimates of prey biomass for honeydew beech forest in parts of the South Island. The distribution of wasp nests was patchy however, and the biomass of prey collected exceeded 10 kg ha-1 in some places.     

Weather-Related Differences in Attractiveness of Protein Foods to

Low acceptance of protein baits by common (Vespula vulgaris) and German (V. germanica) wasps (Hymenoptera: Vespidae) occurred after rain in honeydew beech forest. This corresponded with a sharp decrease in the proportion of natural protein in the diet of V. vulgaris and V. germanica, and a reduction in the concentration of carbohydrate-rich honeydew in the crops of foraging wasps carrying liquid. The reduction of protein foraging most likely results from a change in the efficiency of foraging wasps at gathering high energy foods such as honeydew after rain, because rain reduces honeydew availability. Workers may therefore take longer to meet their own energy requirements before they can forage for protein to feed developing larvae.     

Hemolytic potency and phospholipase activity of some bee and wasp venoms

1. The action of crude venoms of four aculeate species: Apis mellifera, Vespa crabro, Vespula germanica and Vespula vulgaris on human erythrocytes was investigated in order to determine the lytic and phospholipase activity of different aculeate venoms and their ability to induce red blood cell hemolysis. 2. Bee venom was the only extract to completely lyse red blood cells at the concentration of 2-3 micrograms/ml. 3. Phospholipase activity in all of the examined vespid venoms was similar and the highest value was recorded in V. germanica. 4. Vespid venoms exhibited phospholipase B activity, which is lacking in honeybee venom. 5. In all membrane phospholipids but lecithin, lysophospholipase activity of vespid venoms was 2-6 times lower than the relevant phospholipase activity. 6. The incubation of red blood cells with purified bee venom phospholipase A2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cell lysis was approximately 30%.     

Comparison of three liquid lures for trapping social wasps (Hymenoptera: Vespidae)

Two citrus-based sodas and a known wasp attractant were compared in a field trial to assess their attractiveness to local nuisance wasp species. The wasps captured included Vespula germanica (F.), Vespula maculifrons (Buysson), Vespula vulgaris (L.), Vespula flavopilosa Jacobson, Vespula squamosa (Drury), Dolichovespula maculata (L.), Polistes fuscatus (L.), Polistes metricus Say, and Polistes dominulus (Christ). Wasps in the genus Vespula were present in significantly higher numbers in traps than Dolichovespula and Polistes. Both citrus soda products were superior to the isobutanol-acetic acid mixture as attractants for almost all of the wasp species.     

The significance of worker size in the seasonal development of the wasps Vespula vulgaris (L.) and Vespula germanica (F.)

An account is given of the variation of worker size in Vespula vulgaris and V.germanica during the vespine season and of pupal size, as indicative of larval feeding conditions, on the same comb and on different combs in the same nest.     

Vitellogenins Are New High Molecular Weight Components and Allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris Venom

Background/Objectives

Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom.

Methods/Principal Findings

Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants.

Conclusion

With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively.     

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

Attraction of Vespula germanica (Hymenoptera: Vespidae) foragers by conspecific heads

The socialwasp Vespula germanica (F.) is a serious pest in many regions it has invaded. Control programs to reduce its populations are commonly based on the use of poison baits. These baits also attract nonpestiferous invertebrates and vertebrates. In this work we studied the attraction of V. germanica foragers by conspecific worker squashes, comparing the effect of head and abdomen squashes in wasps behavior. We found that head squashes attract V. germanica foragers, elicit landing and transportation to nests. Furthermore, the addition of squashed heads to a protein bait increased attraction. This could be an alternative to improve baiting programs.     

Crystal structure of the major allergen from fire ant venom, Sol i 3

Fire ant venom is an extremely potent allergy-inducing agent containing four major allergens, Sol i 1 to Sol i 4, which are the most frequent cause of hypersensitivity reactions to hymenoptera in the southern USA. The crystal structure of recombinant (Baculovirus) major fire ant allergen Sol i 3 has been determined to a resolution of 3.1 Å by the method of molecular replacement. The secondary-structure elements of Sol i 3 are arranged in an α-β-α sandwich fold consisting of a central antiparallel β-sheet surrounded on both sides by α helices. The overall structure is very similar to that of the homologous wasp venom allergen Ves v 5 with major differences occurring in the solvent-exposed loop regions that contain amino acid insertions. Consequently, the limited conservation of surface chemical properties and topology between Sol i 3 and Ves v 5 may explain the observed lack of relevant cross-reactivity. It is concluded that Sol i 3 recognizes immunoglobulin E antibodies with a distinct set of its own epitopes, which are different from those of Ves v 5. Indeed, the molecular area in Sol i 3 covered by non-conserved residues is large enough to accommodate four unique Sol i 3 epitopes.     

黄唇蜾蠃蜂蜂毒中两个过敏原全长基因的克隆、序列分析和表达

为了进一步研究透明质酸酶的过敏活性,利用昆虫杆状病毒成功地表达了黄唇蜾蠃蜂Rhynchium brunneum蜂毒的透明质酸酶。根据已报道的胡蜂科透明质酸酶和抗原5基因的氨基酸保守序列,设计合成简并引物,利用反转录多聚酶链式反应（RT-PCR）技术扩增了黄唇蜾蠃蜂蜂毒透明质酸酶和抗原5的基因片段,利用RACE技术进一步获得了它们的全长基因（GenBank登录号分别为EU624135和EU624136）。按照过敏原的命名法则,分别命名为Rhy b 2和Rhy b 5。序列比对分析发现,这两个基因与胡蜂科的相应序列高度相似,说明对胡蜂蜂毒过敏的人群也可能对蜾蠃蜂蜂毒有交叉过敏反应。但进一步分析发现,黄唇蜾蠃蜂蜂毒透明质酸酶的B细胞决定表位显著不同,9个保守性氨基酸在蜾蠃蜂中仅保留了3个,而且缺乏两个关键的精氨酸;黄唇蜾蠃蜂蜂毒抗原5序列N端的二级结构和具有过敏活性的常见黄胡蜂Vespula vulgaris蜂毒的抗原5显著不同,有可能因此而缺失依赖其N端二级结构的B细胞决定表位。据此认为,蜾蠃蜂蜂毒透明质酸酶和抗原5很可能是天然弱化的过敏原,在过敏原特异性免疫治疗上具有潜在的应用价值。     

Assessment of prey overlap between a native (Polistes humilis) and an introduced (Vespula germanica) social wasp using morphology and phylogenetic analyses of 16S rDNA

Abstract In newly invaded communities, interspecific competition is thought to play an important role in determining the success of the invader and its impact on the native community. In southern Australia, the native Polistes humilis was the predominant social wasp prior to the arrival of the exotic Vespula germanica (Hymenoptera: Vespidae). Both species forage for similar resources (water, pulp, carbohydrate and protein prey), and concerns have arisen about potential competition between them. The aim of this study was to identify the protein foods that these wasps feed on. As many prey items are masticated by these wasps to the degree that they cannot be identified using conventional means, morphological identification was complemented by sequencing fragments of the mitochondrial 16S rRNA gene. GenBank searches using blast and phylogenetic analyses were used to identify prey items to at least order level. The results were used to construct complete prey inventories for the two species. These indicate that while P. humilis is restricted to feeding on lepidopteran larvae, V. germanica collects a variety of prey of invertebrate and vertebrate origin. Calculated values of prey overlap between the two species are used to discuss the implications of V. germanica impacting on P. humilis. Results obtained are compared to those gained by solely 'conventional' methods, and the advantages of using DNA-based taxonomy in ecological studies are emphasized.     

Resource choice of social wasps: influence of presence, size and species of resident wasps

Summary: The role of visual cues provided by resident wasps on resource choice by yellowjacket and paper wasp foragers was investigated. Large spring queen yellowjackets and small early season yellowjacket foragers (Vespula germanica, Vespula maculifrons, and Vespula vidua) were extracted in hexane to remove odors and posed as though feeding at petri dish feeders bearing daisy-like flower models, equipped with microcapillary feeding tubes, and containing 1:3 honey:water solution. An array of five feeders was presented to foragers at a suburban and a woodland site in Saratoga Springs, New York. The visual cues provided by resident wasps influenced resource choice by approaching social wasp foragers. Vespula germanica, an introduced yellowjacket species that tends to dominate at rich resources, was the only wasp visiting the suburban feeders. Foragers of this species preferentially fed on feeders and flowers with posed wasps and fed most often next to large wasps. Polistes fuscatus foragers at the woodland site similarly preferred to feed on occupied feeders and flowers. Vespula maculifrons and V. consobrina preferentially visited unoccupied feeders. Individual V. maculifrons, V. consobrina and V. vidua foragers that landed on occupied feeders all preferentially visited unoccupied flowers on those feeders. Vespula vidua and V. flavopilosa foragers did not demonstrate a feeder preference based on the presence/absence of posed wasps. Vespula consobrina foragers that visited occupied feeders preferred those occupied by extracted V. maculifrons queens and workers; no other wasps showed species based landing preferences.     

Orchids mimic green-leaf volatiles to attract prey-hunting wasps for pollination

An outstanding feature of orchids is the diversity of their pollination systems [1]. Most remarkable are those species that employ chemical deceit for the attraction of pollinators [2]. The orchid Epipactis helleborine is a typical wasp flower, exhibiting physiological and morphological adaptations for the attraction of pollinating social wasps [3]. As noted by Darwin [1], this species is almost entirely overlooked by other potential pollinators, despite a large nectar reward. Therefore, the mechanism for the attraction of pollinating social wasps was something of a mystery. By using a combination of behavioral experiments, electrophysiological investigations, and chemical analyses, we demonstrate for the first time that the flowers of E. helleborine and E. purpurata emit green-leaf volatiles (GLVs), which are attractive to foragers of the social wasps Vespula germanica and V. vulgaris. GLVs, emitted by damaged plant tissues, are known to guide parasitic wasps to their hosts [4]. Several E. helleborine GLVs that induced response in the antennae of wasps were also emitted by cabbage leaves infested with caterpillars (Pieris brassicae), which are common prey items for wasps [5]. This is the first example in which GLVs have been implicated in chemical mimicry for the attraction of pollinating insects.     

A test of worker policing theory in an advanced eusocial wasp, Vespula rufa

Mutual policing is an important mechanism for maintaining social harmony in group-living organisms. In some ants, bees, and wasps, workers police male eggs laid by other workers in order to maintain the reproductive primacy of the queen. Kin selection theory predicts that multiple mating by the queen is one factor that can selectively favor worker policing. This is because when the queen is mated to multiple males, workers are more closely related to queen's sons than to the sons of other workers. Here we provide an additional test of worker policing theory in Vespinae wasps. We show that the yellowjacket Vespula rufa is characterized by low mating frequency, and that a significant percentage of the males are workers' sons. This supports theoretical predictions for paternities below 2, and contrasts with other Vespula species, in which paternities are higher and few or no adult males are worker produced, probably due to worker policing, which has been shown in one species, Vespula vulgaris. Behavioral observations support the hypothesis that V. rufa has much reduced worker policing compared to other Vespula. In addition, a significant proportion of worker-laid eggs were policed by the queen.     

Abundance of wasps and prey consumption of paper wasps (Hymenoptera, Vespidae : Polistinae) in Northland, New Zealand

Polistine and vespine wasps were captured in Malaise traps in two fire-modified shrubland habitats of varying canopy height and composition at Lake Ohia, Northland, New Zealand. Prey consumption rates were calculated for the Asian paper wasp (Polistes chinensis antennalis) occupying these two areas of shrubland and a home garden in Whangarei, Northland. The sites were systematically searched for nests and wasp prey determined by intercepting foragers returning to nests. The Asian paper wasp predominated in the Malaise trap samples from the low- growing habitat while the German wasp (Vespula germanica) was more common in the taller vegetation type. The Asian paper wasp was more abundant than the German wasp in the samples in February and early March. Only four Australian paper wasps (Polistes humilis) and no common wasps (Vespula vulgaris) were caught. Asian paper wasps collected an estimated 15 000 prey loads per ha per season from one of the shrubland areas, and 478 000 prey loads per ha per season from the second area. These convert to estimates of 31 and 957 g per ha per season of invertebrate biomass removed by paper wasps from each habitat, respectively. The estimate for the garden site was 79 g per ha per season. Wasp nest densities varied between 20 and 210 nests per hectare. The biomass estimates are similar to average figures calculated for vespine wasps in scrubland and pasture. Both Asian paper wasps and Australian paper wasps preyed mainly on lepidopteran larvae. The cabbage white butterfly (Pieris rapae) was the most commonly collected species. Noctuid species were also well represented. Both male and female Asian paper wasps collected nectar in late March and early April.     

An allergen from Blattella germanica cockroaches and its immunological properties

The optimum method for obtaining allergenic preparation with high specific activity and stable physico-chemical characteristics from cockroaches of the species B. germanica was developed. The quantitative content of cockroach allergens Bla g1 and Bla g2 was determined in experimental batches. The age structure of sensitization to cockroaches in 124 bronchial asthma patients (50 adults and 74 children) was studied. Specific IgE antibodies to B. germanica were detected in 34% of the examined adults and 63.5% of the children.