Assessment of the In Vivo and In Vitro Release of Chemical Compounds from Vespa velutina.

Vespa velutina has been rapidly expanding throughout Galicia since 2012. It is causing human health risks and well-known losses in the beekeeping sector. Control methods are scarce, unspecific, and ineffective. Semiochemicals are insect-derived chemicals that play a role in communication and they could be used an integrated pest management tool alternative to conventional pesticides. A previous determination of the organic chemical profile should be the first step in the study of these semiochemicals. HS-SPME in living individuals and the sting apparatus extraction followed by GC-MS spectrometry were combined to extract a possible profile of these compounds in 43 hornets from Galicia. The identified compounds were hydrocarbons, ketones, terpenes, and fatty acid, and fatty acid esters. Nonanal aldehyde appeared in important concentrations in living individuals. While pentadecane, 8-hexyl- and ethyl oleate were mainly extracted from the venom apparatus. Ketones 2-nonanone, 2-undecanone and 7-nonen-2-one, 4,8-dimethyl- were identified by both procedures, as was 1,7-Nonadiene, 4,8-dimethyl-. Some compounds were detected for the first time in V. velutina such as naphthalene, 1,6-dimethyl-4-(1-methylethyl). The chemical profile by caste was also characterized.

Chemical and Biological Characteristics of Antimicrobial α-Helical Peptides Found in Solitary Wasp Venoms and Their Interactions with Model Membranes.

Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an α-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial α-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.

New Mastoparan Peptides in the Venom of the Solitary Eumenine Wasp Eumenes micado.

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic α-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly α-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes.

Proteomic analysis of the venom and venom sac of the woodwasp, Sirex noctilio - Towards understanding its biological impact.

UNLABELLED: The European horntail woodwasp, Sirex noctilio, is an invasive insect that attacks conifer hosts, particularly Pinus species. Venom injected by female S. noctilio, together with its symbiotic fungus, damages the normal physiology of Pinus, leading to death of the tree. To identify the proteinaceous components in the venom and uncover the interplay between venom proteins and tree proteins, clarification of the overall profile of proteins produced in the venom gland apparatus was carried out in this work. The venom sac proteome utilised in-solution digested in either a natural or deglycosylated state, prior to nanoHPLC LTQ-Orbitrap under CID/ETD mode. Here, we report the identification of 1454 and 1225 proteins in venom and sac, respectively, with 410 mutual proteins. Approximately 90 proteins were predicted to be secretory, of which 8 have features characteristic of toxins. Chemosensory binding proteins were also identified. Gene ontology and KEGG pathway analysis were employed to predict the protein functions and biological pathways in venom and sac. Protein-protein interaction (PPI) analysis suggested that one-step responses represent the majority of the Sirex-Pinus PPIs, and the proteins representing network hub nodes could be of importance for the development of pest management strategies. SIGNIFICANCE: The woodwasp Sirex noctilio is an invasive species in many parts of the world, including Australia and North America, where it is considered within the top 10 most serious forest insects. Where they have been introduced, the female woodwasps attack living pine trees, causing significant economic losses. Central to this destruction is the woodwasp's life cycle requirement to bore a hole to deposit eggs and a toxic mucus that disables the tree's network for transporting water and nutrients, yet aids in larval survival. Here we specifically examine the mucus gland apparatus and its contents, revealing the protein components that together with 'noctilisin' facilitate this complex association. The identification of chemosensory binding proteins further supports a role for the woodwasp ovipositor as an instrument for early stages of host tree selection. These findings could provide important clues towards the development of novel control tools against this pest.

Identification of major Toxoneuron nigriceps venom proteins using an integrated transcriptomic/proteomic approach.

Endoparasitoids in the order Hymenoptera are natural enemies of several herbivorous insect pest species. During oviposition they inject a mixture of factors, which include venom, into the host, ensuring the successful parasitism and the development of their progeny. Although these parasitoid factors are known to be responsible for host manipulation, such as immune system suppression, little is known about both identity and function of the majority of their venom components. To identify the major proteins of Toxoneuron nigriceps (Hymenoptera: Braconidae) venom, we used an integrated transcriptomic and proteomic approach. The tandem-mass spectrometric (LC-MS/MS) data combined with T. nigriceps venom gland transcriptome used as a reference database resulted in the identification of a total of thirty one different proteins. While some of the identified proteins have been described in venom from several parasitoids, others were identified for the first time. Among the identified proteins, hydrolases constituted the most abundant family followed by transferases, oxidoreductases, ligases, lyases and isomerases. The hydrolases identified in the T. nigriceps venom glands included proteases, peptidases and glycosidases, reported as common components of venom from several parasitoid species. Taken together, the identified proteins included factors that could potentially inhibit the host immune system, manipulate host physiological processes and host development, as well as provide nutrients to the parasitoid progeny, degrading host tissues by specific hydrolytic enzymes. The venom decoding provides us with information about the identity of candidate venom factors which could contribute to the success of parasitism, together with other maternal and embryonic factors.

Evaluation of the Therapeutic Properties of Mastoparan- and Sifuvirtide- Derivative Antimicrobial Peptides Using Chemical Structure-Function Relationship - in vivo and in silico Approaches.

Antimicrobial peptides, also called body defense peptides, are chemical structures widely distributed across the animal and vegetal kingdoms that have a fundamental role as part of the immune system. These peptides are used against a wide range of pathogens, such as Gram-negative and - positive bacteria, fungi and viruses, etc. Their action spectrum makes them important for the pharmaceutical industry, as they could represent templates for the design of new and more potent structures by using drug design and drug delivery systems. Here we present the antimicrobial activity against Bacillus subtilis (expressed as minimal inhibitory concentration values) for 33 mastoparan analogs and their new derivatives by quantitative structure-activity relationship method (2D, aligned and also non-aligned 3D-QSAR). We establish the contribution to antimicrobial activity of molecular descriptors like hydrophobicity, hydrogen bond donor and steric hindrance, correlated with contributions from the membrane environment (sodium, potassium, chloride ions). Also the studies of HIV-1 fusion inhibitor sifuvirtide and its analogs are presented in context of interaction with lipid structures during fusion and delivery of these drugs.

Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica

Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (4297 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (/)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.

Morphological and histological characterization of production structures, storage and distribution of venom in the parasitic wasp Bracon vulgaris.

It was described the morphology and histological composition of the structures related to production, storage and distribution of Bracon vulgaris venom, a wasp that parasite their hosts after the inoculation of a venom which causes irreversible paralysis. Were found 22 glandular filaments, coated with secretory epithelium associated with a reservoir coated internally by a chitin layer and externally by striated muscular fibers. A valve mediates the passage of the toxin to venom duct towards the parasitoids sting.

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.

Component-resolved diagnosis in vespid venom-allergic individuals

BACKGROUND: Hymenoptera venom-allergic patients frequently present multiple sensitisations. OBJECTIVES: To define the allergic profile by components in wasp allergic patients. To study the usefulness of specific IgE to components in cases of double sensitisation. MATERIALS AND METHODS: Wasp allergic patients who needed Polistes and/or Vespula venom immunotherapy were included. Before immunotherapy and after two years of treatment the following specific IgE (sIgE) levels were measured:Apis mellifera, Vespula spp. Polistes spp., rVes V 5, rPol d 5, nVes V 5, nPol d 5, nVes V 1, nPol d 1, nApi m 1, nApi m 2 and peroxidase. Skin tests with venoms were performed. Based on the sIgE and the skin test results, Polistes and/or Vespula immunotherapy was administered. RESULTS: Thirteen patients were included. Double sensitisation to Polistes/Vespula was detected in eight patients. Sensitisation to rVes V 5 and rPol d 5 was found in two of eight cases, to nVes V 1 and nPol d 1 in eight of 13 cases, and to nVes V 5 and nPol d 5 in 2 of 13 cases. Three patients received double immunotherapy with both wasps. One patient was treated with Vespula and nine with Polistes. sIgE levels decreased after two years of treatment. In patients who showed double sensitisation but were treated with only one venom, sIgE to both venoms decreased. CONCLUSIONS: Components analysis can be useful to study double positivity. In case of doubt, double immunotherapy should be administered. Phospholipase was found to be a major allergen in our population

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In vitro antibacterial effect of wasp (Vespa orientalis) venom

The emergence of antibacterial resistance against several classes of antibiotics is an inevitable consequence of drug overuse. As antimicrobial resistance spreads throughout the globe, new substances will always be necessary to fight against multidrug-resistant microorganisms. Venoms of many animals have recently gained attention in the search for new antimicrobials to treat infectious diseases. Thefore, the present study aimed to study the antibacterial effects of wasp (Vespa orientalis) crude venom. Two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and two gram-negative ones (Escherichia coli and Klesiella pneumonia) were compared for their sensitivity to the venom by determining the inhibition zone (Kirby-Bauer method) and minimum inhibitory concentration (MIC). A microbroth kinetic system based on continuous monitoring of changes in the optical density of bacterial growth was also used for determination of antimicrobial activity.

In vitro antibacterial effect of wasp (Vespa orientalis) venom

The emergence of antibacterial resistance against several classes of antibiotics is an inevitable consequence of drug overuse. As antimicrobial resistance spreads throughout the globe, new substances will always be necessary to fight against multidrug-resistant microorganisms. Venoms of many animals have recently gained attention in the search for new antimicrobials to treat infectious diseases. Thefore, the present study aimed to study the antibacterial effects of wasp (Vespa orientalis) crude venom. Two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and two gram-negative ones (Escherichia coli and Klesiella pneumonia) were compared for their sensitivity to the venom by determining the inhibition zone (Kirby-Bauer method) and minimum inhibitory concentration (MIC). A microbroth kinetic system based on continuous monitoring of changes in the optical density of bacterial growth was also used for determination of antimicrobial activity.

Systematic analysis of a wasp parasitism arsenal.

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus-carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV-mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus-carrying wasp introduces into its host.

Diversity of anti-haemostatic proteins in the salivary glands of Rhodnius species transmitters of Chagas disease in the greater Amazon.

The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.

Chemical and biological characterization of four new linear cationic á-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumeninewasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/ TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry)analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumeninewasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH2) and EMP-EF (FDVMGIIKKIAGAL-NH2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumeninewasp. These sequences have the characteristic features of linear cationic cytolyticpeptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant a-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista.

The phospholipases A(1) (PLA(1) s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1) , combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2) s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Caste differences in venom volatiles and their effect on alarm behaviour in the paper wasp Polistes dominulus (Christ).

Foundresses and workers of Polistes paper wasps show slight morphological and physiological differences. However, after the emergence of the workers, the castes can be readily discriminated by their behaviour: the dominant foundress is the principal egg-layer, whereas workers perform different tasks linked to colony development. Previous studies have demonstrated in this genus that defence of the colony by the workers is more effectively carried out by a collective response elicited by venom volatiles used as alarm pheromones. In the present study, gas chromatography-mass spectrometry analyses of the venom volatiles of foundresses and workers of Polistes dominulus (Christ) show predominantly quantitative differences. Spiroacetals, mainly (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, are significantly higher in the venom volatiles fraction of workers, whereas the amount of N-(3-methylbutyl)acetamide is almost double in foundresses. On the basis of the chemical results, behavioural assays were performed on fifteen field colonies to test the alarm response of the resident wasps to venom extracts from foundresses and workers. Our behavioural results suggest that worker venom has a stronger alarm effect on the colonies than that of the foundresses, which seems unable to elicit the complete alarm response ending with a final attack and sting. The venom volatiles of P. dominulus workers serve mainly to alarm the colony whilst those of foundresses may also be linked to additional functions related to conspecific interactions.

Hypersensitivity to Vespula and Polistes: can we tell the primary sensitization from the clinical history? / Hipersensibilidad a vespula y polistes: ¿se puede determinar la sensibilización predominante a partir de la historia clínica?

Objective: To study the relationship between theprimary sensitization to wasp venoms and the geographicaland seasonal circumstances of the anaphylaxis-induced sting.Methods: We performed a retrospective review of115 patients (age 10-80) who suffered a systemicreaction to a wasp sting. Season and type of locality(urban or rural) at the moment of the sting wererecorded. Serum specific IgE levels to venoms fromVespula and Polistes were measured, and a primarysensitization was determined to whichever genus ofwasp for which the highest class of specific IgE wasobserved. The primary sensitization in relation to thetype of locality and the season was assessed usingthe chi-square test.Results: Most reactions occurred in urban areas(67.8 %), and in the summer season (63.4 %). Mostpatients were sensitized to Vespula venom (94.8 %).Primary sensitization was to Vespula in 56.5 %, toPolistes in 10.4 %, and undetermined in 33 %. Thedistribution of geographical areas did not show significantdifferences in relation to primary sensitization(p > 0.05). Most patients with primary sensitization toVespula suffered the anaphylaxis-induced sting afterthe spring season, with a statistically significant result(p < 0.05).Conclusion: In our population, the probability ofVespula sting is higher than Polistes sting when thereaction occurs after spring. This finding can help usto identify the responsible vespid when the diagnostictests do not provide an accurate result

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Defining the T cell antigen proteome of wasp venom.

BACKGROUND: While modulation of T cell function is believed to be important in the successful acquisition of clinical tolerance during venom immunotherapy, little is known of the role of wasp venom specific T cell antigens. OBJECTIVE: We sought comprehensively to characterize the T cell proteome for wasp venom to facilitate the future development of T cell-based immunotherapeutic approaches. METHODS: Using peripheral blood mononuclear cells from wasp venom-allergic individuals and IL-4 ELISPOT analysis, we characterized T cell responses to whole venom and gel filtration/ion exchange-fractionated venom. Reactive fractions were purified and identified using highly sensitive electrospray ion-trap mass spectrometry. RESULTS: Wasp venom-allergic individuals have detectable whole wasp venom-specific T cells directly ex vivo, which show rapid IL-4 effector function. T cell responses to gel filtration/ion exchange fractionated venom were dominated by responses to phospholipase A(1), hyaluronidase and antigen 5. CONCLUSION: Although it is likely that there are many T cell antigens within wasp venom, the main responses are to proteins coincident with the known IgE-binding proteins.