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.     

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism-related proteins

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.     

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae)

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.     

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.     

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.     

The phylogenetic distribution of sphingomyelinase D activity in venoms of Haplogyne spiders

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31-35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31-32 kDa, African Loxosceles having 32-33.5 kDa and Sicarius having 32-33 kDa molecules.     

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.     

Parasitoid wasp sting: A cocktail of GABA,taurine, and β‐alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal‐giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI‐TOF‐MS revealed high levels of GABA (25 mM), and its receptor agonists β‐alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal‐giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin‐sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: characterization and cross-reactions

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivo in mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatus venom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A(2,) the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of M. pruriens extract. Proteins of E. carinatus venom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.     

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.     

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.     

啮小蜂毒液对寄主亚洲玉米螟蛹发育的影响

假寄生和毒液注射实验表明啮小蜂Tetrastichussp .毒液能永久麻痹亚洲玉米螟蛹Ostriniafurnacalis,较高剂量的毒液对亚洲玉米螟蛹有不同程度的麻痹作用。麻痹的蛹在室温下较长时间内不腐烂和干化。低剂量毒液能延长亚洲玉米螟蛹期 1～ 3d ,导致畸形成虫。啮小蜂初产卵转移寄主实验证实毒液可以帮助啮小蜂卵在亚洲玉米螟蛹体内的发育 ,啮小蜂初产卵在转移寄主体内发育到羽化时间为 2 5d ,比它们在原寄主内的羽化时间增加 8d左右 ,初产蜂卵与 1个毒液储液囊当量的毒液同时注入转移寄主体内 ,初产卵的羽化时间明显缩短 ,死亡率降低     

Wasp venom blocks central cholinergic synapses to induce transient paralysis in cockroach prey

The parasitoid wasp Ampulex compressa induces a set of unique behavioral effects upon stinging its prey, the cockroach. It stings into the first thoracic segment inducing 2 to 3 min of transient flaccid paralysis of the front legs. This facilitates a second sting in the cockroach's head that induces 30 min of excessive grooming followed by a 2 to 5-week long lethargic state. In the present study, we examine the immediate effect of the first sting, which is a transient paralysis of the front legs. Using radiolabeled wasps, we demonstrate that the wasp injects its venom directly into the cockroach's first thoracic ganglion. The artificial injection of milked venom into a thoracic ganglion abolishes spontaneous and evoked responses of the motoneurons associated with leg movements. To investigate the physiological mechanism of action of the venom, we injected venom into the last abdominal ganglion of the cockroach, which houses a well-characterized cholinergic synapse. Injected venom abolishes both sensory-evoked and agonist-evoked postsynaptic potentials recorded in the postsynaptic neuron for 2 to 3 min without affecting action potential propagation. Thus, the venom blocking effect has a postsynaptic component that follows the same time course as the transient paralysis induced by the thoracic sting. Finally, injection of a nicotinic antagonist in the front thoracic ganglion induces paralysis of the front legs. We conclude that the transient paralytic effect of the thoracic sting can be mainly accounted for by the presence of a venom active component that induces a postsynaptic block of central cholinergic synaptic transmission.     

Differences in venom composition between orb-weaving and wandering Hawaiian Tetragnatha (Araneae)

Spider venoms are complex mixtures of toxins that are used primarily for immobilizing prey. There is evidence of chemical variation in spider venoms among close relatives, yet few studies have analysed their evolution within an ecological and phylogenetic framework. On the Hawaiian archipelago, Tetragnatha, a cosmopolitan orb-weaving genus, has undergone a radiation in which a monophyletic lineage has abandoned web-building and become obligately wandering foragers. This study compares venom composition and details of feeding behaviour between orb-weaving and wandering Hawaiian Tetragnatha. Protein gel electrophoresis patterns indicated that relative to orb-weavers, wandering species had a reduced concentration of low molecular weight (<14kDa) components. Both orb-weaving and wandering Tetragnatha captured flying prey (adult lepidopterans, dipterans), but wandering spiders also captured non-flying prey (insect larvae, spiders). There were no distinct differences between orb-weavers and wanderers in prey capture and immobilization sequences, or in the paralytic effects of bites on prey. However, prey bitten by wanderers took longer to be permanently immobilized than prey bitten by orb-weavers. Contrary to predictions, there was no indication that web-loss in this group was associated with an increase in venom potency.     

间斑寇蛛粗毒液的生理生化分析及两种采毒方法的比较

利用两种不同方法采集间斑寇蛛（Latrodectus tredecimguttatus）毒液并对其进行理化和生物学性质的比较分析。结果显示,粗毒液中的蛋白质成分主要是相对分子质量较大（>104）的酸性蛋白质。与毒囊粗毒液相比,电刺激粗毒液中相对分子质量在105左右的蛋白质含量明显高于毒囊粗毒液中的含量,而两者中相对分子质量较小（<104）的蛋白质与多肽的组成非常相似。电刺激粗毒冻干粉和毒囊粗毒冻干粉对小白鼠的LD50值分别为(0.16±0.03) mg/kg和(0.39±0.05)mg/kg体重;对美洲蜚蠊（Periplaneta Americana）的LD50值分别为1.87 μg/g和2.32 μg/g。在浓度为3.2×10-6g/mL时,电刺激粗毒冻干粉能在（25.0±2.2） min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递;毒囊粗毒冻干粉在浓度为6×10-6g/mL时的完全阻断时间为（23.3±2.2） min;粗毒液中的低相对分子质量（<104）部分在10-4g/mL浓度下对标本的神经肌肉接头传递无明显影响。上述结果表明,间斑寇蛛毒液是一种富含大分子量蛋白质的混合物;哺乳动物神经毒性主要基于其中的大的相对分子质量酸性蛋白质成分,而不是低的相对分子质量的多肽;电刺激粗毒液中的活性成分与毒囊粗毒液中的相似,但含量高于毒囊粗毒液。     

Peptide diversity in the venom of the social wasp Polybia paulista (Hymenoptera): A comparison of the intra- and inter-colony compositions

The venoms of the social wasps evolved to be used as defensive tools to protect the colonies of these insects against the attacks of predators. Previous studies estimated the presence of a dozen peptide components in the venoms of each species of these insects, which altogether comprise up to 70% of the weight of freeze-dried venoms. In the present study, an optimized experimental protocol is reported that utilizes liquid chromatography coupled to electrospray ionization mass spectrometry for the detection of peptides in the venom of the social wasp Polybia paulista; peptide profiles for both intra- and inter-colonial comparisons were obtained using this protocol. The results of our study revealed a surprisingly high level of intra- and inter-colonial variability for the same wasp species. We detected 78–108 different peptides in the venom of different colonies of P. paulista in the molar mass range from 400 to 3000 Da; among those, only 36 and 44 common peptides were observed in the inter- and intra-colony comparisons, respectively.     

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.     

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.