Molecular cloning and characterization of acid phosphatase in venom of the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The present study describes cDNA cloning, sequencing, enzyme activity determination and localization, and mRNA expression of acid phosphatase in the venom apparatus of an endoparasitoid, Pteromalus puparum. This is the first report of cloning a venom acid phosphatase gene which has been described in parasitoid wasps. The cDNA consisted of 1378bp with 1215bp open reading frame and encoded a sequence of 405 amino acids. A 23 residues N-terminal signal peptide was followed by a short 15 residues (25-39) histidine acid phosphatases phosphohistidine signature and a long 302 residues (24-325) acid phosphatase family domain. The deduced amino acid sequence shared 32-88% identity to its counterparts from other insects. Enzyme activity was measured by using p-nitrophenyl phosphate (p-NPP) as substrate, and a high level of acid phosphatase activity in venom was detected. Optimal pH and temperature for this enzyme activity was 4.8 and 45 degrees C, respectively. Ultracytochemical analyses further revealed that strong enzyme activity was located in the nuclei and secretory vesicles of the venom gland secretory cells. Expression of the acid phosphatase gene was observed to be regulated at different developmental stages by RT-PCR analysis as it expressed immediately with low abundance after adult emergence, then increased to the high level at 2-4 days, followed by a drop to the low abundance after 4 days. Compared to the mRNA expression, a time-course-related enzyme activity in an individual venom apparatus was also found.     

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.     

Purification, sequencing and structural characterization of the phospholipase A1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae).

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.     

Patterns of venom synthesis and use in the fire ant, Solenopsis invicta.

In the life of the fire ant, Solenopsis invicta, venom plays several important roles, including prey capture, defense, and anti-microbial action. Although this venom's chemistry, pharmacology, and effects on humans have been extensively studied, its patterns of synthesis and use have not. We determined the ability of different-aged workers to synthesize venom, and measured the amount of venom workers delivered per sting. Newly eclosed workers contained little or no venom in their reservoirs. The rate of synthesis was highest in workers aged 1 day after adult eclosion (1.17 microg venom/day for workers with headwidths of 1 mm), declined by 75% in workers aged 15 days (0.30 microg venom/day), and was negligible thereafter. Inducing ants to sting dummies as surrogates for humans, workers were found to deliver an average of 0.66 nl per sting, amounting to 3.1% of the average individual venom supply. Older workers (foragers) delivered less venom per sting than mid-age workers (reserves) and venom dose from nest-defenders was 55% higher in the spring than in the rest of the year. Thus, fire ant venom synthesis is limited to early life, and injected venom dose appears to be modulated. Economic explanations for the observed venom-use patterns are discussed.     

Biochemistry, toxicology and ecology of the venom of the spider Cupiennius salei (Ctenidae).

The venom of Cupiennius salei consists of many low molecular compounds, nine neurotoxic acting peptides (CSTX), at least eight neurotoxic and cytolytic acting peptides (cupiennins), a highly active hyaluronidase, and several hitherto unidentified proteins. The structure of several peptides is given. A synergistic action between three main groups is proposed: injected into the prey tissue, the enzyme hyaluronidase acts as a spreading factor, thus, facilitating a better access of venom neurotoxins to their targets, cupiennins disturb cell membranes and influence cell excitability, through this augmenting the mere neurotoxic effect of CSTX-1 synergistically. The venom glands of an apocrine secretion type provide an average of 12 microl per milking (adult female). Venom sensitivity of arthropods differs between 0.001 and >20nl venom/mg insect. Regeneration time of an empty venom gland is approx. 2 weeks. Consequently, spiders may encounter situations in which they have to decide whether their limited venom storage is sufficient to kill a given prey item. Experiments are presented which show that C. salei knows the actual venom content of its venom glands. It injects no more venom than necessary. This coincides with an experimentally determined LD(50) value in harmless prey items, but C. salei injects more venom in aggressive or otherwise dangerous prey items (quantification of injected venom amounts by monoclonal antibodies). These results indicate that C. salei uses its venom as economically as possible and this supports our venom optimisation hypothesis.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom

Abstract: Eumenine mastoparan‐AF (EMP‐AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP‐AF peptide presented low cytolytic activities in human erythrocytes and in RBL‐2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad‐spectrum inhibitory activity against Gram‐positive and Gram‐negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C‐terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N‐terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.     

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.     

Microvesicles in the venom of Crotalus durissus terrificus (Serpentes, Viperidae).

Microvesicles with electron-dense content are consistently observed by transmission electron microscopy on the luminal face of secretory cells of venom glands of viperid snakes. In this work, we evaluated their presence in Crotalus durissus terrificus venom glands and also in freshly collected venom. Microvesicles were found in the venom glands mainly in regions of exocytosis. They ranged from 40 to 80 nm in diameter. Freeze-fracture replicas of the glands revealed particles on the cytoplasmic leaflet (P-face) of these vesicles, suggesting that they carry transmembrane proteins. Vesicles separated by ultracentrifugation from cell-free venom were similar in size and structure to the microvesicles observed in the glands. A fine fuzzy coat surrounded each microvesicle. The function of these venom vesicles is still unknown, but they may contribute to inactivation of stored venom components, or their activation after the venom is released.     

On the venom system of centipedes (Chilopoda), a neglected group of venomous animals

Centipedes are among the oldest extant terrestrial arthropods and are an ecologically important group of soil and leaf litter predators. Despite their abundance and frequent, often painful, encounters with humans, little is known about the venom and venom apparatus of centipedes, although it is apparent that these are both quite different from other venomous lineages. The venom gland can be regarded as an invaginated cuticle and epidermis, consisting of numerous epithelial secretory units each with its own unique valve-like excretory system. The venom contains several different enzymes, but is strikingly different to most other arthropods in that metalloproteases appear to be important. Myotoxic, cardiotoxic, and neurotoxic activities have been described, most of which have been attributed to high molecular weight proteins. Neurotoxic activities are also unusual in that G-protein coupled receptors often seem to be involved, either directly as targets of neurotoxins or indirectly by activating endogenous agonists. These relatively slow responses may be complemented by the rapid effects caused by histamines present in the venom and from endogenous release of histamines induced by venom cytotoxins. The differences probably reflect the ancient and independent evolutionary history of the centipede venom system, although they may also be somewhat exaggerated by the paucity of information available on this largely neglected group.     

Sexual dimorphism in venom of Bothrops jararaca(Serpentes: Viperidae).

Bothrops jararaca is an abundant snake in Brazil, and its venom has been studied exhaustively. The species exhibits adult size dimorphism in which female are larger. We registered the growth in Snout-Vent Length and weight of one litter (with 11 females and 12 males). We compared growth curves and venom profile between male and female of B. jararaca in order to establish the relationship of those characters and sex. Their venoms were analyzed when they were 36 months old, concerning SDS PAGE, protein content, proteolytic, hyaluronidasic, phospholipasic, blood-clotting, edematogenic, hemorrhagic, myotoxic activities, and lethality. Differences in the growth curves of the females and the males were significantly different after the 12th month of age, with the females growing faster. Females produced five times more venom than males. The electrophoretic patterns were variable: the venom from males had more protein bands than females. Venom composition varied significantly between males and females. Venom from females is more potent for hyaluronidasic, hemorrhagic, and lethality activities, whereas venom from males is more potent for coagulant, phospholipasic, and myotoxic activities. The variability of proteolytic and edematogenic activities were not significant. The important sexual dimorphism in body size and mass, amount of venom produced, and venom composition in B. jararaca may reflect a divergence in niche partitioning.     

A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith).

Wasp is an important venomous animal that can induce human fatalities. Aortic thrombosis and cerebral infarction are major clinical symptoms after massive wasp stings but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnifin, contains phospholipase-like activity and induces platelet aggregation. The cDNA encoding magnifin is cloned from the venom sac cDNA library of the wasp. The predicted protein was deduced from the cDNA with a sequence composed of 337 amino acid residues. Magnifin is very similar to other phospholipase A(1) (PLA(1)), especially to other wasp allergen PLA(1). Magnifin can activate platelet aggregation and induce thrombosis in vivo. The current results proved that PLA(1) in wasp venom could be contributable to aortic thrombosis after massive wasp stings.     

Digestive properties of the venom of the Australian Coastal Taipan, Oxyuranus scutellatus (Peters, 1867).

The digestive properties of Australian elapid snake venoms have not been studied to any great extent. To address this, the in vitro digestive properties of Oxyuranus scutellatus (Australian Coastal Taipan) venom were investigated in a simulation of the in vivo conditions using the parameters reported for the stomach of snakes and representative prey for this species. The amount of soluble protein released was measured over time using a bicinchoninic acid (BCA) assay. Dismembered mouse hindlegs were injected intramuscularly with 0.1 ml O. scutellatus venom (concentration 10 mg/ml) and maintained in a micro-anaerobic, acidic environment (pH approximately 1.2-1.7) at 25 degrees C. The bathing liquid was sampled every 24 h for 7 days, and assayed for soluble protein. Statistical analysis revealed that O. scutellatus venom increased the rate at which proteins were released when compared to a negative control suggesting the potential importance of envenomation in the digestion of whole prey.     

A portable device for the electrical extraction of scorpion venom

An attractive technique to extract scorpion venom is the use of a physiologically stimulating electrical signal across the muscles of the venom gland, resulting in the expression of venom from the aculeus. A Grass™ stimulator is typically used for this purpose, but is difficult to use in the field. The present communication describes a circuit which is battery-powered and simply constructed. Also described is the technique for its construction and housing. The circuit was successfully tested on two species of scorpion. The method for calculating the required values of passive circuit components is given to allow the adaptation and refinement of this circuit for producing different signals, as may be required for use in other species.     

Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae)

The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 ± 0.7 mg of liquid/spider, n = 16; mean ± S.E.M.) than female spiders (25.5 ± 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 ± 0.16 mg/g vs. 2.45 ± 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to ∼12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 ± 2.4 mg/ml (n = 4) and accounted for 16.6 ± 4.7% of the dry venom weight. The venom contained high hyaluronidase activity (275 ± 24 TRU/mg of protein, n = 4), with a molecular mass of ∼45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.     

Agelotoxin: a phospholipase A(2) from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae).

The neotropical wasp Agelaia pallipes pallipes is aggressive and endemic in southeast of Brazil, where very often it causes stinging accidents in rural areas. By using gel filtration on Sephadex G-100, followed by high performance reversed phase chromatography in a C-18 column under acetonitrile/water gradient, the agelotoxin was purified: a toxin presenting phospholipase A(2) (PLA(2)) activity, which occurs under equilibrium of three different aggregation states: monomer (mol. wt 14 kDa), trimer (mol. wt 42 kDa) and pentamer (mol. wt 74 kDa).The enzyme presents high sugar contents attached to the protein chain (22% [w/w]) and a transition of the values of pH optimum for the substrate hydrolysis from 7.5 to 9.0, under aggregation from monomer to pentamer. All the aggregation states present Michaelian steady-state kinetic behavior and the monomer polymerization caused a decreasing of phospholipasic activity due a non-competitive inhibition promoted by the formation of a quaternary structure. The PLA(2) catalytic activity of agelotoxin changes according to its state of aggregation (from 833 to 12533 micromol mg(-1) min(-1)) and both the monomeric and oligomeric forms present lowest activities than the PLA(2) from Apis mellifera venom and hornetin from Vespa basalis. Agelotoxin is also a very potent direct hemolysin; the monomer of agelotoxin presented hemolytic actions until 200 times higher than the PbTx from P. paulista, 740 times higher than the PLA(2) from A. mellifera, 570 times higher than that of neutral PLA(2) from N. nigricolis and about 1250 times than that of cardiotoxin from Naja naja atra venom.     

Identification, expression, and immuno-reactivity of Sol i 2 & Sol i 4 venom proteins of queen red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae)

We report on two low-molecular weight proteins that are stored in the venom of queen red imported fire ants (Solenopsis invicta). Translated amino acid sequences identified one protein to have 74.8% identity with the Sol i 2w worker allergen, and the other protein was found to have 96/97% identity with Sol i 4.01w/4.02w worker allergens. Both Sol i 2 and Sol i 4 queen and worker proteins were expressed using pEXP1-DEST vector in SHuffle™ T7 Express lysY Escherichia coli. Proteins were expressed at significant concentrations, as opposed to the μg/ml amounts by our previous expression methods, enabling further study of these proteins. Sol i 2q protein bound weakly to human IgE, sera pooled from allergic patients, whereas Sol i 2w, Sol i 4.01w, and Sol i 4q proteins bound strongly. Despite Sol i 2w and Sol i 2q proteins having 74.8% identity, the queen protein is less immuno-reactive than the worker allergen. This finding is consistent with allergic individuals being less sensitive to queen than worker venom.     

Characterization of peptide components in the venom of the scorpion Liocheles australasiae (Hemiscorpiidae).

Scorpion venoms are composed of a number of neurotoxic peptides. A variety of toxins have been isolated from the venoms of scorpions of the family Buthidae, however, little interest has been paid to non-Buthidae scorpions. In this study, we examined the toxicity of the venom of Liocheles australasiae (Hemiscorpiidae) to mice and crickets, and characterized the peptide components by HPLC and mass spectrometry. Over 200 components were detected in the L. australasiae venom by LC/MS analysis, with components of molecular masses ranging from 500 to 5000 Da being particularly abundant. A number of peptides contained two to four disulfide bridges, which was estimated based on the mass difference after derivatization of Cys residues. A peptide having a monoisotopic molecular mass of 7781.6 Da and four disulfide bridges was isolated from the venom. The peptide has a primary structure similar in terms of the position of eight Cys residues to those observed in several peptides found from scorpions, ticks and insects, although biological roles of these peptides are unknown.     

Presynaptic neuromuscular activity of venom from the brown-headed snake (Glyphodon tristis).

The brown-headed snake (Glyphodon tristis) inhabits the forest regions of Papua New Guinea, Torres Strait Islands, and far northern Queensland, Australia. Although bites by Glyphodon dunmalli have been reported, G. tristis was regarded as innocuous until 1989 when a healthy 20 year old man was bitten (Sutherland, S.K., Tibballs, J., 2001. Australian Animal Toxins, the Creatures, their Toxins and Care of the Poisoned Patient. University Press, Oxford). Treatment of envenomation by this species is empirical with no specific antivenom available. While no published studies on the venom of G. tristis are available, unpublished studies suggest neurotoxicity as being the main symptom of envenomation. In this study, the in vitro effects of G. tristis venom were examined using the chick biventer cervicis nerve muscle (CBCNM) preparation. Venom (10 microg/ml) inhibited indirect (0.2 ms, 0.1 Hz, supramaximal V) twitches of the CBCNM. This inhibition appeared to be presynaptic in origin as evidenced by the lack of effect of venom on responses to exogenous acetylcholine (1 mM), carbachol (20 microM) and KCl (40 mM) in the non-stimulated CBCNM. Prior addition (10 min) of polyvalent snake antivenom (5 U/ml; CSL Ltd) attenuated twitch inhibition. The venom (10-50 microg/ml) also appears to be myotoxic as indicated by a slowly developing contracture and inhibition of direct (2 ms, 0.1 Hz, supramaximal V, in the presence of tubocurarine 10 microM) twitches. Myotoxicity was confirmed by subsequent histological examination of tissues. This myotoxicity was prevented by the prior addition of polyvalent snake antivenom (30 U/ml). The phospholipase A inhibitor 4-BPB (1.8 mM) significantly attenuated the inhibition of indirect and direct twitches of the CBCNM preparation, indicating the involvement of a PLA2 component in the toxic action of the venom.