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Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.

Analysis of Variance↗

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.

Acetone↗

Characterization of the actions of AvTx 7 isolated from Agelaia vicina (Hymenoptera: Vespidae) wasp venom on synaptosomal glutamate uptake and release.

It has previously been shown that the denatured crude extract of Agelaia vicina wasp venom inhibits glutamate and GABA uptake in rat cerebral cortex synaptosomes. To identify the components responsible for these effects, the neurotoxin AvTx 7 (molecular weight of 1210 Da) was isolated from A. vicina venom and its effects on glutamate neurotransmission investigated. AvTx 7 inhibits glutamate uptake in a dose-dependent and uncompetitive manner. AvTx 7 was found to stimulate the glutamate release in the presence of calcium and sodium channel blockers, suggesting that its action is not mediated through these channels. AvTx 7 potentiates glutamate release in the presence of K(+) channel blockers tetraethylammonium and 4-aminopyridine, indicating that the toxin may act through these drugs-sensible K(+) channels. We suggest that AvTx 7 can be a valuable tool to enhance our understanding of K(+) channels' involvement in the release of glutamate.

Animals↗

Venom apparatus of the endoparasitoid wasp Opius caricivorae Fischer (Hymenoptera: Braconidae): morphology and ultrastructure.

The morphology and ultrastructure of the venom apparatus of the endoparasitoid wasp, Opius caricivorae Fischer (Hymenoptera: Braconidae), were observed using light and electron microscopes. The venom apparatus consists of one venom reservoir and several gland filaments. The gland filaments join together at the end of the reservoir and consist of an outer single layer of secretory cells, a layer of degenerated epidermal cells, and an inner intima that encloses the lumen. The secretory cells are organelle rich, with abundant rough endoplasmic reticulum, mitochondria, and vacuole, in which vesicular organelles secrete the components of venom. The reservoir consists of a muscular sheath, secretory cells, and squamous cells. The intima has an unevenly thickened chitinous coat. The vesicular organelles of the reservoir secretory cells differ from those of the gland filament: the microvilli being much longer and radiating in all directions. The venom reservoir not only serves to store but also secretes the venom. Virus-like particles were discovered in the secretory cells of the gland filaments. The structural features of venom apparatus of this species are discussed in a biological context.

Animals↗

Comparison of the medium molecular weight venom fractions from five species of common social wasps by MALDI-TOF spectra profiling.

The average spectral profiles and the exact mass weight (MW) of biomolecules present in the medium fraction (from 900 to 3000 Da) of the venom of five social wasps (three European and one North American Polistes and the European hornet Vespa crabro) were determined by matrix assisted laser desorption ionization time of flight (MALDI-TOF) MS. Data were obtained analyzing the venom of single specimens (N = 46) and elaborated with the ClinProTools 2.0 (CPT) software to search for differences among the five species examined. Interesting differences in the spectral profiles were found, allowing the discrimination of venoms belonging to the different species, and their possible use as a quality control method in venom immunotherapy (VIT) for allergic patients.

Animals↗

Protolytic properties of polyamine wasp toxin analogues studied by 13C NMR spectroscopy.

Acid-base properties of the natural polyamine wasp toxin PhTX-433 (1) and seven synthetic analogues [PhTX-343 (2), PhTX-334 (3), PhTX-443 (4), PhTX-434 (5), PhTX-344 (6), PhTX-444 (7), and PhTX-333 (8)], each having four protolytic sites, were characterized by 13C NMR spectroscopy. Nonlinear, multiparameter, simultaneous fit of all chemical shift data obtained from the NMR titration curves yielded macroscopic pKa values as well as intrinsic chemical shift data of all differently protonated macrospecies. Analyses of the chemical shift data demonstrated strong interactions between all four sites and provided information about complex relationships between chemical shift values and protonation state. Deprotonation of fully protonated forms starts at the central amino group of the polyamine moiety, and the extent of this trend depends on the distance to the flanking, protonated amino groups. The pKa1 values of 1-8 are in the range 8.2-9.4. Hence, some of the toxins are incompletely protonated at the pH and ionic strength conditions used for assessment of their interactions with ionotropic glutamate and nicotinic acetylcholine receptors, and the degree of protonation is expected to have pharmacological importance in the ion-channel binding event.

Animals↗

Digger wasp versus cricket: mechanisms underlying the total paralysis caused by the predator's venom.

The data presented here describe neurophysiological experiments addressing the question of cellular mechanisms underlying the total paralysis of locomotor behavior in crickets occurring after being stung by females of the digger wasp species Liris niger. The Liris venom effects have been studied by both in vivo recordings from identified neurons of the well-described giant fiber pathway and in vitro recordings from cultured neurons isolated from the terminal ganglion of crickets. The total paralysis of the prey is characterized by a general block of action potential generation as well as by a block of synaptic transmission. Intracellular recordings from neurons in intact ganglia under single electrode voltage-clamp conditions, as well as whole-cell patch-clamp recordings from cultured cricket neurons consistently show that the block of action potential generation by the Liris venom is due to a block of voltage-gated sodium inward currents in neurons of the stung ganglia. Furthermore, our data provide evidence that the Liris venom also blocks calcium currents in identified neurosecretory neurons. On the other hand, outward currents are not affected by the Liris venom. The in vitro recordings suggest that the Liris venom contains active venom components, which, at least for the observed block of inward currents, do not require a metabolic modification. Because venom application does not affect the ACh-induced EPSPs in giant interneurons, the Liris venom does not seem to influence the postsynaptic ACh receptors. The possible pre- and postsynaptic sites of venom action and the functional consequences on synaptic transmission within the giant fiber system are discussed.

Animals↗

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.

Animals↗

Isolation and structure of pompilidotoxins, novel peptide neurotoxins in solitary wasp venoms.

Novel peptide neurotoxins, alpha- and beta-pompilidotoxins (alpha- and beta-PMTXs), were purified from the venoms of the solitary wasps Anoplius samariensis and Batozonellus maculifrons. Their structures were analyzed mostly by MALDI-TOF-MS, which were corroborated by solid-phase synthesis. alpha-PMTX, with 13 amino acid residues and the sequence of Arg-Ile-Lys-Ile-Gly-Leu-Phe-Gln-Asp-Leu-Ser-Lys-Leu-NH2, greatly potentiates synaptic transmission of lobster leg muscle by the presynaptic mechanisms. beta-PMTX, in which the lysine residue at 12 position of alpha-PMTX was replaced with arginine, was more potent than alpha-PMTX.

Animals↗

Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea.

During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom. This complex fluid has toxic properties and recently several venom components were characterized. In addition, it was suggested that venom might be involved in host immune suppression. For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system. Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration. Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro. Injection of 16 microg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined. Hemocyte concentration in sixth instar larvae 32 h after injection with 16 microg of venom was reduced by 56% compared to that in controls. Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P. hypochondriaca venom has cytotoxic properties. In vitro incubation of washed hemocytes for 20 h with 500 ng/microl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact. Treatment with low concentrations of venom (1.6 ng/microl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers. High-resolution microscopy of hemocyte cultures exposed to 320 ng/microl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles. Incubation of hemocytes with 320, 64, or 3.2 ng/microl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes. Treatment with 320 ng/microl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85%. Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.

Animals↗

Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis).

Amino Acid Sequence↗

Are monoaminergic systems involved in the lethargy induced by a parasitoid wasp in the cockroach prey?

The venom of the parasitoid wasp Ampulex compressa induces long-lasting hypokinesia in the cockroach prey. Previous work indicates that the venom acts in the subesophageal ganglion to indirectly affect modulation of thoracic circuits for locomotion. However, the target of the venom in the subesophageal ganglion, and the mechanism by which the venom achieves its effects are as yet unknown. While the stung cockroaches appear generally lethargic, not all behaviors were affected, indicating that the venom targets specific motor systems and not behavior in general. Stung cockroaches were observed "freezing" in abnormal positions. Reserpine, which depletes monoamines, mimics the behavioral effects of the venom. We treated cockroaches with antagonists to dopamine and octopamine receptors, and found that the dopamine system is required for normal escape response. Dopamine injection induces prolonged grooming in normal cockroaches, but not in stung, suggesting that the venom is affecting dopamine receptors, or targets downstream of these receptors, in the subesophageal ganglion. This dopamine blocking effect fades slowly over the course of several weeks, similar to the time course of recovery from hypokinesia. The similarity in the time courses suggests that the mechanism underlying the hypokinesia may be the block of the dopamine receptors.

Adrenergic Uptake Inhibitors↗

Role of lysine residue at 7th position of wasp chemotactic peptides.

Most of chemotactic peptides isolated from various kinds of wasp venom have lysine residue at 7th (or 8th) position, but only a chemotactic peptide from Icaria sp. has no basic amino acid residues in the sequence. The relation of chemotaxis with other biochemical activities such as superoxide generation and lysosomal enzyme release from guinea pig neutrophils was studied by the use of substitution analogs of Icaria chemotactic peptide at 7th position. Findings revealed two distinct ways of chemoattractant signal transduction in neutrophils.

Amino Acid Sequence↗

Venom apparatus of braconid wasps: comparative ultrastructure of reservoirs and gland filaments.

Two types of venom apparatus present in female braconid wasp were examined in nine species and compared ultrastructurally. The reservoir of type 1 venom apparatus has a relatively thick muscular sheath which is innervated, while the longitudinal and circular muscles of the type 2 reservoir consist of scattered fibers which are not innervated. The intima of the reservoir of type 1 venom apparatus is unevenly thickened. In contrast, the reservoir of type 2 venom apparatus has a relatively thinner and more uniform intima. The gland filaments of both types of venom apparatus are ultrastructurally similar. Distinct particles found in the venom apparatus of two of the nine species are described.

Animals↗

Two kinins isolated from an extract of the venom reservoirs of the solitary wasp Megascolia flavifrons.

From an extract of the venom reservoirs of the wasp Megascolia flavifrons two kinins have been isolated. The sequences of amino acids are: Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg (Thr6-bradykinin) and Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-Lys-Ala (Thr6-bradykinin-Lys-Ala). The bradykinin-like effects of the venom on a number of vertebrate smooth muscle preparations can be explained by the actions of these kinins.

Amino Acids↗

Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors.

Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which IC50S were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

Animals↗

Cholinergic antagonists in a solitary wasp venom.

The venom of the solitary wasp Philanthus triangulum contains a cholinergic antagonist of the nicotinic receptor of the rectus abdominis muscle of the frog, Xenopus laevis. The venom of African P. triangulum contains two different cholinergic factors, a competitive and a non-competitive antagonist. The venom of the European P. triangulum may not contain a competitive antagonist of the nicotinic receptor of X. laevis, but only a very strong non-competitive antagonist. The possible non-synonymity of both groups of P. triangulum is discussed.

Acetylcholinesterase↗

The venom of the wasp Campsomeris sexmaculata (F.) blocks synaptic transmission in insect CNS.

1. The action of the venom of the wasp Campsomeris sexmaculata on the insect CNS has been studied using the cercal nerve-giant interneuron preparation of the sixth abdominal ganglion of the cockroach. 2. The venom blocks synaptic transmission either transiently (at low concentration) or for a long time (at higher concentration), and causes a permanent depolarization of the neuron with a delay. 3. The venom does not affect directly the axonal excitability.

Animals↗