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.     

Effects of the venom of Philanthus triangulum F. (Hym. sphecidae) and β- and δ-philanthotoxin on axonal excitability and synaptic transmission in the cockroach CNS

This paper provides answers to the questions which of the toxins present in the venom of the wasp Philanthus triangulum may be responsible for the previously reported blockage of transmission through the sixth abdominal ganglion of the cockroach, and whether this may occur by block of synaptic transmission or by affecting axonal exitability. In current clamp experiments the crude venom induces a slight depolarization of the membrane of the giant axon from the sixth abdominal ganglion of the cockroach and a small and irreversible decrease in the amplitude of the action potential. These marginal effects are not seen with relatively high concentrations of the philanthotoxins β-PTX and δ-PTX. It appears that neither the crude venom nor the toxins significantly affect the excitability of the cockroach giant axon. At a concentration of 20 μg ml^?1 δ-PTX causes a slowly reversible block of synaptic transmission from the cercal nerve XI to a giant interneuron without any change in resting membrane potential, whereas β-PTX is inactive. Iontophoretically evoked acetylcholine potentials of the giant neuron are more sensitive to δ-PTX than excitatory postsynaptic potentials. This suggests that the toxin acts on the postsynaptic membrane.     

The venom of Ampulex compressa--effects on behaviour and synaptic transmission of cockroaches

1. The solitary wasp Ampulex compressa stings a cockroach, Periplaneta americana, twice. 2. The first sting into the ventral thorax results in a transient paralysis. During this paralysis the wasp stings the suboesophageal ganglion, which gradually results in a permanent deactivation. 3. The venom gland is a paired and highly branched organ, with a common ductus venatus. The large lumen is lined with a folded cuticula. No venom reservoir is present. 4. Extract of the venom gland induces a slow contraction of the guinea pig ileum. 5. The agonist present in the venom cannot be identified with a known agonist. 6. Venom gland extract blocks synaptic transmission from the cercal nerve to giant neurons in the sixth abdominal ganglion of the cockroach. 7. The block develops gradually, like the gradual appearance of the effects of the sting into the suboesophageal ganglion on the behaviour of the cockroach.     

Pharmacological characterization and chemical fractionation of the venom of the ponerine ant, Paraponera clavata (F.).

1. The neurotoxic action of the venom of the ponerine ant, Paraponera clavata, was studied using a cascade of mammalian smooth muscle preparations and a preparation for investigating transmission from fibres of the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach. 2. The venom contains three toxic fractions that block synaptic transmission in the insect central nervous system. 3. Two of these fractions have agonistic action on mammalian smooth muscle preparations. 4. One of the later fractions was characterized pharmacologically as containing a kinin. 5. The other, and most active neurotoxic fraction, was rechromatographed, resulting in the purification of a peptide of 25 amino acids residues, called poneratoxin, PoTX: Phe-Leu-Pro-Leu-Leu-Ile-Leu-Gly-Ser-Leu-Leu-Met-Thr-Pro-Pro-Val-Ile-Gln- Ala-Ile-His-Asp-Ala-Gln-Arg-HN2.     

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.     

Threonine6-bradykinin in the venom of the wasp Colpa interrupta (F.) presynaptically blocks nicotinic synaptic transmission in the insect CNS

1. The venom of the solitary scoliid wasp Colpa interrupta (F.) shows a kinin-activity, when tested on a cascade of mammalian smooth muscle preparations, and, in addition, a contraction of the rat colon. 2. The venom also irreversibly blocks the nicotinic synaptic transmission from the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach, Periplaneta americana. 3. The same activities have been found within one HPLC fraction. 4. However, rechromatography of this fraction resulted in four subfractions being active on smooth muscles. 5. One fraction caused contraction of the colon, three other fractions contained kinin-activity. 6. Only the most active kinin fraction blocked synaptic transmission in the insect CNS. 7. This fraction contained threonine-bradykinin. 8. Synthetic Thr-bradykinin causes irreversible presynaptic activation-induced block of transmission in the insect CNS.     

Wasp uses venom cocktail to manipulate the behavior of its cockroach prey

The sting of the parasitoid wasp Ampulex compressa is unusual, as it induces a transient paralysis of the front legs followed by grooming behavior and then by a long-term hypokinesia of its cockroach prey. Because the wasp's goal is to provide a living meal for its newborn larva, the behavioral changes in the prey are brought about by manipulating the host behavior in a way beneficial to the wasp and its offspring. To this end, the wasp injects its venom cocktail with two consecutive stings directly into the host's central nervous system. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis is due to the presence of a venom component that induces a postsynaptic block of central cholinergic synaptic transmission. Following the head sting, dopamine identified in the venom appears to induce 30 min of intense grooming. During the long-term hypokinesia that follows the grooming, specific behaviors of the prey are inhibited while others are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to the thoracic neurons.Abbreviations CNS central nervous system - DA dopamine - GI giant interneuron - PSP postsynaptic potential - SEG sub-esophageal ganglion - TI thoracic interneuron     

The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine.

1. Methods for presenting dose-response data for the ganglionic actions of cholinergic agonists (e.g. carbamylcholine) are compared, using the mannitol-gap technique for electrophysiological recording of synaptic events at the cercal nerve, giant fibre synapse of the sixth abdominal ganglion of the cockroach Periplaneta americana. 2. At concentrations around 10(-5)M, carbamylcholine has no effect on ganglionic polarization but potentiates the monosynaptic EPSP. At 10(-4)M and higher concentrations, ganglionic depolarization is accompanied by a reduction of EPSP. 3. Pretreatment with eserine (10(-6) M) considerably shifts the dose-response curve for acetylcholine so that synaptic transmission is consistently sensitive to 10(-6) M acetylcholine.     

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.     

Actions of amantadine at synaptic and extrasynaptic cholinergic receptors in the central nervous system of the cockroach Periplaneta americana

The effects of amantadine were investigated on cercal afferent, giant interneurone synapses and on the cell body membrane of the fast coxal depressor motoneurone (D_f), in the cockroach Periplaneta americana. Bath-applied amantadine at concentrations above 2.0 × 10^?5 M significantly reduced the amplitude of unitary and compound epsps recorded by sucrose-gap methods from cercal afferent, giant interneurone synapses in the desheathed sixth abdominal ganglion. Complete block of synaptic transmission was achieved at 1.0 × 10^?3 M amantadine. Synaptic blockade, which was not accompanied by changes in resting potential, was almost fully reversed by washing the ganglion in normal saline. From the dose-dependence of the synaptic blocking action, a Hill coefficient of 0.94 was estimated, indicating that there is no co-operativity in the binding of amantadine to its site of action.Bath-application of amantadine (5.0 × 10^?5 M) resulted in a parallel shift to the right of the dose-response curve for the depolarizing postsynaptic actions of acetylcholine. Nevertheless, even at a concentration of 2.0 × 10^?3 M, amantadine failed to protect the synaptic acetylcholine receptor/ion channel complex from the blocking action of α-bungarotoxin (5.0 × 10^?7 M). In addition, the block by amantadine of the acetylcholine-induced current recorded from the cell body membrane of the fast coxal depressor motoneurone (D_f), was strongly dependent on membrane potential in the range ? 120mV to ? 70mV. An action of amantadine at the open acetylcholine receptor/ion channel complex is proposed.     

Block of synaptic transmission in insect CNS by toxins from the venom of the wasp Megascolia flavifrons (Fab.)

1. The effects of the venom and its fractions of Megascolia flavifrons have been studied on synaptic transmission and axonal excitability of the giant interneuron of the cockroach. 2. The venom does not affect axonal excitability, but blocks synaptic transmission, and induces postsynaptic depolarization with a delay. 3. Five different active fractions have been recognized. 4. Three fractions of them contain substances already identified as histamine, Thr6 bradykinin and Thr6 bradykinin-Lys-Ala (megascoliakinin). 5. Three fractions contain activities, which have not yet been chemically identified. 6. All of them, and also bradykinin block synaptic transmission; histamine was not active.     

Mode of action of atracotoxin at central and peripheral synapses of insects

These studies characterized the paralytic and neurophysiological effects of an atracotoxin (ACTX), from the Australian funnel web spider, Hadronyche versuta, and compared it to the established P/Q-type calcium channel blocker, -agatoxin-IVA (-Aga-IVA). ACTX-induced paralysis was of a spastic form in housefly (Musca domestica) larvae, but it was inactive on neuromuscular junction of housefly and tobacco budworm (Heliothis virescens). On cockroach (Periplaneta americana) cercal nerve–giant fiber synapse preparations, both toxins were effective blockers with potencies in the nanomolar range, but some spontaneous, high frequency trains of action potentials were observed with ACTX. In Drosophila melanogaster central nervous preparations, blockage of nerve firing occurred within 20 min when the nerve sheath was intact, demonstrating that the barrier could be breached by ACTX in vitro. There was a potent (pM) excitatory response to ACTX in this tissue, prior to the onset of block at higher concentrations. In contrast, -Aga-IVA was a pure blocker in both cockroach and Drosophila preparations. These studies demonstrate that central synaptic calcium channels underlie the action of ACTX. ACTX-dependent neuroexcitation has a number of possible mechanisms that warrant further study.     

Some observations on the fine structure of the giant synapse in the stellate ganglk of the squid,Doryteuphis bleekeri

Summary The fine structure of the synapse between the second-order giant fibre and the third order-giant fibre of the squid Doryteuphis bleekeri was studied by means of electron microscope. In the synaptic region, the two giant fibres are arranged side by side. Many small processes from the third-order giant fibre penetrate the common sheath which separats the adjacent giant axons making synaptic contact with the second order giant axon.The contact surface consists of opposing two plasma membranes of adjacent axons separated by a narrow space of 20–30 m in width. The synaptic membranes are more electron dense and thicker than the other part of the axon membrane. The synaptic vesicles are concentrated exclusively in the presynaptic axon.The fine structural differences between giant synapse in the stellate ganglion of the squid and the giant-to-motor giant synapse of the crayfish were discussed.This work was supported by Grant Number B-3348 from the National Institutes of Health, United States Public Health Service, Department of Health, Education and Welfare.     

Actions of snake neurotoxins on an insect nicotinic cholinergic synapse

Here we examine the actions of six snake neurotoxins (α-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneurone 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are senstive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes.     

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.     

马氏钳蝎毒昆虫类神经毒素的分离、纯化及性质研究

经Sephadex G-50,sp-Sephadex C-25二步柱层析法,从山东马氏钳蝎(Bu-thus martensii Karch)粗毒中分离出四种对美洲(虫非)蠊有强直麻痹反应的毒性蛋白组份。其中二个组分在SDS聚丙烯酰胺电泳和等电聚焦电泳上均呈现单一区带,命名为BmK IT-Ⅰ,BmK IT-Ⅱ其pI分别为8.2和8.4,分子量分别为8400和7560。同时还分析了二个组份的氨基酸组成。经DABITC/PITC双偶合法测定了BmKIT-Ⅰ和BmK IT-Ⅱ的N端部份氨基酸排列顺序,它们分别为H_2NVal.Arg.Asp.Ala……H_2NVal.Arg.Asp.Gly……。 电生理学研究表明,纯化的BmK IT-I(1×10~(-5)g/ml)对(虫非)蠊腹Ⅵ神经节的突触传递有阻断作用,阻断后用生理溶液洗,则突触传递可恢复。从同一蝎毒粗毒中分离纯化的哺乳动物类神经毒素BmKⅢ在浓度高出100倍(1×10~(-3)g/ml)时也可以阻断(虫非)蠊腹Ⅵ神经节的突触传递,但用生理溶液冲洗没有观察到恢复。     

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 μM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 μM acetamiprid and imidacloprid had a strong effect. 1 and 10 μM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.     

Morphology of soma and dendrites of the giant fiber system in the sixth abdominal ganglion of the cockroach

This study, using the cobalt chloride technique, clarifies the origin of the giant axons in the cockroach, Periplaneta. Each giant axon in the ventral nerve cord arises from a single cell body located in the sixth abdominal ganglion. The position of the soma is always contralateral to the giant axon; it projects anteriorly. In six giant neurons, the axonic and dendritic branches are ipsilateral while the somata are contralateral. In two neurons, both the soma and the dendritic branches are ipsilateral while the axons are contralateral. The dendritic arborizations of the giant neurons form a dense and compact mass of neuropile in each half of the posterior and middorsal part of the ganglion where sensory fibers, primarily from the cercal nerves terminate. The relation of these findings to earlier electrophysiological studies is discussed.     

Wasp manipulates cockroach behavior by injecting venom cocktail into prey central nervous system

The parasitoid wasp Ampulex compressa induces behavioral changes in the cockroach prey by injecting venom into its central nervous system. In contrast to most other venomous predators, the wasp's sting does not induce paralysis. Rather, the two consecutive stings in the thoracic and head ganglia induce three stereotypic behavioral effects. The prey behavior is manipulated in a way beneficial to the wasp and its offspring by providing a living meal for its newborn larva. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis prevents the cockroach from fighting with its front legs, thereby facilitating the second sting in the head. A postsynaptic block of central synaptic transmission mediates this leg paralysis. Following the head sting, dopamine identified in the venom induces 30 minutes of intense grooming that appears to prevent the cockroach from straying until the last and third behavioral effect of hypokinesia commences. In this lethargic state that lasts about three weeks, the cockroach does not respond to various stimuli nor does it initiates movement. However, other specific behaviors of the prey are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to specific thoracic neurons.     

Localization of the site of effect of a wasp's venom in the cockroach escape circuitry

The parasitic wasp Ampulex compressa stings a cockroach Periplaneta americana in the neck, toward the head ganglia (the brain and subesophageal ganglion). In the present study, our aim was to identify the head ganglion that is the target of the venom and the mechanisms by which the venom blocks the thoracic portion of the escape neuronal circuitry. Because the escape responses elicited by a wind stimulus in brainless and sham-operated animals were similar, we propose that the venom effect is on the subesophageal ganglion. Apparently, the subesophageal ganglion modulates the thoracic portion of the escape circuit. Recordings of thoracic interneuron responses to the input from the abdominal giant interneurons showed that the thoracic interneurons receive synaptic drive from these interneurons in control and in stung animals. Unlike normal cockroaches, which use both fast and slow motoneurons for producing rapid escape movements, stung animals activate only the slow motoneuron. However, we show that in stung animals, the fast motoneuron still can be recruited with bath application of pilocarpine, a muscarinic agonist. These results indicate that the descending control from the subesophageal ganglion is presumably exerted on the premotor thoracic interneurons to motoneurons connection of the thoracic escape circuitry. Accepted: 19 December 1998