T. Piek

Myogenic contractions in locust muscle induced by proctolin and by wasp, Philanthus triangulum, venom

Abstract Apparently myogenic slow contractions of the extensor tibiae of Locusta migratoria can be induced by proctolin in a concentration of 10 −10 to 10 −9 mole per liter perfusion fluid. Proctolin in a concentration of 10 −8 mole/l causes a prolonged contraction interrupted by rhythmical relaxations. Higher concentrations of proctolin cause a powerful but irreversible contraction. In some preparations in which proctolin is ineffective in a concentration of 10 −9 mole/l, a short stimulation of nerve 3b can initiate a series of rhythmic contractions. If, however, nerve 3b is stimulated at the peak of such a contraction a rapid relaxation is induced. Administration of the venom of Philanthus triangulum in a concentration which blocks the excitatory and inhibitory neuromuscular transmission, induces similar myogenic contractions. Stimulation of nerve 3b at the peak of such a contraction again causes a relaxation. Similar myogenic contractions can also be induced by administration of a homogenate of a locust.

Neurotoxic kinins from wasp and ant venoms

Kinins are polypeptides of 9-18 amino-acid residues containing a bradykinin-like sequence, in some cases as part of a molecule. The bradykinin-like sequence is either bradykinin, Hyp3-bradykinin or Thr6-bradykinin. Kinins are neurotoxic components of wasp and ant venoms, causing in the insect CNS a presynaptic block of the cholinergic transmission by means of an irreversible depletion, probably caused by a non-competitive inhibition of choline uptake.

δ-philanthotoxin, a semi-irreversible blocker of ion-channels

1. The digger wasp, Philanthus triangulum, which preys on honeybees, produces a paralysing venom possessing a wide variety of activities. 2. In insects, the venom has a central as well as a peripheral effect; the latter effect consists of a presynaptic as well as a postsynaptic block of the skeletal neuromuscular transmission. 3. The presynaptic block is probably caused by an inhibition of the re-uptake of the transmitter. The postsynaptic effect probably consists of a block of open ion channels. 4. The venom contains at least four active toxins called alpha-, beta-, gamma- and delta-philanthotoxin (PTX). alpha-PTX blocks transmission in the cockroach CNS. The other three toxins block neuromuscular transmission. delta-PTX being the most active toxin in blocking glutamate evoked postsynaptic depolarizations. 5. In the junctional, as well as in the extrajunctional, muscle fibre membrane delta-PTX blocks ion channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be semi-irreversible when agonist activation is low (spontaneous release of transmitter and/or leak of glutamate from the pipette). 6. The time constant of blocking is roughly estimated to be in the order of 10 msec, that of unblocking seems to be several hundreds of msec.

Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.

Neurotoxins from venoms of the hymenoptera—Twenty-five years of research in Amsterdam

1. In co-operation with colleagues in Europe, Japan and the U.S.A., 25 years of research in Amsterdam have provided new views on the way some hymenopteran insects incapacitate their prey by a diversity of neurotoxins, resulting in block of synaptic transmission in CNS or neuromuscular junctions, or affecting voltage dependent phenomena in nerve and muscle fibers. 2. Nicotinic synaptic transmission in the insect CNS is irreversibly blocked at the presynaptic side by kinins, or reversibly and postsynaptically blocked by philanthotoxins. 3. Glutamatergic neuromuscular transmission is reversibly blocked by philanthotoxins at the pre- and/or postsynaptic side. 4. A presynaptic block of neuromuscular transmission was found with the Microbracon toxins. 5. An irreversible deactivation, without paralysis, of cockroaches is caused by a sting of Ampulex compressa into the suboesophageal ganglion. 6. Poneratoxin, a 25 amino acid residue polypeptide, isolated from an ant venom, is the first described hymenopteran neurotoxin affecting excitability of nerve and muscle fibres by changing the kinetics of the voltage-dependent sodium channel.

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.

2 – Morphology of the Venom Apparatus

The embedding technique, discovered by Swammerdam, made possible his extensive studies on the chitinous parts of the sting of bees and wasps (Swammerdam, 1672-1673). He described the sting of the honey-bee worker as consisting of three parts, two of them, the lancets, moving inside the sting sheath (see Chapter 1, Fig. 3 and Table I). The stinging apparatus of Aculeata, except that of Sapygidae, Chrysididae and Drynidae, is a transformed ovipositor, and its parts are readily identified with those of the ovipositor of other insects (Bischoff, 1927; Snodgrass, 1956). Detailed descriptions of the chitinous parts and their muscles have been given by several authors (Table I). This table shows the paucity of information on solitary Aculeata. In the honey-bee the entire stinging organ includes two sets of parts that are anatomically and functionally distinct. One is the large basal part, which is the principal motor apparatus, the other is the long shaft, which is the piercing instrument. The sting is protracted and retracted by muscles, described by Snodgrass (1956) (Fig. 1). The mechanism of ejecting venom from the venom reservoir into the sting has been described by Arnhart (1929) and by Maschwitz and Kloft (1971). During protraction of the sting the air present in the bulb of the sting is pressed out. During retraction the venom is sucked up from the reservoir, and renewed protraction drives out the venom. These pumping movements are visible at the extracted sting apparatus. A comparable sting apparatus has been described for the carpenter bee Xylocopa virginica by Hermann and Mullen (1974). This bee is reluctant to

Isolation and some biochemical properties of a paralysing toxin from the venom of the wasp Microbracon hebetor (Say).

Abstract A toxin with paralysing activity was isolated from crude preparations of Microbracon hebetor (Say) venom by ion exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, electrophoresis on polyacrylamide gel and gel chromatography on Sephadex G-75. The active component is very labile. A 28-fold purification was obtained with a recovery of about 2.5% of the original biological activity. The toxin shows one single protein band after disc electrophoresis. The molecular weight was assessed at 61,000 by gel chromatography, and at 62,700 by the sedimentation equilibrium method. The isoelectric point was at pH 6.8. The purified toxin was inactivated by a number of proteolytic enzymes.

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.

Partial purification of components from the paralysing venom of the digger wasp Philanthus triangulum F. (Hym. sphec.) and their action on neuromuscular transmission in the locust

Abstract 1. Using Sephadex G-100, Sephadex G-25 gel chromatography and SP-Sephadex C-25 ion exchange chromatogrraphy, four different biologically active preparations were obtained from the paralysing venom in abdominal extract of the solitary wasp Philanthus triangulum F. 2. Three preparations show paralysing activity in honeybees in a different way and cause, in addition to a presynaptic effect, a postsynaptic block of the glutamatergic neuromuscular transmission in an isolated locust muscle. 3. The fourth preparation causes a block of the cholinergic transmission in the isolated sixth abdominal ganglion of the cockroach (See Piek et al. , subsequent article). 4. Two of the active components could be located on the cellulose thin-layer chromatogram. 5. The toxins have molecular weights probably lower than 700.