Synanceia Trachynis Research Articles

The marine biologist—Bob Endean

Bob Endean was a dedicated marine biologist with an extensive knowledge of coral reef communities in the Great Barrier Reef and fauna in subtropical Queensland waters. He commenced a study of venomous and poisonous marine animals dangerous to man at a time when the field was new, employing a variety of techniques to investigate the venom apparatus, mode of delivery of venom or toxin, mode of toxic action on excitable tissues, and biochemistry of venom or toxin. Determination of the pharmacological properties of crude venom from Conus marine snails advanced characterization of conotoxins by later workers. A study of four types of nematocysts from the box-jellyfish Chironex fleckeri provided information as to their structure, function, and mechanism of discharge; myotoxins T1 and T2 were isolated from microbasic mastigophores. Endean studied poisonous stonefish ( Synanceia trachynis) and, with Ann Cameron, scorpionfish ( Notesthes robusta); investigations of ciguatera and of paralytic shellfish poisoning were initiated. He organized the collection of Australian frogs which led to the isolation of caerulein by Erspamer in Italy. Endean highlighted the ecological danger of the population explosion of the crown-of-thorns starfish ( Acanthaster planci) and provided the impetus for the creation of the Great Barrier Reef Marine Park.

Modulation of intracellular Ca 2+ levels by Scorpaenidae venoms

The crude venoms of the soldierfish ( Gymnapistes marmoratus), the lionfish ( Pterois volitans) and the stonefish ( Synanceia trachynis) display pronounced neuromuscular activity. Since [Ca 2+] i is a key regulator in many aspects of neuromuscular function we sought to determine its involvement in the neuromuscular actions of the venoms. In the chick biventer cervicis muscle, all three venoms produced a sustained contraction (approx 20–30% of 1 mM acetylcholine). Blockade of nicotinic receptors with tubocurarine (10 μM) failed to attenuate the contractile response to either G. marmoratus venom or P. volitans venom, but produced slight inhibition of the response to S. trachynis venom. All three venoms produced a rise in intracellular Ca 2+ (approx. 200–300% of basal) in cultured murine cortical neurons. The Ca 2+-channel blockers ω-conotoxin MVIIC, ω-conotoxin GVIA, ω-agatoxin IVa and nifedipine (each at 1 μM) potentiated the increase in [Ca 2+] i in response to G. marmoratus venom and P. volitans venom, while attenuating the response to S. trachynis venom. Removal of extracellular Ca 2+, replacement of Ca 2+ with La 3+ (0.5 mM), or addition of stonefish antivenom (3 units/ml) inhibited both the venom-induced increase in [Ca 2+] i in cultured neurones and contraction in chick biventer cervicis muscle. Venom-induced increases in [Ca 2+] i correlated with an increased cell death of cultured neurones as measured using propidium iodide (1 μg/ml). Morphological analysis revealed cellular swelling and neurite loss consistent with necrosis. These data indicate that the effects of all three venoms are due in part to an increase in intracellular Ca 2+, possibly via the formation of pores in the cellular membrane which, under certain conditions, can lead to necrosis.

Stonefish (Synanceia trachynis) Antivenom: In Vitro Efficacy and Clinical Use

Certain species of fish have long been recognised as venomous. Although venomous fish do not represent a substantial source of human mortality, they are responsible for a number of envenomations each year that are serious enough to warrant clinical treatment. To the author's knowledge, the only fish antivenom still commercially available is the stonefish antivenom produced by CSL Ltd. in Australia. This antivenom consists of the purified F(ab)2 fragment of equine IgG antibodies raised against the venom of Synanceia trachynis. The antivenom is cheap, and effective in neutralising all known clinical effects of serious S. trachynis envenomation. In addition, there is experimental evidence that stonefish antivenom neutralises the pharmacological effects of other fish venoms, particularly those of the lionfish (Pterois volitans) and the soldierfish (Gymnapistes marmoratus), as well as displaying cross‐reactivity with them in Western immuno‐blotting. The potential therefore exists for the use of stonefish antiv...

Trachynilysin, a Neurosecretory Protein Isolated from Stonefish (Synanceia trachynis) Venom, Forms Nonselective Pores in the Membrane of NG108-15 Cells

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about -3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.

Stonefish ( Synanceia spp.) antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish ( Gymnapistes marmoratus) venom

The soldierfish ( Gymnapistes marmoratus), which is related to the stonefish ( Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish ( Gymnapistes marmoratus). Toxicon 36, 973–872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish ( Synanceja trachynis) venom. Toxicon 38, 391–407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4–12 μg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7–12 V) left atria, G. marmoratus venom extract (40 μg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 μg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 μg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 μg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 μg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Effects of trachynilysin, a protein isolated from stonefish ( Synanceia trachynis) venom, on frog atrial heart muscle

The effects of trachynilysin (TLY), a protein toxin isolated from stonefish ( Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 μg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca 2+ but not when Ca 2+ was replaced by Sr 2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca 2+ influx and to a rise in intracellular Ca 2+ levels which leads to (i) a slowing of the Na +/Ca 2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca 2+-dependent K + current involved in the APD shortening.

Increase of intracellular Ca 2+in cultured mouse hippocampal neurons by trachynilysin, a protein toxin extracted from stonefish (Synanceia trachynis) venom

Increase of intracellular Ca 2+in cultured mouse hippocampal neurons by trachynilysin, a protein toxin extracted from stonefish (Synanceia trachynis) venom

Modification of the electrical activity and of the contraction in frog atrial heart fibres by trachynilysin, a toxic protein purified from Synanceia trachynis venom

Modification of the electrical activity and of the contraction in frog atrial heart fibres by trachynilysin, a toxic protein purified from Synanceia trachynis venom

Selective depletion of clear synaptic vesicles and enhanced quantal transmitter release at frog motor nerve endings produced by trachynilysin, a protein toxin isolated from stonefish (Synanceia trachynis) venom.

Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2-20 micrograms/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.

Effects of trachynilysin, a toxic protein purified from the venom of the stonefish Synanceia trachynis, on frog atrial heart muscle

Effects of trachynilysin, a toxic protein purified from the venom of the stonefish Synanceia trachynis, on frog atrial heart muscle

Effects of stonefish (Synanceia trachynis) venom on murine and frog neuromuscular junctions: A. S. Kreger, 1J. Molgo, 2J. X. Comella, 3B. Hansson4 and S. Thesleff4 ( 1The Musculoskeletal Sciences Research Institute, 2190 Fox Mill Rd, Herndon, VA 22071, U.S.A.; 2Laboratoire de Neurobiologie Cellulaire et Moléculaire, C.N.R.S., 91198 Gif sur Yvette Cedex, France; 3Universitat de Barcelona, Facultad de Medicina de Lleida,

Effects of stonefish (Synanceia trachynis) venom on murine and frog neuromuscular junctions: A. S. Kreger, 1J. Molgo, 2J. X. Comella, 3B. Hansson4 and S. Thesleff4 ( 1The Musculoskeletal Sciences Research Institute, 2190 Fox Mill Rd, Herndon, VA 22071, U.S.A.; 2Laboratoire de Neurobiologie Cellulaire et Moléculaire, C.N.R.S., 91198 Gif sur Yvette Cedex, France; 3Universitat de Barcelona, Facultad de Medicina de Lleida,

Effects of stonefish ( Synanceia trachynis) venom on murine and frog neuromuscular junctions

A. S. Kreger, J. Molgó, J. X. Comella, B. Hansson and S. Thesleff. Effects of stonefish ( Synanceia trachynis) venom on murine and frog neuromuscular junctions. Toxicon 31, 307–317, 1993.—The neuromuscular toxicity of stonefish ( Synanceia trachynis) venom was characterized by electrophysiological and electron microscopic examination of isolated murine and frog nerve-skeletal muscle preparations exposed to various concentrations of venom. Low concentrations of venom (2.5–10 μg/ml) acted presynaptically by causing release and depletion of neurotransmitter from the nerve terminal. The response was Na + channel-independent (resistant to tetrodotoxin), required the presence of either Ca 2+ or Mg 2+, and was observed with botulinum neurotoxin-paralyzed nerve-muscle preparations. Higher concentrations of venom (100–300 μg/ml) acted postsynaptically and presynaptically. They caused irreversible depolarization of muscle cells and microscopically observable muscle and nerve damage. We conclude that the previously observed neuromuscular toxicity of stonefish venom is a consequence of the venom's dose-dependent, presynaptic and postsynaptic actions at the myoneural junction.

Detection of a cytolytic toxin in the venom of the stonefish ( Synanceia trachynis)

A. S. Kreger. Detection of a cytolytic toxin in the venom of the stonefish ( Synanceia trachynis). Toxicon 29, 733–743, 1991.—The venom of the stonefish, Synanceia trachynis, contains a cytolytic toxin which is antigenic and ammonium sulfate-precipitable, and has a pI of ca 5.7 and an M r of ca 158,000. The toxin is a potent but narrow-spectrum cytolysin which is lytic in vitro for rabbit, dog, rat, pig and guinea pig erythrocytes, in that order, but is largely or completely inactive against sheep, cow, human, monkey, mouse, goat, horse, burro and cat erythrocytes. Fractionation of the venom by molecular sieve fast protein liquid chromatography and isoelectric focusing did not separate the haemolytic activity from the venom's lethal and vascular permeability-increasing activities. Also, the three activities were destroyed by heat, proteases, Congo red, potassium permanganate and stonefish antivenoms. The results suggest that the haemolytic, lethal and vascular permeability-increasing activities of stonefish venom are properties of the same molecule, a previously unrecognized, membrane-damaging protein toxin.

Effect of a smooth muscle relaxant from the stonefish, Synanceia trachynis, on KCl-induced responses in the guinea-pig vas deferens

Stonefish skin secretion contains a smooth muscle relaxant, synancein II, that selectively inhibits tonic responses of KCl-induced contractions of the guinea-pig vas deferens. Synancein II (5–50 μg/ml) and nifedipine (10 −8−10 −7 M) reduced tonic responses equaly. Over these concentrations, synancein II inhibited phasic responses more than did nifedipine. Rapid inhibition of phasic responses by synancein II (low concentrations) but its inability to block this response at high concentrations suggests that more than one extracellular and/or intracellular Ca 2+ source may contribute to the generation of the phasic response. Some calcium antagonists selectively inhibit tonic responses induced by KCl in many different organs, whilst others inhibit phasic responses. Whereas we previously reported that synancein II selectively inhibits phasic rather than tonic responses in the guinea-pig ileum, the present result reveals a reversal of this selectivity.

The antispasmogenic action on guinea-pig ileum of a fraction obtained from the toxic skin secretion of the stonefish, Synanceia trachynis

—The toxic skin tubercle gland secretion of the stonefish, Synanceia trachynis, contains a smooth muscle antispasmogen that is readily distinguished from papaverine, verapamil and both α and β adrenoreceptor agonists. At 15 μg/ml, the toxin containing fraction markedly inhibited the phasic response and had a lesser inhibitory effect on the tonic response of acetylcholine- and KCl-induced contractures of guinea-pig ileum, while at 50 μg/ml, the toxin markedly inhibited both responses. Over the same dose range the toxin fraction markedly inhibited BaCl 2-induced contractile responses but did not distinguish between the phasic and tonic components. Whereas the toxin fraction markedly reduced the tone of previously induced supramaximal KCl and acetylcholine responses of guinea-pig ileum, it had little effect on the tone of previously induced BaCl 2 responses. The toxin non-competitively inhibited acetylcholine-induced contractions of guinea-pig ileum and similarly non-competitively inhibited the response induced by Ca 2+ in high-[K +] Ringer. The toxin is not a chelating agent for Ca 2+ or Mg 2+ and it does not affect ATP-induced contracture of glycerinated guinea-pig ileum. The antispasmogenic effect of the toxin contained in the fraction does not result from inhibition of cAMP phosphodiesterase activity.

A crinotoxin from the skin tubercle glands of a stonefish ( Synanceia trachynis)

The milky secretion produced by the skin tubercle glands of the stonefish inhibits mussel gill ciliary movement, is lethal to the ciliate protozoan Blepharisma sp. at a concentration of 1/750 by volume and kills the prawn Metapenaeus bennettae when administered intramuscularly at 5 μl/g. It has negligible protease, haemolytic and haemagglutinating activity and is only mildly toxic to mosquito fishes and blennies immersed in it. The secretion increases the tone of barnacle muscle and enhances its response to direct stimulation. It has no effect on the tone of diaphragm muscle but increases its response to both direct and indirect stimulation. The secretion has a potent musculotropic effect on guinea-pig ileal smooth muscle, inhibiting the response to acetylcholine, 5-hydroxytryptamine and BaCl 2 markedly. The secretion can be separated into at least two active fractions by centrifugation.