A study of the biological activity of toxic material derived from nematocysts of the cubomedusan Chironex fleckeri

doi:10.1016/0041-0101(69)90118-4

Toxicon

Volume 6, Issue 3, February 1969, Pages 179-184, in11-in12, 185-198, in13-in14, 199-204

https://doi.org/10.1016/0041-0101(69)90118-4 Get rights and content

Abstract

Nematocysts of the cubomedusan Chironex fleckeri have been isolated in quantity. Five types of nematocyst were distinguished. Protein, carbohydrate, cystine-containing compounds and 3-indolyl derivatives were detected in all types of nematocyst. Saline extracts of the contents of nematocysts were highly toxic to prawns and fish and were capable of eliciting fatal systemic effects within seconds if administered to rats and mice via the i.v. route.

Saline extracts of toxic material from ruptured nematocysts elicited a powerful contracture of the striated musculature of barnacles and of skeletal, respiratory and extravascular smooth musculature of rats. In each case, the contracture was sustained for a period and then the musculature involved became paralysed in the relaxed condition. When exposed to toxin extracts the perfused heart of the toad and the exposed heart of the rat showed a progressive failure to relax during successive cardiac cycles and the hearts became paralysed in systole. Prior blockade of neuromuscular junctions with d-tubocurarine did not modify the response of rat diaphragm musculature to toxin extracts. Conduction in the sciatic nerve of the toad was not obviously affected by prolonged exposure to toxin extracts.

Preliminary results indicate that C. fleckeri nematocyst toxin is antigenic.

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Jellyfish stings pose a significant threat to humans in coastal areas worldwide, with venomous jellyfish species stinging millions of individuals annually. Nemopilema nomurai is one of the largest jellyfish species, with numerous tentacles rich in nematocysts. N. nomurai venom (NnV) is a complex mixture of proteins, peptides, and small molecules that serve as both prey-capture and defense mechanisms. Yet, the molecular identity of its cardiorespiratory and neuronal toxic components of NnV has not been clearly identified yet. Here, we isolated a cardiotoxic fraction, NnTP (Nemopilema nomurai toxic peak), from NnV using chromatographic methods. In the zebrafish model, NnTP exhibited strong cardiorespiratory and moderate neurotoxic effects. LC-MS/MS analysis identified 23 toxin homologs, including toxic proteinases, ion channel toxins, and neurotoxins. The toxins demonstrated a synergistic effect on the zebrafish, leading to altered swimming behavior, hemorrhage in the cardiorespiratory region, and histopathological changes in organs such as the heart, gill, and brain. These findings provide valuable insights into the mechanisms underlying the cardiorespiratory and neurotoxic effects of NnV, which could be useful in developing therapeutic strategies for venomous jellyfish stings.
An in vitro comparison of venom recovery methods and results on the box jellyfish, Chironex fleckeri
2020, Toxicon
Citation Excerpt :
While effective in adequate volume recovery, the possibility of sample contamination from the amniotic membrane (Endean et al., 1969) prompted initiatives to develop mechanical disruption techniques (Carrette and Seymour, 2004). Subsequent methods of venom extraction included the use of mortar and pestle grinding in ice and filtered seawater (Endean et al., 1969; Endean and Rifkin, 1983), the use of an electrical microgrinder (Endean et al., 1969; Endean and Noble, 1971), tentacle homogenization followed by centrifugation at low temperatures (Turner and Freeman, 1969) and sonication using high sound frequencies to rupture nematocysts (Mustafa et al., 1995; Bloom et al., 1998). Challenges associated with these methods included poor volume recovery and low yields of the toxic venom proteins (Endean and Noble, 1971; Othman and Burnett, 1990), in addition to venom extracts contaminated with extraneous proteins and toxins derived from tentacles, rather than pure nematocyst content (Endean and Noble, 1971; Ramasamy et al., 2005).
The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40–55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction.
The pathology of Chironex fleckeri venom and known biological mechanisms
2020, Toxicon: X
Citation Excerpt :
Chironex fleckeri is a large box jellyfish commonly found in Australia waters, and suspected through out the Indo Pacific region (Fenner and Williamson, 1996; Gershwin et al., 2010; Lippmann et al., 2011; Tibballs, 2006).
The large box jellyfish Chironex fleckeri is found in northern Australian waters. A sting from this cubozoan species can kill within minutes. From clinical and animal studies, symptoms comprise severe pain, welts, scarring, hypotension, vasospasms, cardiac irregularities and cardiac arrest. At present, there is no cure and opioids are used to manage pain. Antivenom is available but controversy exists over its effectiveness. Experimental and combination therapies performed in vitro and in vivo have shown varied efficacy. These inconsistent results are likely a consequence of the different methods used to extract venom. Recent omics analysis has shed light on the systems of C. fleckeri venom action, including new toxin classes that use pore formation, cell membrane collapse and ion channel modulation. This review covers what is known on C. fleckeri pathomechanisms and highlights current gaps in knowledge. A more complete understanding of the mechanisms of C. fleckeri venom-induced pathology may lead to novel treatments and possibly, the discovery of novel cell pathways, novel drug scaffolds and novel drug targets for human disease.
A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues
2012, Toxicology Letters
Citation Excerpt :
By 1969 these findings were extended to show that the venom actions were due to separate protein fractions (Baxter and Marr, 1969). Thereafter, research focused on better defining the features of that cardiovascular toxicity, the chemical nature of the toxins and developing treatment options (Barnes, 1966, 1967; Baxter and Marr, 1969; Endean et al., 1969, 1993; Freeman and Turner, 1969). It was quickly established that the venom caused a biphasic change in blood pressure consisting of an initial hypertensive phase followed by potentially lethal hypotension (Freeman, 1974; Freeman and Turner, 1969, 1972).
The pharmacology of Australian box jellyfish, Chironex fleckeri, unpurified (crude) nematocyst venom extract (CVE) was investigated in rat isolated cardiac and vascular tissues and in anaesthetised rats.
In small mesenteric arteries CVE (0.01–30 μg/ml) caused contractions (EC₅₀ 1.15 ± 0.19 μg/ml) that were unaffected by prazosin (0.1 μM), bosentan (10 μM), CGRP_8–37 (1 μM) or tetrodotoxin (1 μM). Box jellyfish antivenom (5–92.6 units/ml) caused rightward shifts of the CVE concentration–response curve with no change in the maximum. In the presence of l-NAME (100 μM) the sensitivity and maximum response to CVE were increased, whilst MgSO₄ (6 mM) decreased both parameters. CVE (1–10 μg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation.
Left atrial responses to CVE (0.001–30 μg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3 μg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3 μg/ml. These responses were unaffected by 1 μM of propranolol, atropine or CGRP_8–37. Antivenom (54 and 73 units/ml) caused rightward shifts of the CVE concentration–response curve and prevented atrial standstill in left and right atria.
The effects of CVE do not appear to involve autonomic nerves, post-synaptic α₁- or β₁-adrenoceptors, or muscarinic, endothelin or CGRP receptors, but may occur through direct effects on the cardiac and vascular muscle. Box jellyfish antivenom was effective in attenuating CVE-induced responses in isolated cardiac and vascular tissues.
Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye
2010, Toxicon
Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID₅₀ was about 0.6 μg protein/g fish. Toxin samples were stable when kept at −80 ^°C, but after 48 h, an 80% decline in lethality occurred at −20 ^°C. Poor stability of the venom was observed within the range of 65–80 ^°C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60 kDa and 50 kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.
In vivo effects of cnidarian toxins and venoms
2009, Toxicon
Citation Excerpt :
Sublethal doses were neurotoxic in crabs. Experiments with mice and rats showed that i.v. application of the venom caused a rapid cessation of respiration followed by cardiac arrest (Endean et al., 1969; Freeman and Turner, 1969), which was similar to its lethal effects on humans. It seems that in addition to the lethal component there may also be other components present in the venom that are responsible for its neurotoxic, haemolytic/cytolytic, myotoxic, and dermatonecrotic activities.
Cnidarians (Coelenterates), a very old and diverse animal phylum, possess a wide variety of biologically active substances that can be considered as toxins. Anthozoan toxins can be classified into two chemically very different groups, namely polypeptide toxins isolated from sea anemones and diterpenes isolated from octocorals. Cubozoan and scyphozoan protein toxins have been the most elusive cnidarian toxins to investigate – despite a tremendous effort in the past few decades, very few of these large, relatively unstable protein toxins were isolated, but recently this has been achieved for cubozoan venoms. Hydrozoans mainly contain large proteins with physiological mechanisms of action similar to the sea anemone and jellyfish pore-forming toxins. This article will focus on the in vivo physiological effects of cnidarian toxins and venoms; their actions at the cellular level will only be considered to understand their actions at the organ and whole animal levels. An understanding of mechanisms underlying the in vivo toxic effects will facilitate the development of more effective treatments of cnidarian envenomations.

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Abstract

References (28)

Neuromuscular physiology of giant muscle fibers of a barnacle, Balanus nubilis Darwin

Comp. Biochem. Physiol.

The effects of X-irradiation on the biological activity of cottonmouth (Agkistrodon piscivorous) venom

Toxicon

Effects of X-irradiation on the antigenic character of Agkistrodon piscivorus (cottonmouth moccasin) venom

Toxicon

Observations on jellyfish stingings in North Queensland

Aust. med. J.

Das muskuläre Substrat der Bewegungsund Halteleistung des menschlichen Zwerchfells

Acta. anat.

A venomous medusa from Australian waters

Aust. J. Sci.

A deadly poisonous jellyfish

Aust. Mus. Mag.

Injuries to man from marine invertebrates in the Australian region

National Health and Medical Research Council Special Report Series No. 12

Studies on three venomous Cubomedusae

Symposia of London Zoological Society No. 16

Studies on Australian Cubomedusae, including a new genus and species apparently harmful to man

Aust. J. mar. Freshwat. Res.

Isolation of active nematocysts of Metridium senile and their chemical composition

Nature, Lond.

The toxicity of Physalia nematocysts

Biol. Bull. mar. biol. Lab., Woods Hole

Cited by (77)

Identification of cardiorespiratory toxic components of Nemopilema nomurai jellyfish venom using sequential chromatography methods

An in vitro comparison of venom recovery methods and results on the box jellyfish, Chironex fleckeri

The pathology of Chironex fleckeri venom and known biological mechanisms

A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye

In vivo effects of cnidarian toxins and venoms