Box Jellyfish Chironex Research Articles

Real-time PCR assay for detection of Environmental DNA from <i>Chironex yamaguchii</i>

The box jellyfish Chironex yamaguchii is a highly venomous cubomedusa, also known as “habu-kurage” in Japan. It is a significant threat to marine activities in Okinawa in southern Japan. Annually, envenomation of the species causes nearly 100 to 200 injuries in the Okinawa Prefecture. The present study developed a species-specific real-time PCR assay to analyze environmental DNA (eDNA) for rapid detection of jellyfish in water samples. Primers and probe were designed based on the sequences of the mitochondrial cytochrome c oxidase subunit I region using tissue samples of nine specimens obtained from the Okinawa Prefecture. The specificity of the real-time PCR assay was confirmed by amplifying the DNA samples of 10 different box jellyfish species. A practical assay for eDNA detection was successful on water samples obtained from the natural habitat of C. yamaguchii. In addition, the degradation rate of eDNA in the tank experiment was 65% of the initial concentration at 1 h, 22% at 12 h, and 3% at 48 h.

Bay watch: Using unmanned aerial vehicles (UAV's) to survey the box jellyfish Chironex fleckeri.

Biological investigations on free ranging marine species are regarded as challenging throughout the scientific community. This is particularly true for ‘logistically difficult species’ where their cryptic natures, low abundance, patchy distributions and difficult and/or dangerous sampling environments, make traditional surveys near impossible. What results is a lack of ecological knowledge on such marine species. However, advances in UAV technology holds potential for overcoming these logistical difficulties and filling this knowledge gap. Our research focused on one such logistically difficult species, the Australian box Jellyfish (Chironex fleckeri), and we investigated the capacity of consumer grade UAV technology to detect this, highly venomous, target species in the inshore waters of Northern Queensland Australia. At two sites in the Weipa area, we utilized video analysis, visual count comparisons with a netted animal tally, and evaluated the role of associated environmental conditions, such as wind speed, water visibility and cloud cover on jellyfish detection rates. In total fifteen, 70 meter transects were completed between two sites, with 107 individuals captured. Drone success varied between the two sites with a significant difference between field and post-field (laboratory) counts. Animal size and cloud cover also had significant effects on detection rates with an increase in cloud cover and animal size enhancing detection probability. This study provides evidence to suggest drone surveys overcome obstacles that traditional surveys can’t, with respect to species deemed logistically difficult and open scope for further ecological investigations on such species.

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

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.

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.

Molecular dissection of box jellyfish venom cytotoxicity highlights an effective venom antidote

The box jellyfish Chironex fleckeri is extremely venomous, and envenoming causes tissue necrosis, extreme pain and death within minutes after severe exposure. Despite rapid and potent venom action, basic mechanistic insight is lacking. Here we perform molecular dissection of a jellyfish venom-induced cell death pathway by screening for host components required for venom exposure-induced cell death using genome-scale lenti-CRISPR mutagenesis. We identify the peripheral membrane protein ATP2B1, a calcium transporting ATPase, as one host factor required for venom cytotoxicity. Targeting ATP2B1 prevents venom action and confers long lasting protection. Informatics analysis of host genes required for venom cytotoxicity reveal pathways not previously implicated in cell death. We also discover a venom antidote that functions up to 15 minutes after exposure and suppresses tissue necrosis and pain in mice. These results highlight the power of whole genome CRISPR screening to investigate venom mechanisms of action and to rapidly identify new medicines.

Identification of Allergens in the Box Jellyfish Chironex yamaguchii That Cause Sting Dermatitis

Background: Jellyfish stings cause painful, papular-urticarial eruptions due to the immediate allergic, acute toxic and persistent inflammatory responses. In spite of many marine accidents and their economic impact, modes of first-aid treatment remain conventional and specific allergen and medical treatment are not yet available. The purpose of this study was to define the specific allergen of the box jellyfish Chironex yamaguchii and to study the precise mechanism of the resulting dermatitis. Methods: We comprehensively studied the immunoglobulin-binding molecules from the box jellyfish C. yamaguchii with a purification procedure and Western blotting, using sera from 1 patient and from several controls. Results: From the nematocyst wall and spine, we detected IgG-binding acidic glycoprotein (of 66 and 30 kDa) as determined by Western blot and ion-exchange chromatography. In addition, the 66-kDa protein was found to be an asparagine residue-coupled N-linked glycoprotein and the epitope resided in the protein fraction. We found that CqTX-A, the major toxic protein of the nematocyst, is also a heat-stable IgE-binding allergen. This was confirmed as a 45-kDa protein by Western blot from both nematocyst extracts and purified CqTX-A. Conclusions: The detection of these proteins may, in part, explain the combined immediate allergic-toxic and persistent allergic responses. Hopefully, our findings will lead to the development of specific venom immunotherapy for marine professional workers and tourists for jellyfish-sting dermatitis and anaphylaxis.

First record of association of copepods with highly venomous box jellyfish Chironex, with description of new species of Paramacrochiron (Cyclopoida: Macrochironidae).

Paramacrochiron chironecicola n. sp. (Copepoda: Cyclopoida: Macrochironidae) is described from the highly venomous box jellyfish Chironex sp. collected from Malampaya Sound, Palawan Island, The Philippines. This is the first record of copepods associated with cubozoan medusae, although other cnidarian groups such scyphozoans, hydrozoans, and anthozoans are common hosts for symbiotic copepods. The infection sites were on the subumbrella, pedalium, and rhopalium, but also rarely on the adradial furrow. The new species is distinguished from other congeners by a combination of the following features: (1) the fifth pedigerous somite dorsally covering the anterior part of the female genital double-somite; (2) the fine structures of the antenna (relative lengths of segments) and maxilliped (positions of terminal elements) of the female; (3) the relatively long outer spines on the exopodal segments of legs 1-4; (4) the relatively long and thick female leg 5 bearing a long protopodal seta which reaches to the distal margin of the exopod; (5) the relatively short caudal ramus in the female; and (6) the plump prosome and short urosome in the male. Since members of the genus typically parasitize scyphozoans, especially rhizostomes, the association of this parasitic copepod on cubozoans may reflect the relatively close phylogenetic relationship between cubozoans and scyphozoans.

Chironex fleckeri (Box Jellyfish) Venom Proteins: EXPANSION OF A CNIDARIAN TOXIN FAMILY THAT ELICITS VARIABLE CYTOLYTIC AND CARDIOVASCULAR EFFECTS

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

Cardiotoxic effects of venom fractions from the Australian box jellyfish Chironex fleckeri on human myocardiocytes

An investigation into the cardiotoxic effects in human cardiomyocytes of different fractions (as produced from an FPLC) of the venom from Chironex fleckeri showed that whole venom caused cardiac cell death in minutes, measured as cell detachment using xCELLigence technology. However, only one fraction of the venom was responsible for this effect. When all extracted venoms were recombined a similar result was seen for the toxic fraction, however these effects were slower than unfractionated venom alone even though the concentrations were similar. The difference in the results between fractioned and unfractionated venom may have been caused by compounds remaining in the FPLC column, which may interact with the toxic fraction to cause rapid cell detachment or death.

The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1–100μg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1μM), but significantly attenuated by tetrodotoxin (TTX; 0.1μM), propranolol (1μM), Mg2+ (6mM) or calcitonin gene-related peptide antagonist (CGRP8–37; 1μM). CVE caused no change in right atrial rate until 100μg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP8–37. In the presence of Mg2+, CVE 30–100μg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC50 1.03±0.07μg/mL) that were unaffected by prazosin (0.3μM), ω-conotoxin GVIA (0.1μM) or Mg2+ (6mM). There was a 2-fold increase in sensitivity in the presence of CGRP8–37 (3μM). TTX (0.1μM), box jellyfish Chironex fleckeri antivenom (92.6U/mL) and benextramine (3μM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (−22%) or benextramine (−49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri

Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal δ-endotoxins Cry1Aa, Cry3Bb and Cry3A.

An in vivo examination of the stability of venom from the Australian box jellyfish Chironex fleckeri

We have previously characterised the pharmacological activity of a number of jellyfish venoms with a particular emphasis on the profound cardiovascular effects. It has been suggested that jellyfish venoms are difficult to work with and are sensitive to pH, temperature and chemical changes. The current study aimed to examine the working parameters of the venom of the Australian box jellyfish Chironex fleckeri to enable fractionation and isolation of the toxins with cardiovascular activity. C. fleckeri venom was made up fresh each day and subjected to a number of different environments (i.e. a pH range of 5–9 and a temperature range of 4–30 °C). In addition, the effect of freeze drying and reconstituting the venom was investigated. Venom (50 μg/kg, i.v.) produced a transient hypertensive response followed by cardiovascular collapse in anaesthetised rats. This biphasic response was not significantly effected by preparation of the venom at a pH of 5, 7 or 9. Similarly, venom (50 μg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30 °C for 1.5 h. However, the cardiovascular activity was abolished by boiling the venom. Freeze drying, and then reconstituting, the venom did not significantly affect its cardiovascular activity. However, repeated freeze drying and reconstituting of extracted venom resulted in a significantly loss of activity. This study provides a more detailed knowledge of the parameters in which C. fleckeri venom can be used and, while supporting some previous studies, contradicts some of the perceived problems of working with the venom.

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.

Variation in lethality and effects of two Australian chirodropid jellyfish venoms in fish

The North Queensland chirodropid box jellyfish Chironex fleckeri and Chiropsalmus sp. share similar nematocyst composition and the same prey of Acetes australis shrimps in their early medusa stages; however, as C. fleckeri individuals reach larger size, the animals add fish to their diet and their complement of nematocyst types changes, allowing larger doses of venom to be delivered to prey. This study demonstrated that the venoms of the two species differ as well: despite similar effects previously documented in crustacean prey models, the two had widely different cardiac and lethal effects in fish, with C. fleckeri being substantially more potent in its ability to cause death. Comparisons between the venom delivery abilities of the two species showed that the change in nematocysts of C. fleckeri cannot alone account for its ontogenetic shift to prey fish; instead, its prey ecology clearly necessitates it having venom capable of acting efficiently to cause death in fish. Although this venom is almost certainly produced at greater metabolic cost to the animal than the less-lethal venom of Chiropsalmus sp., owing to its greater molecular protein complexity, it confers the advantage of increased caloric intake from fish prey, facilitating larger size and potentially greater reproductive output of C. fleckeri over Chiropsalmus sp.

Phospholipase A2 in Cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.

The Trial Intensive Management Area for Crocodiles: A Crocodile Removal Zone in Queensland, Australia

The Estuarine Crocodile (Crocodylus porosus) is the world's largest living crocodile, and in Queensland it inhabits tropical coastal wetlands and waterways. From 1990 to 2001, there have been nine substantiated crocodile attacks on people in Queensland, resulting in one death and eight serious injuries. Several crocodile attacks occurred in the Cairns area during these years, and Cairns's popular swimming beaches have been closed on a number of occasions because of crocodiles. Human safety from crocodile attack is an issue of great public and political concern in Queensland. From May 1998 to June 2001, the Queensland Parks and Wildlife Service operated a trial crocodile removal program in the Cairns area, with a removal zone that extended 70 km along the coast. The program was named the Trial Intensive Management Area for Crocodiles (TIMAC), and its primary purpose was to protect the Cairns's popular swimming beaches from crocodiles. The program was expanded to provide a problem crocodile response service throughout north Queensland and to train additional rangers in crocodile management skills. During the three years of the TIMAC program, 80 crocodiles were captured in north Queensland: 46 crocodiles from within the removal zone, and 34 from outside the removal zone. There were no crocodile attacks in the removal zone during the three-year trial program. At the request of local governments, the program became permanent in July 2001. In effect, Cairns is the only tropical city whose popular swimming beaches are protected by a combination of a lifeguard service, a shark control program, netted enclosures (seasonal) for protection from dangerous Box Jellyfish (Chironex fleckeri), and a crocodile removal program.

The in vivo cardiovascular effects of box jellyfish Chironex fleckeri venom in rats: efficacy of pre-treatment with antivenom, verapamil and magnesium sulphate

Using a new technique to extract venom from the nematocysts, the efficacy of CSL box jellyfish antivenom (AV) and adjunct therapies, verapamil and magnesium sulfate (MgSO 4), were investigated against the in vivo cardiovascular effects of Chironex fleckeri venom in anaesthetised rats. C. fleckeri venom (30 μg/kg; i.v.) produced a transient hypertensive response followed by hypotension and cardiovascular collapse within 4 min of administration. Prophylactic treatment of anaesthetised rats with CSL box jellyfish AV (3000 U/kg; i.v.) did not have any effect on the venom-induced pressor response, but prevented cardiovascular collapse in four out of 10 animals. Administration of verapamil (20 mM@0.25 ml/min; i.v.) either alone or in combination with AV, did not have any effect on the C. fleckeri venom-induced pressor response nor the consequent hypotension or cardiovascular collapse of animals. However, the administration of verapamil negated the partially protective effects of AV. Concurrent artificial respiration of animals with the above treatments did not attenuate the C. fleckeri venom-induced cardiovascular effects. MgSO 4 (0.05–0.07 M@0.25 ml/min; i.v.) alone did not have any effect on the venom-induced pressor response nor the consequent cardiovascular collapse of animals. However, although combined AV and MgSO 4 administration could not inhibit the transient pressor effect following the administration of C. fleckeri venom, it prevented cardiovascular collapse in all animals. We show for the first time, the cardiovascular effects of a C. fleckeri venom sample free of tentacular contamination and the potential of MgSO 4 as an adjunct therapy for the treatment of potentially fatal C. fleckeri envenomings.

The use of pressure immobilization bandages in the first aid management of cubozoan envenomings

This study is aimed to evaluate whether the application of pressure results in additional release of venom from naturally discharged, vinegar soaked nematocysts of the box jellyfish Chironex fleckeri. The results show that large quantities of venom are expressed with the application of pressures similar to that applied by compression immobilization bandages. The volume of venom expressed by this pressure was similar to the quantity expressed upon initial natural discharge of the nematocysts. The current recommended practice of applying PIB to cubozoan stings might worsen the envenomation. As the existing data now show that PIB may be detrimental to victims envenomed by cubozoans, we suggest that the current practice of the use of PIB in cubozoan envenomings be discarded until there is direct experimental evidence to support its use.

Clinical toxicology: a tropical Australian perspective.

Tropical Australia has an amazing diversity of venomous fauna, from "the world's most venomous creature," the multi-tentacled (chirodropid) box jellyfish Chironex fleckeri, to aggressive spiders whose venom remains to be characterized. All genera of highly venomous Australasian elapid snakes are present, except for tiger snakes. Most notable is the taipan (Oxyuranus scutellatus), with the most efficient "snap-release" biting mechanism of any snake and venom components causing the full constellation of clinical envenoming features: coagulopathy from fibrinogen depletion (procoagulant), neurotoxicity (predominantly presynaptic neurotoxin) and rhabdomyolysis (myotoxin). Brown snakes (Pseudonaja textilis and P. nuchalis) now account for most snake bite fatalities in Australia, as a result of severe coagulopathy and a poorly defined early scenario of collapse, postulated to be caused by profound hypotension caused by transient myocardial dysfunction associated with prothrombin activation. Other venomous entities include paralyzing ticks, the blue-ringed octopus, stone fish and other marine animals with venomous spines, paralyzing cone shells, and a wide range of jellyfish including Carukia barnesi and possibly other four-tentacled (carybdeid) box jellyfish causing the Irukandji syndrome.

The mechanism underlying the cardiotoxic effect of the toxin from the jellyfish Chironex fleckeri.

We have investigated the mechanisms underlying the cardiac effects of the toxin from the box jellyfish Chironex fleckeri. Papillary muscles isolated from the hearts of ferrets and ventricular myocytes isolated from the hearts of ferrets and rats were used. Force, intracellular [Ca2+], and membrane potential were monitored in the papillary muscles; contraction, intracellular [Ca2+], intracellular [Na+], and membrane currents were monitored in the isolated myocytes. Application of the toxin to these preparations resulted in a large increase in intracellular [Ca2+] and the adverse symptoms of Ca2+ overload (aftercontractions, spontaneous contractions, a decrease in developed force, and an increase in resting force). The response of papillary muscles to the toxin was not inhibited by blockers of Ca2+ or Na+ channels or by inhibitors of the sarcoplasmic reticulum, Na+/K+ ATPase, or Na+/H+ exchange. The response to the toxin was, however, blocked by prior exposure to a solution which contained no Na+ and by Ni2+. In the isolated myocytes, as well as an increase in intracellular [Ca2+], the toxin also caused an increase in intracellular [Na+] and the appearance of a current which was inward at negative potentials and reversed at about -10 mV. These data can be explained by the toxin increasing Na+ influx into the cell. The increase in intracellular [Na+] will then increase intracellular [Ca2+] via the Na+/Ca2+ exchange mechanism, thus producing the observed Ca2+ overload.