Jellyfish Cyanea Research Articles

Mitochondrial dysfunction contributes to the cytotoxicity induced by tentacle extract from the jellyfish Cyanea capillata in rat renal tubular epithelial NRK-52E cells

Our previous studies have shown that tentacle extract (TE) from the jellyfish Cyanea capillata could induce a delayed jellyfish envenomation syndrome with severe multiple organ dysfunctions, among which renal injury with tubular necrosis seemed to be most serious. So, in this study, we aimed to explore the toxic effect of TE on rat renal tubular epithelial NRK-52E cells. Based on the previous findings that TE could cause oxidative damage in erythrocytes, the effects of TE on cell oxidative stress conditions, including ROS production and lipid peroxidation, and mitochondrial dysfunction associated with cell death were investigated in NRK-52E cells. The results showed that TE caused cell morphological change and decreased cell viability through induction of apoptosis and necrosis in NRK-52E cells. Meanwhile, ROS overproduction and mitochondrial membrane potential decrease were found before the cell death occurred. It was concluded that TE could induce cytotoxicity, especially apoptosis and necrosis, in NRK-52E cells, and mitochondrial dysfunction and ROS overproduction might play important roles in the process of cell injury and death.

Lipid Peroxidation Is another Potential Mechanism besides Pore-Formation Underlying Hemolysis of Tentacle Extract from the Jellyfish Cyanea capillata

This study was performed to explore other potential mechanisms underlying hemolysis in addition to pore-formation of tentacle extract (TE) from the jellyfish Cyanea capillata. A dose-dependent increase of hemolysis was observed in rat erythrocyte suspensions and the hemolytic activity of TE was enhanced in the presence of Ca2+, which was attenuated by Ca2+ channel blockers (Diltiazem, Verapamil and Nifedipine). Direct intracellular Ca2+ increase was observed after TE treatment by confocal laser scanning microscopy, and the Ca2+ increase could be depressed by Diltiazem. The osmotic protectant polyethylenglycol (PEG) significantly blocked hemolysis with a molecular mass exceeding 4000 Da. These results support a pore-forming mechanism of TE in the erythrocyte membrane, which is consistent with previous studies by us and other groups. The concentration of malondialdehyde (MDA), an important marker of lipid peroxidation, increased dose-dependently in rat erythrocytes after TE treatment, while in vitro hemolysis of TE was inhibited by the antioxidants ascorbic acid—Vitamin C (Vc)—and reduced glutathione (GSH). Furthermore, in vivo hemolysis and electrolyte change after TE administration could be partly recovered by Vc. These results indicate that lipid peroxidation is another potential mechanism besides pore-formation underlying the hemolysis of TE, and both Ca2+ channel blockers and antioxidants could be useful candidates against the hemolytic activity of jellyfish venoms.

Multiple organ dysfunction: A delayed envenomation syndrome caused by tentacle extract from the jellyfish Cyanea capillata

The delayed jellyfish envenomation syndrome (DJES) with serious multiple organ dysfunction or systemic damages, generally developed 2 h after jellyfish stings, deserves special attention for it is very meaningful to the clinical interventions. To set up a DJES model as well as to obtain more details about its process, an integrative approach, including clinical chemistry, pathology and immunohistochemistry, was conducted to simultaneously monitor the effects of tentacle extract (TE) from the jellyfish Cyanea capillata on the vital target organs (heart, lung, liver and kidney). Our results showed that the TE from C. capillata could induce diverse toxic effects on these organs, among which the liver and kidney injuries seemed to be more serious than cardiopulmonary injuries and might be the leading causes of death in rats with DJES. In summary, we have established a DJES model with multiple organ dysfunction, which could facilitate the research on its underlying mechanism as well as the development of specific prevention or therapy strategies against jellyfish envenomation. The application of this model suggested that the possible mechanism of DJES might be attributed to the synergy of cytotoxicity, vasoconstriction effect and other specific target organ toxicities of jellyfish venom.

Cardiovascular Effect Is Independent of Hemolytic Toxicity of Tentacle-Only Extract from the Jellyfish Cyanea capillata

Our previous studies have confirmed that the crude tentacle-only extract (cTOE) from the jellyfish Cyanea capillata (Cyaneidae) exhibits hemolytic and cardiovascular toxicities simultaneously. So, it is quite difficult to discern the underlying active component responsible for heart injury caused by cTOE. The inactivation of the hemolytic toxicity from cTOE accompanied with a removal of plenty of precipitates would facilitate the separation of cardiovascular component and the investigation of its cardiovascular injury mechanism. In our research, after the treatment of one-step alkaline denaturation followed by twice dialysis, the protein concentration of the treated tentacle-only extract (tTOE) was about 1/3 of cTOE, and SDS-PAGE showed smaller numbers and lower density of protein bands in tTOE. The hemolytic toxicity of tTOE was completely lost while its cardiovascular toxicity was well retained. The observations of cardiac function, histopathology and ultrastructural pathology all support tTOE with significant cardiovascular toxicity. Blood gas indexes and electrolytes changed far less by tTOE than those by cTOE, though still with significant difference from normal. In summary, the cardiovascular toxicity of cTOE can exist independently of the hemolytic toxicity and tTOE can be employed as a better venom sample for further purification and mechanism research on the jellyfish cardiovascular toxic proteins.

Vertical and horizontal movement patterns of scyphozoan jellyfish in a fjord-like estuary

Despite their important functional role in marine ecosystems, we lack much information about jellyfish, including basic research on their swimming behavior. Here we used acoustic teleme- try to obtain detailed behavioral data on 2 scypho- zoans, lion's mane jellyfish Cyanea capillata and fried-egg jellyfish Phacellophora camtschatica, in Hood Canal, Puget Sound, Washington, USA. Indi- vidual variation was high in both the short-term (hours) and long-term (days) data, although several patterns of behavior emerged. Lion's mane jellyfish performed diel vertical migrations over the longer time period, but their depth did not vary with tidal stage. Additionally, horizontal swimming speeds var- ied with diel period and tidal stage for both lion's mane and fried-egg jellyfish. Lion's mane jellyfish swam faster during the night than day, whereas fried-egg jellyfish swam faster during the day. Both species had the highest swimming rates during flood tides. Jellyfish clearly exhibited active swimming behaviors and were not passively planktonic. Net and cumulative distances traveled suggest that Hood Canal may be a sink for jellyfish, potentially exacer- bating effects of population blooms on the ecosystem. Our findings provide mixed support for previous studies reporting diel vertical migrations in other jelly fish species, and provide evidence that jellyfish are capable of more advanced swimming behaviors than often thought.

Effect of Sub-Lethal Exposure to Ultraviolet Radiation on the Escape Performance of Atlantic Cod Larvae (Gadus morhua)

The amount of ultraviolet (UV) radiation reaching the earth's surface has increased due to depletion of the ozone layer. Several studies have reported that UV radiation reduces survival of fish larvae. However, indirect and sub-lethal impacts of UV radiation on fish behavior have been given little consideration. We observed the escape performance of larval cod (24 dph, SL: 7.6±0.2 mm; 29 dph, SL: 8.2±0.3 mm) that had been exposed to sub-lethal levels of UV radiation vs. unexposed controls. Two predators were used (in separate experiments): two-spotted goby (Gobiusculus flavescens; a suction predator) and lion's mane jellyfish (Cyanea capillata; a “passive" ambush predator). Ten cod larvae were observed in the presence of a predator for 20 minutes using a digital video camera. Trials were replicated 4 times for goby and 5 times for jellyfish. Escape rate (total number of escapes/total number of attacks ×100), escape distance and the number of larvae remaining at the end of the experiment were measured. In the experiment with gobies, in the UV-treated larvae, both escape rate and escape distance (36%, 38±7.5 mm respectively) were significantly lower than those of control larvae (75%, 69±4.7 mm respectively). There was a significant difference in survival as well (UV: 35%, Control: 63%). No apparent escape response was observed, and survival rate was not significantly different, between treatments (UV: 66%, Control: 74%) in the experiment with jellyfish. We conclude that the effect and impact of exposure to sub-lethal levels of UV radiation on the escape performance of cod larvae depends on the type of predator. Our results also suggest that prediction of UV impacts on fish larvae based only on direct effects are underestimations.

Occurrence and public perception of jellyfish along the German Baltic coastline

Jellyfish accumulations are often problematic for tourism and fisheries both along European coastlines and overseas. They damage the reputation of several seaside resorts and cause serious economic losses. On the German Baltic coast, jellyfish accumulations are well known, too. Besides population increase due to anthropogenic effects, normal hydrodynamic processes are notable key factors when it comes to jellyfish accumulations. Along German Baltic coastlines, moon jellyfish (Aurelia aurita) occur most frequently but also stinging lion’s mane jellyfish (Cyanea capillata) appear regularly, however not as abundant. The objectives of our study were a) to screen when, where, how many and which jellyfish species occur on German Baltic coasts, b) to find the causes for jellyfish accumulations and c) to evaluate how beach visitors perceive native medusae and whether information can influence their perception. Different methods where combined: systematic jellyfish observations in cooperation with lifeguards, investigations of jellyfish abundance in shallow water zones, and interviews with beach visitors. Our results suggest that jellyfish occurrence along the German Baltic coast correlates with offshore wind or shore-parallel wind, which cause upwelling events. In contrast, there is no evidence that frequency of jellyfish occurrence was dependent on water temperature in summer. In regard to tourism, we found that beach visitors who received information about jellyfish stated to feel significantly less bothered by medusae. Overall, this article addresses different methods to learn more about jellyfish accumulations and it shows information strategies in terms of improved beach management.

Direct Cardiac Toxicity of the Tentacle-Only Extract From the Jellyfish Cyanea capillata Demonstrated in Isolated Rat Heart

Previous studies in our laboratory have shown that the cardiotoxicity is the main reason for rat death caused by tentacle-only extract from jellyfish Cyanea capillata. However, the direct cardiotoxicity in vitro and its mechanisms of toxic action remain unclear. The current studies were performed by using the Langendorff-perfused isolated heart model, which showed a dose-dependent hemodynamic and electrocardiogram changes. Heart injury-related enzymes increased. Histopathological analysis showed early ischemic damage in the myocardium. The Ca channel blockers nifedipine and verapamil led to a marked improvement in recovery of cardiac function, including heart rate, left ventricular developed pressure, positive and negative first derivatives of intraventricular pressure, coronary flow, left ventricular end-diastolic pressure, and electrocardiogram changes. Tentacle-only extract-induced cardiac dysfunction could be partly improved by the pretreatments of both propranolol and phentolamine, but not by either atropine or neostigmine at all. In conclusion, we have verified the direct cardiotoxicity of tentacle-only extract from jellyfish C. capillata by the Langendorff isolated heart model, which consisted of 3 separate parts: sinoatrial node malfunction, cardiomyocyte injury, and coronary spasm. The potential mechanism might be attributed to the overactivation of L-type Ca channel, β- and α-adrenergic receptors, but not cholinergic receptors.

Jellyfish Support High Energy Intake of Leatherback Sea Turtles (Dermochelys coriacea): Video Evidence from Animal-Borne Cameras

The endangered leatherback turtle is a large, highly migratory marine predator that inexplicably relies upon a diet of low-energy gelatinous zooplankton. The location of these prey may be predictable at large oceanographic scales, given that leatherback turtles perform long distance migrations (1000s of km) from nesting beaches to high latitude foraging grounds. However, little is known about the profitability of this migration and foraging strategy. We used GPS location data and video from animal-borne cameras to examine how prey characteristics (i.e., prey size, prey type, prey encounter rate) correlate with the daytime foraging behavior of leatherbacks (n = 19) in shelf waters off Cape Breton Island, NS, Canada, during August and September. Video was recorded continuously, averaged 1:53 h per turtle (range 0:08–3:38 h), and documented a total of 601 prey captures. Lion's mane jellyfish (Cyanea capillata) was the dominant prey (83–100%), but moon jellyfish (Aurelia aurita) were also consumed. Turtles approached and attacked most jellyfish within the camera's field of view and appeared to consume prey completely. There was no significant relationship between encounter rate and dive duration (p = 0.74, linear mixed-effects models). Handling time increased with prey size regardless of prey species (p = 0.0001). Estimates of energy intake averaged 66,018 kJ•d−1 but were as high as 167,797 kJ•d−1 corresponding to turtles consuming an average of 330 kg wet mass•d−1 (up to 840 kg•d−1) or approximately 261 (up to 664) jellyfish•d-1. Assuming our turtles averaged 455 kg body mass, they consumed an average of 73% of their body mass•d−1 equating to an average energy intake of 3–7 times their daily metabolic requirements, depending on estimates used. This study provides evidence that feeding tactics used by leatherbacks in Atlantic Canadian waters are highly profitable and our results are consistent with estimates of mass gain prior to southward migration.

Cytotoxicity of the venom from the nematocysts of jellyfish Cyanea nozakii Kishinouye

In this article, the cytotoxicity of the venom from the nematocysts of jellyfish Cyanea nozakii Kishinouye on human hepatoma cells (Bel-7402, SMMC-7721) and human colon cancer cells (H630) was investigated first. Of the three kinds of cells, the venom had the strongest cytotoxicity on H630 cells with the 50% lethal concentration (IC(50)) of 5.1 μg/ml. However, the IC(50) on Bel-7402 and SMMC-7721 was approximate 17.9 and 24.3 μg/ml, respectively. The cytotoxicity of the venom was affected by pH, temperature and storage conditions. At the pH 4.5-8.5, the venom displayed obvious cytotoxicity and the percentage of survival was about 50%. When pre-incubated at temperatures over 60°C for as short as 10 min, the percentage of survival sharply improved from 4.6% up to 80%. The venom was stored in a more stable condition at -80°C and in lyophilized state compared to other storage conditions used in this study. Lactate dehydrogenase release assay performed on H630 cells indicated that exposure to the venom could result in damage to the cell membrane.

Large-scale sampling reveals the spatio-temporal distributions of the jellyfish Aurelia aurita and Cyanea capillata in the Irish Sea

At-sea distributions of large scyphozoan jellyfish across the Irish Sea were studied using visual surface counts from ships of opportunity. Thirty-seven surveys were conducted along two >100 km long transects between Ireland and the UK from April to September in 2009 and 2010. Five species were recorded but only Aurelia aurita and Cyanea capillata were frequently observed. The first formal description of the seasonal changes in the abundances and distributions of these two species in the study area is provided. The highest densities of these species were more likely to be found ~30 km offshore, but large aggregations were present both in coastal and offshore waters. Evidence for aggregations of medusae along physical discontinuities was provided by coupling jellyfish observations with simultaneous records of environmental parameters. The value of surveys from ships of opportunity as cost-effective semi-quantitative tools, to develop local knowledge on jellyfish abundance, distribution, and phenology is discussed.

Comparison of haemolytic activity of tentacle-only extract from jellyfish Cyanea capillata in diluted whole blood and erythrocyte suspension: Diluted whole blood is a valid test system for haemolysis study

In this paper, we utilized two different test systems to compare the haemolysis of tentacle-only extract (TOE) devoid of nematocysts from jellyfish Cyanea capillata, the 1% whole blood and 0.45% erythrocyte suspension approximately with the same erythrocyte concentration from the blood samples of sheep, rabbit, mouse, rat and human, respectively. Without exception, the haemolytic activity of TOE was dose-dependent in both test systems from all the five kinds of blood samples, while it was generally stronger in erythrocyte suspension than that in diluted whole blood at the relatively high concentration of TOE. When various aliquots of plasma were added into the erythrocyte suspension test system, the haemolytic activity of TOE was declined with the plasma quantity increasing, and dropped to about 20% at the presence of two aliquots of plasma. If serum albumin of 0.5 mg/ml, approximately the same albumin content in 1% whole blood, was added into the erythrocyte suspension test system instead, the haemolysis of TOE was similarly inhibited. The effects of GSH, ascorbic acid and protease inhibitor on the haemolytic activity of TOE were detected in the erythrocyte suspension and diluted whole blood simultaneously, and the test results were coincident between the two systems. These results suggested that the inconsistency of TOE haemolysis between the erythrocyte suspension and diluted whole blood is a universal occurrence in the mammals, and blood plasma plays a dose-dependent protective role against haemolysis which may be due to serum albumin. Diluted whole blood is a valid and convenient test system for haemolysis study in vitro.

The Acute Toxicity and Hematological Characterization of the Effects of Tentacle-Only Extract from the Jellyfish Cyanea capillata

To investigate the hematologic changes and the activities of jellyfish venoms other than hemolytic and cardiovascular toxicities, the acute toxicity of tentacle-only extract (TOE) from the jellyfish Cyanea capillata was observed in mice, and hematological indexes were examined in rats. The median lethal dose (LD50) of TOE was 4.25 mg/kg, and the acute toxicity involved both heart- and nervous system-related symptoms. Arterial blood gas indexes, including pH, PCO2, HCO3−, HCO3std, TCO2, BEecf and BE (B), decreased significantly. PO2 showed a slight increase, while SO2c (%) had no change at any time. Na+ and Ca2+ decreased, but K+ increased. Biochemical indexes, including LDH, CK, CK-MB, ALT, AST and sCr, significantly increased. Other biochemical indexes, including BUN and hemodiastase, remained normal. Lactic acid significantly increased, while glucose, Hct% and THbc showed slight temporary increases and then returned to normal. These results on the acute toxicity and hematological changes should improve our understanding of the in vivo pathophysiological effects of TOE from C. capillata and indicate that it may also have neurotoxicity, liver toxicity and muscular toxicity in addition to hemolytic and cardiovascular toxicities, but no kidney or pancreatic toxicity.

Two-step purification and in vitro characterization of a hemolysin from the venom of jellyfish Cyanea nozakii Kishinouye

Hemolysin is one of the most hazardous components in the venom of Cyanea nozakii Kishinouye. Here we describe the purification and in vitro characterization of the hemolysin, which we named CnPH. The CnPH was isolated by anion-exchange and size-exclusion chromatography from the nematocyst venom. Two protein bands with molecular masses of 20kDa, 60kDa respectively were shown in the reducing SDS–PAGE analysis of the CnPH. And Approximately 5μg/mL of the CnPH resulted in 50% hemolysis of the erythrocyte suspension. The hemolytic activity of the CnPH was both temperature and pH dependent. Moreover, it was significantly inhibited in the presence of divalent metal cations, including Cu2+, Mg2+, Mn2+, Zn2+ and Ca2+, but enhanced in the presence of EDTA. However, how CnPH performs its hemolytic activity is not yet clear, therefore the mechanism of the hemolytic activity of the CnPH is under research.

Intraguild predation between the native North Sea jellyfish Cyanea capillata and the invasive ctenophore Mnemiopsis leidyi

Native predators may be important in controlling populations of the invasive ctenophore Mnemiopsis leidyi. We experimentally quantified predation rates of the scyphozoan Cyanea capillata on M. leidyi. Cyanea capillata with 74.8 ± 13.3 mm diameter feeding on 28.7 ± 5.5 mm (total length) M. leidyi in 380 L experimental containers had a maximum clearance rate of 40 ± 11 L ind.−1 h−1 (AV ± SD). Ingestion rate increased linearly through the observed range of prey concentrations (5.3–42.1 m−3). Over 90% of encounters ended in escape, but the escaping M. leidyi suffered damage in the process.

남해 연안에 분포하는 해파리(Aurelia aurita, Cyanea nozakii)의 복수 개체에 의한 음향 표적강도 특성

Understanding the abundance and distribution of massive jellyfish is necessary to forecast where or when their blooms will happen. The acoustic technique is one of the most useful methods of obtaining information if the acoustic characteristics of the targets are known. This study was conducted to determine the acoustic target strength (TS, dB) of two jellyfish species, Aurelia aurita and Cyanea nozakii, in the southern coast of Korea. For the ex situ measurements, 120, 200, and 420 kHz split beam transducers were used, and jellyfish with various bell lengths were arranged to prepare multiple jellyfish. Under 2 vertical individuals, the mean TS for multiple A. aurita at 120, 200, and 420 kHz was -72.7, -71.7, and -68.2 dB, respectively. In the case of 5 vertical individuals, the mean TS of the species was -71.3, -68.2, and -62.0 dB. Finally, the mean TS of C. nozakii at 120, 200, and 200 kHz was -62.0, -60.3, and -58.2 dB under 2 individuals and -58.1, -57.4, and -54.0 dB under 4 individuals, respectively. For both species, higher numbers of jellyfish resulted in a higher TS. In addition, higher frequencies were associated with a higher TS for the same jellyfish. These TS results for two species can be used as essential data for the acoustic detection of jellyfish in an open ocean or coastal area.

Selective toxin–lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin–membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore ® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.

Jellyfish blooms in China: Dominant species, causes and consequences

Three jellyfish species, Aurelia aurita, Cyanea nozakii and Nemopilema nomurai, form large blooms in Chinese seas. We report on the distribution and increasing incidence of jellyfish blooms and their consequences in Chinese coastal seas and analyze their relationship to anthropogenically derived changes to the environment in order to determine the possible causes. A. aurita, C. nozakii and N. nomurai form blooms in the temperate Chinese seas including the northern East China Sea, Yellow Sea and Bohai Sea. N. nomurai forms offshore blooms while the other two species bloom mainly in inshore areas. Eutrophication, overfishing, habitat modification for aquaculture and climate change are all possible contributory factors facilitating plausible mechanisms for the proliferation of jellyfish blooms. In the absence of improvement in coastal marine ecosystem health, jellyfish blooms could be sustained and may even spread from the locations in which they now occur.

Partial characterization of the hemolytic activity of the nematocyst venom from the jellyfish Cyanea nozakii Kishinouye

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the hemolytic activity of Cyanea nozakii Kishinouye nematocyst venom on chicken erythrocytes. Venom extract caused a significant concentration-dependent hemolytic effect. The extract could retain its activity at −80 °C but was unstable when kept at 4 °C and −20 °C for 2 days. The hemolytic activity was inhibited by heating within the range of 37–100 °C. The extract was active over a pH range of 5.0–8.63 and the pH optima for the extract was 7.8. Incubation of the venom with sphingomyelin specially inhibited hemolytic activity by up to 70%. Cu 2+ and Mn 2+ greatly reduced the hemolytic activity while Mg 2+, Sr 2+ and Ba 2+ produced a relatively low inhibiting effect on the hemolytic activity. Treatment with Ca 2+ induced a concentration-dependent increase in the hemolytic activity. In the presence of 5 mM EDTA, all the hemolytic activity was lost, however, the venom containing 1.5 mM EDTA was stable in the long-term storage. PLA 2 activity was also found in the nematocyst venom of C. nozakii. These characteristics provide us a fundamental knowledge in the C. nozakii nematocyst venom which would benefit future research.

In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata

To approach the real haemolytic process of jellyfish toxins, both in vitro and in vivo haemolysis of tentacle-only extract (TOE) from jellyfish Cyanea capillata has been studied. Dose–response curves of the haemolytic activity of TOE in vitro were sigmoid shaped in both erythrocyte suspension and diluted whole blood, with the former more sensitive to TOE. The in vivo haemolysis increased sharply in the first 10min and was followed by a gradual increase in the following 3h, with increasing blood potassium and lactic acid accordingly. SC5b-9 complexes were significantly up-regulated in vitro, but not in vivo. These results showed that the haemolysis of TOE in diluted whole blood and in vivo is not totally consistent with that in the erythrocyte suspension, and blood plasma might play a protective role against haemolysis. Thus we suggested that erythrocyte suspension can be used to test the damage of toxin on erythrocyte membrane, while the diluted whole blood may be more suitable to test the haemolysis of toxins.