A pharmacological and biochemical examination of the geographical variation of Chironex fleckeri venom

Toxicology Letters 192 (2010) 419–424

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A pharmacological and biochemical examination of the geographical variation of Chironex fleckeri venom Kelly L. Winter a , Geoffrey K. Isbister a,b , Sheena McGowan c , Nicki Konstantakopoulos a , Jamie E. Seymour d , Wayne C. Hodgson a,∗ a

Monash Venom Group, Department of Pharmacology, Monash University, Victoria, Australia Tropical Toxicology Unit, Menzies School of Health Research, Northern Territory, Australia Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia d School of Marine and Tropical Biology, James Cook University, Queensland, Australia b c

a r t i c l e

i n f o

Article history: Received 15 October 2009 Received in revised form 19 November 2009 Accepted 22 November 2009 Available online 27 November 2009 Keywords: Venom Box jellyfish Geographical variation Cytotoxic Cardiovascular Envenoming

a b s t r a c t Chironex fleckeri (box jellyfish) are found in the northern tropical waters of Australia. Although C. fleckeri have a wide geographical distribution and are able to swim large distances, adults tend to stay in small restricted areas. Clinical data shows that deaths from envenoming have not been recorded in Western Australia, yet numerous fatalities have occurred in Northern Territory and Queensland waters. One explanation for this discrepancy is a geographical variation in venom composition. This study examined the pharmacological and biochemical profiles of C. fleckeri venom from different geographical locations and seasons. Venoms were screened for cytotoxicity using a rat aortic smooth muscle cell line (A7r5). While all venoms caused concentration-dependent cytotoxicity, differences were seen in the potency of venoms from Mission Beach and Weipa, when collected in different seasons, as indicated by IC50 values. Similarly venoms collected within the same season, from different locations around Australia, displayed marked differences in venom composition as shown by size exclusion HPLC and SDS-PAGE profiles which indicated the absence or reduced quantity of ‘peaks’ in some venoms. Based on IC50 data obtained from the cell assay, the effects of the most potent (i.e. from Weipa in 2006) and the least potent (i.e. from Broome in 2007) venoms were examined in anesthetised rats. Both venoms at 10 ␮g/kg (i.v.) caused a transient hypertensive phase followed by cardiovascular collapse. However, at 4 ␮g/kg (i.v.) venom from Weipa 2006 caused a transient hypertensive phase followed by a transient decrease in MAP while venom from Broome 2007 only caused a small transient increase in MAP. This study demonstrates that there is considerable geographical variation in the composition of C. fleckeri venoms which is most distinct between specimens from western and eastern Australia and may explain the geographical variation in reported deaths. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Chironex fleckeri (box jellyfish) are found in the northern tropical waters of Australia. Systemic envenoming may result in the rapid onset of life-threatening hypotension and cardiac arrest (Currie and Jacups, 2005). Recent animal studies indicate that death is most likely to be due to lethal toxins acting on the cardiovascular system (Ramasamy et al., 2004, 2005; Winter et al., 2007a,b, 2009). Variation in the composition of venom has been established for many species of venomous animals and includes geographical, intergenic, interfamilial, inter- and intra-species, and even individual variation (Chippaux et al., 1991; Minton and Weinstein, 1986; Wickramaratna et al., 2003; Yang et al., 1991). In particu-

∗ Corresponding author. Tel.: +61 3 99054861; fax: +61 3 99052547. E-mail address: [email protected] (W.C. Hodgson). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.11.019

lar, ontogenic differences have been reported in the composition of C. fleckeri nematocysts as the jellyfish mature (Carrette et al., 2002; Kintner et al., 2005). This is associated with a change in their preferred diet (i.e. invertebrate versus vertebrate prey source) and a change in the number and type of mastigaphores (i.e. venom containing nematocysts) on their tentacles (Carrette et al., 2002; Kintner et al., 2005). This is in contrast to Chiropsella bronzie (previously known as Chiropsalmus sp.) which does not appear to change nematocyst ratio or food preference with maturation (Carrette et al., 2002). However, similar venom composition changes have been reported for the ‘Irukandji’ jellyfish Carukia barnesi (Underwood and Seymour, 2007). In this later study, it was also shown that tentacle and bell wart structure showed developmental changes (Underwood and Seymour, 2007). Collection records over a ten-year period suggest that size at maturity in C. fleckeri is directly correlated with geographical location. Specimens from the western coast of Australia are small

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compared with specimens from Darwin which are small compared to those collected from the coastline of Queensland (Seymour, personal communication). Despite a wide geographical distribution, all deaths from C. fleckeri envenoming have occurred along the Northern Territory or Queensland coasts (Currie and Jacups, 2005). One possible explanation for this discrepancy would be geographical variation in venom composition resulting in a reduction in the ‘lethality’ of venom from the specimens found along the west coast. It would therefore be useful to understand any geographical or seasonal variation in venom composition, particularly the variation in lethal effects. The aim of the present study was to elucidate whether there are any differences in the biochemical composition and pharmacological activity of venom from specimens of C. fleckeri collected from different geographical locations or from the same geographical location but from different seasons. 2. Materials and methods

the protein content. Samples were read at 562 nm in a Fusion ␣ microplate reader (Packard Bioscience). 2.3. Drugs and solutions The following drugs and solutions were used: 30% acrylamide-base (BioRad); ␤-mercaptoethanol (Bio-Rad); bromophenol blue (Sigma), Coomassie G250 (Bio-Rad), fetal calf serum (CSL Ltd., Melbourne, Australia), Dulbecco’s minimal media high glucose (Sigma), phosphate buffered saline (PBS) (Sigma), heparin sodium (porcine mucosa) (Sigma); Laemmeli’s sample buffer (Bio-Rad); Pierce BCA protein assay kit (Pierce Biotechnology, IL, USA); Penicillin/streptomycin (Trace Scientific, Melbourne, Australia); trypsin (Trace Scientific, Melbourne, Australia); sodium dodecyl sulphate (SDS; Bio-Rad); sodium pentobarbital (Jurox Pty Ltd); Cell titer 96 Aqueous One Solution Cell Proliferation Assay (MTS assay) (Promega,Melbourne, Australia), TEMED (N,N,N ,N -tetramethylethylenediamine, Sigma); trisma–hydrochloride (Sigma). Unless otherwise indicated all drugs were made up in distilled water as were subsequent dilutions. 2.4. Cells The rat aorta smooth muscle cell line, A7r5, was purchased from the American Type Culture Collection (ATCC Virginia, USA).

2.1. Collection of jellyfish Mature specimens of C. fleckeri were collected from the following sites in the years indicated: Weipa (north Queensland; November/December of 2006 and 2007); Mission Beach (north Queensland; December 2004/January 2005 and January 2007); Karumbra (north Queensland; December 2007) and Broome (north Western Australia, December 2007). Tentacle extract, devoid of nematocysts, was obtained from Weipa specimens (November/December 2007) (Fig. 1). For all specimens, tentacles were removed and stored immediately in sea water. The nematocysts were isolated from the tentacles as described by Bloom et al. (1998). In brief, the tentacles were stored and refrigerated in sea water for four days. Each of the containers was vigorously shaken daily. Nematocysts were separated from the tentacles by filtering the sea water in which they were contained through a fine sieve. The filtrate was washed again with fresh sea water and allowed to settle for 3 h. The sediment (i.e. nematocysts) was then lyophilised and stored at −20 ◦ C until use. 2.2. Venom extraction and preparation Using a technique developed by Carrette and Seymour (2004), venom was extracted from the freeze-dried nematocysts. In brief, nematocysts were weighed into aliquots of approximately 20 mg in screw top vials. Glass beads (approximately 8000, 0.5 mm in diameter) and 1.5 ml of distilled water were added to the vials. Samples were then shaken four times in a mini bead mill at 5000 rpm for 10 s, and placed on ice for 1 min in between each cycle. The supernatant (i.e. venom) was separated from the pellet (i.e. nematocysts debris) using a pipette and stored on ice. It was then centrifuged at 4 ◦ C for 5 min at 12,000 rpm. If further purification from nematocyst debris was required (e.g. gel filtration) the filtrate was filtered through a micro (0.2 ␮m) syringe filter. A Pierce BCA protein assay kit was used to determine

2.4.1. Cell culture A7r5 cells were cultured in 75 cm2 flasks (Greiner Bio-One; Frickenhausen, Germany) using the following culture medium, Dulbecco’s minimal medium, high glucose, supplemented with 5% (v/v) FCS and 1% (v/v) penicillin/streptomycin (media). Cells were incubated at 37 ◦ C in an atmosphere of 5% CO2 and the medium was replaced every second day until cells were approximately 80% confluent (assessed by eye using light microscopy). 2.4.2. Standard procedure Cells were lifted using trypsin and a cell count was performed using a haemocytometer. Cells were resuspended in media and seeded at 50,000 cells per well in a 96-well microtitre tissue culture plate (Greiner Bio-One; Frickenhausen, Germany) and incubated for 48 h at 37 ◦ C in an atmosphere of 5% CO2 . Media were removed from wells and the plate was washed three times with pre-warmed (37 ◦ C) PBS. Venom stock was diluted to a final concentration of 2 ␮g/ml and then serially diluted 1.5-fold a further fifteen times (2–0.004 ␮g/ml). Treatment medium (100 ␮l) was added to wells in the plate in quadruplicate. Control (cells and media, with no venom) and media blank (no cells) were also run in parallel. The plate was incubated at 37 ◦ C in an atmosphere of 5% CO2 for a further 24 h. The plate was removed from the incubator and MTS solution (20 ␮l) added to each well. The plate was then incubated at 37 ◦ C in an atmosphere of 5% CO2 for 3 h, to allow the development of colour to occur. Absorbance was then measured at 492 nm on a fusion ␣ plate reader (Packard Bioscience; CT, USA). 2.5. Anesthetised rat preparation All procedures were approved by the School of Biomedical Sciences (SOBS)B Animal Ethics Committee, Monash University. Male Sprague–Dawley rats were anesthetised with pentobarbitone sodium (60–100 mg/kg; i.p.) which was supplemented as required. A midline incision was made in the cervical region and cannulae inserted into the trachea for artificial respiration if needed, and jugular vein and carotid artery, for administration of drugs/venom and measurement of blood pressure, respectively. Arterial blood pressure was measured using a Gould Statham P23 pressure transducer connected to a PowerLab system (ADInstruments). 2.6. Size exclusion chromatography Venom was prepared as previously described (Section 2.2). Size exclusion chromatography was run on a high performance liquid chromatography system (Shimadzu LC10AT Class VP System). Phosphate buffered saline (150 mM NaCl; 2 mM EDTA; pH 7.4) was used as the mobile phase. Venom samples of 250 ␮g total protein was applied to a Superdex S-200 column (13 ␮M, 10 mm × 300 mm) and equilibrated with the appropriate buffer. Samples were eluted at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm. 2.7. SDS-PAGE

Fig. 1. Map of Australia indicating the locations from where specimens were collected.

C. fleckeri venom samples were prepared, as described above (Section 2.2), for electrophoresis prior to the addition of the reducing agent (5% ␤-mercaptoethanol in Laemmli’s sample buffer (62.5 mM Trisma–HCl, 25% glycerol, 2% sodium dodecyl sulphate (SDS), 0.01% bromophenol blue)). Electrophoresis was performed according to Laemmli (1970) using 12% polyacrylamide gel with 4% stacking gel. Samples were incubated at 95 ◦ C for 5 min then cooled to room temperature on ice. Seven (7) ␮g of venom (total protein load) was loaded on the gel and electrophoresed at room temperature for 45 min at 200 V. The molecular weight standard (Kalieidoscope Pre-stained standards, Bio-Rad) was also run in parallel. Gels were placed in Coomassie G250 brilliant blue solution and left incubating for 20 min on an orbital

K.L. Winter et al. / Toxicology Letters 192 (2010) 419–424 shaker for staining. Gels were transferred into destain solution (10% acetic acid, 40% methanol) which was changed regularly until background was reduced and protein bands could be visualised clearly. The gel image was captured utilising Gel Doc Imaging System (UVP, Cambridge). 2.8. Statistics One-way analysis of variance (ANOVA) followed by a Bonferonni post hoc test was used to compare treatments. When one-way ANOVA’s were unable to be preformed unpaired t-tests were used to compare treatments. In all cases, statistical significance was indicated by P < 0.05. All values were expressed as mean ± standard error of the mean.

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Table 1 IC50 values for C. fleckeri venom from different geographical locations, including year of collection, on cell viability. Collection site

IC50 (␮g/ml)

Weipa 2006* Weipa 2007* Mission Beach 2004/2005* Mission Beach 2007* Karumbra 2007* Broome 2007

0.03 0.04 0.1 0.05 0.2 0.7

* Significantly different than venom from least potent venom (i.e. Broome 2007), P < 0.05, One-way ANOVA.

3. Results 3.2. Anesthetised rat 3.1. Effect of venom in cells Incubation of A7r5 cells with C. fleckeri venom (0.004–2 ␮g/ml) from each of the geographical locations and different seasons resulted in a concentration-dependent inhibition of cell proliferation (Fig. 2a). The IC50 value of venom from Broome 2007 was significantly lower than all other venoms (P < 0.05, One-way ANOVA, Table 1) indicating that the venom was less cytotoxic. Tentacle material, devoid of nematocysts, had no significant inhibitory effect on cell viability (Fig. 2c). For comparative analysis of geographical variation, venoms collected in the same season from different locations were examined. While all venoms caused concentration-dependent inhibition of cell proliferation, venoms from Weipa 2007, Mission Beach 2007 and Karumbra 2007 were significantly more potent than the venom from Broome 2007 as indicated by IC50 values (P < 0.05, One-way ANOVA, Table 1, Fig. 2d).

Based on IC50 values from the cell-based assay, the most potent venom (i.e. Weipa 2006), least potent venom (i.e. Broome 2006) and tentacle extract were screened in the anesthetised rat to examine their effects on blood pressure. Venom from Weipa (2006; 10 ␮g/kg, i.v.) caused an initial pressor response (43 ± 3 mmHg, n = 3, Fig. 3a) followed by cardiovascular collapse. Similarly, venom from Broome (2007; 10 ␮g/kg, i.v.) caused an initial pressor response (38 ± 2 mmHg, n = 3, Fig. 3b) followed by cardiovascular collapse. There was no significant difference in the initial pressor response when comparing venom from Weipa (2006) and Broome (2007) at 10 ␮g/kg (i.v.) and both venoms caused cardiovascular collapse. In contrast, at a lower dose (i.e. 4 ␮g/kg, i.v.), venom from Weipa (2006) caused an initial pressor response (25 ± 6 mmHg, n = 3, Fig. 3b) followed by a decrease in MAP (−77 ± 13 mmHg, n = 3) that gradually returned to basal levels. Venom from Broome (2007) caused an initial pressor response (10 ± 0.5 mmHg, n = 3, Fig. 3c)

Fig. 2. Effect of C. fleckeri venom (0.004–2 ␮g/ml) from (a) Weipa (2006) and Weipa (2007) (b) Mission Beach (MB; 2004/2005) and Mission Beach (MB; 2007), and (c) Broome (2007) and Karumbra (2007) on cell viability. (d) Comparative effect on cell viability of C. fleckeri venom (0.004–2 ␮g/ml) from samples that were collected in 2007 from Weipa, Mission Beach, Broome and Karumbra.

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Fig. 3. (a) Effect of C. fleckeri venom (10 ␮g/kg, i.v.) from Weipa (2006), Karumbra (2007) or tentacle extract on the initial pressor response on the mean arterial pressure (MAP) of anesthetised rats. *P < 0.05, significantly different from venom. Traces showing the effect of C. fleckeri venom (4 ␮g/kg, i.v.) from (b) Weipa (2006) or (c) Broome (2007) on the mean arterial pressure (MAP) of the anesthetised rat.

and a minimal decrease in MAP after the initial hypertensive phase (−10 ± 1 mmHg, n = 3). Neither venom caused cardiovascular collapse at the lower concentration. Tentacle extract (10 ␮g/kg, i.v.) caused no significant change in MAP (3 ± 1 mmHg, n = 3, one-way ANOVA, Fig. 3c) in the anesthetised rat. 3.3. Size exclusion chromatography Size exclusion chromatography profiles of venoms were obtained to compare venom composition. Venoms collected from Weipa (2006) and Weipa (2007) displayed similar profiles however the venom collected at Weipa in 2006 contained considerably less of peak 1 in terms of total venom protein (Fig. 4a). Further to this, we observed that peak 5 was a single peak in venom from Weipa (2006) however in venom from Weipa (2007) this was clearly two separate peaks (Fig. 4a). Similarly, venom collected from Mission Beach (2007) showed this change in peak 5 (Fig. 4b), while venom

Fig. 4. Gel filtration profiles of C. fleckeri venom, numbers represent fractions identified. (a) Weipa (2006) and Weipa (2007), (b) Mission Beach (MB; 2004/2005) and Mission Beach (MB; 2007), (c) Broome (2007), Karumbra (2007) and tentacle extract. (d) Gel filtration profiles of C. fleckeri venom collected in 2007 from Weipa, Mission Beach, Broome and Karumbra.

from the Mission Beach (2004/2005) only had a single peak 5. Mission Beach (2004/2005) showed further changes with the complete loss of peak 3 and profile shifts in the elution times of peaks 4, 5 and 6. When comparing venoms collected in the same year from different geographical locations, venom from Karumbra (2007) showed a marked increase in the amount of peak 1 present in the venom as well as a marked difference in the height and width of peak 3. Peak 4 was smaller and peak 5 was seen as a single peak. Venom from Mission Beach (2007) and Weipa (2007) showed almost identical traces with only a small difference in peak heights seen in fractions 3, 4, 5 and 6 (Fig. 4c). Venom from Broome (2007) did show changes in the peak heights and width across the whole profile with peaks 2, 3 and 6 showing marked reductions in peak height. Peak 4 is completely absent in the profile and peak 5 is seen as a single peak (Fig. 4c). The sample of tentacle material, devoid of nematocysts, had a completely difference profile from nematocyst-derived venom, confirming previously published observations (Ramasamy

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Fig. 5. SDS-PAGE analysis of the venoms on 12% polyacrylamide gel.

et al., 2005) that venom and tentacle extract are different in their proteinaceous components. 3.4. SDS-PAGE analysis SDS-PAGE analysis of the venoms shows that there were differences in both the abundance and occurrence of proteins. The most dominant protein bands in all samples occurred at approximately 40 kDa. However, the samples from Mission Beach (2004/2005) and Broome (2007) show variances in the intensity of these major bands as well as the abundance of other proteins. There are also notable differences in the presence and intensity of bands found at approximately 60 and 75 kDa. Similarly tentacle extract shows that it is devoid of the major dominant bands which are present in the profile of the other venoms showing a profile that is vastly different to the whole venoms (Fig. 5). 4. Discussion In the present study, we have shown that variation in venom composition occurs in the venoms of the Australian box jellyfish C. fleckeri. It appears that this variation is clinically important because there is variation in the effects of the different venoms on a cell-based assay and differences in the cardiovascular effects as reflected by blood pressure changes in an in vivo animal model. These results also support the pattern of mortality over the last 100 years where most deaths have occurred in far north Queensland and the Northern Territory (Currie and Jacups, 2005), and none in northern Western Australia. Unfortunately we did not have venom to compare from the Northern Territory. Venom collected in the same season (i.e. 2007) from different parts of Northern Australia differed significantly in their effects on cell viability and in vivo cardiovascular activity. The most potent venoms were from far north Queensland (Weipa and Mission Beach) compared to venom from specimens collected in north Western Australia (Broome). These differences were also evident on the SDS-PAGE and size exclusion HPLC profiles that where there were a number of differences in the composition of the venoms.

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Although there were obvious differences between the HPLC traces of the venoms from the same location, but different seasons, and different locations within north Queensland, there was not a significant difference in the activity of these venoms. Although the venom profiles were similar there were marked differences in peak heights and widths from the size exclusion profiles and in the intensities of the protein bands see in the SDS-PAGE profiles. This suggests that the differences seen in the SDS-PAGE and size exclusion chromatography studies are not solely the components in the venoms that affect cell viability. The use of a cell-based assay enables the venom to be studied over varying concentrations in the same assay/experiment which is not possible in whole animal studies (Konstantakopoulos et al., 2009). This allowed us to rapidly compare a large number of venoms at different concentrations to find potentially minor differences in potency. However, each cell-based assay is limited to a single cell type with a single end point and may not reflect toxin-mediated effects in vivo. We therefore undertook whole animal experiments to determine if the differences found with the cell-based assay might be clinically important. The most potent (Weipa, 2006) and least potent (Broome, 2007) venoms were examined in the anesthetised rat confirming the results of the cell-based assay and suggesting that venom variation includes the component responsible for mediating cardiovascular collapse. It is also possible that the size of the specimens is related to the differences in venom potency. Animals collected from Broome are significantly smaller than those collected in Queensland. As has been previously shown (Carrette et al., 2002; Kintner et al., 2005), the number of nematocysts (i.e. mastigaphores) that contain the venom, changes as C. fleckeri mature and increase in size. This is associated with a change in prey preference which is thought to drive a shift in nematocyst ratio as more venom is needed to neutralise vertebrate prey (Carrette et al., 2002; Kintner et al., 2005). The difference in venom composition and potency may account for the differences seen in the ability of CSL box jellyfish antivenom to neutralise venoms effects. In a recent study we demonstrated that at an appropriately high dose antivenom was not able to prevent cardiovascular collapse and the initial transient hypertension in 100% of rats (Winter et al., 2009). However, in a previous study by our group antivenom was able to prevent cardiovascular collapse in 40% of the rats, and was not able to reduce the transient hypertensive phase (Ramasamy et al., 2004). Venom used in the earlier study (Ramasamy et al., 2004) was from Mission Beach (2004/2005) compared to venom from Weipa 2006 in the more recent study (Winter et al., 2009). Hence the difference in these studies is more likely due to the variation in the venom samples rather than the effectiveness of the antivenom. However, results from the SDS-PAGE and size exclusion profiles suggest that the variation seen in the venoms is not restricted to the lethal component(s), with the most notable differences occurring between Queensland and Western Australian animals. Although C. fleckeri is capable of travelling large distances, mature adults seem to stay in small restricted areas (Gordon and Seymour, 2008) thus resulting in isolated populations. The variation in venom components may reflect differences in feeding ecology between the populations, but this hypothesis requires further examination. Conflict of interest None. Acknowledgements This research was funded in part by an NHMRC Project Grant (ID436606) awarded to WCH, GKI and JES, and funding to JES

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from the Irukandji Task force. KLW is funded by an NHMRC Dora Lush and a Monash University Postgraduate Publications Award. GKI is funded by an NHMRC Clinical Career Development Award (ID300785). Thanks also go to Avril Underwood for help in collecting venom samples. References Bloom, D.A., Burnett, J.W., Alderslade, P., 1998. Partial purification of box jellyfish (Chironex fleckeri) nematocyst venom isolated at the beachside. Toxicon 36, 1075–1085. Carrette, T., Alderslade, P., Seymour, J., 2002. Nematocyst ratio and prey in two Australian cubomedusans, Chironex fleckeri and Chiropsalmus sp. Toxicon 40, 1547–1551. Carrette, T., Seymour, J.E., 2004. A rapid and repeatable method for venom extraction from Cubozoan nematocysts. Toxicon 44, 135–139. Chippaux, J.P., White, J., Williams, V., 1991. Snake venom variability: methods of study results and interpretation. Toxicon 29, 1279–1303. Currie, B., Jacups, S.P., 2005. Prospective study of Chironex fleckeri and other box jellyfish stings in the “Top End” of Australia’s Northern Territory. Med. J. Aust. 183, 631–636. Gordon, M.R., Seymour, J.E., 2008. Quantifying movement of the tropical Australian cubozoan Chironex fleckeri using acoustic telemetry. Hydrobiologia 616, 87–97. Kintner, A., Seymour, J.E., Edwards, S.L., 2005. Variation in lethality and effects of two Australian chirodropid jellyfish venoms in fish. Toxicon 46, 699–708. Konstantakopoulos, N., Isbister, G.K., Seymour, J.E., Hodgson, W.C., 2009. A cell-based assay for screening of antidotes to, and antivenom against Chironex fleckeri (box jellyfish) venom. J. Pharmacol. Toxicol. Methods 59, 166–170.

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