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In this paper Atlantic blue marlin (ABM) optimization algorithm, Boops optimization (BO) algorithm, Chironex fleckeri search optimization (CSO) algorithm, general practitioner-sick person (PS) optimization algorithm are applied for solving factual power loss reduction problem. Natural actions of Atlantic blue marlin are emulated to design the Atlantic blue marlin (ABM) optimization algorithm and populace in the examination space is capriciously stimulated. Boops optimization (BO) algorithm is designed by imitating the stalking physiognomies of Boops. CSO is based on the drive and search behavior of Chironex fleckeri. A general practitioner will treat a sick person with various procedures which have been imitated to model the Projected PS algorithm. Inoculation, medicine and operation are the procedures considered in the PS algorithm. Atlantic blue marlin (ABM) optimization algorithm, Boops optimization (BO) algorithm, Chironex fleckeri search optimization (CSO) algorithm, general practitioner – sick person (PS) optimization algorithm validated in IEEE 57, 300 systems and 220 KV network. Factual power loss lessening, power divergence restraining, and power constancy index amplification have been attained. [ABSTRACT FROM AUTHOR]

Introduction: Box jellyfish stings are painful and may be life-threatening. The venom of Chironex fleckeri contains a variety of bioactive proteins as well as two of the most abundant proteins, namely CfTX-1 and CfTX-2 which cannot be isolated easily using electrophoresis or chromatography techniques. Recombinant expression technology may offer an alternative to the isolation of native C.fleckeri venom protein. This study aimed at expressing C-CfTX1-STxB protein in Escherichia coli and assessing its antigenicity in Syrian mice. Materials & Methods: Synthesis of the artificial CfTX1complete gene was prepared in plasmid pUC57. The C-cftx1 was cloned using a polymerase chain reaction (PCR) and subcloned with BamHI and SalI restriction enzyme sites in pET28a-stxB expression vector and transformed into E.coli. Gene expression was artificially induced by Isopropyl β- d-1-thiogalactopyranoside. After the purification of the protein and its injection into the Syrian mice, the amount of produced antibody was measured in the serum. The rats were also challenged by the venom of the jellyfish (i.e., Rhopilema nomadic). Findings: In this experimental study, the C-CfTX1-STxB gene was cloned in the expression vector pET28a (+), sequenced by PCR, and analyzed by enzymatic analysis. Moreover, the produced recombinant protein was confirmed by Western blotting. The produced antibody in the serum was quantified using an enzyme-linked immunosorbent assay. Discussion & Conclusions: After 60 days, the immunized mice tolerated 50x LD50 of jellyfish venom. Considering the ineffectiveness of cardiotoxicity and neurotoxicity of the recombinant protein, this produced protein can be suggested as a jellyfish venom vaccine candidate in Syrian mice or at a later stage of a clinical trial in humans.

Lethal jellyfish envenoming is a very rare event. Two illustrative cases of fatalities following envenomation from the multi‐tentacled box jellyfish, Chironex fleckeri, are reported that involved a six‐year‐old boy and a three‐year‐old girl, both of whom had been playing in shallow beach waters in remote Northern Territory communities (Australia). A recommended approach to autopsy in suspected cases is described, which should include examination of the clothing and skin surfaces for the fine tentacles of Chironex fleckeri. In addition, the combined length and maximum width of tentacle marks should be measured/calculated and collection of unfixed tentacles with adhesive tape onto a glass slide should be undertaken to check for nematocysts. Such rare cases show that children are at higher risk of a lethal outcome, particularly if they are resident in remote coastal tropical communities. [ABSTRACT FROM AUTHOR]

Background and Objectives: Chironex. fleckeri is the most toxic and dangerous marine species. CfTX-2 is one of the antigenic proteins of jellyfish venom that has been suggested for stimulation of the immune system, for treatment, and as a candidate for vaccine. The purpose of this study was designing, optimization, synthesis, and expression of the N-CfTX-2 gene in E. coli and investigation of the production of antibodies and immunization in mice. Methods: In this experimental study, the complete sequence of N-CfTX-2 gene was obtained from NCBI (with accession number AFQ00677.1). After optimizing the codon of E. coli host, other bioinformatics studies needed for better gene expression were performed, such as protein stability and mRNA structure. The gene sequence was ordered for synthesis and replication in the pET21a (+) vector. The recombinant vector was transformed into E. coli BL21 and protein expression was induced by IPTG. After purification of the protein, the obtained antigen was injected into the mice in four steps and the amount of antibody produced in their serum, was measured. Then, the produced antibody was isolated from the serum and confirmed by ELISA assay. Sixty days after the first injection, the mice were challenged by the Chironex fleckeri jellyfish venom. Results: The N-CfTX-2 gene sequence cloned in the expression vector pET21a (+) was confirmed by PCR, and the produced recombinant protein was confirmed by SDS-PAGE and Western blot. Moreover, the immunized mice tolerated jellyfish venom 50 x LD50. Given the antigenic properties of N-CfTX-2 and antibody production in laboratory animal, this protein can be used as a vaccine candidate in future studies.

Background: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX-1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice. Methods: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata. Results: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD50[1] of jellyfish venom. Conclusion: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.

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.

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. • Venom extracted via ethanol had a different venom profile than bead mill extraction. • Venom extracted via ethanol lacked cardiotoxicity and repeatability. • Venom yield was lower in ethanol extracted venom. • Venom profile was less complex in ethanol extracted venom. [ABSTRACT FROM AUTHOR]

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or “venom” recovery, we utilized both top-down and bottom-up transcriptomics–proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

Background The effectiveness of the currently available box jellyfish (Chironex fleckeri) antivenom has been subject of debate for many years. To assess whether the box jellyfish antivenom has the ability to attenuate venom-induced damage at cellular level, the present study analyzed the dose and time dependence of the antivenom in a cell-based assay. Methods Different doses of antivenom were added to venom and subsequently administered to cells and the cell index was measured using xCelligence Technology (ACEA Biosciences). Similarly, antivenom and venom were incubated over different time periods and cell survival measured as stated above. For both experiments, the cell index was plotted as a measure of cell survival against the dose or incubation time and significance was determined with the use of a one-way ANOVA with a LSD post hoc test. Results Increasing concentrations of antivenom significantly augmented cell survival, with a concentration of approximately five times the currently recommended dose for human envenomation, causing the first significant increase in cell survival compared venom alone. Further, cell survival improved with increasing incubation time of venom and antivenom prior to addition to the cells, indicating that box jellyfish antivenom requires approximately 70 minutes to neutralize C. fleckeri venom. Conclusion The presented results suggest that the currently recommended dose of antivenom requires adjustment, and more importantly, a human trial to test the effects of higher concentrations is also necessary. Further, antivenom has delayed neutralizing effects (i.e. after 70 minutes) which underlines the eminence of immediate and prolonged cardiopulmonary resuscitation in victims suffering from a C. fleckeri venom-induced cardiovascular collapse.

Cnidarian envenomations are the leading cause of severe and lethal human sting injuries from marine life. The total amount of venom discharged into sting-site tissues, sometimes referred to as "venom load", has been previously shown to correlate with tentacle contact length and sequelae severity. Since <1% of cnidae discharge upon initial tentacle contact, effective and safe removal of adherent tentacles is of paramount importance in the management of life-threatening cubozoan stings. We evaluated whether common rinse solutions or scraping increased venom load as measured in a direct functional assay of venom activity (hemolysis). Scraping significantly increased hemolysis by increasing cnidae discharge. For Alatina alata, increases did not occur if the tentacles were first doused with vinegar or if heat was applied. However, in Chironex fleckeri, vinegar dousing and heat treatment were less effective, and the best outcomes occurred with the use of venom-inhibiting technologies (Sting No More® products). Seawater rinsing, considered a "no-harm" alternative, significantly increased venom load. The application of ice severely exacerbated A. alata stings, but had a less pronounced effect on C. fleckeri stings, while heat application markedly reduced hemolysis for both species. Our results do not support scraping or seawater rinsing to remove adherent tentacles. [ABSTRACT FROM AUTHOR]

Extracted venoms from two species of cubozoan, Chironex fleckeri (C. fleckeri)and Chiropsalmus sp., were injected into the freshwater crayfish Cherax quadricarinatus (C. quadricarinatus). By means of a Vascular Doppler, the cardiac parameters heart rate and contraction amplitude were recorded, with the theoretical total heart output calculated from these two recordings. Cherax quadricarinatus reflects prey items utilized by these two species in the wild and so comparisons of venom action on this animal have greater ecological relevance than those of previous mammalian models used. While the results of this study reflect previous research showing that the overall potency of C. fleckeri venom is greater than that of Chiropsalmus sp. venom, the action of the venoms on the heart muscle is found to be different. Chironex fleckeri,which has the most potent venom out of these two species, is actually less effective in reducing cardiac activity than Chiropsalmus sp., which may be a direct reflection of the variation in primary prey utilized by each species.

The venoms of jellyfish cause toxic effects in diverse biological systems that can trigger local and systemic reactions. In this study, the cytotoxic and cytolytic effects of Chrysaora quinquecirrha and Chironex fleckeri venoms were assessed and compared using three in vitro assays. Venoms from both species were cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic in sheep erythrocytes. Both venoms decreased cell viability in a concentration-dependent manner; however, the greatest difference in venom potencies was observed in the fish gill cell line , wherein C. fleckeri was 12.2- ( P = 0.0005) and 35.7-fold ( P < 0.0001) more potently cytotoxic than C. quinquecirrha venom with 30 min and 120 min cell exposure periods, respectively. Gill cells and rat cardiomyocytes exposed to venoms showed morphological changes characterised by cell shrinkage, clumping and detachment. The cytotoxic effects of venoms may be caused by a group of toxic proteins that have been previously identified in C. fleckeri and other cubozoan jellyfish species. In this study, proteins homologous to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii were identified in C. quinquecirrha venom using tandem mass spectrometry. The presence and relative abundance of these proteins may explain the differences in venom potency between cubozoan and scyphozoan jellyfish and may reflect their importance in the action of venoms. [ABSTRACT FROM AUTHOR]

Cnidarian envenomations are the leading cause of severe and lethal human sting injuries from marine life. The total amount of venom discharged into sting-site tissues, sometimes referred to as “venom load”, has been previously shown to correlate with tentacle contact length and sequelae severity. Since

Background: The box jellyfish, Chironex fleckeri, is the largest and most dangerous cubozoan jellyfish to humans. It produces potent and rapid-acting venom and its sting causes severe localized and systemic effects that are potentially life-threatening. In this study, a combined transcriptomic and proteomic approach was used to identify C. fleckeri proteins that elicit toxic effects in envenoming. Results: More than 40,000,000 Illumina reads were used to de novo assemble ~ 34,000 contiguous cDNA sequences and ~ 20,000 proteins were predicted based on homology searches, protein motifs, gene ontology and biological pathway mapping. More than 170 potential toxin proteins were identified from the transcriptome on the basis of homology to known toxins in publicly available sequence databases. MS/MS analysis of C. fleckeri venom identified over 250 proteins, including a subset of the toxins predicted from analysis of the transcriptome. Potential toxins identified using MS/MS included metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins and a turripeptide-like protease inhibitor. Nine novel examples of a taxonomically restricted family of potent cnidarian pore-forming toxins were also identified. Members of this toxin family are potently haemolytic and cause pain, inflammation, dermonecrosis, cardiovascular collapse and death in experimental animals, suggesting that these toxins are responsible for many of the symptoms of C. fleckeri envenomation. Conclusions: This study provides the first overview of a box jellyfish transcriptome which, coupled with venom proteomics data, enhances our current understanding of box jellyfish venom composition and the molecular structure and function of cnidarian toxins. The generated data represent a useful resource to guide future comparative studies, novel protein/peptide discovery and the development of more effective treatments for jellyfish stings in humans. (Length: 300). [ABSTRACT FROM AUTHOR]

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts. [ABSTRACT FROM AUTHOR]

Currently the Australian Resuscitation Council (ARC) recommends dousing with vinegar followed by ice as first aid for jellyfish stings in tropical Australia, with limited evidence to support this recommendation (Li et al., 2013). We report our successful experience in using hot water immersion as first aid in treating two people stung by venomous tropical Australian jellyfish, one by Chironex fleckeri and one by Carukia barnesi . [ABSTRACT FROM AUTHOR]

Abstract: The vertebrate cardiotoxic components of the venom produced by the Australian box jellyfish, Chironex fleckeri, have not previously been isolated. We have uncovered for the first time, three distinct cytotoxic crude fractions from within the vertebrate cardiotoxic peak of C. fleckeri venom by monitoring viability of human muscle cells with an impedance based assay (ACEA xCELLigence system) measuring cell detachment as cytotoxicity which was correlated with a reduction in cell metabolism using a cell proliferation (MTS) assay. When the effects of the venom components on human cardiomyocytes and human skeletal muscle cells were compared, two fractions were found to specifically affect cardiomyocytes with distinct temporal profiles (labelled Crude Toxic Fractions (CTF), α and β). A third fraction (CTF-γ) was toxic to both muscle cell types and therefore not cardio specific. The vertebrate, cardio specific CTF-α and CTF-β, presented distinct activities; CTF-α caused rapid but short term cell detachment and reduction in cell metabolism with enhanced activity at lower concentrations than CTF-β. This activity was not permanent, with cell reattachment and subsequent increased metabolism of heart muscle cells observed when exposed to all but the highest concentrations of CTF-α tested. The cytotoxic effect of CTF-β took twice as long to act on the cells compared to CTF-α, however, the activity was permanent. Furthermore, we showed that the two fractions combined have a synergistic effect causing a much stronger and faster cell detachment (death) when combined than the sum of the individual effects of each toxin. These data presented here improves the current understanding of the toxic mechanisms of the Australian box jellyfish, C. fleckeri, and provides a basis for in vivo research of these newly isolated toxic fractions. [Copyright &y& Elsevier]

Abstract: Although Chironex fleckeri and Carukia barnesi cause significant human envenomation, research into their effects in human models or human cells has been limited. In this in vitro study we have presented data that shows that although C. fleckeri is highly cytotoxic to human cardiac and skeletal muscle cells, C. barnesi is not cytotoxic at all concentrations tested to both cardiac and skeletal muscles cells. We also demonstrate that in vitro C. fleckeri venom cardiocytotoxic activity is significantly attenuated when heated to 44 °C for 20 min. There is a similar attenuation with skeletal cells at 46 °C. [Copyright &y& Elsevier]

Abstract: This is the first study to explore venom and cnidome variation of individual cubomedusae, Chironex fleckeri, of different ages and from different regional locations in relation to feeding ecology. As medusae matured the proportion of mastigophores (those nematocysts containing the lethal venom component) in the cnidome increased, along with proportion of the vertebrate toxic fraction, in the venom profile. This switch in cnidome and venom occurred at the seven to ten tentacle stage. Whole venom was found to be toxic specifically to vertebrate cardiac cells, as opposed to vertebrate skeletal cells, and dose dependent, along with the vertebrate toxic fraction. The venom and cnidome ontogeny, along with venom toxicity, is correlated with C. fleckeri''s known feeding ecology. Large and mature C. fleckeri feed predominantly on vertebrates, and have a greater proportion of mastigophores in their cnidome along with more vertebrate toxic fraction in their venom, compared to when they are young and small feeding on invertebrates. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 (0.01–30μg/ml) caused contractions (EC50 1.15±0.19μg/ml) that were unaffected by prazosin (0.1μM), bosentan (10μM), CGRP8–37 (1μM) or tetrodotoxin (1μM). Box jellyfish antivenom (5–92.6units/ml) caused rightward shifts of the CVE concentration–response curve with no change in the maximum. In the presence of l-NAME (100μM) the sensitivity and maximum response to CVE were increased, whilst MgSO4 (6mM) decreased both parameters. CVE (1–10μg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation. Left atrial responses to CVE (0.001–30μg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3μg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3μg/ml. These responses were unaffected by 1μM of propranolol, atropine or CGRP8–37. Antivenom (54 and 73units/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. [Copyright &y& Elsevier]

The discovery that Chironex fleckeri occurs annually in northern Australia occurred long ago, possibly before written records were available by local indigenous people, however, to date the precision of this yearly phenomenon is still not fully understood. Sea surface temperature (SST) appears to be a determining factor signaling the “arrival” of C. fleckeri each year. Anthropogenic climate change modeling predicts global rises in SST. Rises in SST may result in an earlier “arrival” of C. fleckeri during the dry season, possibly necessitating extension of the official Northern Territory stinger season to commence in September (currently October through to June). This short report presents data to support this hypothesis, facilitating policy makers with an environmental cue upon which changes to current stinger season can be based—prior to painful and potentially life-threatening presentations. [ABSTRACT FROM AUTHOR]

Abstract: Introduction : Chironex fleckeri is a large box jellyfish that has been labelled the ‘most venomous animal’ in the world. We have recently shown that the primary effect of C. fleckeri venom in vivo is cardiovascular collapse. This study utilised a cell-based assay to examine the effects of C. fleckeri venom on the proliferation of a rat aortic smooth muscle cell line. In addition, the ability of CSL box jellyfish antivenom and/or various potential treatment strategies to neutralise the effects of the venom was examined. Methods : A7r5 cells were cultured in media containing venom. The effect of CSL box jellyfish antivenom (5 U/mL), CSL polyvalent snake antivenom (5 U/mL), lanthanum (5 µM), MgSO4 (50 mM), verapamil (5 µM) or felodipine (5 µM) was examined. Cell viability was determined using a Cell titer 96 AQueous One Solution cell proliferation assay. Results : Incubation of A7r5 cells with serially diluted venom (2–0.004 µg/mL) caused a concentration-dependent inhibition of cell proliferation with an IC50 value of 0.056 µg/mL. This response was not affected by the absence of calcium or the presence of lanthanum in the media. Box jellyfish antivenom (5 U/mL) prevented the inhibition of cell proliferation caused by the venom. Verapamil (5 µM) had no significant effect on the inhibition. In contrast, felodipine (5 µM) or MgSO4 (50 mM) potentiated the effects of the venom and partially negated the protective effect of the antivenom. Discussion : This study displayed the ability to utilise a cell-based assay to determine the effects of C. fleckeri venom on vascular cell viability. It showed that CSL box jellyfish can neutralise the effects of the venom but only if added prior to the venom. In addition, potential adjunct therapies verapamil, felodipine and MgSO4 were found to be ineffective, with felodipine and MgSO4 potentiating the detrimental effects of the venom. [Copyright &y& Elsevier]

Abstract: Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of ∼370 and 145kDa, and one minor cytolysin (∼70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits (∼43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins (∼39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase (∼6–7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom. [Copyright &y& Elsevier]

Abstract: 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 45kDa, 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. [Copyright &y& Elsevier]

Abstract: 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 (50μg/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 (50μg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30°C for 1.5h. 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. [Copyright &y& Elsevier]

Abstract: Clinical observations suggest a primary cardiotoxic role in fatal Chironex fleckeri stings. The limited research available indicates that Chiropsella bronzie venom acts in a similar manner although appears to be less potent. The aim of the present study was to elucidate the vascular effects of C. fleckeri and C. bronzie venoms using rat isolated aorta. Both venoms produced a sustained contraction of endothelium-denuded aorta which was not significantly affected by prazosin or box jellyfish antivenom. Felodipine significantly reduced the contractile response to C. fleckeri venom but not C. bronzie venom. Both venoms produced an initial relaxation (Phase 1), followed by a sustained contraction (Phase 2), in pre-contracted endothelium-intact aorta. Removal of the endothelium significantly inhibited both phases of the response. NOLA significantly inhibited Phase 1, but not Phase 2, of the response to both venoms. Atropine, HOE 140 or BQ 123 did not have any significant inhibitory effect on either phase. In conclusion, neither C. fleckeri nor C. bronzie venoms appear to contain components with activity at α1-adrenoceptors. Antivenom was ineffective in reversing the effects of the venom suggesting it is incapable of completely neutralising nematocyst-derived venom. Determining the mechanism of action of these venoms will allow for the development of better treatment strategies. [Copyright &y& Elsevier]

Summary: A 35-year-old male presented on the beach, having been stung on the leg by a box jellyfish, whilst swimming at Mission Beach, Qld. Appropriate diagnosis, the use of vinegar and pressure immobilisation bandaging and immediate pain relief helped to provide a positive outcome for this patient. The aim of this discussion paper is to present this case study and a review of the current and recommended assessment and initial management of such envenomation. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Using a new technique to extract venom from the nematocysts of jellyfish, the in vivo cardiovascular effects of Chiropsalmus sp. venom were investigated in anaesthetized rats. Chiropsalmus sp. venom (150μg/kg, i.v.) produced a transient hypertensive response (44±4mmHg; n=6) followed by hypotension and cardiovascular collapse. Concurrent artificial respiration or pretreatment with Chironex fleckeri antivenom (AV, 3000U/kg, i.v.) did not have any effect on the venom-induced hypertensive response nor the subsequent cardiovascular collapse. The cardiovascular response of animals receiving venom after the infusion of MgSO4 (50–70mM @ 0.25ml/min, i.v.; n=5) alone, or in combination with AV (n=5), was not significantly different from rats receiving venom alone. Prior administration of prazosin (50μg/kg, i.v.; n=4) or ketanserin (1mg/kg, i.v.; n=4) did not significantly attenuate the hypertensive response nor prevent the cardiovascular collapse induced by venom (50μg/kg, i.v.). In contrast to previous work examining C. fleckeri venom, administration of AV alone, or in combination with MgSO4, was not effective in preventing cardiovascular collapse following the administration of Chiropsalmus sp. venom. This indicates that the venom of the two related box jellyfish contain different lethal components and highlights the importance of species identification prior to initiating treatment regimes following jellyfish envenoming. [Copyright &y& Elsevier]

Abstract: Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the in vivo cardiovascular effects of Chironex fleckeri venom and tentacle extract (devoid of nematocysts). In anaesthetised rats, venom (10μg/kg, i.v.) produced a transient pressor response (23±4mmHg) followed, in two of five animals, by cardiovascular collapse. Tentacle extract (100μg/kg, i.v.) produced a more prolonged hypertensive effect (31±3mmHg) without cardiovascular collapse. Prazosin (50μg/kg, i.v.) did not have any significant effect on the cardiovascular effects produced by venom. However, prazosin significantly attenuated the pressor response produced by tentacle extract. Ketanserin (1mg/kg, i.v.) did not have any significant effect on the cardiovascular response of the anaesthetised rat to venom (10μg/kg, i.v.; 25±1mmHg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to compare the two jellyfish samples used in the present study. In addition to ensuring reproducibility of future studies and allow comparison with previous research. We show, for the first time, that a pure venom sample extracted from C. fleckeri nematocysts and a tentacle extract have cardiovascular effects in the anaesthetised rat which are different and pharmacologically distinct. [Copyright &y& Elsevier]

Abstract: Cnidarian venoms produce a wide spectrum of envenoming syndromes in humans ranging from minor local irritation to death. Here, the effects of Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana venoms on ventricular myocyte cytosolic Ca2+, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca2+ in myocytes as measured using the Ca2+ sensitive fluorescent probe Indo-1. The L-type Ca2+ channel antagonist verapamil had no effect on Ca2+ influx whilst La3+, a non-specific channel and pore blocker, inhibited the effect. Haemolytic activity was observed for all venoms, with C. xaymacana venom displaying the greatest activity. These activities are consistent with the presence of a pore-forming toxin existing in the venoms which has been demonstrated by transmission electron microscopy in the case of C. fleckeri. The venom of C. fleckeri was found to be more lethal against Artemia sp. than the venom of the other species, consistent with the order of known human toxicities. This suggests that the observed lytic effects may not underlie the lethal effects of the venom, and raises the question of how such potent activities are dealt with by envenomed humans. [Copyright &y& Elsevier]

Various comparative studies into the biological activity and relative toxicity of cubozoan venoms have been investigated, in particular the venom from the potentially lethal cubozoan Chironex fleckeri. Efficient and reliable extraction of venom from nematocysts is essential before any research into venom toxicity can be conducted and previous cited methods of extraction have varied greatly, each with their own associated problems. A new standardised technique for the recovery of venom from nematocysts of cubozoans is investigated to decrease the variation displayed between authors due to differing extraction techniques. The use of a mini bead mill beater, as investigated in this trial, allows for the rapid extraction of venom from nematocysts and is devoid of the previously isolated problems experienced with other methods of venom isolation, such as excessive heat build up. [Copyright &y& Elsevier]

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 (MgSO4), 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. MgSO4 (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 MgSO4 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 MgSO4 as an adjunct therapy for the treatment of potentially fatal C. fleckeri envenomings. [Copyright &y& Elsevier]

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 μg/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1 U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5 U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5 U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 μg/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 μg/ml). Antivenom (5 U/ml) added 1 h after the addition of C. fleckeri venom (50 μg/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 μg/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin.Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically. [Copyright &y& Elsevier]

This study examined differences in the nematocyst ratios between two species of Australian cubozoans. In Chiropsalmus sp., a species that feeds exclusively on shrimp, no changes in the ratio of the three groups of nematocyst present in the cnidome were detected with size of the individual animals. In Chironex fleckeri, the ratio of different types of nematocysts in the cnidome for small animals (less than 40 mm) was similar to that of Chiropsalmus sp. However, with an increase in body size in C. fleckeri, the nematocyst ratio changed, with mastigophores (nematocysts believed to hold the lethal venom component for prey) increasing in proportion. The change in cnidome ratio is correlated with a change in the prey of C. fleckeri with increased size. Small C. fleckeri appeared to feed exclusively on prawns, medium sized animals fed on fish and prawns and large animals fed predominantly on fish. An increase in the proportion of mastigophores (and presumably the lethal venom component) in the cnidome of C. fleckeri may also be responsible for why this species has caused numerous human fatalities, while the Australian Chiropsalmus sp. has not. [Copyright &y& Elsevier]