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Concept: Chironex fleckeri

191

Chironex fleckeri (Australian box jellyfish) stings can cause acute cardiovascular collapse and death. We developed methods to recover venom with high specific activity, and evaluated the effects of both total venom and constituent porins at doses equivalent to lethal envenomation. Marked potassium release occurred within 5 min and hemolysis within 20 min in human red blood cells (RBC) exposed to venom or purified venom porin. Electron microscopy revealed abundant ∼12-nm transmembrane pores in RBC exposed to purified venom porins. C57BL/6 mice injected with venom showed rapid decline in ejection fraction with progression to electromechanical dissociation and electrocardiographic findings consistent with acute hyperkalemia. Recognizing that porin assembly can be inhibited by zinc, we found that zinc gluconate inhibited potassium efflux from RBC exposed to total venom or purified porin, and prolonged survival time in mice following venom injection. These findings suggest that hyperkalemia is the critical event following Chironex fleckeri envenomation and that rapid administration of zinc could be life saving in human sting victims.

Concepts: Blood, Red blood cell, Cnidaria, Potassium chloride, Box jellyfish, Chironex fleckeri, Cubozoa, Chirodropidae

168

The nematocyst is a complex intracellular structure unique to Cnidaria. When triggered to discharge, the nematocyst explosively releases a long spiny, tubule that delivers an often highly venomous mixture of components. The box jellyfish, Chironex fleckeri, produces exceptionally potent and rapid-acting venom and its stings to humans cause severe localized and systemic effects that are potentially life-threatening. In an effort to identify toxins that could be responsible for the serious health effects caused by C. fleckeri and related species, we used a proteomic approach to profile the protein components of C. fleckeri venom. Collectively, 61 proteins were identified, including toxins and proteins important for nematocyte development and nematocyst formation (nematogenesis). The most abundant toxins identified were isoforms of a taxonomically restricted family of potent cnidarian proteins. These toxins are associated with cytolytic, nociceptive, inflammatory, dermonecrotic and lethal properties and expansion of this important protein family goes some way to explaining the destructive and potentially fatal effects of C. fleckeri venom. Venom proteins and their post-translational modifications (PTMs) were further characterized using toxin-specific antibodies and phosphoprotein/glycoprotein-specific stains. Results indicated that glycosylation is a common PTM of the toxin family while a lack of cross-reactivity by toxin-specific antibodies infers there is significant divergence in structure and possibly function among family members. This study provides insight into the depth and diversity of protein toxins produced by harmful box jellyfish and represents the first description of a cubozoan jellyfish venom proteome.

Concepts: Protein, Proteomics, Cnidaria, Jellyfish, Toxin, Cnidocyte, Box jellyfish, Chironex fleckeri

75

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.

Concepts: Cnidaria, Jellyfish, Cnidocyte, Box jellyfish, Chironex fleckeri, Chirodropidae

27

Directional swimming in the box jellyfish Tripedalia cystophora (cubozoa, cnidaria) is controlled by the shape of the velarium, which is a thin muscular sheet that forms the opening of the bell. It was unclear how different patterns of visual stimulation control directional swimming and that is the focus of this study. Jellyfish were tethered inside a small experimental tank, where the four vertical walls formed light panels. All four panels were lit at the start of an experiment. The shape of the opening in the velarium was recorded in response to switching off different combinations of panels. We found that under the experimental conditions the opening in the velarium assumed three distinct shapes during a swim contraction. The opening was (1) centred or it was off-centred and pocketed out either towards (2) a rhopalium or (3) a pedalium. The shape of the opening in the velarium followed the direction of the stimulus as long as the stimulus contained directional information. When the stimulus contained no directional information, the percentage of centred pulses increased and the shape of the off-centred pulses had a random orientation. Removing one rhopalium did not change the directional response of the animals, however, the number of centred pulses increased. When three rhopalia were removed, the percentage of centred pulses increased even further and the animals lost their ability to respond to directional information.

Concepts: Animal, Cnidaria, Jellyfish, Ctenophora, Cnidocyte, Box jellyfish, Chironex fleckeri, Medusa

21

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.

Concepts: Protein, Serine protease, Protease, Cnidaria, Jellyfish, Cnidocyte, Box jellyfish, Chironex fleckeri

11

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.

Concepts: Proteome, Jellyfish, Cnidocyte, Box jellyfish, Chironex fleckeri, Cubozoa, Chirodropidae, Venomous animals

1

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterisation of two C. fleckeri venom proteins, CfTX-A (≈40 kDa) and CfTX-B (≈42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/-2 (25µg kg(-1)) caused profound effects on the cardiovascular system of anaesthetised rats whereas CfTX-A/-B elicited only minor effects at the same dose. Conversely, the haemolytic activity of CfTX-A/-B (HU50 = 5ng mL(-1)) was at least 30 times greater than that of CfTX-1/-2. Structural homology between the cubozoan toxins and insecticidal 3d-Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, while structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

Concepts: DNA, Protein, Protein structure, Molecular biology, Cnidaria, Cnidocyte, Box jellyfish, Chironex fleckeri

0

To investigate the effectiveness of hot water immersion for relieving the pain of major box jellyfish (Chironex fleckeri) stings.Design, interventions: Open label, randomised controlled trial comparing the effects of hot water immersion (45°C) and icepacks.Setting, participants: 42 patients with suspected C. fleckeri stings treated in the emergency department of the Royal Darwin Hospital during September 2005 - October 2008.

Concepts: Hospital, Randomized controlled trial, Box jellyfish, Chironex fleckeri

0

Box jellyfish cause human fatalities and have a life cycle and habit associated with shallow waters (<5 m) in mangrove creeks, coastal beaches, embayments. In north-western Australia, tow video and epibenthic sled surveys discovered large numbers (64 in a 1500 m tow or 0.05 m(-2)) of Chironex sp. very near to the benthos (<50 cm) at depths of 39-56 m. This is the first record of a population of box jellyfish closely associated with the benthos at such depths. Chironex were not widespread, occurring only in 2 of 33 tow videos and 3 of 41 epibenthic sleds spread over 2000 km(2). All Chironex filmed or captured were on low to medium relief reefs with rich filter feeder communities. None were on soft sediment habitat despite these habitats comprising 49% of all sites. The importance of the reef habitat to Chironex remains unclear. Being associated with filter feeder communities might represent a hazard, and other studies have shown C. fleckeri avoid habitats which represent a risk of entanglement of their tentacles. Most of our observations were made during the period of lowest tidal current flow in the morning. This may represent a period favourable for active hunting for prey close to the seabed.

Concepts: Habitat, Jellyfish, Cnidocyte, Box jellyfish, Chironex fleckeri, Medusa, Cubozoa, Chirodropidae

0

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.

Concepts: Fish, Cytotoxicity, Cnidaria, Jellyfish, Toxin, Scyphozoa, Box jellyfish, Chironex fleckeri