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Toxicon xxx (2014) 1e12

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Toxicon

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Bioactive toxins from stinging jellyfish

Sophie Badr�e*

Prevor, Moulin de Verville, 95760 Valmondois, France

a r t i c l e i n f o

Article history:Received 26 June 2014Received in revised form 19 September 2014Accepted 25 September 2014Available online xxxx

Keywords:JellyfishToxinsBiological activitiesHuman envenomation

* Tel.: þ33 1 34 08 95 38.E-mail address: sbadre@prevor.com.

http://dx.doi.org/10.1016/j.toxicon.2014.09.0100041-0101/© 2014 Elsevier Ltd. All rights reserved.

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a b s t r a c t

Jellyfish blooms occur throughout the world. Human contact with a jellyfish induces a localreaction of the skin, which can be painful and leave scaring. Systemic symptoms are alsoobserved and contact with some species is lethal. A number of studies have evaluated thein vitro biological activity of whole jellyfish venom or of purified fractions. Hemolytic,cytotoxic, neurotoxic or enzymatic activities are commonly observed. Some toxins havebeen purified and characterized. A family of pore forming toxins specific to Medusozoanshas been identified. There remains a need for detailed characterization of jellyfish toxins tofully understand the symptoms observed in vivo.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Cnidarians are found in seas and oceans throughout theworld. They are characterized by their specialized cells, thecnidocytes, which are used for prey capture, defense, andlocomotion (Anderson and Bouchard, 2009). The phylum isdivided into two clades, Anthozoa, which includes seaanemones and corals, and Medusozoa, comprising theclasses Staurozoa, Hydrozoa, Schyphozoa, and Cubozoa(Daly et al., 2007). During their lifecycle, most Medoso-zoans take the form of a free-floating medusa, which iscommonly named jellyfish (Tibballs, 2006).

Jellyfish swarms can significantly impact human activ-ities and ecosystems because of their venomous andgelatinous nature (Brotz et al., 2012). They affect tourism,fishing, and aquaculture industries as well as other usersthat rely on coastal water pumping. Human envenomationby jellyfish induces a large variety of symptoms, rangingfrom a slight local effect to life-threatening symptoms(Fenner, 1998; Cegolon et al., 2013). Additionally, somepeople are susceptible to allergic reactions (Tibballs et al.,2011). Jellyfish are relatively common in the

., Bioactive toxins from

Mediterranean area or in Australia (Mariottini and Pane,2010; Tibballs, 2006). The most dangerous species arefound in this last country (Tibballs, 2006). Conversely,blooms of hazardous jellyfish species constitute anemerging risk in other areas, such as the Aquitaine coast ofFrance or in China (Labadie et al., 2012; Kang et al., 2014).

Like all cnidarians, jellyfish produce venom for defenseand prey capture, neutralization, and digestion (Mebs,2002). Cnidarian venom is a mix of toxins with a widerange of biological activities (�Suput, 2009). Neurotoxic,cytolytic, and enzymatic (proteases, phospholipases) toxinshave been described in the phylum (Fraz~ao et al., 2012;Mariottini and Pane, 2013). The complex mix of toxic sub-stances is injected into the prey by nematocysts, which areproduced by nematocytes, a subtype of cnidocytes. Sometoxins have also been found in other types of tissues or cellsin cnidarians (Moran et al., 2012; Zhang et al., 2003).

Understanding and treating the symptoms observedafter human envenomation relies partly on in vitro char-acterization of the biological activities of those toxins. Thisreview will focus on the toxins or partially purified bioac-tive fractions that have been isolated from jellyfish venomto date. First, the jellyfish species of interest will be intro-duced, along with a brief summary of their impact onhuman health. Then, the general principles for jellyfishtoxin purification will be presented, and the toxins and

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

S. Badr�e / Toxicon xxx (2014) 1e122

in vitro biological activities of jellyfish belonging to theclasses Cubozoa, Schyphozoa, and Hydrozoa will bedescribed. Last, the observed biological activities will besummarized. The jellyfish species cited in this paper, andtheir phylogenic relationship, are given in Fig. 1.

2. Human envenomation

2.1. Class cubozoa

In the order Chirodropida, the Australian box jellyfishChironex fleckeri is known to be the most dangerous jelly-fish in the world (Tibballs, 2006). Contact with its tentacleinduces a local cutaneous inflammatory reaction which isvery painful and can leave permanent scarring (Brinkmanand Burnell, 2009). Systemic symptoms can also beobserved, including excruciating pain, impaired con-sciousness, hypertension, hypotension, and cardiac andrespiratory failure (Brinkman and Burnell, 2009). Death canoccur within minutes after the envenomation, most prob-ably because of cardiac and respiratory effects (Tibballs,2006).

Other species from the order Chirodropida can inducelocal pain, cutaneous inflammation, and scarring (Baileyet al., 2005; Brinkman and Burnell, 2009; Cegolon et al.,2013). Chiropsalmus quadrigatus and Chiropsalmus quad-rumanus venoms can be harmful to the cardiovascular andrespiratory systems, and can be fatal to humans (Nagaiet al., 2002; Cegolon et al., 2013).

Cnidaria

Anthozoa

Sea anemones and corals

Cubozoa

Sem

Carybdeida

AlatinidaeAlatina mordens(Alatina moseri)Carybdea alata(Alatina alata)

CarukiidaeCarukia barnesi

Malo kingiMalo maxima

CarybdeidaeCarybdea rastonii

Carybdea marsupialis

Chirodropida

ChirodropidaeChironex fleckeri

ChiropsalmidaeChiropsalmus quadrigatus(Chiropsoides quadrigatus) Chiropsalmus quadrumanus

Fig. 1. Summary of the jellyfish species included in this review, classified by phylum(WoRMS), consulted on July 18, 2014 http://www.marinespecies.org/index.php. Speaccepted names of the species in the WoRMS.

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In the order Carybdeida, contact with jellyfish from thefamily Carybdeidae typically induces local cutaneousinflammation and pain (Brinkman and Burnell, 2009;Cegolon et al., 2013; Peca et al., 1997; Nagai et al., 2000a).Contact with other families in this order can induce theIrukandji syndrome (Gershwin et al., 2013; Bentlage et al.,2010). The biology and ecology of jellyfish causing Iru-kandji was recently reviewed by Gershin and coworkers(Gershwin et al., 2013). A large number of species maycause Irukandji or Irukandji-like symptoms. Most of thesespecies belong to the order Carybdeida, the main generabeing Alatina, Carukia, Malo, and Morbakka. Symptomssimilar to Irukandji have also been described after contactwith species from other classes of cnidarians (Gershwinet al., 2013; Carrette et al., 2012).

The Irukandji syndrome is described as a set of delayedsystemic effects that appears on average 25e40 min aftercontact with the jellyfish and can last from a few hours toseveral days (Carrette et al., 2012; Tibballs et al., 2012). Itwas first described in Australia, but has been reported atlatitudes from 53�N to 38�S. The sting is hardly noticed andproduces an erythema, which heals without scarring. Thesystemic symptoms can include vomiting, nausea, intensemuscle pain, cramps, lower back pain, headache, sweating,agitation, distress, and hypertension. Severe cases involvecardiac dysfunction (Carrette et al., 2012; Tibballs et al.,2012). Current knowledge links this syndrome to anexcess of circulating catecholamines, including adrenalineand noradrenaline (Gershwin et al., 2013).

Medusozoa

Scyphozoa Hydrozoa

Siphonophorae Limnomedusae

PhysaliidaePhysalia physalis

OlindidaeOlindias

sambaquiensis

Rhizostomeae

RhizostomatidaeRhopilema nomadica

Rhopilema esculentumNemopilema nomurai

StomolophidaeStomolophus meleagris

aeostomemeae

CyaneidaeCyanea capillataCyanea nozakii

Cyanea lamarckii

PelagiidaePelagia noctiluca

UlmaridaeAurelia aurita

, clade, class, order, family, genus. Source: World register of marine speciescies name are those used in the publication. Names between brackets are the

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S. Badr�e / Toxicon xxx (2014) 1e12 3

2.2. Class scyphozoa

Although jellyfish from the class scyphozoa are lessdangerous to humans than species of the class cubozoa(Cegolon et al., 2013) (Mariottini and Pane, 2010), contactwith Cyanea capillata can lead to systemic symptoms(Cegolon et al., 2013), as can contact with Stomolophusmeleagris (Li et al., 2013).

Cyanea capillata is a pelagic jellyfish from the cold re-gions of the Atlantic and Pacific oceans. It is very commonin the North and Baltic sea, and it is also present in Australia(Mebs, 2002; Lassen et al., 2011). The diameter of the bellvaries from 30 cm to 2 m for the largest specimen. Contactwith this jellyfish produces pain, edema, and erythema.Systemic symptoms are unusual, but include musclecramps, sweating, dizziness, and nausea (Mebs, 2002;Cegolon et al., 2013).

Pelagia noctiluca is found worldwide, in both tropicaland cold waters (Cegolon et al., 2013), and is very commonin the Mediterranean (Mariottini and Pane, 2010). Enven-omation induces local and painful cutaneous reactions(Mariottini and Pane, 2010; Mariottini et al., 2008)including blistering, edema, and erythema. Lesions can healwithin 2 weeks but scarring and hyperpigmentation canremain. Delayed or relapsing eruptions are sometimesobserved at the site of contact. Systemic effects are veryrare. Allergic reactions have been observed, with possiblecross reactivity with other jellyfish.

Aurelia aurita is distributed in temperate and temper-ateecold waters around the world (Mariottini and Pane,2010). It is known to be harmless, but cases of local cuta-neous reaction, associated with pain, have been reported(Burnett et al., 1988; Segura-Puertas et al., 2002; Tibballs,2006; Mariottini and Pane, 2010).

Rhopilema esculentum is found along the Chinesecoastline (Feng et al., 2009). Rhopilema nomadica is nativeto the Indian and Pacific oceans, but has also been observedin the eastern Mediterranean sea since the 70s (Mariottiniand Pane, 2010). Stings from Rhopilema nomadica inducelocal burning pain and erythematous eruptions. Delayedcutaneous reactions and systemic symptoms such as fever,fatigue, and muscular aches were also described (Gusmaniet al., 1997; Mariottini and Pane, 2010).

Nemopilema nomurai is a large jellyfish with a bell sizeup to 2 m that is found in the Yellow Sea, East China Sea,and East Sea. Blooms have been more frequent in recentyears. Burning pain and erythematous eruption with smallvesicles has been described after contact with this jellyfish(Kang et al., 2014).

Also abundant along the coasts of China, Stomolophusmeleagris can cause local cutaneous reaction such as itchingand edema, but also more severe symptoms like myalgia,dyspnea, hypotension, and shock. Death can occur aftercontact with this jellyfish (Li et al., 2013).

2.3. Class hydrozoa

Physalia physalis belongs to the order Siphonophorae,thus, this ‘jellyfish’ is actually a colony of organismsspecialized for movement, prey capture and digestion, andreproduction (Mapstone, 2014). It has a cosmopolitan

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distribution between the latitudes 51�N and 38�S(Mapstone, 2014). The float height can be as high as 30 cmwith tentacles up to 30 m in length (Cegolon et al., 2013).Small specimens with one tentacle may be described asPhysalia utriculus in the Indo-Pacific ocean and in Australia,but a recent review of Siphonophorae updated the sys-tematics of the order and confirmed that there is only onePhysaliidae species (Mapstone, 2014).

Contact with Physalia physalis induces acute pain, and alocal cutaneous reaction with erythema and inflammation(Labadie et al., 2012; Cazorla-Perfetti et al., 2012). Long-lasting dermal marks at the site of contact with thetentacle can be observed (Labadie et al., 2012; Haddad et al.,2013). Systemic symptoms are also observed, includingrespiratory distress, neurological, musculoskeletal, anddigestive signs. The prevalence of generalized symptomsvaried between 15 and 20% on the French Atlantic coast, inVenezuela, and in Brazil with a peak of 52% in 2010 inFrance (Labadie et al., 2012; Cazorla-Perfetti et al., 2012;Haddad et al., 2013). Stings from Physalia physalis are themost common type of jellyfish stings in Australia, but sys-temic symptoms are rarely observed (Tibballs, 2006).Deaths by respiratory arrest and cardiovascular collapsehave been reported (Elston, 2007).

Olindias sambaquiensis is found in the South AtlanticOcean. It is responsible for the majority of jellyfish stings inBrazil. It produces moderate to severe cutaneous reactions,withpossible cardiovascularcomplication (Junioret al., 2014).

The local and systemic symptoms observed after contactwith jellyfish are summarized in Table 1. A more or lesssevere cutaneous reaction is observed at the site of contactwith the jellyfish, which can be painful and leave scaring. Awider range of systemic symptoms is observed.

3. Strategies of toxin purification and identification

3.1. Venom localization

Cnidarians discharge nematocysts during the process ofcapturing prey. Several categories of nematocysts havebeen described based on morphological criteria (Kass-Simon and Scappaticci, 2002). Nematocysts consist of atubule, covered with venom, which is coiled inside a pro-teinaceous capsule. Nematocyst discharge, i.e., release ofthe tubule, is triggered by mechanical and chemical stimuliof the nematocyte and surrounding tissues, and the tubuleinjects the venom by penetration into the prey (€Ozbeket al., 2009; Anderson and Bouchard, 2009).

In anthozoans, toxins can be produced by nematocystsor by ectodermal gland cells (Fraz~ao et al., 2012; Moranet al., 2012). There is evidence that bioactive products arealso present in the surrounding tissues in medusozoans.For instance, hydralysin is a noncnidocystic neurotoxinfrom the hydra Chlorohydra viridissima (Zhang et al., 2003).Likewise, the antimicrobial peptide aurelin was extractedand characterized from the mesoglea of Aurelia aurita(Ovchinnikova et al., 2006; Shenkarev et al., 2012).Nematocyst-free tentacle extract from Chironex fleckeri hada cardiotoxic effect in anaesthetized rats, which differedfrom the effect of nematocyst extract (Ramasamy et al.,2005a).

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

Table 1Local and systemic symptoms observed after contact with jellyfish.

Class Species Localsymptoms

Systemic symptoms

Cubozoa Chironex fleckeri Cutaneousreaction

Pain

Pain Impaired consciousnessScarring Cardiac and respiratory

failureDeath

Chiropsalmusquadrigatus

Cutaneousreaction

Cardiac and respiratoryfailure

(Chiropsoidesquadrigatus)

Scarring Death

Chiropsalmusquadrumanus

Cutaneousreaction

Cardiac and respiratoryfailure

Scarring DeathCarybdea rastonii Cutaneous

reactione

PainCarybdeamarsupialis

Cutaneousreaction

e

PainAlatina mordens Erythema Irukandji(Alatina moseri)Carybdea alata(Alatina alata)Carukia barnesiMalo kingiMalo maxima

Scyphozoa Cyanea capillata Cutaneousreaction

Muscle cramps

Pain SweatingImpaired consciousness

Pelagia noctiluca Cutaneousreaction

Allergy

PainScarring

Aurelia aurita Cutaneousreaction

e

PainRhopilemanomadica

Cutaneousreaction

Delayed cutaneousreaction

Pain FeverMuscular aches

Nemopilemanomurai

Cutaneousreaction

e

PainStomolophusmeleagris

Cutaneousreaction

MyalgiaCardiac and respiratoryfailureDeath

Hydrozoa Physaliaphysalis

Cutaneousreaction

Respiratory distress

Pain Neurological signScarring Musculoskeletal sign

Digestive signDeath

Olindiassambaquiensis

Cutaneousreaction

Cardiovascularcomplication

S. Badr�e / Toxicon xxx (2014) 1e124

3.2. Venom extraction

Because nematocyst discharge is the primary method ofprey capture, the majority of studies on jellyfish toxicityfocus on nematocyst proteic content. Nematocysts aremicrometric capsules embedded in the jellyfish epidermisof the tentacles or of other parts of the body. Venom can besampled directly from the whole tissue or from a

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suspension of nematocysts (Xiao et al., 2009). Nematocystsare isolated by tissue autolysis (in sea water or in distilledwater) and centrifugation (Bloom et al., 1998; Lee et al.,2011; Morabito et al., 2012). To extract venom from thenematocysts, discharge is induced by mechanical stimula-tion in most protocols (Yanagihara and Shohet, 2012).Grinding with micrometer-size glass beads (Carrette andSeymour, 2004) or sonication (Helmholz et al., 2007;Morabito et al., 2012) are used. The sample is then centri-fuged to remove capsules, tubules and potential tissuedebris. Depending on the protocol, the venom samplecontains proteins and peptides from nematocysts, withvarying amounts of protein from the tentacle debris.

3.3. Venom purification

Bioactive toxins or fractions from the venom are isolatedby chromatographic separation. Common techniquesinclude size exclusion chromatography, ion exchangechromatography, reversed phase high performance liquidchromatography, affinity chromatography, or some com-bination thereof (Nagai et al., 2000a; Helmholz et al., 2008;Lassen et al., 2011; Ponce et al., 2013; Brinkman et al., 2014).Effective purification is guided by assessing the bioactivityof each of the fractions (Lassen et al., 2011, 2012).

3.4. Venomics

Amino acid sequences are available for some purifiedtoxins (Brinkman et al., 2014; Lassen et al., 2011, 2012; Liet al., 2012). DNA and RNA sequencing have been con-ducted for Carukia barnesi, Malo kingi (Avila Soria, 2009),and Aurelia aurita. Putative toxins were identified by pro-teomic analysis of nematocysts of Chironex fleckeri(Brinkman et al., 2012), Olindias sambaquiensis (Westonet al., 2013), and Stomolophus meleagris (Li et al., 2014).

3.5. Potential origins of variation in experimental results

Handling and storage of the venom can modify its bio-logical activity. For example, aggregation and adhesion topreparative surfaces is known to impair purification(Bloom et al., 1998; Brinkman and Burnell, 2009). Loss ofactivity after heating above 45 �C was reported for Chironexfleckeri (Pereira and Seymour, 2013), Cyanea capillata (Xiaoet al., 2009), Rhopilema nomadica (Gusmani et al., 1997), orRhopilema esculentum (Yu et al., 2007). Conversely, hemo-lytic activity of Pelagia noctiluca venom remained stableafter treatment at 100 �C (Marino et al., 2007). Storagetemperature, pH, and buffer composition can also affectvenom activity (Marino et al., 2006; Xiao et al., 2009).In vitro cardiotoxicity of Chironex fleckeri venom on rats waslost after more than two freeze-drying cycles (Winter et al.,2007).

Changes in the venom composition within the samespecies are frequently observed in venomous animals. Thechanges are linked to variation of food intake duringdevelopment and between geographic locations(McClounan and Seymour, 2012). An SDS-PAGE profile canbe used to assess differences in the composition of thevenom, along with measurement of biological activity. In

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

S. Badr�e / Toxicon xxx (2014) 1e12 5

jellyfish, the collection location, the time of collection, thematurity of the specimen collected, and the tissue origin(bell or tentacle) can also lead to differences in venomcomposition (Radwan et al., 2001; Winter et al., 2010;Helmholz et al., 2010; Underwood and Seymour, 2007;Avila Soria, 2009).

Different types of nematocysts can also have differentactivities or toxic potentials, in accordance with the mul-tiple physiological roles of those organelles. Chironexfleckeri venoms, extracted from nematocysts sorted by flowcytometry, were separated by liquid chromatography(McClounan and Seymour, 2012). The relative proportion ofeach bioactive fraction varied with the type of nematocyst.In another study, samples obtained from nematocysts ofPhysalia physalis sorted by size had different toxicities to-wards chick embryonic cardiocytes (Burnett et al., 1986).

4. Studies on jellyfish venom

4.1. Class cubozoa

4.1.1. Chironex fleckeriLethal, hemolytic, cardiotoxic, dermonecrotic, pore

forming, and cytotoxic activities were observed using crudevenom or partially purified fractions (Brinkman andBurnell, 2009; Saggiomo and Seymour, 2012; Pereira andSeymour, 2013; Yanagihara and Shohet, 2012). Four poreforming toxins have been identified and purified fromvenom extracted from nematocysts: CfTX-1 and CfTX-2(Brinkman and Burnell, 2007, 2008) and CfTX-A and CfTX-B (Brinkman et al., 2014). The molecular weight,measured by SDS-PAGE, is: CfTX-1: 43 kDA, CfTX-2: 45 kDA,CfTX-A: 40 kDA and CfTX-B: 42 kDA. A purified fractioncontaining CfTX-1 and -2, given by IV injection, inducedcardiovascular collapse in anaesthetized rats within 1 min,whereas the effect was minor with purified CfTX-A and -Bat the same protein concentration. Conversely, the hemo-lytic activity of CfTX-1/-2 is 30 times lower than the one ofCfTX-A/-B. Furthermore, antibodies raised against CfTX-1/-2 do not react with CfTX-A/-B (Brinkman and Burnell, 2008;Brinkman et al., 2014). Homologies in the amino-acidsequence were found within this group of toxins and be-tween Cf-TXs and other jellyfish pore-forming toxins.

Cell-based assays have been used to study the toxicity ofthe venom and to test potential antidotes. The crude venomand purified CfTX-s are cytotoxic to the A7r5 cell line (rataorta smooth muscle) in a dose-dependent way(Konstantakopoulos et al., 2009; Brinkman et al., 2014).Human cells from skeletal and cardiac muscles have alsobeen used to evaluate the cardiotoxicity of the venom(Saggiomo and Seymour, 2012; Pereira and Seymour, 2013;Chaousis et al., 2014). Using size exclusion chromatography,researchers isolated one fraction that was toxic to car-diomyocytes (Saggiomo and Seymour, 2012). Three crudetoxic fractions with distinct activities were identified in thisfirst fraction: CTF-alpha, beta and gamma (Chaousis et al.,2014). CTF-gamma toxicity was not specific to cardiaccells. CTF-alpha and beta were specifically toxic to car-diomyocytes but with distinct mechanisms: CTF-alphaacted faster and at a lower dose than CTF-beta, but cell

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recovery was observedwith a low dose of CTF-alpha, whichwas not the case for CTF-beta.

A proteomic study of the venom of Chironex fleckeriwaspublished in 2012 (Brinkman et al., 2012). CfTX-1 and CfTX-2were identified as well as homologues of the toxins CqTX-A from Chiropsalmus quadrigatus, CaTX-A from Carybdeaalata, CrTX-A from Carybdea rastoni and Cytotoxin A iso-form 1 from Malo kingi. These proteins are homologouspore-forming toxins. Isoforms of neprilysin andendothelin-converting factor were also present in thisproteome. Those proteases are putative toxic componentsof the venom since neprilysin is a neurotoxic metal-lopeptidase found in snake venom and the endothelin-converting factor can be found in wasp venom.

4.1.2. Irukandji syndromeStudies on the venom of species inducing Irukandji

syndrome are summarized in the recent review on jellyfishcausing this syndrome (Gershwin et al., 2013). Pharmaco-logical studies were performed on unfractionated venomfrom Carukia Barnesi, Alatina Mordens,Malo Kingi, andMaloMaxima to understand the cardiac dysfunction described inthe syndrome. The in vitro effect of the venom from CarukiaBarnesiwas described in a more recent publication (Pereiraand Seymour, 2013). The cell metabolism of human car-diomyocytes and skeletal cells was similar to the controlwithout venom, at all the doses tested. This result isconsistent with a hypothesis that the cardiac effect asso-ciated with the syndrome is an indirect effect of the toxin.SDS-PAGE profiles were published, with bands between 10and 200 kDa, but no protein was purified (Gershwin et al.,2013).

Carybdeida jellyfish have a box shaped bell with a singletentacle attached to each corner of the bell. The bell heightof the mature specimens ranges from 1 to 15 cm for mostspecies (Gershwin et al., 2013). Because of the small size ofthese jellyfish, it is difficult to detect and collect them andthen extract sufficient amounts of venom (Gershwin et al.,2013; Ramasamy et al., 2005b). To overcome the lack ofavailable specimens, a transcriptomic analysis was per-formed on Carukia barnesi and Malo kingi (Avila Soria,2009). cDNA libraries were developed for both speciesand expressed sequence tags (EST) were generated forMalokingi. Putative toxic proteins with protease or neurotoxicactivity were identified. The neurotoxins genes have ShKtoxin motifs, a potassium channel blocking toxin from thesea anemone Stichodactyla helianthus (Casta~neda et al.,1995). In Carukia barnesi, a recombinant neurotoxin wasexpressed from this gene and was active in cockroaches.Two putative cytolysins were extracted from the EST libraryof M. kingi. They belong to the same family of medusozoantoxins as Cf-TX1/2 of C. Fleckeri. The author suggested thattoxin(s) of the same family are also found in C. barnesivenom, as the SDS-PAGE profile of the venom has bands atthe same molecular weight (~45 kDa) that are antigenic toCf-TX1/2 and CSL box jellyfish antivenoms.

4.1.3. Other cubozoansPore forming toxins specific to medusozoans were first

described in three cubozoan species in 2000 and 2002(Nagai, 2003; Brinkman and Burnell, 2009). They were

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

S. Badr�e / Toxicon xxx (2014) 1e126

named CrTX-A and CrTX-B from Carybdea rastoni (Nagaiet al., 2000a), CaTX-A from Carybdea alata (Nagai et al.,2000b), and CqTX-A from Chiropsalmus quadrigatus(Nagai et al., 2002). The toxins were isolated by chroma-tography, and the corresponding amino acid and cDNAsequences were then determined. The three toxins arebasic proteins with apparent SDS-PAGE molecular weightsbetween 40 and 45 kDa. They are lethal to crayfish andinduce hemolysis of sheep red blood cells (Nagai, 2003).Homologous domains were identified between each aminoacid sequence, but no similarities were found with otherknown toxins (Nagai, 2003). Since then, other toxins of thesame family have been identified in medusozoan species(Brinkman et al., 2014).

CAH-1 is a basic 42 kDa hemolysin isolated from thevenom of C. alata (Chung et al., 2001). Comparison of the N-terminal amino acid sequence from CAH-1 to the sequenceof CaTX-A suggest that it is the same toxin (Brinkman andBurnell, 2009).

Cytolysins have also been found in Carybdea marsupialisvenom: a hemolytic toxin with a molecular weight of102e107 kDawas identified in specimens from the Adriaticsea (Rottini et al., 1995). More recently, three cytolysinswith apparent molecular weights of 220 kDa, 139 kDa, and36 kDa were purified from samples collected in theMexican Caribbean north coast (S�anchez-Rodríguez et al.,2006). In the same study, a neurotoxin was also identi-fied. The cytolysins and the neurotoxinwere isolated from adistinct chromatographic fraction.

4.2. Class scyphozoa

4.2.1. Cyanea sp.Within the genus Cyanea, the best-described venom is

from Cyanea capillata. This venom has hemolytic, cytotoxic,cardiotoxic, neurotoxic, and phospholipase A activities(Helmholz et al., 2007, 2010; Lassen et al., 2010; Xiao et al.,2011). Unfractionated venomwas toxic to rainbow trout gillcell (ATCC CRL-2523) (Helmholz et al., 2010), mouse neu-roblastoma (Neuro 2A) (Helmholz et al., 2012), humanhepatocytes (HepG2) (Helmholz et al., 2007), and rat renaltubular epithelial NRK-52E cells (Wang et al., 2013a). Thehemolytic activity was consistent with the presence of apore-forming toxin but lipid peroxidation was alsoobserved (Wang et al., 2013b). Oxidative stress was alsoinduced by the venom in rat renal tubular epithelial NRK-52E cells (Wang et al., 2013a). Acute toxicity in rats wasattributed to cardiac and neurological symptoms (Xiaoet al., 2011). Two mechanisms were observed, dependingon the dose. At the lowest lethal concentrations, death wasobserved within a few hours, associated with neurologic,heart and lung damage. At higher doses, death occurredwithin 15 min with only neurological symptoms.

An SDS-PAGE analysis suggests the venom is a mix ofproteins with molecular weights in the range 6e200 kDa(Helmholz et al., 2010; Liang et al., 2012). More than oneprotein is responsible for the biological activities of thevenom. For example, venom extracted directly from ten-tacles was purified by alkaline denaturation followed bydialysis (Liang et al., 2012). Before purification, the venomhad hemolytic and cardiotoxic activities, but after

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treatment, only the cardiotoxicity remained. Purification ofthe venom, guided by toxicity bioassays, led to the isolationand characterization of two toxins. The first one, namedCcTX-1, was isolated in the fraction that is cytotoxic to thehuman hepatocyte cell line HepG2 (Lassen et al., 2011).Homologies with the pore-forming toxins CaTX-1 andCrTX-1 were detected in the amino-acid sequence. Thesecond toxin, CcNT, was purified based on its toxicity tomouse neuroblastoma (Lassen et al., 2012). It is a neuro-toxin that targets voltage-gated sodium channels.

Cyanea nozakii can be observed in warm and temperatewaters, especially along the coasts of China (Li et al., 2011).The venom from this jellyfish was lethal to the grass carp,Ctenopharyngodon idellus. Neurotoxic, hemolytic, andcytotoxic activities were observed (Feng et al., 2010a,b; Liet al., 2011; Lee et al., 2011; Cuiping et al., 2012), as wellas phospholipase and proteolytic enzymatic activities (Fenget al., 2010b; Lee et al., 2011). A lethal and a hemolyticfraction were isolated by chromatography (Feng et al.,2010a; Li et al., 2011). The lethal activity of the crudevenom and hemolysis by the purified hemolysin wereinactivated by heat above 50 �C and were also pH sensitive(Feng et al., 2010a; Li et al., 2011).

The venom from Cyanea lamarckii was cytotoxic tohepatoma cell line HepG2, lytic to human red blood cells,and presented PLA2-like activity (Helmholz et al., 2007). Aglycoprotein, ClGp1, was isolated from the venom by lectin-affinity chromatography (Helmholz et al., 2008). This toxinwas cytotoxic to HepG2.

4.2.2. Pelagia noctilucaNeurotoxic, hemolytic, and cytotoxic activities of the

crude venom have been studied. A neurotoxic lethal effectwas observed in the shore crab Ocypode quadrata (S�anchez-Rodríguez and Lucio-Martínez, 2011). Hemolysis wasobserved in human, rabbit, chicken, and fish erythrocytes(Marino et al., 2007, 2008; S�anchez-Rodríguez and Lucio-Martínez, 2011; Maisano et al., 2013). The activity wasdose and time dependent (Marino et al., 2007). It wasstrongly inhibited by osmotic protectants and the cationsBa2þ and Cu2þ. Antioxidants had a lesser effect and otherdivalent cations, proteases, and carbohydrates did not haveany significant effect (Marino et al., 2008). Hemolytic ac-tivity on rabbit erythrocytes was reduced by the phos-pholipid sphingomyelin but not by phosphatidylserine orphosphatidylethanolamine (Maisano et al., 2013). Gluta-thione (GSH) levels in a suspension of fish erythrocytes didnot significantly change in the presence of crude venom(Maisano et al., 2013). These results suggest that hemolysisis due to pore formation on the red blood cell membrane.The toxicity of a nematocyst suspension was assayed onV79 Chinese hamster lung fibroblasts (Mariottini et al.,2002), and on Vero cells (kidney epithelial cells fromgreen monkey) (Ayed et al., 2012a, 2013). Crude venomextracted from isolated nematocysts was tested on an SH-SY5Y human neuroblastoma cell line (Morabito et al.,2012), a HCT116 human colon cancer cell line (Ayed et al.,2011), and a U87 human glioblastoma cell line (Ayedet al., 2012b). Cytotoxicity was observed in all of thesestudies, with evidence of oxidative stress and induction ofapoptosis. Oxidative stress was also observed in human red

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

S. Badr�e / Toxicon xxx (2014) 1e12 7

blood cells treated with non-hemolytic doses of venom(Morabito et al., 2013).

The SDS-PAGE profiles of venom extracted from Medi-terranean and Caribbean specimens (S�anchez-Rodríguezand Lucio-Martínez, 2011; Ayed et al., 2012c; Maisanoet al., 2013) revealed protein bands between 4 and150 kDa, with the most intense bands at an apparent mo-lecular weight higher than 80 kDa (S�anchez-Rodríguez andLucio-Martínez, 2011; Ayed et al., 2012c).

Very few studies have focused on the isolation ofbioactive component from the venom. A purification ofvenom from specimen collected in the Caribbean Sea wasperformed but the biological activities of the fractions werenot detailed (S�anchez-Rodríguez and Lucio-Martínez,2011). Venom from Mediterranean specimens was frac-tionated by size exclusion chromatography to find bioactivecompounds of pharmacological interest (Ayed et al.,2012b,c). In a first study, the two main fractions wereused to test the analgesic activity at low doses and the in-hibition of butyrylcholineesterase activity. The latter couldbe linked to the neurotoxic effects of the venom (Ayed et al.,2012c). In a second study, four fractionswere isolated (Ayedet al., 2012b). The first and the third fractionwere cytotoxicto human glioblastoma (U87 cell line), whereas the secondand the fourth were not. The effect on U87 cell proliferationwas tested at a protein concentration of 10 mg/mL. Thiscorresponds to 5% of the dose of crude venom that inducesa decrease in cell viability by 50%. The first two fractionshad an important inhibitory effect on cell proliferation, thethird had a minimal effect and the last had no effect. In themost recent study, the venomwas fractionated according tomolecular weight by HPLC and the hemolytic activity of thefractions was measured (Maisano et al., 2013). Four of theseven fractions were hemolytic to rabbit erythrocytes. Thefirst fraction was the most potent. It contained proteinswith the highest molecular weight.

4.2.3. Aurelia auritaDespite having low activity in humans, the in vitro toxic

action of the venom from Aurelia aurita has received someattention. Lethality and dermonecrosis were observed inmice, with variation in the potency depending on the originof the specimen (Radwan et al., 2001). Mortality was alsoobserved in shrimps (Artemia salina) (Segura-Puertas et al.,2002) and in crabs with symptoms of neurotoxicity(Segura-Puertas et al., 2002; Ponce et al., 2013). The venomis hemolytic to human, sheep, and bovine red blood cells(Radwan et al., 2001; Segura-Puertas et al., 2002; Rastogiet al., 2012). Protease and phospholipase A activities wererecorded (Nevalainen et al., 2004; Lee et al., 2011).Zymography experiments were performed with gelatin,casein, or fibrin as substrates (Lee et al., 2011). Severalproteins bands were able to lyse each substrate. In the samestudy, hyaluronidase activity was not detected, but A. auritavenomwas cytotoxic to NIH 3T3mouse fibroblasts in a dosedependent manner. All these activities were inhibited by o-phenantroline, a metalloprotease inhibitor. Cytotoxicity torainbow trout gill cell (ATCC CRL-2523) was also docu-mented (Helmholz et al., 2010).

SDS-PAGE electrophoresis revealed that the venomcontains several bands between 200 and 6 kDa (Rastogi

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et al., 2012). The venom was purified by chromatographyand the neurotoxic activity of some fractions was charac-terized (Segura-Puertas et al., 2002; Ponce et al., 2013). Inthe most recent study, two fractions (n�4 and 5) wereneurotoxic to adult ghost crabs in vivo and one fraction(n�4), inhibited the activation of muscle nicotinic acetyl-choline receptors (nAChRs) by acetylcholine in a reversibleand dose dependent manner. Phospholipase A activity wasdetected in the unfractionated venom, but not in theseneurotoxic fractions. Action on nAChRs and lack of PLAactivity suggest the neurotoxin targets the post-synapticside of the neuromuscular junction.

4.2.4. Rhopilema sp.The venom from Rhopilema nomadica was purified by

size exclusion chromatography (Gusmani et al., 1997). Ityielded four bioactive fractions: the first one was hemo-lytic, the second and the third contained proteases, and thelast exhibited PLA2 activity. Each fraction had only one typeof biological activity. Hemolysis was attributed to a pore-forming toxin. A protein with PLA2 activity was alsosequenced (Lotan et al., 1995). In the same genus, proteo-lytic, cytotoxic and hemolytic activities were observed inthe venom of Rhopilema esculentum (Li et al., 2005; Leeet al., 2011; Yu et al., 2007). For both species, the hemo-lytic activity was reduced when the temperature increased(Gusmani et al., 1997; Yu et al., 2007).

4.2.5. Nemopilema nomuraiThe venom induced hypotension and bradycardia in rats

(Kim et al., 2006) and induced dermonecrotic skin lesions inrabbits (Kang et al., 2013). Gelatinolytic, caseinolytic andfibrinolytic activities were observed by zymography forseveral proteins of the venom (Lee et al., 2011). The venomwas cytotoxic to HaCaT cell line (human keratinocyte),NIH3T3 cell line (mouse fibroblast), C2C12 cell line (ratskeletal myoblast) and H9C2 cell line (rat heart myoblast)(Kang et al., 2013, 2009, 2014). The venomwasmore toxic toheartmyoblast than to skeletalmyoblast andwas inactivatedby heating over 40 �C (Kang et al., 2009). Hemolysis was alsoobservedwith cat, dog, human, rabbit and rat red blood cells(Kang et al., 2009). Proteolysis and cytotoxicitywas inhibitedby two metalloprotease inhibitors, o-phenantroline andtetracycline (Lee et al., 2011, Kang et al., 2013).

A polyclonal antibody against the venom of Nemopilemanomurai was recently produced by Kang et al. (2014). Pre-incubation of the venom with this antibody inhibitedcytotoxicity, hemolysis, and lethality towards mice. Theantibody was used in a proteomic analysis to select venomspots in a 2D-electrophoresis gel before mass spectroscopyanalysis. A total of 18 proteins were identified, including amatrix-metalloproteinase-14 (MMP-14) and an astacin-likemetalloprotease toxin 3 precursor. Suchmetalloproteinasesare found in the venom of brown spider and snake, wherethey induce local effects similar to those observed for N.nomurai.

4.2.6. Stomolophus meleagrisThe venom extracted from nematocysts of S. meleagris

was fractionated by chromatography. Using different sep-aration strategies, researchers first obtained a hemolytic

stinging jellyfish, Toxicon (2014), http://dx.doi.org/10.1016/

Table 2Toxins purified from jellyfish venom. Names in bold letters correspond tohomologous proteins. CbTX-1 is a recombinant protein. All other toxinswere purified by chromatography.

Species Name Mw

(kDa)aMw

(kDa)bBiological activity

Chironex fleckeri CfTX-1 43 CardiotoxicCytotoxicHemolytic

CfTX-2 45 CardiotoxicCytotoxicHemolytic

CfTX-A 40 CytotoxicHemolytic

CfTX-B 42 CytotoxicHemolytic

Carukia barnesi CbTX-I 21 21.67 NeurotoxicCarybdea rastonii CrTX-A 43 Hemolytic/lethal

to crayfishCrTX-B 46Carybdea alata CaTX-A 43 Hemolytic/lethal

to crayfish(Alatina alata) CaTX-B 45Chiropsalmus

quadrigatusCqTX-A 44 Hemolytic/lethal

to crayfish(Chiropsoides

quadrigatus)Carybdea

marsupialis220 Cytolytic102e107 Hemolytic139 Cytolytic36 Neurotoxic

Cyanea capillata CcTX-1 31.173 CytotoxicCcNT 8.22 Neurotoxic

Cyanealamarckii

ClGP-1 27 25.7 Cytotoxic

Rhopilemanomadica

PLA2

Stomolophusmeleagris

SmP90 90 Radicalscavenging

Physalia physalis Physalitoxin 220 HemolyticP1 220 NeurotoxicP3 85 NeurotoxicPpV9.4 0.55 Action on

insulinsecretingcells

PpV19.3 4.72 Action oninsulinsecreting cells

Olindiassambaquiensis

Oshem1 3.013 HemolyticOshem2 3.376 Hemolytic

a Molecular weight estimated by SDS-PAGE in kDa.b Molecular weight measured by mass spectroscopy in kDa.

S. Badr�e / Toxicon xxx (2014) 1e128

fraction and then a protein that exhibited superoxide anionradical-scavenging activity (Li et al., 2013, 2012). This lastprotein was named SmP90. Results of peptide massfingerprinting and N-terminal amino acid sequence couldnot be matched with any known protein (Li et al., 2012). Aproteomic and transcriptomic analysis of the venom is alsoavailable (Li et al., 2014). Several putative toxins wereidentified by homology to proteins from bacteria andvenomous and non-venomous animals. They were classi-fied as serine protease inhibitors, phospholipase A2, po-tassium channel inhibitors, C-type lectins, hemolysins, orother type of toxins. The sequences of the putative hemo-lysins of S. meleagriswere compared to the sequences of thepore-forming toxins CaTX-A, CrTX-A, CqTX-A and CfTX-1/2,by multiple sequence alignment of the unigenes. The au-thors did not find any significant match between the he-molysins from S. meleagris and from the other cubozoanspecies.

4.3. Class hydrozoa

4.3.1. Physalia physalisThe venom of this species has hemolytic, neurotoxic and

cardiotoxic activities (Tamkun and Hessinger, 1981) (�Suput,2009). It induced calcium influx into several types ofcultured cells: chick embryo cardiocytes, GH4C1 cells fromrat pituitary gland, L-929 mouse fibroblasts and fetal ratlung cells (Edwards et al., 2000, 2002; Edwards andHessinger, 2000). Analysis of the mechanism of action ledto the conclusion that the venom contains a pore-formingtoxin.

Five toxins were isolated from the venom by chroma-tography. Physalitoxin was a 220 kDa glycosylated protein,which was hemolytic and lethal to mice (Tamkun andHessinger, 1981). Among the five toxins, two neurotoxinswere identified: P1 had a molecular weight of 220 kDa andblocked nicotinic cholinergic post-synaptic receptors in anon-competitive way (Men�endez et al., 1990). P3 was a85 kDa toxin acting as a reversible glutamate antagonist(Mas et al., 1989). Two low-molecular weight toxins, PpV9.4and PpV19.3, were purified based on their action on insulinsecreting beta cells (Diaz-Garcia et al., 2012).

4.3.2. Olindias sambaquiensisOshem1 (3.013 kDa) and Oshem2 (3.375 kDa), two hy-

drophilic low-molecular weight peptides were purifiedfrom the sole hemolytic fraction of the venom of this jel-lyfish (Junior et al., 2014). These peptides exhibited he-molytic, myonecrotic, and cytotoxic activities. The N-terminal amino acid sequences were compared to the se-quences of toxins from sea anemones with similar activ-ities. They shared a 50% amino acid identity with the toxinUpI of Urticina piscivora. A proteomic analysis of the nem-atocysts was also recently published (Weston et al., 2013)that identified 29 putative toxins. These toxins are similarto metallo and serine proteases, lipid degrading enzymes,neurotoxins, ion channel blockers, and hemolysins found insnake, spider, scorpion, sea cone, hymenoptera or seaanemone venoms. The two putative hemolysins share theclosest homologies with pore-forming toxins from the seaanemones Actineria villosa (AvTX-60A) and Phyllodiscus

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semoni (PsTX-60A). Both toxins belong to the family ofmembrane attack complex/perforin (MACPF) cytolysins(Anderluh et al., 2011).

5. Biological activities of jellyfish toxins

The SDS-PAGE profiles of jellyfish venom include severalbands between 3 and 200 kDa. Table 2 lists the toxins pu-rified to date. Biological activities commonly associatedwith venomous toxicities have been described.

The best described activity is cell lysis by pore-formingtoxins. Most of the known toxins belong to a family ofpore forming toxins, first identified in the class Cubozoa(Brinkman and Burnell, 2009). Homologous toxins werealso detected in the meduzosoan species Cyanea capillata,Aurelia aurita and Hydra magnipapillata. 3D-structure

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S. Badr�e / Toxicon xxx (2014) 1e12 9

modeling predicted that the N-terminal side of the toxins isdominated by a-helices and that CfTX like toxins arestructural analogues of the 3d-Cry toxins from the bacteriaBacillus thuringiensis, which are toxic to insect cells(Brinkman and Burnell, 2009; Brinkman et al., 2014). Thegroup was divided into two types of toxins: CfTX-1/2 andCqTX-A are type I toxins and CfTX-A/B and CaTX-A andCrTX-A belong to the type II group. The efficacy of thetoxins differs depending on the biological target (Brinkmanet al., 2014). With respect to the biological activities ofChironex fleckeri toxins, type I toxins have a stronger car-diovascular effect and a lower hemolytic power than type IItoxins. Studies on CrTX-A, CaTX-A and CqTX-A, also suggestthat type II toxins have a greater hemolytic effect comparedto type I toxins. Type II toxins are also more toxic to crayfishthan the type I toxin CqTX-A (Nagai, 2003).

The venoms from Stomolophus meleagris, Olindias sam-baquiensis, Pelagia noctiluca, and Physalia physalis alsocontain hemolysins. The toxins from Olindias sambaquiensisshare their closest homologies with sea anemones toxins.Hemolysis by pore forming toxins was observed in thevenom of Pelagia noctiluca and Physalia physalis, but theactive componentof thevenom is currently uncharacterized.

Neurotoxins have been isolated from some jellyfishspecies. Neurotoxic and hemolytic activities are usuallyfound in different chromatographic fractions. A number ofneurologic targets have been described, including ionicchannels and neurotransmitter receptors.

Other biological activities have been observed in jelly-fish venom, but the corresponding toxins have not yet beenfully described. Metalloproteases, serine proteases, andphospholipases A have been detected in jellyfish venoms,either by direct activity assay or by proteomic analysis.Cytotoxicity mediated by oxidative damage has been sug-gested for the venoms of Cyanea capillata and Pelagia noc-tiluca. Additionally, there are reports of toxicity specific tocardiac cells and, for C. fleckeri venom, at least two mech-anisms of toxicity to human cardiomyocytes have beenidentified (Chaousis et al., 2014).

The in vivo mode of action of jellyfish venoms is not yetunderstood. These venoms contain hemolysins that act aspore-forming toxins, but hemolysis is not observed inhumans. These toxins are also toxic to other cell types,suggesting different targets in vivo. Evidence of additionaltoxicity, induced by other types of toxins, is observed inmany species but their role in the envenomation has notbeen explored.

Because of the specificity of the venomous apparatus ofjellyfish, the tissue origin of a purified toxin should betaken into account in the analysis of its role in human en-venomation. Upon contact, the content of the nematocystsis injected into the skin, but the surface of the epidermis isalso in contact with surrounding tissues from the jellyfish.Depending on the extraction protocol, the venom extrac-tion can yield proteins from only the nematocysts or fromboth nematocysts and surrounding tissues and both typesof samples can contain bioactive molecules. For example,the activities and extraction methods of partially purifiedbioactive proteins from Chironex fleckeri are summarized inBrinkman and Burnell (2009), revealing a wide range ofresults.

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6. Conclusion

The biological activities observed in jellyfish venoms aresimilar to those observed in other venomous species.However, specimen availability can be an issue for biolog-ical and structural studies, and detailed knowledge on thestructure of the toxins and of their mode of action is stillfragmentary. Some jellyfish toxins have been purified andsequenced, and most of them belong to a family of poreforming toxins described only in Medusozoans. Themechanisms underlying the most severe human symptomsare still under debate. There are large gaps in our knowl-edge about the toxins and their specificities.

Conflict of interest

The author has no conflict of interest to declare.

Transparency document

Transparency document related to this article can befound online at http://dx.doi.org/10.1016/j.toxicon.2014.09.010.

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