Chiropsalmus Quadrigatus Research Articles

Real-time PCR assay for detection of Environmental DNA from <i>Chironex yamaguchii</i>

The box jellyfish Chironex yamaguchii is a highly venomous cubomedusa, also known as “habu-kurage” in Japan. It is a significant threat to marine activities in Okinawa in southern Japan. Annually, envenomation of the species causes nearly 100 to 200 injuries in the Okinawa Prefecture. The present study developed a species-specific real-time PCR assay to analyze environmental DNA (eDNA) for rapid detection of jellyfish in water samples. Primers and probe were designed based on the sequences of the mitochondrial cytochrome c oxidase subunit I region using tissue samples of nine specimens obtained from the Okinawa Prefecture. The specificity of the real-time PCR assay was confirmed by amplifying the DNA samples of 10 different box jellyfish species. A practical assay for eDNA detection was successful on water samples obtained from the natural habitat of C. yamaguchii. In addition, the degradation rate of eDNA in the tank experiment was 65% of the initial concentration at 1 h, 22% at 12 h, and 3% at 48 h.

New Records of Cubozoan and Scyphozoan Jellyfish from Sabah Waters, Malaysia

Jellyfish play a vital role in the ocean’s ecosystem, acting as a nursery for young fishes, crabs, and brittle stars, as well as a source of food for certain charismatic megafauna, such as sunfish and sea turtles. They also pose a threat to human activities, with jellyfish blooms negatively impacting fisheries, power generation, and tourism. However, very little information is available on the biodiversity of jellyfish within the waters of Borneo. Here, we present new records of jellyfish found along the coast of Sabah, Malaysia, located in northern Borneo, bordering the megadiverse region of the Coral Triangle. A total of six species belonging to two classes and six families hitherto not recorded to the state are reported, Chironex yamaguchii, Acromitus maculosus, Crambione mastigophora, Linuche aquila, Netrostoma sp., and Phyllorhiza punctata. Of these, two species (C. yamaguchii and L. aquila) are harmful to humans, with C. yamaguchii capable of causing human fatalities. Reports of harmful jellyfish are useful to the medical and tourism industry, as some of these species may inflict stings and adverse reactions to humans ranging from rashes and skin irritation to fatal envenomation. A checklist has also been provided for all collected jellyfish species from Sabah waters of Borneo, Malaysia.

Exumbrellar Surface of Jellyfish: A Comparative Fine Structure Study with Remarks on Surface Reflectance

The exumbrellar surfaces of six pelagic cnidarians from three classes were ultra-structurally compared to reveal their structural diversity in relation to their gelatinous, transparent bodies. We examined two hydrozoans (Diphyes chamissonis and Colobonema sericeum), a cubozoan (Chironex yamaguchii), and three scyphozoans (Atolla vanhöffeni, Aurelia coerulea, and Mastigias papua). The exumbrellar surfaces of the mesoglea in D. chamissonis, Ch. yamaguchii, Au. coerulea, and M. papua were covered with a simple epidermis; the shapes of the epidermal cells were remarkably different among the species. The epidermal cells of Ch. yamaguchii and M. papua possessed an array of microvilli on the apical side. The array possibly reduced light reflectance and provided some other surface properties, as seen for the cuticular nipple array in tunicates, considering the length, width, and pitch of the microvilli. The reduction of light reflectance on the array of microvilli was supported by the simulation with rigorous coupled wave analysis (RCWA). Microvilli were sparse and did not form an array in metephyrae of Au. coerulea. The mesoglea matrix beneath the basal side of the epidermis was loose in all of the species. The exumbrellar side of the mesoglea was exposed only in the mesopelagic species, At. vanhöffeni and Co. sericeum, and electron-dense layer(s) covered the surface of the mesoglea. It is uncertain whether the exumbrellar epidermis is absent in these species or the epidermal cells are completely exfoliated during the sampling and handling processes. In the latter case, the electron-dense layer(s) on the mesoglea surface might originally underlie the epidermis.

Isolation, Structure Determination, and Synthesis of Cyclic Tetraglutamic Acids from Box Jellyfish Species Alatina alata and Chironex yamaguchii.

Cubozoan nematocyst venoms contain known cytolytic and hemolytic proteins, but small molecule components have not been previously reported from cubozoan venom. We screened nematocyst extracts of Alatina alata and Chironex yamaguchii by LC-MS for the presence of small molecule metabolites. Three isomeric compounds, cnidarins 4A (1), 4B (2), and 4C (3), were isolated from venom extracts and characterized by NMR and MS, which revealed their planar structure as cyclic γ-linked tetraglutamic acids. The full configurational assignments were established by syntheses of all six possible stereoisomers, comparison of spectral data and optical rotations, and stereochemical analysis of derivatized degradation products. Compounds 1–3 were subsequently detected by LC-MS in tissues of eight other cnidarian species. The most abundant of these compounds, cnidarin 4A (1), showed no mammalian cell toxicity or hemolytic activity, which may suggest a role for these cyclic tetraglutamates in nematocyst discharge.

Clinical characteristics of jellyfish stings in Japan.

AimJellyfish known as “habu‐kurage” (Chironex yamaguchii) inhabit the waters surrounding Okinawa and Amami, Japan, and jellyfish stings are limited to areas outside the Japanese main island. However, the shifts promoted by global warming and increasingly intensive interactions with people have led to concerns regarding the possibility of increased jellyfish stings on the main island of Japan. Similar concerns are being raised all over the world. However, studies examining clinical characteristics of jellyfish stings have been limited to Australia, the USA, Europe, and South‐East Asia. Thus, this study aimed to examine for the first time the clinical characteristics of jellyfish stings in Japan.MethodsWe undertook retrospective questionnaire surveys from January 2013 to December 2017 to determine patient characteristics, treatment, and clinical outcomes. We compared patient characteristics between tourists and non‐tourists. The primary end‐point of the present study was to clarify the clinical characteristics of jellyfish stings.ResultsOver the 5‐year study period, 204 patients were identified from eight hospitals based on the basic questionnaire, and 35 patients with moderate‐to‐severe jellyfish stings were also identified. All patients recovered well, including five patients with severe jellyfish stings. Antivenom was not given. The time intervals from the occurrence of jellyfish stings to hospital arrival significantly differed between tourists and non‐tourists (P = 0.049), and all tourists visited the emergency department (P = 0.009).ConclusionsApproximately 40 cases of jellyfish stings occurred annually in Japan between 2013 and 2017. Patients recovered well without the use of antivenom.

In vitro angiotensin I converting enzyme inhibition by a peptide isolated from Chiropsalmus quadrigatus Haeckel (box jellyfish) venom hydrolysate

The anti-angiotensin I converting enzyme activity of box jellyfish, Chiropsalmus quadrigatus Haeckel venom hydrolysate was studied. The venom extract was obtained by centrifugation and ultrasonication. Protein concentration of 12.99 μg/mL was determined using Bradford assay. The pepsin and papain hydrolysate was tested for its toxicity by Limit test following the OECD Guideline 425 using 5 female Sprague-Dawley rats. Results showed that the hydrolysate is nontoxic with an LD50 above 2000 mg/kg. In vitro angiotensin I converting enzyme (ACE) inhibitory activity was determined using ACE kit-WST. Isolation of ACE inhibitory peptides using column chromatography with SP-Sephadex G-25 yielded 8 pooled fractions with fraction 3 (86.5%) exhibiting the highest activity. This was followed by reverse phase - high performance liquid chromatography (RP-HPLC) with an octadecyl silica column (Inertsil ODS-3) using methanol:water 15:85 at a flow rate of 1.0 mL/min. Among the 13 fractions separated with the RP-HPLC, fraction 3.5 exhibited the highest ACE inhibitory activity (84.1%). The peptide sequence ACPGPNPGRP (IC50 2.03 μM) from fraction 3.5 was identified using Matrix-assisted laser desorption/ionization with time-of-flight tandem mass spectroscopy analysis (MALDI–TOF/MS).

Comparative study of the toxic effects of Chrysaora quinquecirrha (Cnidaria: Scyphozoa) and Chironex fleckeri (Cnidaria: Cubozoa) venoms using cell-based assays

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.

Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish.

A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.

Identification of Allergens in the Box Jellyfish Chironex yamaguchii That Cause Sting Dermatitis

Background: Jellyfish stings cause painful, papular-urticarial eruptions due to the immediate allergic, acute toxic and persistent inflammatory responses. In spite of many marine accidents and their economic impact, modes of first-aid treatment remain conventional and specific allergen and medical treatment are not yet available. The purpose of this study was to define the specific allergen of the box jellyfish Chironex yamaguchii and to study the precise mechanism of the resulting dermatitis. Methods: We comprehensively studied the immunoglobulin-binding molecules from the box jellyfish C. yamaguchii with a purification procedure and Western blotting, using sera from 1 patient and from several controls. Results: From the nematocyst wall and spine, we detected IgG-binding acidic glycoprotein (of 66 and 30 kDa) as determined by Western blot and ion-exchange chromatography. In addition, the 66-kDa protein was found to be an asparagine residue-coupled N-linked glycoprotein and the epitope resided in the protein fraction. We found that CqTX-A, the major toxic protein of the nematocyst, is also a heat-stable IgE-binding allergen. This was confirmed as a 45-kDa protein by Western blot from both nematocyst extracts and purified CqTX-A. Conclusions: The detection of these proteins may, in part, explain the combined immediate allergic-toxic and persistent allergic responses. Hopefully, our findings will lead to the development of specific venom immunotherapy for marine professional workers and tourists for jellyfish-sting dermatitis and anaphylaxis.

Jellyfish sting injuries

Jellyfish are distributed worldwide; they cause local skin injuries upon contact which are often followed by systemic signs of envenoming. Which jellyfish species are of medical importance, which skin reactions and systemic symptoms occur, which first-aid measures and treatment options exist? Review of the medical literature and discussion of first-aid and therapeutic options. Jellyfish capable of causing skin injuries occur in almost all oceans. Several jellyfish species may cause severe, potentially lethal, systemic symptoms; they include the Portuguese man-of-war (Physalia physalis) and box jellyfish (Chironex fleckeri, Carukia barnesi, Chiropsalmus quadrigatus). Among the injuries and envenoming symptoms caused by marine organisms, jellyfish dermatitis should not be underestimated. Skin reactions may not only a dermatological problem, but also be accompanied by complex systemic toxic symptoms which are a challenge for internists.

Clarifying the identity of the Japanese Habu-kurage, Chironex yamaguchii, sp. nov. (Cnidaria: Cubozoa: Chirodropida)

Here we describe the new species Chironex yamaguchii (Cnidaria: Cubozoa) from the Ryukyu Archipelago, Japan. This highly venomous cubomedusa, commonly referred to as Habu-kurage in Japan, is the culprit for several fatalities in Japanese waters. The scientific name adopted for this species in the literature is Chiropsalmus quadrigatus, but our taxonomic investigations show that this represents a case of mistaken identity. In fact, Habu-kurage is a close relative of Chironex fleckeri, which is particularly interesting because the latter has a reputation for being the most venomous animal known. Differences in the shape of the pedalial canal bend, the number of tentacles, and bell size distinguish the two species. In addition to Japan, Chironex yamaguchii is widely distributed in the Philippines.

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri

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 45 kDa, 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.

Comments on Chiropsalmus (Cnidaria: Cubozoa: Chirodropida): a preliminary revision of the Chiropsalmidae, with descriptions of two new genera and two new species

The nomenclature and identity of Chiropsalmus quadrigatus have been extensively confused. Originally described from Rangoon, Burma, based on an immature and badly damaged specimen, subsequent Philippine and Australian redescriptions do not match primary characters of the holotype. This has led to widespread messiness in identification and classification of this and related species. In order to clarify the nomenclature of the Chiropsalmus-type cubomedusae, a preliminary revision of the Chiropsalmidae is given. Chiropsoides quadrigatus comb. nov. is proposed, in order to reflect the close relationship of this taxon with Chiropsoides buitendijki, based on the shared character of linear-branching pedalia. A common Australian form, often attributed to Chiropsalmus quadrigatus, is herein described as a new genus and species, Chiropsella bronzie. This new form differs from other Chirodropida in having sessile, solid, smooth gastric saccules, whereas in other Chirodropida these structures are pendant whether they are smooth or branched. This new species is not dangerous to humans. Another form, Chiropsalmus alipes n. sp., is described from the Pacific coast of southern Mexico; this new species differs from others in having long, blade-like pedalia similar to those of the Carybdeida, only four tentacles at maturity per pedalium, each branching in a different direction, and a warty body; comments are made regarding its apparent relationship to the enigmatic Chiropsalmus zygonema. The recently described Chiropsalmus maculatus is moved to a new genus, Chirodectes in the family Chirodropidae.

Cardiovascular effects and lethality of venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rats

Haemodynamic effects of saline-extracted venom from nematocysts isolated from Chiropsalmus quadrigatus (Habu-kurage) were studied in anaesthetized rats. Intravenous administration of venom (0.2–5 μg protein/kg) produced immediately dose-dependent hypertension and bradycardia. Femoral blood flow transiently increased but calculated femoral vascular conductance decreased. Changes caused by 1 μg/kg of venom were reproducible, and were not affected by prazosin, atropine or BQ123 (ET A receptor antagonist) but were significantly attenuated by nicardipine. At doses over 2 μg/kg, hypotension and a decrease in pulse pressure were observed subsequent to transient hypertension. In 5 of 8 rats received 5 μg/kg venom and 6 of 6 rats at 10 μg/kg, death due to irreversible cardiac arrest occurred within 30 min after intravenous injection. However, during nicardipine infusion, venom (10 μg/kg) exerted only modest effects and the rats survived. Heating venom (50 °C for 10 min) before injection practically abolished the haemodynamic effects of 10 μg/kg venom, indicating its thermolability. Data show that C. quadrigatus venom has both vasoconstrictor and cardiodepressive effects in rats, and suggest that a calcium channel blocker can protect against the cardiovascular and lethal effects of the venom.

Haemodynamic effects of the crude venom from nematocysts of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) in anaesthetized rabbits

Haemodynamic effects of saline-extracted venom from nematocysts isolated from tentacles of the box-jellyfish Chiropsalmus quadrigatus (Habu-kurage) were investigated. In anaesthetized rabbits, i.v. injections of the venom produced hypotension following a transient hypertension. Mean femoral arterial blood flow markedly decreased immediately after the injection and femoral vascular resistance increased. Left ventricular d P/d t remarkably decreased after a transient and small increase, and heart rate decreased. Left ventricular end-diastolic pressure markedly elevated. All of the above changes by 0.2–5 μg/kg of the venom expressed as the amount of protein were seen dose-dependently and occurred without tachyphylaxis. In five of seven animals received an injection of the venom at 10 μg/kg, irreversible cardiac arrest occurred. Changes produced by 1 or 2 μg/kg of the venom were significantly attenuated either by heating the venom at 40 °C for 10 min or by pretreatment with diltiazem. These results indicate that the venom from Habu-kurage has both vasoconstrictor and cardiodepressive effects, and suggest that these thermolabile actions may be due partly to activation of voltage-dependent calcium channels and probably subsequent calcium-overload.

Recent Progress in Jellyfish Toxin Study

Recently, we have reported the chemical properties of some jellyfish proteinaceous toxins. These were the first chemical characterizations of jellyfish protein toxins to be reported. The isolation of the proteinaceous toxins in their active forms was the key step in the studies. We isolated the toxins from three box jellyfish (Cubozoa) species [Carybdea rastoni (C. rastoni), Carybdea alata (C. alata), and Chiropsalmus quadrigatus (C. quadrigatus)]. These toxins showed lethal toxicity to crustaceans and hemolytic activity. Furthermore, the full-length cDNAs and the deduced amino acid sequences of the toxins were clarified. All of these toxins have a molecular weight of around 45 kDa and their amino acid sequences showed homology with each other. The box jellyfish toxins represent a novel bioactive protein family.

Jellyfish Antivenoms: Past, Present, and Future

If snake antivenoms are considered orphan drugs, then jellyfish antivenoms are the poorest of the orphans. Despite the diversity, ubiquity and toxicity of the venomous cnidarians, only a single antivenom is available for jellyfish stings worldwide. That antivenom, an ovine whole IgG product, is directed against the ‘box’ jellyfish, Chironex fleckeri, and is manufactured by CSL Limited (Melbourne, Australia). It also neutralises the venom of closely related cubozoans such as Chiropsalmus quadrigatus. The recognition of the life‐threatening effects of various other jellyfish demonstrates the need for broadening the specificity of the existing product and/or developing additional specific jellyfish antivenoms. These emerging threats include the irukandji syndrome, due to Carukia barnesi and other carybdeids, as well as those from scyphozoans such as Stomolophus spp. The role of ancillary drug therapy, in addition to, or instead of, antivenoms remains controversial. This review will consider the development o...

A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus.

The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50 = 80 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50 = 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

Apoptosis induced by box jellyfish ( Chiropsalmus Quadrigatus) toxin in glioma and vascular endothelial cell lines

This study was made to investigate whether Chiropsalmus Quadrigatus toxins (CqTX), which isolated from box jellyfish C. Quadrigatus venom, could induce apoptosis in human U251 and rat C6 malignant glioma cells and transformed vascular endothelial ECV 304 cell lines. Cell viability was estimated by MTT assay. Apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labeling (TUNEL) method and DNA gel electrophoresis. Furthermore, the expression of p53 protein was examined immunohistochemically in the U251 cells. After the CqTX treatment, the growth of all cell lines was inhibited, the fragmented DNA was observed and some cells became TUNEL positive. The expression of p53 protein was increased in the tested U251 cells. The results suggested that CqTX induced apoptosis in these cell lines. The promotion of the p53 expression might be one mechanism of apoptosis induced by CqTX in the glioma cells.

Sym.2 沖縄県におけるハブクラゲ等海洋危険生物 : 2. 毒

Sym.2 沖縄県におけるハブクラゲ等海洋危険生物 : 2. 毒