Evaluation of a Guest Lecture on Medical Emergency of Box Jellyfish Envenomation Pathophysiology and First Aid for Medical Students.

Introduction: Lethal box jellyfish envenomation has been reported in Indonesia and other countries; therefore, medical students should be equipped with related knowledge. The aim of this study was to evaluate the results of summative exams by student cohort and gender and determine the factors that contribute to success in the summative exams after novel intensive instruction in box jellyfish envenomation pathophysiology and first aid in undergraduate medical students in Surabaya, Indonesia. Methods: This study used explanatory sequential mixed methods, consisting of a cross-sectional study and interviews. A total population sampling of 203 sixth-semester students was employed. Student cohort, gender, previous semester grade point average (GPA), and English proficiency test (EPT) were considered. All statistical tests were carried out using IBM® SPSS® Statistics version 24.0 for Macintosh. The study was complemented by interviews conducted with 20 students. Results: The one-way ANOVA test showed that students from the 2016 cohort had significantly higher mean scores in the exam than the 2015 and 2014 cohorts (p=0.002). Independent samples t-test showed that such differences were not gender-specific (p=0.249). In the binary logistic regression, the GPA in the previous semester was the only factor that contributed to success in the summative exam (OR 3.031, 95% CI: 1.520-6.044). All students commented that the lecture and practicum were interesting and beneficial. However, some considered that the language barrier might have prevented them from understanding the topic well. Conclusion: Results of the summative exam differed by the student cohorts, and previous semester GPA was a predictor of success in the summative exam.

Small-molecule mimicry hunting strategy in the imperial cone snail, Conus imperialis.

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Conus imperialis Using the model polychaete worm Platynereis dumerilii, we demonstrate that C. imperialis venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, C. imperialis uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.

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.

Revision of the genus Carybdea (Cnidaria: Cubozoa: Carybdeidae): clarifying the identity of its type species Carybdea marsupialis.

While records of Carybdea marsupialis in the literature suggest a worldwide distribution of this species, the validity of some of these records has been questioned recently, as has the validity of some nominal Carybdea species. We inspected material of all known species of Carybdea from multiple locations (i.e. Spain, Algeria, Tunisia, Puerto Rico, California, Hawaii, Australia, South Africa, and Japan) using morphological and genetic tools to differentiate Carybdea species as well as understand their evolutionary relationships. We observed morphological differences between adult medusae of Mediterranean and Caribbean C. marsupialis; the most obvious differences were the structure of the phacellae, the structure of the pedalial canal knee bend, and the number and structure of the velarial canals. The characters of the adult Mediterranean specimens agree with the description provided by Claus (1878) for individuals of C. marsupialis from the Adriatic Sea (Italy); specimens from the Caribbean (Puerto Rico) agreed with the description of C. xaymacana by Conant (1897). Significant differences between both species were also observed in the newly released medusa stage. Further, we resolved a discord about the undefined polyp culture originating from Puerto Rico that was long considered Carybdea marsupialis but should be referred to as C. xaymacana. Although C. marsupialis is currently considered the only species of Cubozoa to occur in the Mediterranean, specimens collected in Algeria and Tunisia suggest that species of Alatinidae may also be present in the Mediterranean. Our investigations indicate that Carybdea spp. are more restricted in their geographical distribution than has been recognized historically. These findings confirm that Carybdea arborifera Maas, 1897 from Hawaii, Carybdea branchi, Gershwin Gibbons, 2009 from South Africa, Carybdea brevipedalia Kishinouye, 1891 from Japan, Carybdea confusa Straehler-Pohl, Matsumoto Acevedo, 2017 from California, Carybdea marsupialis Linnaeus, 1758 from the European Mediterranean Sea, Carybdea rastonii Haacke, 1886 from South Australia, and Carybdea xaymacana, Conant, 1897 from the Caribbean Sea are valid names representing distinct species, rather than synonyms. A taxonomic key for all valid species is provided, and a neotype for C. marsupialis is designated.

Evaluation of Cyanea capillata Sting Management Protocols Using Ex Vivo and In Vitro Envenomation Models.

Lion's mane jellyfish (Cyanea capillata) stings cause severe pain and can lead to dangerous systemic effects, including Irukandji-like syndrome. As is the case for most cnidarian stings, recommended medical protocols in response to such stings lack rigorous scientific support. In this study, we sought to evaluate potential first aid care protocols using previously described envenomation models that allow for direct measurements of venom activity. We found that seawater rinsing, the most commonly recommended method of tentacle removal for this species, induced significant increases in venom delivery, while rinsing with vinegar or Sting No More® Spray did not. Post-sting temperature treatments affected sting severity, with 40 min of hot-pack treatment reducing lysis of sheep's blood (in agar plates), a direct representation of venom load, by over 90%. Ice pack treatment had no effect on sting severity. These results indicate that sting management protocols for Cyanea need to be revised immediately to discontinue rinsing with seawater and include the use of heat treatment.

Assessing the Efficacy of First-Aid Measures in Physalia sp. Envenomation, Using Solution- and Blood Agarose-Based Models.

Stings from the hydrozoan species in the genus Physalia cause intense, immediate skin pain and elicit serious systemic effects. There has been much scientific debate about the most appropriate first aid for these stings, particularly with regard to whether vinegar use is appropriate (most current recommendations recommend against vinegar). We found that only a small percentage (≤1.0%) of tentacle cnidae discharge during a sting event using an ex vivo tissue model which elicits spontaneous stinging from live cnidarian tentacles. We then tested a variety of rinse solutions on both Atlantic and Pacific Physalia species to determine if they elicit cnidae discharge, further investigating any that did not cause immediate significant discharge to determine if they are able to inhibit cnidae discharge in response to chemical and physical stimuli. We found commercially available vinegars, as well as the recently developed Sting No More® Spray, were the most effective rinse solutions, as they irreversibly inhibited cnidae discharge. However, even slight dilution of vinegar reduced its protective effects. Alcohols and folk remedies, such as urine, baking soda and shaving cream, caused varying amounts of immediate cnidae discharge and failed to inhibit further discharge, and thus likely worsen stings.

Cubozoan Sting-Site Seawater Rinse, Scraping, and Ice Can Increase Venom Load: Upending Current First Aid Recommendations.

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.

Box Jellyfish Alatina alata Has a Circumtropical Distribution.

Species of the box jellyfish (Cubozoa) genus Alatina are notorious for their sting along the beaches of several localities of the Atlantic and Pacific. These species include Alatina alata on the Caribbean Island of Bonaire (the Netherlands), A. moseri in Hawaii, and A. mordens in Australia. Most cubozoans inhabit coastal waters, but Alatina is unusual in that specimens have also been collected in the open ocean at great depths. Alatina is notable in that populations form monthly aggregations for spermcast mating in conjunction with the lunar cycle. Nominal species are difficult to differentiate morphologically, and it has been unclear whether they are distinct or a single species with worldwide distribution. Here we report the results of a population genetic study, using nuclear and mitochondrial sequence data from four geographical localities. Our analyses revealed a general lack of geographic structure among Alatina populations, and slight though significant isolation by distance. These data corroborate morphological and behavioral similarities observed in the geographically disparate localities, and indicate the presence of a single, pantropically distributed species, Alatina alata. While repeated, human-mediated introductions of A. alata could explain the patterns we have observed, it seems more likely that genetic metapopulation cohesion is maintained via dispersal through the swimming medusa stage, and perhaps via dispersal of encysted planulae, which are described here for the first time in Alatina.

Heated Debates: Hot-Water Immersion or Ice Packs as First Aid for Cnidarian Envenomations?

Cnidarian envenomations are an important public health problem, responsible for more deaths than shark attacks annually. For this reason, optimization of first-aid care is essential. According to the published literature, cnidarian venoms and toxins are heat labile at temperatures safe for human application, which supports the use of hot-water immersion of the sting area(s). However, ice packs are often recommended and used by emergency personnel. After conducting a systematic review of the evidence for the use of heat or ice in the treatment of cnidarian envenomations, we conclude that the majority of studies to date support the use of hot-water immersion for pain relief and improved health outcomes.

Experimental Assays to Assess the Efficacy of Vinegar and Other Topical First-Aid Approaches on Cubozoan (Alatina alata) Tentacle Firing and Venom Toxicity.

Despite the medical urgency presented by cubozoan envenomations, ineffective and contradictory first-aid management recommendations persist. A critical barrier to progress has been the lack of readily available and reproducible envenomation assays that (1) recapitulate live-tentacle stings; (2) allow quantitation and imaging of cnidae discharge; (3) allow primary quantitation of venom toxicity; and (4) employ rigorous controls. We report the implementation of an integrated array of three experimental approaches designed to meet the above-stated criteria. Mechanistically overlapping, yet distinct, the three approaches comprised (1) direct application of test solutions on live tentacles (termed tentacle solution assay, or TSA) with single image- and video-microscopy; (2) spontaneous stinging assay using freshly excised tentacles overlaid on substrate of live human red blood cells suspended in agarose (tentacle blood agarose assays, or TBAA); and (3) a "skin" covered adaptation of TBAA (tentacle skin blood agarose assay, or TSBAA). We report the use and results of these assays to evaluate the efficacy of topical first-aid approaches to inhibit tentacle firing and venom activity. TSA results included the potent stimulation of massive cnidae discharge by alcohols but only moderate induction by urine, freshwater, and "cola" (carbonated soft drink). Although vinegar, the 40-year field standard of first aid for the removal of adherent tentacles, completely inhibited cnidae firing in TSA and TSBAA ex vivo models, the most striking inhibition of both tentacle firing and subsequent venom-induced hemolysis was observed using newly-developed proprietary formulations (Sting No More™) containing copper gluconate, magnesium sulfate, and urea.

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription.

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms.

Ancient Venom Systems: A Review on Cnidaria Toxins.

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or "venom" that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom.

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.

Long-term fluctuations in circalunar Beach aggregations of the box jellyfish Alatina moseri in Hawaii, with links to environmental variability.

The box jellyfish Alatina moseri forms monthly aggregations at Waikiki Beach 8-12 days after each full moon, posing a recurrent hazard to swimmers due to painful stings. We present an analysis of long-term (14 years: Jan 1998- Dec 2011) changes in box jellyfish abundance at Waikiki Beach. We tested the relationship of beach counts to climate and biogeochemical variables over time in the North Pacific Sub-tropical Gyre (NPSG). Generalized Additive Models (GAM), Change-Point Analysis (CPA), and General Regression Models (GRM) were used to characterize patterns in box jellyfish arrival at Waikiki Beach 8-12 days following 173 consecutive full moons. Variation in box jellyfish abundance lacked seasonality, but exhibited dramatic differences among months and among years, and followed an oscillating pattern with significant periods of increase (1998-2001; 2006-2011) and decrease (2001-2006). Of three climatic and 12 biogeochemical variables examined, box jellyfish showed a strong, positive relationship with primary production, >2 mm zooplankton biomass, and the North Pacific Gyre Oscillation (NPGO) index. It is clear that that the moon cycle plays a key role in synchronizing timing of the arrival of Alatina moseri medusae to shore. We propose that bottom-up processes, likely initiated by inter-annual regional climatic fluctuations influence primary production, secondary production, and ultimately regulate food availability, and are therefore important in controlling the inter-annual changes in box jellyfish abundance observed at Waikiki Beach.

Redescription of Alatina alata (Reynaud, 1830) (Cnidaria: Cubozoa) from Bonaire, Dutch Caribbean.

Here we establish a neotype for Alatina alata (Reynaud, 1830) from the Dutch Caribbean island of Bonaire. The species was originally described one hundred and eighty three years ago as Carybdea alata in La Centurie Zoologique-a monograph published by René Primevère Lesson during the age of worldwide scientific exploration. While monitoring monthly reproductive swarms of A. alata medusae in Bonaire, we documented the ecology and sexual reproduction of this cubozoan species. Examination of forty six A. alata specimens and additional archived multimedia material in the collections of the National Museum of Natural History, Washington, DC revealed that A. alata is found at depths ranging from surface waters to 675 m. Additional studies have reported it at depths of up to 1607 m in the tropical and subtropical Atlantic Ocean. Herein, we resolve the taxonomic confusion long associated with A. alata due to a lack of detail in the original description and conflicting statements in the scientific literature. A new cubozoan character, the velarial lappet, is described for this taxon. The complete description provided here serves to stabilize the taxonomy of the second oldest box jellyfish species, and provide a thorough redescription of the species.

Cubozoan venom-induced cardiovascular collapse is caused by hyperkalemia and prevented by zinc gluconate in mice.

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.

First complete mitochondrial genome sequence from a box jellyfish reveals a highly fragmented linear architecture and insights into telomere evolution.

Animal mitochondrial DNAs (mtDNAs) are typically single circular chromosomes, with the exception of those from medusozoan cnidarians (jellyfish and hydroids), which are linear and sometimes fragmented. Most medusozoans have linear monomeric or linear bipartite mitochondrial genomes, but preliminary data have suggested that box jellyfish (cubozoans) have mtDNAs that consist of many linear chromosomes. Here, we present the complete mtDNA sequence from the winged box jellyfish Alatina moseri (the first from a cubozoan). This genome contains unprecedented levels of fragmentation: 18 unique genes distributed over eight 2.9- to 4.6-kb linear chromosomes. The telomeres are identical within and between chromosomes, and recombination between subtelomeric sequences has led to many genes initiating or terminating with sequences from other genes (the most extreme case being 150 nt of a ribosomal RNA containing the 5' end of nad2), providing evidence for a gene conversion-based model of telomere evolution. The silent-site nucleotide variation within the A. moseri mtDNA is among the highest observed from a eukaryotic genome and may be associated with elevated rates of recombination.

Immunological and toxinological responses to jellyfish stings.

Just over a century ago, animal responses to injections of jellyfish extracts unveiled the phenomenon of anaphylaxis. Yet, until very recently, understanding of jellyfish sting toxicity has remained limited. Upon contact, jellyfish stinging cells discharge complex venoms, through thousands of barbed tubules, into the skin resulting in painful and, potentially, lethal envenomations. This review examines the immunological and toxinological responses to stings by prominent species of jellyfish including Physalia sp (Portuguese Man-o-War, Blue-bottle), Cubozoan jellyfish including Chironex fleckeri, several Carybdeids including Carybdea arborifera and Alatina moseri, Linuche unguiculta (Thimble jellyfish), a jellyfish responsible for Irukandji syndrome (Carukia barnesi) and Pelagia noctiluca. Jellyfish venoms are composed of potent proteinaceous porins (cellular membrane pore-forming toxins), neurotoxic peptides, bioactive lipids and other small molecules whilst the tubules contain ancient collagens and chitins. We postulate that immunologically, both tubular structural and functional biopolymers as well as venom components can initiate innate, adaptive, as well as immediate and delayed hypersensitivity reactions that may be amenable to topical anti-inflammatory-immunomodifier therapy. The current challenge for immunotoxinologists is to deconstruct the actions of venom components to target therapeutic modalities for sting treatment.