Angel A. Yanagihara

Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by D-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS-PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.

Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels

Cnidarian envenomations cause a burning‐pain sensation of which the underlying mechanisms are unknown. Activation of TRPV1, a non‐selective cation channel expressed in nociceptive neurons, leads to cell depolarisation and pain. Here, we show in vitro and in vivo evidence for desensitization‐dependent TRPV1 activation in cnidarian envenomations. Cnidarian venom induced a nociceptive reactivity, comparable to capsaicin, in laboratory rats, which could be reduced by the selective TRPV1 antagonist, BCTC. These findings are the first to explain at least part of the symptomology of cnidarian envenomations and provide insights into the design of more effective treatments for this global public health problem.

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.

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.

Ultrastructure of a novel eurytele nematocyst of Carybdea alata Reynaud (Cubozoa, Cnidaria).

Abstract. The ultrastructural characteristics of nematocysts from the cubozoan Carybdea alata Reynaud, 1830 (Hawaiian box jellyfish) were examined using light, scanning and transmission electron microscopy. We reclassified the predominant nematocyst in C. alata tentacles as a heterotrichous microbasic eurytele, based on spine, tubule and capsule measurements. These nematocysts exhibited a prominent and singular stylet, herein referred to as the lancet. Discharged nematocysts from fixed tentacle preparations displayed the following structures: a smooth shaft base, lamellae, a hemicircumferential fissure demarking the proximal end of a stratified lancet, and a gradually tapering tubule densely covered with large triangularly shaped spines. The lancet remained partially adjoined to the shaft base in a hinge-like fashion in rapidly fixed, whole-tentacle preparations. In contrast, this structure was not observed in discharged nematocyst preparations which involved multiple transfer steps prior to fixation. Various approaches were designed to detect this structure in the absence of fixative. Detached lancets were located in proximity to discharged tubules in undisturbed coverslip preparations of fresh tentacles. In addition, examination of embedded nematocysts from fresh tentacles laid on polyacrylamide gels revealed still-attached lancets. To examine the function of this structure in prey capture, Artemia sp. laden tentacles were prepared for scanning electron microscopy. While carapace exteriors exhibited structures proximal to the lancet, i.e., the nematocyst capsule and shaft base, neither tubule nor lancet structures were visible. Taken together, the morphological data suggested a series of events involved in the discharge of a novel eurytele from C. alata.

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.

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.

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.

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.

The ultrastructure of nematocysts from the fishing tentacle of the Hawaiian bluebottle, Physalia utriculus (Cnidaria, Hydrozoa, Siphonophora)

Fishing tentacle nematocysts of the hydrozoan, Physalia utriculus, from Hawaiian coastal waters, were examined by light microscopy, as well as by scanning and transmission electron microscopy (SEM and TEM). Hawaiian P. utriculus has been assumed to be conspecific with the Australian morph based on general macroscopic descriptions. Ultrastructural examination using SEM and TEM revealed that the cnidome was composed of two sizes of heterotrichous anisorhizas as well as rare rhopoloids (less than one observed per 1000 nematocysts). Beyond classification of the cnidome of Hawaiian P. utriculus, an unprecedented morphological structure was observed. Specifically, SEM revealed fibers (<1 μm in diameter) along the spiral crest of spines of some discharged tubules of P. utriculus anisorhizas. A hypothetical model of spine morphogenesis was formulated based on comparative analysis of hundreds of discharged tubules.