Rapid and Accurate Species-Specific PCR for the Identification of Lethal Chironex Box Jellyfish in Thailand.

Box jellyfish are extremely potent venom-producing marine organisms. While they have been found worldwide, the highest health burden has been anticipated to be the tropical Indo-Pacific of Southeast Asia (SEA). At least 12 Cubozoan species have now been documented in Thai waters, and many of them inflict acutely lethal strings, especially those under the order Chirodropida. Our previous study has successfully differentiated species of box jellyfish using DNA sequencing to support the morphological study. In this study, we specifically designed polymerase chain reaction (PCR) primers for the 16S ribosomal RNA (rRNA) gene and the mitochondrial DNA cytochrome oxidase subunit I (COI) gene of lethal Thai Chironex species. The SYBR green-based real-time PCR panel was performed for rapid species identification. The sensitivity and specificity of the panel were determined by testing samples of different species. Moreover, we applied the panel to the tentacle sample from a real patient, which helped confirm the animal-of-cause of envenomation. Our results show a success for species identification of box jellyfish using 16S rRNA and COI PCR panel, which revealed congruence between molecular and morphological identification. Furthermore, the panel worked very well with the unknown samples and jellyfish tissue from the real envenomation case. The results demonstrated that molecular panels were able to identify three species of Chironex box jellyfish both rapidly and accurately, and can be performed without having a complete specimen or morphological study.

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.

Statolith Morphometrics Can Discriminate among Taxa of Cubozoan Jellyfishes.

Identification of potentially harmful cubomedusae is difficult due to their gelatinous nature. The only hard structure of medusae, the statolith, has the potential to provide robust measurements for morphometric analysis. Traditional morphometric length to width ratios (L: W) and modern morphometric Elliptical Fourier Analysis (EFA) were applied to proximal, oral and lateral statolith faces of 12 cubozoan species. EFA outperformed L: W as L: W did not account for the curvature of the statolith. Best discrimination was achieved with Canonical Discriminant Analysis (CDA) when analysing proximal + oral + lateral statolith faces in combination. Normalised Elliptical Fourier (NEF) coefficients classified 98% of samples to their correct species and 94% to family group. Statolith shape agreed with currently accepted cubozoan taxonomy. This has potential to assist in identifying levels of risk and stock structure of populations in areas where box jellyfish envenomations are a concern as the severity of envenomation is family dependent. We have only studied 12 (27%) of the 45 currently accepted cubomedusae, but analyses demonstrated that statolith shape is an effective taxonomic discriminator within the Class.

Phylogenomic Analyses Support Traditional Relationships within Cnidaria.

Cnidaria, the sister group to Bilateria, is a highly diverse group of animals in terms of morphology, lifecycles, ecology, and development. How this diversity originated and evolved is not well understood because phylogenetic relationships among major cnidarian lineages are unclear, and recent studies present contrasting phylogenetic hypotheses. Here, we use transcriptome data from 15 newly-sequenced species in combination with 26 publicly available genomes and transcriptomes to assess phylogenetic relationships among major cnidarian lineages. Phylogenetic analyses using different partition schemes and models of molecular evolution, as well as topology tests for alternative phylogenetic relationships, support the monophyly of Medusozoa, Anthozoa, Octocorallia, Hydrozoa, and a clade consisting of Staurozoa, Cubozoa, and Scyphozoa. Support for the monophyly of Hexacorallia is weak due to the equivocal position of Ceriantharia. Taken together, these results further resolve deep cnidarian relationships, largely support traditional phylogenetic views on relationships, and provide a historical framework for studying the evolutionary processes involved in one of the most ancient animal radiations.

Early life history of Alatina cf. moseri populations from Australia and Hawaii with implications for taxonomy (Cubozoa: Carybdeida, Alatinidae).

The early life stages of the cubomedusa Alatina cf. moseri from Osprey Reef (North Queensland, Australia) and Waikiki (Oahu, Hawaii) were studied using laboratory-based culturing conditions. Spawning populations from both regions were observed with reliable periodicity allowing polyp cultures from these locations to be collected and established under laboratory conditions. The polyps of this species were successfully reared from spawning adults. Polyps of Alatina cf. moseri were cultured at temperatures of 23-28 °C, developed up to 19 tentacles and reached up to 1.70 mm in height. The balloon-shaped hypostomes possessed 4 well-defined lips. The polyps increased their numbers by means of formation of either sedentary polyp buds or creeping-polyp buds, which attached after 2-3 days. Metamorphosis occurred at temperatures of 25-28 °C. Development of polyps and medusae were achieved for the first time within the genus Alatina and allowed comparisons of early life history between these and other species of the Carybdeida families. The metamorphosis and young medusa of this genus showed characters that differed distinctly from those noted for other Carybdeida species, but are very similar to the one described from Puerto Rico by Arneson and Cutress in 1976 for Alatina sp. (named by them Carybdea alata). Based on this evidence, the discrepancies in original specimen descriptions and the previous genetic comparisons, we support the suggestion that the two previously described species of Alatina from Australia and Hawaii (Alatina mordens and Alatina moseri) appear to represent artificial taxonomic units and may in fact be the same as the original Carybdea alata species named from Puerto Rico. Further taxonomic studies are desperately needed in order to clarify the various species and description discrepancies that exist within this newly proposed genus.

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.

Evolution of box jellyfish (Cnidaria: Cubozoa), a group of highly toxic invertebrates.

Cubozoa (Cnidaria: Medusozoa) represents a small clade of approximately 50 described species, some of which cause serious human envenomations. Our understanding of the evolutionary history of Cubozoa has been limited by the lack of a sound phylogenetic hypothesis for the group. Here, we present a comprehensive cubozoan phylogeny based on ribosomal genes coding for near-complete nuclear 18S (small subunit) and 28S (large subunit) and partial mitochondrial 16S. We discuss the implications of this phylogeny for our understanding of cubozoan venom evolution, biogeography and life-history evolution. Our phylogenetic hypothesis suggests that: (i) the last common ancestor of Carybdeida probably possessed the mechanism(s) underlying Irukandji syndrome, (ii) deep divergences between Atlantic and Indo-Pacific clades may be explained by ancient vicariant events, and (iii) sexual dimorphism evolved a single time in concert with complex sexual behaviour. Furthermore, several cubozoan taxa are either para- or polyphyletic, and we address some of these taxonomic issues by designating a new family, Carukiidae, a new genus, Copula, and by redefining the families Tamoyidae and Tripedaliidae. Lastly, cubozoan species identities have long been misunderstood and the data presented here support many of the recent scientific descriptions of cubozoan species. However, the results of a phylogeographic analysis of Alatina moseri from Hawai'i and Alatina mordens from Australia indicate that these two nominal species represent a single species that has maintained metapopulation cohesion by natural or anthropogenic dispersal.

Biochemical and molecular characterisation of cubozoan protein toxins.

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as alpha-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.

Preliminary observations on the response of Chironex fleckeri (Cnidaria: Cubozoa: Chirodropida) to different colors of light.

Cubozoans are well known for their attraction to light and light-colored objects. Two highly venomous types are a public safety concern in Australian waters and elsewhere: Chironex fleckeri, long considered the world's deadliest animal and colloquially called the box jellyfish; and the irukandjis, a group of at least 10 species that cause various degrees of debilitating illness. We were asked by the tourism industry whether there might be a color of light that box jellyfish and irukandjis are not attracted to, such that nighttime diving activities might pose less risk of being stung. Our preliminary trials with Chironex fleckeri indicated a marked positive response to lights of white, red, yellow, green, orange, and blue. All colors elicited a strong and directed attraction to light; however, medusae slowed down their pulsation rate, streamed out their tentacles, and performed a series of figure-eight patterns back and forth through the lighted area when exposed to blue light, which we interpreted as feeding behavior. This compares curiously with a report subsequent to our testing, in which the small, mangrove-inhabiting cubomedusa Tripedalia cystophora and the beach-dwelling Chiropsella bronzie demonstrate a peak sensitivity to blue-green light in the region of 500 nm, and that the former is behaviorally attracted to blue and green light, but ignores red. This leaves open the possibility that Irukandji species, which are more closely related to Tripedalia than to Chironex, may be blind to red.

Medusozoan phylogeny and character evolution clarified by new large and small subunit rDNA data and an assessment of the utility of phylogenetic mixture models.

A newly compiled data set of nearly complete sequences of the large subunit of the nuclear ribosome (LSU or 28S) sampled from 31 diverse medusozoans greatly clarifies the phylogenetic history of Cnidaria. These data have substantial power to discern among many of the competing hypotheses of relationship derived from prior work. Moreover, LSU data provide strong support at key nodes that were equivocal based on other molecular markers. Combining LSU sequences with those of the small subunit of the nuclear ribosome (SSU or 18S), we present a detailed working hypothesis of medusozoan relationships and discuss character evolution within this diverse clade. Stauromedusae, comprising the benthic, so-called stalked jellyfish, appears to be the sister group of all other medusozoans, implying that the free-swimming medusa stage, the motor nerve net, and statocysts of ecto-endodermal origin are features derived within Medusozoa. Cubozoans, which have had uncertain phylogenetic affinities since the elucidation of their life cycles, form a clade-named Acraspeda-with the scyphozoan groups Coronatae, Rhizostomeae, and Semaeostomeae. The polyps of both cubozoans and hydrozoans appear to be secondarily simplified. Hydrozoa is comprised by two well-supported clades, Trachylina and Hydroidolina. The position of Limnomedusae within Trachylina indicates that the ancestral hydrozoan had a biphasic life cycle and that the medusa was formed via an entocodon. Recently hypothesized homologies between the entocodon and bilaterian mesoderm are therefore suspect. Laingiomedusae, which has often been viewed as a close ally of the trachyline group Narcomedusae, is instead shown to be unambiguously a member of Hydroidolina. The important model organisms of the Hydra species complex are part of a clade, Aplanulata, with other hydrozoans possessing direct development not involving a ciliated planula stage. Finally, applying phylogenetic mixture models to our data proved to be of little additional value over a more traditional phylogenetic approach involving explicit hypothesis testing and bootstrap analyses under multiple optimality criteria. [18S; 28S; Cubozoa; Hydrozoa; medusa; molecular systematics; polyp; Scyphozoa; Staurozoa.].