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.

Mechanisms of cnidocyte development in the moon jellyfish Aurelia.

Stinging cells called cnidocytes are a defining trait of the cnidarians (sea anemones, corals, jellyfish, and their relatives). In hydrozoan cnidarians such as Hydra, cnidocytes develop from interstitial stem cells set aside in the ectoderm. It is less clear how cnidocytes develop outside the Hydrozoa, as other cnidarians appear to lack interstitial stem cells. We addressed this question by studying cnidogenesis in the moon jellyfish (Aurelia) through the visualization of minicollagen-a protein associated with cnidocyte development-as well as transmission electron microscopy. We discovered that developing cnidoblasts are rare or absent in feeding structures rich in mature cnidocytes, such as tentacles and lappets. Using transmission electron microscopy, we determined that the progenitors of cnidocytes have traits consistent with epitheliomuscular cells. Our data suggests a dynamic where cnidocytes develop at high concentrations in the epithelium of more proximal regions, and subsequently migrate to more distal regions where they exhibit high usage and turnover. Similar to some anthozoans, cnidocytes in Aurelia do not appear to be generated by interstitial stem cells; instead, epitheliomuscular cells appear to be the progenitor cell type. This observation polarizes the evolution of cnidogenesis, and raises the question of how interstitial stem cells came to regulate cnidogenesis in hydrozoans.

[Reason to split the genus Aurelia. Mesoglein from two populations differ].

The medusa, Aurelia aurita (Scyphozoa, Cnidaria), is considered to be a cosmopolitan species with a worldwide distribution in most seas from the poles to the tropics. Cnidarian is thought to possess two tissue layers: endoderm (gastroderm) and ectoderm, which are separated by huge mesoglea in medusa. The basic morphology of medusa is similar in different populations. Previously we have determined a new protein "mesoglein" as one of the main components of mesoglea. Deduced amino acid sequence of mesoglein contains Zona Pellucida (ZP) domain. In this paper, we have comparied of mesoglein and its gene in medusa from three habitats (White Sea (WsA), Black Sea (BsA), Japonic Sea (JsA)). The set of the mesoglea protein bands after SDS-PAGE is similar in all samples. Nevertheless, JsA mesogleins' M(r) is 53-55 kDa, while WsA and BsA mesogleins have M(r) of 47 kDa. Antibodies raised against WsA mesoglein recognize only mesogleins with M(r) of 47 kDa, but not 53-55 kDa, both on immunoblot and immunocytochemistry. Mesogleal cells and elastic fibrils are stained intensively in the mesoglea both from WsA and BsA but not from JsA. The possibility of gene divergency was checked by PCR with primers specific for WsA mesoglein gene. PCR products of expected length obtained on polyA-cDNA template from mesogleal cells of WsA and BsA medusa but not on cDNA of JsA medusa. Our results evidence that there are two different species in genus Aurelia: Aurelia aurita inhabits White and Black Seas while Aurelia sp. inhabits Japonic Sea. This is consistent with findings of other recept molecular biological studies.

Ulcerative umbrellar lesions in captive moon jelly (Aurelia aurita) medusae.

Over a period of 6 months, dozens of moon jelly (Aurelia aurita) medusae from a single-species exhibit at the California Science Center (CSC) developed exumbrellar ulcers. Ulcers were progressive, causing umbrellar creases that expanded radially to the bell rim and occasional adoral erosions that extended into gastrovascular cavities. Husbandry interventions, including addition of ultraviolet light sterilizers, repopulation with fresh cultures, and enclosure disinfection, did not arrest the recurrence of lesions. Biopsies or whole specimens representing 17 medusae (15 affected and 2 grossly unaffected) from CSC and 2 control medusae from Aquarium of the Pacific were submitted to a private diagnostic laboratory and processed for light and electron microscopy. Microscopic lesions were present in all CSC medusae and were not observed or negligible in control medusae. Lesions included ulceration, necrosis, and hyperplasia in all umbrellar layers, with most severe lesions in the exumbrella and amoebocyte infiltration in the underlying mesoglea. Special stains, electron microscopy, and fungal culture did not associate microorganisms with the lesions. Bacterial cultures from the CSC population consistently grew Shewanella and Vibrio spp, both of which were considered commensal. Trauma and environmental stress are proposed as possible causes for the ulcers.

Micro- and macrorheology of jellyfish extracellular matrix.

Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish.

Cassiosomes are stinging-cell structures in the mucus of the upside-down jellyfish Cassiopea xamachana.

Snorkelers in mangrove forest waters inhabited by the upside-down jellyfish Cassiopea xamachana report discomfort due to a sensation known as stinging water, the cause of which is unknown. Using a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, we describe C. xamachana stinging-cell structures that we term cassiosomes. These structures are released within C. xamachana mucus and are capable of killing prey. Cassiosomes consist of an outer epithelial layer mainly composed of nematocytes surrounding a core filled by endosymbiotic dinoflagellates hosted within amoebocytes and presumptive mesoglea. Furthermore, we report cassiosome structures in four additional jellyfish species in the same taxonomic group as C. xamachana (Class Scyphozoa; Order Rhizostomeae), categorized as either motile (ciliated) or nonmotile types. This inaugural study provides a qualitative assessment of the stinging contents of C. xamachana mucus and implicates mucus containing cassiosomes and free intact nematocytes as the cause of stinging water.

Embryonic development and metamorphosis of the scyphozoan Aurelia.

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of "secondary" gastrulation during early metamorphosis.

Early development, pattern, and reorganization of the planula nervous system in Aurelia (Cnidaria, Scyphozoa).

We examined the development of the nervous system in Aurelia (Cnidaria, Scyphozoa) from the early planula to the polyp stage using confocal and transmission electron microscopy. Fluorescently labeled anti-FMRFamide, antitaurine, and antityrosinated tubulin antibodies were used to visualize the nervous system. The first detectable FMRFamide-like immunoreactivity occurs in a narrow circumferential belt toward the anterior/aboral end of the ectoderm in the early planula. As the planula matures, the FMRFamide-immunoreactive cells send horizontal processes (i.e., neurites) basally along the longitudinal axis. Neurites extend both anteriorly/aborally and posteriorly/orally, but the preference is for anterior neurite extension, and neurites converge to form a plexus at the aboral/anterior end at the base of the ectoderm. In the mature planula, a subset of cells in the apical organ at the anterior/aboral pole begins to show FMRFamide-like and taurine-like immunoreactivity, suggesting a sensory function of the apical organ. During metamorphosis, FMRFamide-like immunoreactivity diminishes in the ectoderm but begins to occur in the degenerating primary endoderm, indicating that degenerating FMRFamide-immunoreactive neurons are taken up by the primary endoderm. FMRFamide-like expression reappears in the ectoderm of the oral disc and the tentacle anlagen of the growing polyp, indicating metamorphosis-associated restructuring of the nervous system. These observations are discussed in the context of metazoan nervous system evolution.

A simple visual system without neurons in jellyfish larvae.

Earlier detailed studies of cnidarian planula larvae have revealed a simple nervous system but no eyes or identifiable light sensing structures. Here, we describe the planula of a box jellyfish, Tripedalia cystophora, and report that these larvae have an extremely simple organization with no nervous system at all. Their only advanced feature is the presence of 10-15 pigment-cup ocelli, evenly spaced across the posterior half of the larval ectoderm. The ocelli are single cell structures containing a cup of screening pigment filled with presumably photosensory microvilli. These rhabdomeric photoreceptors have no neural connections to any other cells, but each has a well-developed motor-cilium, appearing to be the only means by which light can control the behaviour of the larva. The ocelli are thus self-contained sensory-motor entities, making a nervous system superfluous.

Separation of two myotoxins from nematocysts of the box jellyfish (Chironex fleckeri).

Two myotoxins, both lethal to mice by i.v. injection, were obtained by chromatography on Sephadex G-200 of material released from isolated microbasic mastigophores of C. fleckeri. Both toxins elicit contractures of skeletal (diaphragm) musculature of the rat and of smooth (ileum and vas deferens) and atrial musculature of the guinea-pig, although consistent differences in the parameters of the contractures of each muscle type elicited by the two toxins are shown. Moreover, one toxin, with a molecular weight of approximately 150,000, also elicits activity on crustacean (barnacle) musculature, whilst the other toxin, with a molecular weight of approximately 600,000, elicits no activity in barnacle musculature at the concentrations tested. The toxins are labile when released from nematocysts and they lose all myotoxic activity within 3 days at 5 degrees C. They can also be isolated chromatographically from crude extracts of the contents of mixed nematocysts of C. fleckeri. They are considered to be the principal toxins injected by C. fleckeri during nematocyst discharge and appear to be different from the C. fleckeri toxins described by other workers.

Hidden in plain sight: the ecology and physiology of organismal transparency.

Despite the prevalence and importance of transparency in organisms, particularly pelagic species, it is a poorly understood characteristic. This article reviews the current state of knowledge on the distribution, ecology, and physical basis of biological transparency. Particular attention is paid to the distribution of transparent species relative to their optical environment, the relationship between transparency and visual predation, the physics of transparency, and what is known about the anatomical and ultrastructural modifications required to achieve this condition. Transparency is shown to be primarily a pelagic trait, uncommon in other aquatic habitats and extremely rare on land. Experimental and theoretical studies in terrestrial, freshwater, and marine ecosystems have shown that transparency is a successful form of camouflage, and that several visual adaptations seem to counter it. The physical basis of transparency is still poorly understood, but anatomical observations and mathematical models show that there are various routes to transparency. Future avenues for research include examination of the ultrastructure and optical properties of transparent tissue, exploring the link between transparent species and special visual modifications in the species they interact with, and analysis of the evolution of transparency using comparative methods.

Graviceptor development in jellyfish ephyrae in space and on Earth.

Graviceptor (rhopalium) development in Aurelia aurita ephyrae which developed on Earth and in space during the nine-day NASA SLS-1 mission was compared. The space-developed ephyrae made graviceptors which were morphologically similar to those of their ground-based controls. Rhopalia of both groups developed statocysts with statoliths, ocelli, ciliated mechanoreceptor cells, and immature touch-plates with one type of hair cell. The number of rhopalia formed per arm of ephyrae of both groups revealed no significant differences. The number of statoliths formed per rhopalium was statistically higher in ephyrae which were induced to form in space with iodine than in L(Launch)+8h controls. Statolith numbers were not significantly different between Earth-formed control ephyrae and those formed from polyps induced on Earth and then sent into space 24h and 48h later. Statolith loss from rhopalia was significantly enhanced in the space-maintained ephyrae in ASW as compared to their controls. Ephyrae formed through thyroxine treatment and those maintained in thyroxine in space had statolith numbers comparable to thyroxine-treated controls. Pulsing abnormalities seen in some space-developed ephyrae suggest that some space-formed ephyrae may have developed abnormal rhopalia because normal rhopalia development and function is necessary for normal pulsing.

Development studies of Aurelia (jellyfish) ephyrae which developed during the SLS-1 mission.

Aurelia polyps (scyphistomae) and ephyrae were exposed to microgravity for nine days aboard the space shuttle during the SLS-1 mission. During strobilation, polyps segment transversely and each segment develops into an ephyra. Polyps were induced to strobilate at 28 degrees C, using iodine or thyroxine, at L(Launch)-48h, L-24h, and L+8h. Ephyrae developed in the groups tested in space and on Earth. The number of ephyrae formed per polyp was slightly higher in the L+8h groups as compared with those induced at L-24h and L-48h. On Earth, iodine is used by jellyfish to synthesize jellyfish-thyroxine (Jf T4), needed for ephyra production. Since iodine-treated polyps strobilated and formed ephyrae in space, it appears that jellyfish can synthesize Jf-T4 in space. Indeed, two groups of polyps not given inducer formed ephyrae [correction of ephryae] in space, presumably due to enhanced Jf-T4 synthesis, utilization or accumulation. Some ephyrae that formed in space were also fixed in space on Mission Day (MD) 8; others were fixed post-flight. Examination of living ephyrae with the light microscope and fixed ones with the Scanning and Transmission Electron Microscopes revealed that those which developed in space were morphologically very similar to those which developed on Earth. Quantitation of arm numbers determined that there were no significant differences between space and Earth-developed ephyrae. Pulsing abnormalities, however, were found in greater numbers (18.3%) in space-developed ephyrae than in Earth-developed controls (2.9%). These abnormalities suggest abnormal development of the graviceptors, the neuromuscular system, or a defect in the integration between these systems in apparently microgravity-sensitive animals.

A fast centrifuge method for nematocyst isolation from Pelagia noctiluca Forskal (Cnidaria: Scyphozoa).

Nematocyst isolation from surrounding tissue is an important step to characterize Cnidarian venom. Although several protocols have been used to extract venoms from cnidarian tissues, the complete isolation of nematocysts from tissue is still difficult. The goal of the present work was to evaluate the effectiveness of three different media, Percoll, Ficoll and Methylcellulose in isolating nematocysts from Pelagia noctiluca tentacles by centrifugation. The complete sedimentation of nematocysts and tissue fragments to the bottom of the test tubes was observed in Ficoll and Methylcellulose suspensions. The best result was obtained using a discontinuous density gradient of Percoll: three types of nematocysts were concentrated in three different fractions along the density gradient. Protein assay and preliminary chromatographic analyses confirmed these results.

[Shape conservatism and shaping variability. The comparative analysis of Hydrozoa and Scyphozoa early development]. / Konservativnost' formy--variabel'nost' morfogeneza. Sravnitel'nyi analiz rannego razvitiia Hydrozoa i Scyphozoa.

The morphogenetic pathways based on the self-organization take an important part in the early development of White Sea Cnidarians--Dynamena pumila (Hydrozoa) and Aurelia aurita (Scyphozoa). Comparative analysis of their early development revealed two patterns of embryonic spatial structure reproduced in the morphogenesis of both species in spite of the differences of morphogenetic paths. These are toroidal and bilaterally symmetrical shapes. It is possible that these shapes correspond to the equilibrium states of developing system and their stable reproduction is a result of epigenetic rather than genetic program.

Fluorescence imaging in the last two decades.

In commemoration of the 20th anniversary of the molecular cloning of the gene for the green fluorescent protein from the jellyfish Aequorea victoria, I would like to reflect on the development of new fluorescence imaging technology in the last two decades. As this technology has become increasingly diversified, it has become more and more of a challenge to come up with a comprehensive and exhaustive review of it. Here I will focus on optogenetics and large-scale, three-dimensional reconstruction. Those two technological innovations have been achieved in the neuroscience community owing to the combined efforts of molecular biologists and light microscopists. In addition, modern fluorescence imaging has indeed improved our understanding of the spatiotemporal regulation of fundamental biological functions at cellular level. As an example, I will introduce some findings we made regarding the movement of biomolecules across the nuclear membrane. The above-mentioned imaging approaches are possible today but were impossible two decades ago.

Aurelia aurita (Cnidaria) oocytes' contact plate structure and development.

One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the "contact plate". Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high M(r) bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides M(r) corresponds to that of mesogleal cells, the other ones' M(r) is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida.

Fine structure, histochemistry, and morphogenesis during excystment of the podocysts of the giant jellyfish Nemopilema nomurai (Scyphozoa, Rhizostomeae).

Production of podocysts is the exclusive form of asexual reproduction by polyps of the giant jellyfish Nemopilema nomurai, which has been recurrently blooming in the East Asian seas in the last decade. Podocycts consist of a dome-shaped chitinous capsule with laminated structure that encapsulates a mass of cyst cells filled with granules containing nutrient reserves such as proteins, carbohydrates, and lipids. Mitochondria, rough endoplasmic reticulum, and Golgi complexes are scarce in the cytoplasm of these cells, and the staining reaction for RNA is weak, indicating very low metabolic activity. Podocysts are capable of dormancy for at least 5 years without significant change of internal structure or nutrient reserves. Integrated information about spontaneous and artificially induced metamorphosis suggests that the following processes occur during excystment: (1) nematocyst formation in the internal cell mass, (2) stratification of the cell mass into endoderm and ectoderm, (3) extrusion of the cell mass through a gradual opening of the capsule, (4) formation of primordial polyp mouth and tentacles, and (5) metamorphosis to a polyp. We morphologically confirmed that N. nomurai podocysts have the capacity for long-term dormancy, an ability that should contribute to the periodic nature of the massive blooms of medusae of this species.

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye.

Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID(50) was about 0.6microg protein/g fish. Toxin samples were stable when kept at -80( degrees )C, but after 48h, an 80% decline in lethality occurred at -20( degrees )C. Poor stability of the venom was observed within the range of 65-80( degrees )C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60kDa and 50kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.

Nerves in the endodermal canals of hydromedusae and their role in swimming inhibition.

N eoturris breviconis (Anthomedusae) has a nerve plexus in the walls of its endodermal canals. The plexus is distinct from the ectodermal nerve plexuses supplying the radial and circular muscles in the ectoderm and no connections have been observed between them. Stimulation of the endodermal plexus evokes electrical events recorded extracellularly as "E" potentials. These propagate through all areas where the plexus has been shown by immunohistology to exist and nowhere else. When Neoturris is ingesting food, trains of "E" potentials propagate down the radial canals to the margin and cause inhibition of swimming. This response is distinct from the inhibition of swimming associated with contractions of the radial muscles but both may play a part in feeding and involve chemoreceptors. Preliminary observations suggest that the "E" system occurs in other medusae including Aglantha digitale (Trachymedusae) where the conduction pathway was previously thought to be an excitable epithelium.