Organelle survival in a foreign organism: Hydra nematocysts in the flatworm Microstomum lineare.

Nematocysts are characteristic organelles of the phylum cnidaria. They are designated kleptocnidae when sequestered in animals that feed on cnidaria. Kleptocnidae are known for more than a century. Nevertheless it is still enigmatic how selected nematocyst types survive in the predator and how they reach their final destination in the foreign body. In the free-living Platyhelminth Microstomum lineare the fate of nematocysts of the prey Hydra oligactis was analyzed at the ultrastructural level and by fluorescence microscopy using hydra polyps that had been stained in vivo with the fluorescent dyes TROMI and TRITC. M. lineare digested hydra tissue in its intestine within 30 min and all nematocyst types were phagocytosed without adherent cytoplasm by intestinal cnidophagocytes. Desmoneme and isorhiza nematocysts were digested whereas cnidophagocytes containing the venom-loaded stenotele nematocysts started to migrate out of the intestinal epithelia through the parenchyma to the epidermis thereby traversing the subintestinal and subepidermal muscle layer. Within one to two days, M. lineare began to form a muscle layer basolateral around epidermal cnidophagocytes. Epidermal stenoteles survived in M. lineare for at least four weeks. The ability of epidermal stenotele nematocysts to discharge suggest that this hydra organelle preserved its physiological properties in the new host.

Evidence of Cnidarians sensitivity to sound after exposure to low frequency underwater sources.

Jellyfishes represent a group of species that play an important role in oceans, particularly as a food source for different taxa and as a predator of fish larvae and planktonic prey. The massive introduction of artificial sound sources in the oceans has become a concern to science and society. While we are only beginning to understand that non-hearing specialists like cephalopods can be affected by anthropogenic noises and regulation is underway to measure European water noise levels, we still don't know yet if the impact of sound may be extended to other lower level taxa of the food web. Here we exposed two species of Mediterranean Scyphozoan medusa, Cotylorhiza tuberculata and Rhizostoma pulmo to a sweep of low frequency sounds. Scanning electron microscopy (SEM) revealed injuries in the statocyst sensory epithelium of both species after exposure to sound, that are consistent with the manifestation of a massive acoustic trauma observed in other species. The presence of acoustic trauma in marine species that are not hearing specialists, like medusa, shows the magnitude of the problem of noise pollution and the complexity of the task to determine threshold values that would help building up regulation to prevent permanent damage of the ecosystems.

First Description of Sulphur-Oxidizing Bacterial Symbiosis in a Cnidarian (Medusozoa) Living in Sulphidic Shallow-Water Environments.

BACKGROUND: Since the discovery of thioautotrophic bacterial symbiosis in the giant tubeworm Riftia pachyptila, there has been great impetus to investigate such partnerships in other invertebrates. In this study, we present the occurrence of a sulphur-oxidizing symbiosis in a metazoan belonging to the phylum Cnidaria in which this event has never been described previously. METHODOLOGY/PRINCIPAL FINDINGS: Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) observations and Energy-dispersive X-ray spectroscopy (EDXs) analysis, were employed to unveil the presence of prokaryotes population bearing elemental sulphur granules, growing on the body surface of the metazoan. Phylogenetic assessments were also undertaken to identify this invertebrate and microorganisms in thiotrophic symbiosis. Our results showed the occurrence of a thiotrophic symbiosis in a cnidarian identified as Cladonema sp. CONCLUSIONS/SIGNIFICANCE: This is the first report describing the occurrence of a sulphur-oxidizing symbiosis in a cnidarian. Furthermore, of the two adult morphologies, the polyp and medusa, this mutualistic association was found restricted to the polyp form of Cladonema sp.

Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.

The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene's conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoan Trichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes. HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan's expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N. vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions.

Dendrogramma, new genus, with two new non-bilaterian species from the marine bathyal of southeastern Australia (Animalia, Metazoa incertae sedis)--with similarities to some medusoids from the Precambrian Ediacara.

A new genus, Dendrogramma, with two new species of multicellular, non-bilaterian, mesogleal animals with some bilateral aspects, D. enigmatica and D. discoides, are described from the south-east Australian bathyal (400 and 1000 metres depth). A new family, Dendrogrammatidae, is established for Dendrogramma. These mushroom-shaped organisms cannot be referred to either of the two phyla Ctenophora or Cnidaria at present, because they lack any specialised characters of these taxa. Resolving the phylogenetic position of Dendrogramma depends much on how the basal metazoan lineages (Ctenophora, Porifera, Placozoa, Cnidaria, and Bilateria) are related to each other, a question still under debate. At least Dendrogramma must have branched off before Bilateria and is possibly related to Ctenophora and/or Cnidaria. Dendrogramma, therefore, is referred to Metazoa incertae sedis. The specimens were fixed in neutral formaldehyde and stored in 80% ethanol and are not suitable for molecular analysis. We recommend, therefore, that attempts be made to secure new material for further study. Finally similarities between Dendrogramma and a group of Ediacaran (Vendian) medusoids are discussed.

Early Cambrian pentamerous cubozoan embryos from South China.

BACKGROUND: Extant cubozoans are voracious predators characterized by their square shape, four evenly spaced outstretched tentacles and well-developed eyes. A few cubozoan fossils are known from the Middle Cambrian Marjum Formation of Utah and the well-known Carboniferous Mazon Creek Formation of Illinois. Undisputed cubozoan fossils were previously unknown from the early Cambrian; by that time probably all representatives of the living marine phyla, especially those of basal animals, should have evolved. METHODS: Microscopic fossils were recovered from a phosphatic limestone in the Lower Cambrian Kuanchuanpu Formation of South China using traditional acetic-acid maceration. Seven of the pre-hatched pentamerous cubozoan embryos, each of which bears five pairs of subumbrellar tentacle buds, were analyzed in detail through computed microtomography (Micro-CT) and scanning electron microscopy (SEM) without coating. RESULTS: The figured microscopic fossils are unequivocal pre-hatching embryos based on their spherical fertilization envelope and the enclosed soft-tissue that has preserved key anatomical features arranged in perfect pentaradial symmetry, allowing detailed comparison with modern cnidarians, especially medusozoans. A combination of features, such as the claustrum, gonad-lamella, suspensorium and velarium suspended by the frenula, occur exclusively in the gastrovascular system of extant cubozoans, indicating a cubozoan affinity for these fossils. Additionally, the interior anatomy of these embryonic cubozoan fossils unprecedentedly exhibits the development of many new septum-derived lamellae and well-partitioned gastric pockets unknown in living cubozoans, implying that ancestral cubozoans had already evolved highly specialized structures displaying unexpected complexity at the dawn of the Cambrian. The well-developed endodermic lamellae and gastric pockets developed in the late embryonic stages of these cubozoan fossils are comparable with extant pelagic juvenile cubomedusae rather than sessile cubopolyps, whcih indicates a direct development in these fossil taxa, lacking characteristic stages of a typical cnidarian metagenesis such as planktonic planula and sessile polyps.

[On the nervous system of a parasitic cnidarian Polypodium hydriforme].

Nerve cells in a parasitic cnidarian Polypodium hydriforme at the parasitic and free-living stages of the life cycle have been localized immunocytochemically using antibodies to FMRF-amide, and their ultrastructure has been described. Ganglion cells form a net under epidermis consisting of bi- and tripolar neurons which cross the mesoglea and usually contact muscle cells and cnidocytes. Fusiform sensory and neurosecretory cells, especially characteristic to sensory tentacles, are interspersed among epidermal cells. All three types of nerve cells have dense cored vesicles about 80-120 nm in diameter. The sensory cells demonstrate a sensory flagellum-like immobile structure. Neurosecretory and sensory cells form septate junctions with epidermal cells. Ganglion cells show gap junctions between them. A centriole encircled by a fragment of nuclear envelope which is a marker of ectodermal lineage cells in Polypodium has been described in the cytoplasm of a sensory cell, thus proving the ectodermal nature of the nervous system.

Structural diversity, systematics, and evolution of cnidae.

Cnidae are secreted by the Golgi apparatus of all cnidarians and only cnidarians. Of the three categories of cnidae (also called cnidocysts), nematocysts occur in all cnidarians, and are the means by which cnidarians defend themselves and obtain prey; spirocysts and ptychocysts are restricted to a minority of major taxa. A cnida discharges by eversion of its tubule; venom may be associated with the tubule of a nematocyst. About 30 major morphological types of nematocysts are recognized, but no single nomenclature for them is accepted. Function seems not to correlate tightly with morphology--nematocysts of at least some types are used both offensively and defensively. Similarly, it is not clear if morphology correlates with toxicity. Some types of nematocysts are taxonomically diagnostic whereas others are widespread. Nonetheless, an inventory of types of cnidae (the cnidom), with their distribution and size, is an essential component of most taxonomic descriptions. Complicating the taxonomic value of cnidae are the facts that not all members of a species may have the same types of cnidae, even at the same life-cycle stage, and size of nematocysts of a species may vary geographically and with size of individual. The diversity of nematocysts is so great and the features within each major type are so variable that homologies have not been determined. Nematocyst complement, morphology, and size likely reflect both phylogeny and biology; the feedback between the two may confound analysis. Although cnidae are valuable in taxonomy of at least some groups, more understanding of the forces that affect them is needed for their systematic and phylogenetic value to be understood and their potential as indicators of evolution to be realized.

Light and electron microscopic study on Henneguya suprabranchiae Landsberg, 1987 (Myxozoa: Myxosporea) infecting Oreochromis niloticus, a new host record.

Out of 58 live tilapia fish, five Oreochromis niloticus were found to be naturally infected with Henneguya suprabranchiae (8.62%). Such infection was recorded only during winter season from Bahr Shebin, a tributary of the River Nile at Menoufia Governorate, Nile Delta, Egypt. Based on the structure and measurements of fresh spores, this parasite was identified as H. suprabranchiae. Spores are oval in shape and they measure 15 (13-16) x 5 (4-6) microm length by width. It has two polar capsules inside and they measure 4 (5-7) x 1 (2-3) microm length by width. Each polar capsule has spirally coiled (7-9 turns) polar filament. The plasmodia as well as all other parasitic stages were described using light and transmission electron microscopy and discussed regarding to those of other fish hosts especially those of Africa.

Ultrastructure of Enteromyxum leei (Diamant, Lom, & Dyková, 1994) (Myxozoa), an enteric parasite infecting gilthead sea bream (Sparus aurata) and sharpsnout sea bream (Diplodus puntazzo).

The ultrastructure of the developmental stages of the myxozoan Enteromyxum leei parasitizing gilthead seabream (Sparus aurata) intestine and sharpsnout sea bream (Diplodus puntazzo) intestine and gallbladder are described. The earliest stage observed was a small dense trophozoite located among enterocytes. Proliferative stages, observed intercellularly in the epithelium of the intestine and gallbladder as well as in the lumen, possessed the typical cell-in-cell configuration throughout their development. Secondary cells were seen undergoing division within a common vacuolar membrane that also encompassed pairs of tertiary cells. Cytochemical studies showed that primary cells stored mainly lipids whereas secondary cells stored abundant beta-glycogen granules. Sporogonic development resembled that described for other disporous myxozoans. Within sporogonic stages, nonsporogonic secondary cells were observed accompanying two developing spores. Mature spores had a binucleated sporoplasm in which glycogen stores were abundant and no sporoplasmosomes were found. Our observations are discussed in relation to our knowledge on other myxozoans of the genus Enteromyxum.

Detection of carbohydrate terminals in the enteric parasite Enteromyxum scophthalmi (Myxozoa) and possible interactions with its fish host Psetta maxima.

The existence and localisation of carbohydrate terminals in Enteromyxum scophthalmi stages was investigated at light (LM) and transmission electron microscopes (TEM) using lectin histochemistry techniques, with the aim of contributing to elucidate the participation of carbohydrate-lectin interactions in the parasite invasion and relationships with the fish host. The presence of abundant mannose and/or glucose residues was demonstrated by the intense staining by concanavalin A at both LM and TEM. The staining pattern obtained with soybean agglutinin and Bandeiraea simplicifolia I (BSI) indicated the abundance of N-acetyl-galactosamine and D-galactose at a lesser extent. The lectins wheat germ agglutinin, BSI and Ulex europaeus agglutinin produced weaker marks. Most lectins recognised structures present in both pre-sporogonic and sporogonic stages, though the glycosidic pattern and/or staining intensity varied between developmental stages. No staining was obtained with Sambucus nigra agglutinin. The TEM studies demonstrated glucose-mannose, N-acetyl-glucosamine, N-acetyl-galactosamine and alpha-D-galactose as dominant structures at the parasite membrane and host-parasite interface, suggesting a role in host-parasite interactions. All these terminals were also detected in the mitochondria of P cells and were scant in the S cells and nuclei. In turbot intestine, mannose-glucose terminals and N-acetyl-glucosamine were labelled on the epithelial brush border and in the mucous cells and rodlet cells. The relevance of these findings in relation to the host-parasite interaction is discussed.

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria).

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.

Cnidarian chemical neurotransmission, an updated overview.

The ultrastructural, histochemical, immunocytochemical, biochemical, molecular, behavioral and physiological evidence for non-peptidergic and peptidergic chemical neurotransmission in the Anthozoa, Hydrozoa, Scyphozoa and Cubozoa is surveyed. With the possible exception of data for the catecholamines and peptides in some animals, the set of cumulative data - the evidence from all methodologies - is incomplete. Taken together, the evidence from all experimental approaches suggests that both classical fast (acetylcholine, glutamate, GABA, glycine) and slow (catecholamines and serotonin) transmitters, as well as neuropeptides, are involved in cnidarian neurotransmission. Ultrastructural evidence for peptidergic, serotonergic, and catecholaminergic synaptic localization is available, but the presence of clear and dense-cored synaptic vesicles also suggests both fast and slow classical transmission. Immunocytochemical studies, in general, reveal a continuous, non-localized distribution of neuropeptides, suggesting a neuromodulatory role for them. Immunocytochemical and biochemical studies indicate the presence of glutamate, GABA, serotonin, catecholamines (and/or their receptors), RFamides, nitric oxide and eicosanoids in cnidarian neurons and tissues. Gene sequences for peptidergic preprohormones have been reported; putative gene homologies to receptor proteins for vertebrate transmitters have been found in Hydra. Behavioral and physiological studies implicate classical transmitters, neuropeptides, eicosanoids and nitric oxide in the coordination of the neuroeffector systems.

Synchrotron X-ray tomographic microscopy of fossil embryos.

Fossilized embryos from the late Neoproterozoic and earliest Phanerozoic have caused much excitement because they preserve the earliest stages of embryology of animals that represent the initial diversification of metazoans. However, the potential of this material has not been fully realized because of reliance on traditional, non-destructive methods that allow analysis of exposed surfaces only, and destructive methods that preserve only a single two-dimensional view of the interior of the specimen. Here, we have applied synchrotron-radiation X-ray tomographic microscopy (SRXTM), obtaining complete three-dimensional recordings at submicrometre resolution. The embryos are preserved by early diagenetic impregnation and encrustation with calcium phosphate, and differences in X-ray attenuation provide information about the distribution of these two diagenetic phases. Three-dimensional visualization of blastomere arrangement and diagenetic cement in cleavage embryos resolves outstanding questions about their nature, including the identity of the columnar blastomeres. The anterior and posterior anatomy of embryos of the bilaterian worm-like Markuelia confirms its position as a scalidophoran, providing new insights into body-plan assembly among constituent phyla. The structure of the developing germ band in another bilaterian, Pseudooides, indicates a unique mode of germ-band development. SRXTM provides a method of non-invasive analysis that rivals the resolution achieved even by destructive methods, probing the very limits of fossilization and providing insight into embryology during the emergence of metazoan phyla.

Light and electron microscopic study of the myxosporean, Henneguya friderici n. sp. from the Amazonian teleostean fish, Leporinus friderici.

A new histozoic species of myxosporean was found to infect the gill filaments, gut, kidney and liver of the freshwater teleost Leporinus friderici, collected from the estuarine region of the Amazon, near the city of Belém, Brazil. The plasmodia show asynchronous development, at any one time composed of mature spores and all sporogonic stages. The ellipsoidal spore body, measuring 10.4 microm long and 5.7 microm wide, consists of 2 equal shell valves adhering together along the straight suture line. Each valve has a caudal process measuring 23.3 microm in length. There are 2 symmetric polar capsules, without intercapsular appendix, measuring 5.0 microm x 2.1 microm, and each has a polar filament with 7-8 coils. In general, ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. Some ultrastructural aspects such as cellular alterations of the pericyte in the different organs infected and characterization of the sporoplasmosomes during the sporoplasm maturation are described. This parasite was studied under light and electron microscope and compared with others species of the genus Henneguya, considering also host specificity. From our observations we propose the creation of a new species, Henneguya friderici n. sp.

[Amoebocytes in mesoglea of Polypodium hydriforme]. / Amebotsity v mezoglee u Polypodium hydriforme.

Mesogleal amoebocytes of free-living Polypodium hydriforme have been studied with transmission electron microscope. The amoebocytes have numerous processes and contain cytoplasmic vacuoles with fibrous material of different density. The phenomenon of cell death (apoptosis) of mesogleal amoebocytes is described. Chromatin of dying cells becomes condensed forming picnotic "caps" in the nucleus. No mitotic cells were encountered among mesogleal amoebocytes. The origin and functions of mesogleal amoebocytes of P. hydriforme are discussed.

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

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.

Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals.

AIMS: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia). METHODS AND RESULTS: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases. CONCLUSIONS: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.

[Structural characteristics of nematocyst stinging threads from the parasitic cnidarian Polypodium hydriforme]. / Osobennosti stroeniia strekatelnykh nitei nematotsist paraziticheskoi knidarii Polypodium hydriforme.

The structure of discharged nematocyst stinging threads present in free-living individuals of Polypodium hydriforme was studied by scanning and transmission electron microscopy. Not all cnidae of P. hydriforme proved to be atrichous isorhizas (as previously was accepted), but only one of the five nematocyst categories studied. A unique feature of P. hydriforme nematocysts was revealed: their stinging threads possess two strands of spines, rather than 5 or 3, as in Narcomedusae and other Cnidarians, respectively. This fact supplements the evidence in favour of P. hydriforme being a rather isolated branche in the phylogenetic tree of Cnidaria.