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.     

Acetylcholinesterase activity in Clytia hemisphaerica (Cnidaria)

Cholinesterase activity is known in representatives of all living organisms phyla but the origin of the cholinergic system as known in bilaterian animals is still undeciphered. In particular the implication of cholinesterases in the nervous system of non-bilaterian Metazoa is not well known. We thus chose to investigate this activity in the Clytia hemisphaerica (Cnidaria) medusa. In toto histochemical staining revealed an acetylcholinesterase activity in the tentacle bulbs but not in the nervous system. Sequences homologous to acetylcholinesterase were searched within Clytia ESTs and compared to other sequences found in public databases.     

A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica

Regionalised activation of canonical Wnt signalling via beta-catenin stabilisation is a key early step in embryonic patterning in many metazoans, including the basally diverging cnidarians, but the upstream maternal cues appear surprisingly variable. In Clytia, regionalised beta-catenin stabilisation defining a presumptive 'oral' territory is determined by two maternally coded Frizzled family Wnt receptors of opposite localisation and function. We have identified a maternally coded ligand, CheWnt3, the RNA of which is localised to the animal cortex (future oral side) of the egg. Antisense morpholino oligonucleotide experiments showed that CheWnt3 is required maternally for regionalised oral beta-catenin stabilisation in the early embryo, being only the second clear example of a maternally required Wnt ligand after Xenopus Xwnt11. In line with the determinant role of the maternally localised Frizzleds, CheWnt3 overexpression by RNA injection initially had little effect on establishing the oral domain. Subsequently, however, overexpression had dramatic consequences for axis development, causing progressive expansion of beta-catenin stabilisation to yield spherical 'oralised' larvae. Upregulation of both CheFz1 and CheFz3 RNAs in CheWnt3 morpholino embryos indicated that CheWnt3 participates in an active axial patterning system involving reciprocal downregulation of the receptors to maintain oral and aboral territories. Localised introduction of CheWnt3 RNA induced ectopic oral poles in CheWnt3 morpholino embryos, demonstrating its importance in directing oral fate. These findings suggest that the complete ligand-dependent Wnt signalling cascade is involved in axial patterning in ancestral eumetazoans. In Clytia, two variant Frizzled receptors and one Wnt ligand produced from localised RNAs cooperate to initiate regionalised Wnt pathway activation.     

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.     

Antimicrobial activity in the common seawhip, Leptogorgia virgulata (Cnidaria: Gorgonaceae)

Antimicrobial activity was examined in the gorgonian Leptogorgia virgulata (common seawhip) from South Carolina waters. Extraction and assay protocols were developed to identify antimicrobial activity in crude extracts of L. virgulata. Detection was determined by liquid growth inhibition assays using Escherichia coli BL21, Vibrio harveyii, Micrococcus luteus, and a Bacillus sp. isolate. This represents the first report of antimicrobial activity in L. virgulata, a temperate/sub-tropical coral of the western Atlantic Ocean. Results from growth inhibition assays guided a fractionation scheme to identify active compounds. Reverse-phase HPLC, HPLC-mass spectrometry, and 1H and 13C NMR spectroscopy were used to isolate, purify, and characterize metabolites in antimicrobial fractions of L. virgulata. Corroborative HPLC-MS/NMR evidence validated the presence of homarine and a homarine analog, well-known emetic metabolites previously isolated from L. virgulata, in coral extracts. In subsequent assays, partially-purified L. virgulata fractions collected from HPLC-MS fractionation were shown to contain antimicrobial activity using M. luteus and V. harveyii. This study provides evidence that homarine is an active constituent of the innate immune system in L. virgulata. We speculate it may act synergistically with cofactors and/or congeners in this octocoral to mount a response to microbial invasion and disease.     

Character evolution in Hydrozoa (phylum Cnidaria)

The diversity of hydrozoan life cycles, as manifested in the wide range of polyp, colony, and medusa morphologies, has been appreciated for centuries. Unraveling the complex history of characters involved in this diversity is critical for understanding the processes driving hydrozoan evolution. In this study, we use a phylogenetic approach to investigate the evolution of morphological characters in Hydrozoa. A molecular phylogeny is reconstructed using ribosomal DNA sequence data. Several characters involving polyp, colony, and medusa morphology are coded in the terminal taxa. These characters are mapped onto the phylogeny and then the ancestral character states are reconstructed. This study confirms the complex evolutionary history of hydrozoan morphological characters. Many of the characters involving polyp, colony, and medusa morphology appear as synapomorphies for major hydrozoan clades, yet homoplasy is commonplace.     

Acetylcholinesterase activity in chronic renal failure

Twenty healthy subjects and 39 Chronic Renal Failure patients (CRF-patients) maintained on chronic hemodialysis were used in this investigation to study the changes in acetylcholinesterase (AChE) activity of red blood cells (RBCs). The CRF-patients were all undergoing hemodialysis treatment. AChE activity from the CRF-patients was determined before and after dialysis. An additional objective was to study the effect of chronic renal failure on human red blood cell aging. Blood samples were drawn from controls and CRF-patients in tubes containing EDTA or sodium heparin as an anticoagulant. Red blood cells were purified to avoid interference with monocytes, reticulocytes and leukocytes. The purified RBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinous percoll gradient. The mean +/- SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB), purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) in CRF-patients were 31.2+/-3.43, 29.3+/-3.26, 30.4+/-3.91, 25.1+/-5.25, 17.1+/-6.02 in females and 29.8+/-5.39, 28.8+/-5.29, 28.7+/-5.29, 23.7+/-5.39 and 16.0+/-5.60 in males. AChE activity from CRF-patients were higher than that found in the control subjects. The aging of human RBCs in both the controls and CRF-patients showed a progressive reduction in AChE activity. AChE activity of RBCs from female CRF-patients were significantly higher (p < 0.05) than that of the female control subjects. The RBCs isolated from male CRF-patients showed a higher AChE activity than control males, but a significant difference was only observed with the mid-age-cells. These studies further indicate that AChE activity remained insignificantly different in the various density based age subfractions of RBCs of both CRF-patients and controls.     

Cholinergic mechanism in Liriope tetraphylla (Cnidaria, Hydrozoa)

Crude whole body homogenates of Liriope tetraphylla exhibit a cholinesterase particularly active on acetylthiocholine but not on butyrylthiocholine. The acetylthiocholine hydrolysis is completely blocked by neostigmine. The Michaelis-Menten constant for acetylthiocholine is 0.14 mM. The pharmacological analysis of the responses to the choline esters nicotine and atropine suggests the involvement in Liriope tetraphylla of a cholinergic mechanism in the pointing reflex. Butyrylcholine, nicotine and atropine (but not muscarinic agonists) caused the contraction of the subumbrellar radial muscles. The effects of atropine were dose-dependent and were depressed in competition with muscarinic agonists. MgCl2 interfered with the action of atropine. The results were explained by suggesting the existence, at least at the neuromuscular junction, of excitatory (nicotinic) and inhibitory (muscarinic) pre-synaptic receptors modulating the release of the (unknown) transmitter acting post-synaptically.     

New Cnidaria species in Saratov oblast waterbodies (Povolzhye)

Cnidaria species that were earlier unknown in the waterbodies of Saratov oblast have been found in this region in the last decade. Colonies of Cordylophora caspia (Pallas, 1771) were found on stems of Phragmites australis (Cav.) Trin. ex Steud. in the basin cooler of the Balakovo nuclear power plant (Saratov water reservoir), while the freshwater jellyfish Craspedacusta sowerbii Lankaster, 1880 was found in the Saratovka River (Volgograd water reservoir). It is possible that these species penetrate into waterbodies with foreign plants or are introduced by birds. Another possibility is that their discovery is due to a more thorough examination of the waterbodies’ biota.     

Heat shock proteins in Aurelia (Cnidaria, Scyphozoa)

Heat shock proteins (hsp) in Aurelia identified by one-dimensional SDS-PAGE are of sizes 93,83,70,68,45, and 39 kD, the most rapidly labeled being hsp 70 in all developmental stages. Labeled hsp in the polyp are found mostly in the epidermis; gastrodermal nuclei are also labeled. The minimum temperature for induction of the proteins is about the same (27 degrees to 28 degrees C), regardless of whether polyps have been cultured at 15 degrees or 24 degrees C. Adults and planulae taken from natural water at 28 degrees C do not show accumulation of hsp 70. Induction of strobilation by raising polyps from 15 degrees to 25 degrees C is not associated with appreciable labeling of hsp. Polyps transferred to higher or lower salinity have decreased protein synthesis but do not synthesize stress proteins.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Metamorphosis of Hydractinia echinata (Cnidaria) is caspase-dependent

Apoptotic cell death plays an important role in many developmental pathways in multicellular animals. Here, we show that metamorphosis in the basal invertebrate Hydractinia echinata (Cnidaria) depends on the activity of caspases, the central enzymes in apoptosis. Caspases are activated during metamorphosis and this activity can be measured with caspase-3 specific fluorogenic substrates. In affinity labelling experiments 23/25 kDa bands were obtained, which represented active caspase. Specific inhibition of caspase activity with caspase-3 inhibitors abolished metamorphosis completely, reversibly and in a dose-dependent manner. This suggests that caspase activity is indispensable for metamorphosis in Hydractinia echinata.     

Modes of reproduction in sea anemones (Cnidaria,Anthozoa)

The data on different modes of reproduction in sea anemones are generalized. These animals can reproduce sexually in an ordinary way or by parthenogenesis. Asexual reproduction occurs in various forms, such as transverse and longitudinal fission, pedal laceration, or autotomy of tentacles. Specific features of different variants of sexual and asexual reproduction and their combinations in sea anemones from different habitats of the World Ocean are discussed.     

Isoenzymes and Taxonomy in Scandinavian Hydroids (Cnidaria, Campanulariidae)

Acid phosphatases, esterases and leucine aminopeptidases were studied in Campanularia integra, Clytia hemisphaerica (the johnstoni form), Laomedea ftexuosa, Gonothyraea loveni, Obelia geniculata, O. longissima and O. dichotoma , using polyacrylamide gel electrophoresis. Species-specific patterns were obtained, notably regarding acid phosphatase, despite strong individual variation. The electrophoretic results combined with differences in morphology and cnidom indicate that O. longissima and O. dichotoma are separate species. The acid phosphatase patterns of the two species and of O. geniculata showed basic similarities, supporting their assignment to a common genus. The profound differences in the acid phosphatase patterns of C. integra and L. flexuosa , may in conjunction with morphological distinction justify their assignment to separate genera. C. integra also included specimens of caliculata morphology: however, these did not deviate in their band patterns. Electrophoretograms of the typical O. dichotoma resembled those of the dubia and plicata forms. Moreover, O. gelatinosa with free Obelia medusae (sensu Hincks 1868) is suggested to be conspecific with O. longissima . The present study indicates that electrophoretic analyses, particularly of acid phosphatases presenting highly reproducible patterns, is a valuable complement to morphological characters when studying the taxonomy in hydroids.     

Acetylcholinesterase activity in developing rat brain during undernutrition

The effect of low protein diet on rat brain AChE activity has been studied during gestation, lactation and postweaning periods. There was decrease in enzyme activity of pups undernourished either during gestation and lactation or lactation alone, the decrease being maximum in 18-day-old pups. In postweaning rats, a significant decrease was observed after 2 and 4 weeks of undernutrition compared to the control. However, the effect of undernutrition was annuled by 2-week rehabilitation, thereby indicating that imposed undernutrition only delays the normal level of the enzyme. Moreover, it appears that the enzyme activity depends both on the nutritional status and the development age.     

Actiniaria and Ceriantharia of the Azores (Cnidaria Anthozoa)

The common shallow water species of sea anemones (Actiniaria) and tube anemones (Ceriantharia) of the Azores are listed. Eight species of sea anemones are mentioned, the species Cereus pedunculatus and Sagartia affinis being new records for the archipelago. Both species of Ceriantharia, namely Arachnanthus nocturnus and Pachycerianthus solitarius, are recorded from the Azores for the first time. Arachnanthus nocturnus is also recorded from the Cape Verde Islands and from Madeira for the first time. Communicated by H.-D. Franke     

Benthic hydroids (Cnidaria,Hydrozoa) from the Balleny Islands (Antarctica)

Twenty-two species of benthic hydroids, belonging to ten families and 14 genera, were found in a hydroid collection obtained in the Balleny Islands during the BioRoss expedition with the NIWA research vessel Tangaroa in 2004. Twenty of those species constitute new records for the Balleny Islands, raising the total number of known species in the area to 25. Most are members of the subclass Leptothecata, although the subclass Anthoathecata is also relatively well represented. Kirchenpaueriidae and Sertulariidae constitute families with the greatest numbers of species in the collection, with five species (20%) each. Oswaldella with five species (20%) and Staurotheca with four (16%), were the most diverse genera. Twelve species (63%) are endemic to Antarctic waters, most of them with a circum-Antarctic distribution, and 17 (89%) are restricted to Antarctic or Antarctic/sub-Antarctic waters. Although the Balleny Islands hydroid fauna seems to be a typical Antarctic assemblage, it has some striking peculiarities, namely the absence or low representation of some typical and widespread Antarctic genera (Antarctoscyphus and Schizotricha/Symplectoscyphus, respectively).