Cardiosporidium cionae (Apicomplexa), from the ascidian Ciona intestinalis L., is redescribed with novel ultrastructural, phylogenetic and prevalence data. Ultrastructural analysis of specimens of C. intestinalis collected from the Gulf of Naples showed sporonts and plasmodia of C. cionae within the host pericardial body. Several merogonic stages and free merozoites were found in the pericardial body, together with sexual stages. All stages showed typical apicomplexan cell organelles, i.e. apicoplasts, rhoptries and subpellicular microtubules. Merogonic stages of C. cionae were also produced inside haemocytes. A fragment of the rSSU gene of C. cionae was amplified by PCR using DNA extracted from the pericardial bodies. The amplified product showed closest affinity with other apicomplexan representatives and a 66 bp unique insertion, specific for C. cionae, at position 1644. Neighbour-joining phylogenetic analysis placed C. cionae in a clade with other piroplasm genera, including Cytauxzoon, Babesia and Theileria spp. The parasite was found in different populations of C. intestinalis with highest prevalence in October–November. Ultrastructural and DNA data showed that the organism, described in 1907 from the same host but not illustrated in detail, is a member of a novel marine apicomplexan radiation of tunicate parasites.

Different stages of the embryonic development of the ascidians, Ciona intestinalis and Phallusia mammillata, were observed by confocal microscopy after treating embryos with polyclonal antibody raised against C. intestinalis hatching enzyme and after staining with FTTC-conjugated second antibody. in both species fluorescence is localized, at the gastrula stage, in the ectoderm. At the subsequent neurula and tail bud stages, in C. intestinalis, the enzyme is localized in the anterior region and tail region, while in P. mammillata it is only present in the anterior region.

Summary Different stages of the embryonic development of the ascidians, Ciona intestinalis and Phallusia mammillata, were observed by confocal microscopy after treating embryos with polyclonal antibody raised against C. intestinalis hatching enzyme and after staining with FITC-conjugated second antibody. In both species fluorescence is localized, at the gastrula stage, in the ectoderm. At the subsequent neurula and tail bud stages, in C. intestinalis, the enzyme is localized in the anterior region and tail region, while in P. mammillata it is only present in the anterior region. Key words: Ascidianhatching enzyme antibodyimmunofluorescenceconfocal microscopy

Most recent studies of vanadium in ascidian blood indicate the signet ring cell as the cell type that stores vanadium. However, in Phallusia fumigata the situation is less clear, with contradictory reports. Therefore, the blood cells of the ascidian P. fumigata were stained with the vanadium(III) specific ligand 2,2′-bipyridine. The presence of vanadium was revealed in the vacuolated amoebocyte, the second compartment cell type or pluri-vacuolated cell, as well as the bivacuolated cell and the signet ring cell. Staining was not observed in either the morula cells or the compartment cells, the predominant cell types in the mature animal.

Mouse erythroleukemia (Mel) cells have a cell cycle-dependent high sensitivity to chemical and physical mutagens. This report shows that a 5 h exposure to 0.1 or 0.01 microg/ml metavanadate during the initial period of erythroid differentiation induction was sufficient to permanently damage the ability of treated Mel cells and their progeny to undergo erythroid differentiation, without affecting cell viability and proliferation. Conversely, a 5 h pulse of metavanadate at 1 or 10 microg/ml inhibited both differentiation and cell proliferation. The cell cycle-dependent period of mutagenesis was essential for fixation of damage in the cell genome and the progeny of the cells treated with 0.1 or 0.01 microg/ml metavanadate stably inherited an impaired capacity to differentiate. The efficiency of the DNA repair synthesis machinery during the specific period of exposure of Mel cells seemed directly involved in damage fixation. In fact, the mutagenic effects of a 0.1 microg/ml metavanadate pulse was further increased in the presence of 1 mM hydroxyurea, an inhibitor of DNA repair synthesis. In contrast, 5 microg/ml vanillin, an antimutagenic agent that stimulates repair, completely restored the capacity of progeny of cells treated with 0.1 microg/ml metavanadate to complete differentiation. Determination of [(3)H]deoxythymidine in acid-insoluble DNA indicated that incorporation was stimulated by metavanadate alone and was further increased by metavanadate plus vanillin; conversely, incorporation of thymidine was reduced in the presence of hydroxyurea. The capacity of metavanadate to permanently damage Mel cell erythroid differentiation appeared to depend on the cell cycle-related efficiency of the DNA repair systems, activated to correct the induced alteration, rather than on a specific concentration.

The gregarine Lankesteria ascidiae (Lankester 1872) Mingazzini, 1891 was studied within specimens of the ascidian Ciona intestinalis L. collected from the Bay of Naples. Examinations of parasitized ascidiae by light microscopy showed trophozoites inserted in the host's stomach epithelium or free in the lumen. The gregarine induced an hypertrophic reaction in the host's epithelium cells, and trophozoites free in the stomach were surrounded by cilia of gastric cells. The host cells flanking infected cells appeared laterally compressed and a niche formed as an invagination of the gastric wall covering the trophozoite. Transmission electron microscopy of C. intestinalis gastric epithelium showed mononucleated trophozoites with large mitochondria frequently arranged in peripheral clusters. Maturing trophozoites showed a mucron filled by a dense fibre matrix; these fibres extended in a root-like formation through the whole trophozoite cell up to its periphery and appeared to occupy a separate cytoplasmic compartment enclosed in a membrane. The membrane covering the compact mucron was in continuous close contact with the membrane of the host cell. The trophozoite's cytoplasm was rich in paraglycogen-like inclusions and electron-dense lipid deposits. The surface showed rows of electron-dark knobs protruding at regular intervals from a basal layer. These rows were cross-connected by underlying perpendicular bundles of filaments.

Proteolytic activity was studied in the fractionated blood cells of the vanadium accumulating ascidian P. mammillata by separating the cells before measuring their activity. Cells were separated to avoid vanadocyte breakdown and subsequent vanadium diffusion into the assay medium. Our study revealed activity in the morula cell extract that was obtained by sonication and Centricon concentration. After removing part of the extract for enzyme activity assay the remainder was kept at 0°C; it was later found that much of the protein in this latter fraction formed a sediment whereas the protease remained in solution. The serine-protease substrate specificity of the enzyme was measured and the results are discussed in relation to serine protease involvement in immune defense.

The "Henze solution", derived originally from the aqueous extraction of pelleted whole blood from the ascidian Phallusia mammillata, was examined using spectral studies. The aqueous extraction of fractionated blood cells including compartment cells, signet ring cells. and morula cells obtained using cell separation techniques were also examined. It was found that this Henze solution, and the Henze precipitate itself derived from this solution, emanated solely from the morula cells. Furthermore, it was found that this solution is formed independently of the vanadium metal ions otherwise associated with the vanadocytes. Observation of the Henze precipitate by light microscopy shows that this material partially forms crystallites or microglasses.

When the blood cells of ascidians Phallusia mammillata are stained with the ligand 2,2'-bipyridine, those cells which contain vanadium(III), in an easily sequestered form, take up the stain producing in situ, a purple complex. This material extracted displays spectral characteristics consistent with the formation of an oxo-bridge vanadium(III) bipyridine dimer. The staining is localized in the signet ring cell, a bivacuolated cell, a cell type with numerous darkly staining compartments, and also by the vacuolated amoebocyte. The possible ramifications of these observation are discussed in relation to the delineation of the signet ring cell lineage.

Summary The vanadocytes of living juvenile Phallusia mammillata ascidians were examined by utilising cytochemical localisation of vanadium(III) in situ, to help determine the commencement of vanadium assimilation. The first visual indication of accumulation of vanadium(III) within the vanadocytes occurs at five days after fertilisation, shortly after the siphons of the developing larvae have opened. At nine days a number of different cell types, including vacuolated amoebocytes, a cell type which possess numerous compartments, together with bivacuolated cells and signet ring cells, all stain positively for vanadium(III) with the ligand 2.2′-bipyridine.

A polyclonal antibody raised against the hatching enzyme of Ciona intestinalis (D'Aniello et al., 1997) was used on larvae of different ages in whole mount immunofluorescence experiments in order to localize the cells secreting the enzyme. After staining with FITC-conjugated second antibody, the larvae were observed by confocal microscopy. Larvae just before hatching (9-10 hours after fertilization) showed the presence of the enzyme in the peripheral cells of the adhesive papillae. The newly hatched larvae showed fluorescence also in the epidermal cells of the tip of the tail. Higher magnification confocal images of the papillae revealed bright fluorescence both in peripheral cells of the papillae and in the cavity between the tunic and the apex of the papillae (hyaline cap).The swimming larvae maintain the fluorescence in the peripheral cells and in the hyaline cap for some hours until the beginning of metamorphosis, whereas the fluorescence of the tip of the tail disappears.Following application of the antibody to Phallusia mamillata, the peripheral cells of the papillae of the newly hatched larvae were fluorescent and a bright fluorescence was also present between the two layers of the tunic above the papillae and the anterior part of the cephalenteron. We never observed fluorescence in the cells of the epidermis of the tail.Retinoic acid (RA) treatment has been used to confirm the localization on the papillae of the cells secreting the hatching enzyme. The larvae of Ciona intestinalis were able to hatch because the cells of the tip of the tail positively reacted to immunofluorescence stain with anti-hatching enzyme antibody. On the contrary Phallusia mamillata larvate failed to hatch and did not show anti-hatching enzyme reaction in the tail.

We have purified a 34 kDa hatching enzyme from the water in which the embryos of the sea-squirt Ciona intestinalis hatch. This enzyme was obtained in homogeneous form as judged from SDS-PAGE and HPLC gel filtration. The enzyme possesses proteolytic activity and is able to digest the chorion of the egg of C. intestinalis. It is a metalloproteinase and contains one atom of Zn per molecule. The optimum pH is 8.5. The enzyme shows hydrolytic activity towards the -CO-NH- bonds, which are hydrolyzed by the members of the serine proteinase family. It has a trypsin-like activity in that it cuts the bond of Arg and Lys at P1 position of the scissile bond -P1-P1', but it differs from trypsin insofar as it hydrolyzes the peptide bond on either side of Arg and Lys. The purified enzyme is inhibited by the common metal-chelators and by the classical trypsin proteinase inhibitors. The apparent K(m) values at 37 degrees C and pH 8.5 toward tosyl-Gly-Pro-Arg-NHNap, tosyl-Gly-Pro-Lys-NHNap and Bz-Arg-Gly-Arg-NHNap were 0.125, 0.5 and 2.5 mM, respectively. The results obtained in this study suggest that the hatching enzyme from C. intestinalis exhibits both trypsin-like activity and metalloproteinase activity.

A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.

The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates. (C) 1997 Published by Elsevier Science Ltd on behalf of The Royal Swedish Academy of Sciences.

The cells of the body cavity of Phallusia mammillata larvae were examined by transmission electron microscopy before hatching, after it and at the beginning of tail retraction. The results revealed the presence - both before and after hatching - of cells with small electron-lucent vacuoles, which might be the precursors of the monovacuolated cells observed at the beginning of tail retraction. These cells share morphological characteristics with the vanadium-accumulating signet ring cells of the blood of adults.

Proteolytic activity on azocasein was studied in the hemocytes of vanadium-accumulating ascidians (Phallusia mammillata and Ciona intestinalis) and iron-accumulating ascidians (Halocynthia papillosa and Microcosmus sulcatus). A possible inhibitory role of vanadium on proteolytic enzymes was detected.The following results were obtained:1. In the neutral range M. sulcatus and C. intestinalis had optimal activity while H. papillosa showed only an activity shoulder, its activity being strongest at alkaline pH. P. mammillata displayed no proteolytic activity at all.Two types of individuals, with high and low activities, were present in H. papillosa at both pHs, and in M. sulcatus at alkaline pH. In C. intestinalis only individuals active at pH 6.5 were observed.2. In the blood cell sonicate of H. papillosa and M. sulcatus the proteolytic activity was inhibited by ethilene-diaminetetraacetate (EDTA) and N ethylmaleinimide (NEM).3. P. mammillata blood cells sonicate inhibited proteolytic activity of H. papillosa and M. sulcatus blood cells: this is attributed to the presence of large quantities of vanadium in the blood cells of P. mammillata.4. Metavanadate, orthovanadate and vanadyl (IV) sulfate inhibited proteolytic activity of H. papillosa and M. sulcatus blood cells. The inhibitory effect showed dependence on the metal ion species, the pH, and the ascidian species assayed.

The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.

Peptides containing beta-(3,4,5-trihydroxyphenyl)-L-alanine and beta-(3,4-dihydroxyphenyl)-L-alanine with C-terminal styrylamides form the tunichromes of the vanadium concentrating Phallusia mammillata from the subspecies of the tunicates (Urochorda). The structure of 1, the main tunichrome from P. mammillata, was elucidated with mass spectrometry (the fragmentation pattern is shown on the right). The native tunichromes are not bound to vanadium. The equimolar relationship of tunichrome and vanadium in the blood cells of tunicates suggests that tunichromes are responsible for their efficient enrichment of vanadium.

Summary The intracellular compartmentation of P, S, Cl, K, Fe, Br, and I in several V non-accumulating blood cells of the ascidian Phallusia mammillata was studied by X-ray microanalysis of freeze-dried cryosections in the scanning transmission electron microscope.