Silvia Scippa

Scanning X-ray Microscopy of Living and Freeze-Dried Blood Cells in Two Vanadium-Rich Ascidian Species, Phallusia mammillata and Ascidia sydneiensis samea

Abstract Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 107 times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchrotron Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

Ultrastructure of vegetative and sporulation stages of Haplosporidium ascidiarum from the ascidian Ciona intestinalis L.

Summary Specimens of the ascidian Ciona intestinalis L. collected in the Bay of Naples showed the occurrence of an eukaryotic microparasite characterized by sporogonial plasmodia with internal sporulation stages. The microorganism was identified as Haplosporidium ascidiarum and was located in the hosts stomach lumen. Transmission electron microscopy examinations were performed on specimens of C. intestinalis collected during spring and summer. Monoand binucleated cells characterized by enlarged mitochondria and by the occurrence of reticles and vesicular organelles were frequently observed. Plasmodia of H. ascidiarum appeared abundant and measured 6–10 × 8–14 urn. They cleaved internally producing 6–8 asynchronous sporoblasts. The anterior-posterior cell arrangement was observed at the beginning of the sporulation phase within the plasmodia. The sporoblasts and immature spores showed a complex sequence of spore wall formation, characterized by the sequential assemblage of fibrous components producing the spore wall. The spherular complex and lid opercule formed prior to the spore wall completion. Organelles and electron dense bodies were also observed in the cytoplasm of early vegetative stages or in the maturing spores, frequently clustered in the anterior region. The sporogonial plasmodia were the most frequent stages observed in the host.

Ultrastructure of trophozoites of the gregarine Lankesteria ascidiae (Apicomplexa: Eugregarinida) parasitic in the ascidian Ciona intestinalis (Protochordata)

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 hosts stomach epithelium or free in the lumen. The gregarine induced an hypertrophic reaction in the hosts 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. intestinalisgastric 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 trophozoites 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.

Redescription of Cardiosporidium cionae (Van Gaver and Stephan, 1907) (Apicomplexa: Piroplasmida), a plasmodial parasite of ascidian haemocytes

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 66bp 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.

Cytochemical localization of vanadium (III) in blood cells of ascidian Phallusia mammillata Cuvier, and its relevance to hematic cell lineage determination

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.

Protease activity in fractionated blood cells of the vanadium accumulating ascidian Phallusia mammillata

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 degrees 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.

Observations on an apicomplexan microparasite from the pericardic body of Ciona intestinalis L. (Protochordata)

Summary Specimens of the ascidia Ciona intestinalis L. collected in the Gulf of Naples showed the occurrence within the pericardic body of a microorganism characterized by sporogonial plasmodia and complex sporulation stages. An ultrastructural study revealed several sporonts and plasmodia. The latter showed asynchronous merogonic stages, characterized by merozoites budding at their periphery. These stages were abundant and aggregated at the periphery of the pericardic body among haematic cells, cell debris and other host degenerative material. The merogonic plasmodia had a coccidian-like structure and were the most frequent stages observed in the C. intestinalis pericardic bodies. The microorganism was considered as parasitic and represents a possible re-discovery of Cardiosporidium cionae , a sporozoan described early this century from the same host. Infection by the microparasite appeared implicated in the origin of the pericardic body, considered as an host response to infection.

Analysis of the Henze precipitate from the blood cells of the ascidian Phallusia mammillata

The Henze precipitate, a peculiar blue-green microparticulate obtained by lysis of the blood cells of the ascidian Phallusia mammillata (Protochordata), was investigated with atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray microanalysis. The precipitate was collected from the Henze solution, an unstable red-brown product obtained by treating blood with distilled water, whose degradation yields a characteristic blue-green product. The microparticulates measured 50–100xa0µm in diameter and appeared irregular in shape. SEM examination showed smooth, roughly round boundaries. The microparticulate surface examined with AFM appeared as an irregular matrix formed by 70–320-nm-wide mammillate composites, including and embedding small (500–800xa0nm wide) crystal-like multilayered formations. X- ray analysis showed that the elements present in these same precipitates were mainly C, Si, Al and O. The microparticulate composition appeared close to those of natural waxes or lacquers, embedding amorphous silicates and/or other Si–Al components. The unusual occurrence of Si in ascidian blood and its role are discussed.

PROTEOLYTIC ACTIVITY AND VANADIUM INHIBITION IN ASCIDIAN BLOOD CELLS

Abstract 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. 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. 2. In the blood cell sonicate of H. papillosa and M. sulcatus the proteolytic activity was inhibited by ethilenediaminetetraacetate (EDTA) and N-ethylmaleinimide (NEM). 3. 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. 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.