The localization of inorganic elements,particularly vanadium and sulphur,in haemolymph from the ascidians Ascidia mentula (Müller) and Ascidiella aspersa (Müller)

Vanadium and sulphur were found by X-ray microanalysis in three different cell types, morula cells, signet-ring cells, and granular amoebocytes, from each of the ascidians Ascidia mentula (Müller) and Ascidiella aspersa (Müller). In addition to the cells containing vanadium and sulphur a few vacuolar cells containing high concentrations of iron, magnesium and calcium were also found. After fixation in the presence of strontium or barium chlorides another cell type containing large amounts of precipitated sulphur was also found, confirming our previous findings that many haemolymph cells in these species contain large amounts of sulphate only. The presence of these various cell types is discussed in relation to the question of the acidity of ascidian blood cells.     

The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.     

Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

Scanning x-ray microscopy of living and freeze-dried blood cells in two vanadium-rich ascidian species,Phallusia mammillata and Ascidia sydneiensis samea

Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 10(7) 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 Synchroton 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.     

The fine structure of granular amoebocytes from the gonads of the sea anemoneActinia fragacea (Cnidaria: Anthozoa)

Summary The structure of granular amoebocytes of the intertidal sea anemoneActinia fragacea (Cnidaria: Anthozoa) has been investigated using the electron microscope. Cells from the gonads of large, intact individuals were studied in most detail, but other regions of the anemone were also examined. The amoebocytes are cells of variable appearance which are widely distributed both in the mesogloea and in the epithelial cell layers. They contain numbers of characteristic dense granules, which may enclose spherical cores of greater or lesser electron density. They also contain rough endoplasmic reticulum, Golgi apparatus and a range of inclusions, some of which may have lysosomal origins. They may contain extensive deposits of glycogen, and usually smaller quantities of lipid droplets. They may take on a variety of forms, depending partly on their location within the various types of mesogloea and epithelia. The amoebocytes appear to be motile and phagocytic, and may also be involved in the storage and transport of glycogen. They are involved with gametogenesis, both during the development of the oocytes and spermatogenic cysts and during the resorption of degenerating gametes. Their possible role in the secretion or maintenance of the mesogloea remains uncertain. No evidence of amoebocytes differentiating into other cell types was obtained.     

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

Summary Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.     

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.     

Localisation of Vanadium, Sulphur and Bromine Within the Vanadocytes of Ascidia mentula Müller: A Quantitative Electron Probe X-Ray Microanalytical Study

Levels of vanadium, sulphur and bromine within the vanadopohores of Ascidia mentula vanadocytes have been determined by quantitative X-ray microanalytical techniques. Values obtained for vanadophore vanadium in thin sections correspond to 2.5-3.25% or 0.5-0.65 molal aqueous solutions in vivo. Quantitation of sulphur in thin sections gives levels an order of magnitude lower than for vanadium, but it is likely that major losses of sulphur have occurred, since analysis of unfixed, air dried vanadocytes gives V/S atomic ratios close to unity. This 1: 1 ratio may have implications for the structure of the vanadium complex. Quantitation of bromine is less reliable, but levels of this halogen in vanadocytes are significant. The possibility that bromine, in the form of brominated metabolites, together with vanadium and acid, contribute to a multicomponent chemical defense role in ascidians is discussed.     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

Accumulation of vanadium, manganese, and nickel in Antarctic tunicates

The vanadium, manganese, and nickel contents of nine species of Antarctic tunicates were determined by atomic absorption spectroscopy. The Antarctic species Distaplia cylindrica contained significantly more vanadium (1,445 ppm dry weight) than the other Antarctic tunicates investigated. Antarctic Ascidia sp. was also shown to accumulate considerable amounts of vanadium (567 ppm). Low levels of bioaccumulated manganese (<50 ppm) and nickel (<15 ppm) were observed in all tunicates examined.     

The blood cells of the sea squirt, Ciona intestinalis: Morphology,differential counts,and in vitro phagocytic activity

The sea squirt, Ciona intestinalis, contains several types of blood cells: stem cells, hyaline, granular, and refractile amoebocytes, signet ring cells, morula cells, small and large compartment cells, and orange cells. Of these cell types, only the hyaline and granular amoebocytes are capable of phagocytosing formalized sheep erythrocytes in vitro. After the addition of erythrocytes to blood cell monolayers, the attachment and ingestion of these particles occurs rapidly. The interrelationships of the various blood cell types are discussed.     

The characterization of granular amoebocytes and their possible roles in the asexual reproduction of the polystyelid ascidian,Polyzoa vesiculiphora

The blood cells in the bud and the zooid of the polystyelid ascidian, Polyzoa vesiculiphora, were examined by means of light and electron microscopy to identify the cells that have been named trophocytes. The large blood cells were abundant in the mesenchymal space of the bud, but not in that of the functional zooid. They contained glycogen particles, lipid droplets, large protein granules and autophagosomes in their cytoplasm and were identified as granular amoebocytes. The majority of these cells were specifically phagocytized by phagocytes during bud development and disappeared. These results indicate that the granular amoebocytes virtually represent trophocytes in Polyzoa and may participate in bud development via nutrient supply to the developing tissues.     

Cellular cadmium distribution in the common winkle,Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Summary Various tissues of common winkles,Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Differences in molybdenum uptake by micro-organisms from the rhizosphere ofraphanus sativus L. grown in two soils of similar origin

Summary Differences have been shown in molybdenum uptake by microorganisms from the rhizosphere and soil sampled away from the roots, of the radish,Raphanus sativus L., grown in market garden soils from Napier and Hastings (New Zealand).The organisms from the rhizosphere of plants in Hastings soil concentrated up to 55 ppm of molybdenum dry weight when grown in a liquid medium made from Hastings soil extract and supplemented with carbon, phosphorus, nitrogen, sulphur and molybdenum. The growth from an inoculum of pooled fungal isolates from the rhizosphere has been shown to contain a higher concentration of molybdenum than growth from pooled bacterial or streptomycete isolates. The growth from a combined bacterial and streptomycete inoculum contained a higher concentration of molybdenum than the growth from either group alone.Organisms from the rhizosphere and soil sampled away from the roots of radishes grown in Napier soil did not contain such high concentrations of molybdenum.No significant differences in the frequency of morphological types were found in the isolates from either soil.     

Ultrastructure of the Coelomocytes in the Tentacular Coelom of Thysanocardia nigra Ikeda, 1904 (Sipuncula)

Free-floating coelomocytes in the tentacular coelomic cavity of the sipunculan Thysanocardia nigra Ikeda, 1904, were studied using light interference contrast microscopy and scanning and transmission electron microscopy. The following coelomocyte types were distinguished: hemerythrocytes, amoebocytes, and two morphological types of granular cells. No clusters of specialized cells that had been reported to occur in the trunk coelom of Th. nigra were found in the tentacular coelom. The corresponding types of coelomocytes from the tentacular and trunk coelomic cavities were shown to differ in size. These two coeloms are completely separated in sipunculans.     

Amoebocytes in mesoglea of 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.     

Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons

Substrate-bound and soluble factors regulate neurite outgrowth and synapse formation during development, regeneration, and learning and memory. We report that sheath cells from CNS connectives and arterial cells from the anterior aorta of the sea slug, Aplysia californica, enhance neurite outgrowth from co-cultured Aplysia neurons. Sheath and arterial cell cultures contain several cell types, including fibrocytes, myocytes, and amoebocytes. When compared to controls (neurons with defined growth medium alone), the percentage of neurons with growth and the average neurite lengths are significantly enhanced by sheath and arterial cells at 48 h after plating of the neurons; these parameters are comparable to those of neurons cultured in medium containing hemolymph. Our results indicate that sheath cells produce substrate-bound factor(s) and arterial cells produce diffusible factor(s) that promote growth. These growth factors likely promote neuron survival and neurite outgrowth during neural plasticity exhibited in the adult CNS. Electronic Publication     

RESPONSES OF FRESHWATER ALGAE TO INHIBITORY VANADIUM CONCENTRATIONS: THE ROLE OF PHOSPHORUS1

We found that species-specific differences exist among a variety of freshwater algae and cyanobacteria in the extent to which growth and photosynthesis are inhibited by vanadium. A major factor controlling the degree of inhibition by vanadium was the phosphorus state (P-sufficient vs. P-deficient) of the organisms. In P-sufficient cultures, vanadium was inhibitory when the vanadium concentration exceeded the phosphate concentration. In P-deficient cultures, the depression of photosynthesis by vanadium increased with increasing phosphorus deficiency. Our conclusion that vanadium competed with phosphate for uptake sites was supported by the following three observations: 1) the decreased influx of ³²P-PO ₄ into P-deficient cells in the presence of vanadium, 2) the amelioration of vanadium inhibition of photosynthesis by the addition of phosphate, and 3) the accumulation of vanadium by cells. At vanadium concentrations that severely inhibited growth, the cells of Scenedesmus obliquus (Turp.) Kruger were larger than normal and contained more vacuoles, lipid, and starch bodies than normal cells. Four-celled coenobia were replaced by unicells. Scenedesmus acutusf: alternans Hortobagyi cells from vanadium-inhibited cultures had 7.5 times more vanadium per cell than control cultures and contained numerous granules that did not stain for polyphosphate and may be composed of condensed vanadate molecules. The cellular P quota and turnover time of PO₄in the medium are important regulators of the extent of inhibition by vanadium.     

Changes of Relative Weight and Cell Cycle,and Lesions of Bursa of Fabricius Induced by Dietary Excess Vanadium in Broilers

The purpose of this 42-day study was to investigate the effects of dietary excess vanadium on immune function by determining the morphological changes and cell cycle of bursa of Fabricius, and the serum Ig contents. A total of 420 one-day-old avian broilers were divided into six groups and fed on a corn–soybean basal diet as control diet, or the same diet amended to contain 5, 15, 30, 45, and 60 ppm vanadium supplied as ammonium metavanadate. When compared with that of control group, the relative weight of bursa was significantly increased in the 15 ppm group from 14 to 35 days of age and increased in the 5 ppm group at 42 days of age, and significantly decreased in the 60 ppm group from 14 to 42 days of age and decreased in 30 and 45 ppm groups from 35 to 42 days of age. Pathological lesions progressed as the dietary vanadium increased. The gross lesions of bursa showed obvious atrophy with decreased volume and pale color in 45 and 60 ppm groups. Histopathologically, widened cortex and increased number of lymphocytes appeared in 5 and 15 ppm groups, and reduced lymphocytes and connective tissue hyperplasia appeared in 45 and 60 ppm groups. The bursal cells in static phase (G₀/G₁) were decreased, and those in the mitotic phase (G₂ + M) and the proliferating index (PI) were increased in 5 and 15 ppm groups. However, bursal cells in the G₀/G₁ phase were increased, and those in G₂ + M phase, synthesis phase (S) and the PI were decreased in 45 and 60 ppm groups. Also, the serum IgG and IgA contents were increased in 5 and 15 ppm groups, and the serum IgG, IgA, and IgM contents were decreased in 45 and 60 ppm groups. These results suggested that dietary excess vanadium (45 and 60 ppm) could inhibit growth of bursa of Fabricius and impair humoral immunity in chicken.