In vitro recalcification of the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The role of the amoebocytes in the calcification process of the shell-repair membrane of the snail, Helix pomatia, was investigated in vitro. The shell-repair membranes were demineralized with 0.5 M EDTA at pH 7.4. For the recalcification of the demineralized membranes two substrates were chosen: (i) Tris-buffered Helix pomatia-saline, pH 7.4, and (ii) Helix pomatia-saline supplemented with 5 mM CaCl₂ and 5 mM NaHCO₃. The membranes were incubated in 2 ml substrate at 37° C and examined after 2 h, 24 h, and 3, 5 and 7 days. Calcium deposition and crystal formation were observed within the membrane incubated in the salt-supplemented substrate. The control membranes were either heat-inactivated or deprived of lipids. No calcium precipitation was observed in control membranes. The experiments show that the recalcification of the shell-repair membrane is under strict cellular control and that the granules released from the amoebocytes serve as sites for calcium deposition. The role of phospholipids in the calcification process is discussed.     

Defensive mechanisms of holothuroids (Echinodermata): Formation,role, and fate of intracoelomic brown bodies in the sea cucumber Holothuria tubulosa

Brown bodies are pigmented aggregates of amoebocytes found in the coelomic cavities of most holothuroids (sea cucumbers). Brown body formation was induced by injection of carmine particles into the perivisceral coelom of Holothuria tubulosa. Formation begins with release of a fibrillar material by the spherulocytes. This fibrillar material acts as an extracellular matrix upon which amoebocytes and carmine particles collect. Amoebocytes develop an extensive pseudopodial network and progressively condense into aggregates with varying degrees of compactness. While condensing, amoebocytes either phagocytose or encapsulate carmine particles. A destructive process begins once particle aggregation is complete, resulting in numerous intracellular residual bodies and extracellular residual body-like structures, depending upon whether the carmine particles were phagocytosed or encapsulated. Induced bodies have the same ultrastructural features as naturally occurring ones. Brown bodies are progressively eliminated to the outside through coelo-rectal canaliculi, and the body cavity is essentially cleared of all induced bodies within seven days following injection.     

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.     

Glycosaminoglycans and fibrillar collagen in priapulida: a histo- and cytochemical study

The distribution of glycosaminoglycans and fibrillar collagen was studied in various tissues of priapulids, which represent an ancient group of mFine metazoa. Sulphated glycosaminoglycans, as demonstrated at the electron microscopical level by Cupromeronic blue, were predominantly found in the cuticle, in basement membranes and also in the narrow connective tissue space below epidermis and anterior intestine. On the basis of their morphology the Cupromeronic blue precipitates could be divided into several groups. Fibrillar collagen occurred in the connective tissue under the epidermis and the epithelium of the anterior intestine. The spatial interrelationship between fibrillar collagen and glycosaminoglycans lacked with some exceptions, the high regularity found in connective tissues of other invertebrates and of vertebrates. This might be related to the special skeletal system of priapulids, consisting mainly of a strong extracellular cuticle and the turgor of the fluid-filled body cavity. In such a system the usual supportive structures seem to be of less functional significance.     

Glycosaminoglycans and fibrillar collagen in Priapulida: a histo- and cytochemical study.

The distribution of glycosaminoglycans and fibrillar collagen was studied in various tissues of priapulids, which represent an ancient group of marine metazoa. Sulphated glycosaminoglycans, as demonstrated at the electron microscopical level by Cupromeronic blue, were predominantly found in the cuticle, in basement membranes and also in the narrow connective tissue space below epidermis and anterior intestine. On the basis of their morphology the Cupromeronic blue precipitates could be divided into several groups. Fibrillar collagen occurred in the connective tissue under the epidermis and the epithelium of the anterior intestine. The spatial interrelationship between fibrillar collagen and glycosaminoglycans lacked with some exceptions, the high regularity found in connective tissues of other invertebrates and of vertebrates. This might be related to the special skeletal system of priapulids, consisting mainly of a strong extracellular cuticle and the turgor of the fluid-filled body cavity. In such a system the usual supportive structures seem to be of less functional significance.     

Ultrastructure of Holothuria polii encapsulating body

The coelomic cavity of freshly collected Holothuria polii specimens contains a variable number of brown pigmented and unpigmented encapsulating structures. They are composed of nodules with entrapped parasites and an internodular mass which comprises a number of nodules. Both brown and unpigmented bodies occur in different size classes depending upon the number of nodules accumulated in a complete body. The unpigmented bodies probably represent an early unmelanized stage of the brown ones.
The nodule was ultrastructurally constituted by foreign bodies surrounded by a fibrous, electron-dense, non-cellular layer, probably melanin, followed by a layer of elongated and extremely flattened amoebocytes. Nodules were assembled in an internodular mass formed by amoebocytes type I, II and III spherule cells. As for the amoebocytes constituting the nodule, those present in the spaces between nodules did not develop junctional complexes. It is conceivable that the intricate network established among the cell processes could represent the mechanical force maintaining the whole structure.
As suggested by our results, two functional amoebocyte populations seem to be responsible for the organization of the scavenger body: 1. encapsulating amoebocytes, characteristically non phagocytosing, elongated cells; and 2, phagocytosing amoebocytes. The former organize the nodules, the latter constitute the internodular mass of several nodules.
Most probably, the double scavenger activity justifies the considerable complexity of the H. polii encapsulating structure compared to other invertebrates.
Spherule cells participate only in constituting the internodular mass.     

Zur feinstruktur integumentaler bildungen bei priapuliden (Halicryptus spinulosus und Priapulus caudatus)

Zusammentassung Die Integumente der Priapuliden Halicryptus spinulosus und Priapulus caudatus sind ähnlich aufgebaut. Die miteinander verzahnten und über lange septierte Desmosomen verbundenen Epithelzellen tragen apikal eine amorphe, in Abhgngigkeit von der Häutungsphase± geschichtete Kutikula. Ähnliche Kutikulastrukturen finden sich bei Nematoden. Der Panzer der Halicryptus-Larve ist in seiner Ultrastruktur deutlich verschieden von der Kutikula adulter Tiere and zeigt auch keine Übereinstimmung mit dem Panzer der Kinorhynchen.Die epithelialen Stacheln der Art Halicryptus spinulosus enthalten mehrere Sekretzelltypen. Die epithelialen Differenzierungen am Rumpfende von Priapulus caudatus sind durch ihre Ultrastruktur ebenfalls als sezernierende Zellkomplexe gekennzeichnet.Der Feinbau des distalen Anhangsorganes von Priapulus caudatus entspricht der Ultrastruktur von Zellen mit aktivem Ionentransport. AuBerdem ist dieses Organ Träger von Mechanorezeptoren.

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Fine structure of integumental features in priapulids (Halicryptus spinulosus and Priapulus caudatus)

Summary The integuments of the priapulids Halicryptus spinulosus and Priapulus caudatus exhibit a similar ultrastructure. The epithelial cells, the neighbouring lateral plasma membranes of which are frequently highly interdigitated, are connected by long septate desmosomes and are apically covered by an amorphous cuticle. The latter shows a stratification, the pattern of which is dependent on the moulting phase. Similar cuticular structures are to be found in nematodes. The cuticle of the larvae of Halicryptus differs markedly from that of the adult animals and also does not show agreements with the armour of kinorhynchs. It is threelaminated and contains in the middle layer canalicular structures. The epithelial spines of Halicryptus spinulosus contain various types of secretory cells. The epithelial differentiations at the end of the body of Priapulus caudatus also represent, according to their ultrastructure, secretory cells. The fine structure of the appendicular organs of Priapulus caudatus corresponds to that of cells engaged in ion transport. In addition this organ possesses mechanoreceptors. Both priapulids carry, on the integumental papillae and within the epithelium of the body and pharynx, characteristically constructed receptors with an apical cilium surrounded by seven microvilli. They are interpreted to represent mechanoreceptors.

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Für die Überlassung eines Arbeitsplatzes im Institut far Pharmakognosie Kiel danke ich Herrn Prof. Dr. D. Frohne. Alit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The macro‐ and microfossil record of the Cambrian priapulid Ottoia

The stem‐group priapulid Ottoia Walcott, 1911, is the most abundant worm in the mid‐Cambrian Burgess Shale, but has not been unambiguously demonstrated elsewhere. High‐resolution electron and optical microscopy of macroscopic Burgess Shale specimens reveals the detailed anatomy of its robust hooks, spines and pharyngeal teeth, establishing the presence of two species: Ottoia prolifica Walcott, 1911, and Ottoia tricuspida sp. nov. Direct comparison of these sclerotized elements with a suite of shale‐hosted mid‐to‐late Cambrian microfossils extends the range of ottoiid priapulids throughout the middle to upper Cambrian strata of the Western Canada Sedimentary Basin. Ottoiid priapulids represented an important component of Cambrian ecosystems: they occur in a range of lithologies and thrived in shallow water as well as in the deep‐water setting of the Burgess Shale. A wider survey of Burgess Shale macrofossils reveals specific characters that diagnose priapulid sclerites more generally, establishing the affinity of a wide range of Small Carbonaceous Fossils and demonstrating the prominent role of priapulids in Cambrian seas.     

Mitogenomics and phylogenomics reveal priapulid worms as extant models of the ancestral Ecdysozoan

Research into arthropod evolution is hampered by the derived nature and rapid evolution of the best-studied out-group: the nematodes. We consider priapulids as an alternative out-group. Priapulids are a small phylum of bottom-dwelling marine worms; their tubular body with spiny proboscis or introvert has changed little over 520 million years and recognizable priapulids are common among exceptionally preserved Cambrian fossils. Using the complete mitochondrial genome and 42 nuclear genes from Priapulus caudatus, we show that priapulids are slowly evolving ecdysozoans; almost all these priapulid genes have evolved more slowly than nematode orthologs and the priapulid mitochondrial gene order may be unchanged since the Cambrian. Considering their primitive bodyplan and embryology and the great conservation of both nuclear and mitochondrial genomes, priapulids may deserve the popular epithet of "living fossil." Their study is likely to yield significant new insights into the early evolution of the Ecdysozoa and the origins of the arthropods and their kin as well as aiding inference of the morphology of ancestral Ecdysozoa and Bilateria and their genomes.     

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.     

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.     

The occurrence of inflammatory granulomas in the ctenidial marsupium of Corbicula fluminea (mollusca: Bivalvia): A consequence of larval incubation

A number of tumor-like cysts in the ctenidia of the freshwater bivalve Corbicula fluminea are described. The cysts result from the encapsulation of incubated larvae within the female inner demibranchs which are modified to form a marsupium. Corbicula fluminea is a protandric consecutive hermaphrodite, and larval incubation commences with sex reversal and the release of the first eggs. For some reason a few of the larvae are not released, and their retention and subsequent death results in the mobilization of the innate cellular defensive mechanism of the parent. This process involves, initially, an invasion of epithelioid cells and amoebocytes (granulocytes), resulting in hyperplasia. This is followed by structural changes to the epithelium bordering the interlamellar spaces (within which the larvae are incubated) to form ultimately the cyst wall. The epithelium is surrounded externally by layers of fibrocytes which eventually form a thick capsule. The autolyzed larval tissues are themselves invaded by fibrocytes, epithelioid cells, and amoebocytes and, in one specimen, had formed a three-layered capsule within the surrounding capsule. The amoebocytes probably reabsorb the larval cellular debris. This unusual example of a molluscan cellular defensive mechanism may assist in the diagnosis and separation of hyperplastic injury responses from neoplasmic conditions in invertebrates.     

Structure of the body cavity of the sea spider <Emphasis Type="Italic">Nymphon brevirostre</Emphasis> Hodge, 1863 (Arthropoda: Pycnogonida)

The microscopic anatomy and ultrastructure of the body cavity and adjacent organs in the sea spider Nymphon brevirostre Hodge, 1863 (Pycnogonida, Nymphonidae) were examined by transmission electron microscopy. The longitudinal septa subdividing the body cavity are described: (1) Dohrn’s horizontal septum, (2) lateral heart walls, and (3) paired ventral septa consisting of separate cellular bands. The body cavity is a hemocoel, it has no epithelial lining and is only bordered by a basal lamina. The epidermis, heart, and Dohrn’s septum are not separated from each other by basal laminae and may have a common origin. The cellular bands forming the longitudinal ventral septa are not covered with the basal lamina and presumably derive from cells belonging to the hemocoel. The roles of the morphological structures studied for the circulation of hemolymph are discussed. The gonad lies inside Dohrn’s septum, it is covered with its own basal lamina and surrounded by numerous lacunae of the hemocoel entering the septum. The gonad wall is formed with a single layer of epithelium. The same epithelial cells form the gonad stroma. The gonad cavity is not lined with the basal lamina; muscle cells are present in the gonad wall epithelium, thus rendering the lumen similar to a coelomic cavity. Freely circulating cells of two types are found in the hemocoel: small amebocytes containing electronic-dense granules that are similar to granulocytes of other arthropods, as well as hemocytes with large vacuoles of varying structure that are comparable with plasmatocytes; however some of these may be activated granulocytes.     

Antioxidant Enzymatic Activity of Coelomocytes of the Far East Sea Cucumber Eupentacta fraudatrix

The basal activity of superoxide dismutase (SOD), glutathione reductase (GR), and catalase in a pool of coelomocytes as well as in the fraction of amoebocytes and the mixed fraction of amoebocytes and morula-like cells of the sea cucumber Eupentacta fraudatrix is studied. For SOD and catalase, pH optima are in the range of values of pH optimum for tissues of mammals, the pH optimum for GR is shifted to a more acidic region in comparison with the latter enzymes. Temperature optima for all studied enzymes are higher than the usual temperature values of the sea cucumber habitation. A pronounced temperature dependence of all three enzymes is revealed. In coelomocytes, the activities of SOD and catalase, but not of GR, are lower than in the fraction of amoebocytes, but higher than in the mixed fraction of amoebocytes and morula-like cells. The rate of production of active forms of oxygen (AFO) is three times higher in amoebocytes than in the fraction relatively enriched in morula-like cells. Apparently, the main part of the SOD and catalase activities, as well as AFO production in coelomocytes is located in amoebocytes, which confirms the existence of cytophagic function in the latter cells as well as argues in favor of functional differentiation between individual types of coelomocytes.     

Ultrastructure of erythrocytes and leucocytes of Priapulus caudatus (de Lamarck) (Priapulida)

The marine priapulid Priapulus caudatus has a voluminous body cavity filled with a blood-like fluid containing erythrocytes and leucocytes (amoebocytes). The hematocrit of animals weighing 0.5–14 gm was 2–10%. The erythrocytes contain a hemerythrin blood pigment. The structure of the coelomocytes was studied by light and electron microscopy. The erythrocytes are nucleated and contain marginal bands, vacuoles and occasionally crystals. The cytoplasm has few organelles. The leucocytes are amoeboid motile cells, the cytoplasm of which contains numerous organelles. The most conspicuous of these are oval particles, probably representing developmental stages of lysosomes. Most of these organelles contain tubules stretching from one pole to another. In the hind part of the animal, certain tissues, primarily the posterior warts contain large numbers of coelomocytes. The histological picture is complicated, showing some resemblance to the lymphoepithelial tissues of vertebrates.     

The mode of action of acute and chronic concentrations of waterborne Cd in the digestive gland of the acclimated infested freshwater crab (<Emphasis Type="Italic">Potamonautes warreni</Emphasis>)

Cadmium (Cd) uptake, transport and accumulation were investigated in the digestive gland of the freshwater crab, Potamonautes warreni, acclimated in its natural habitat to stresses, such as microbial gill infestations, Cd(2+) and NH(4)(+), and subsequently exposed to increasing concentrations of Cd in the laboratory for up to 21 days. Cd exposure (0.2 mg l(-1)) for 7-14 days led to Cd permeating cell membranes in a particulate form; it was adsorbed intracellularly to endocytotic circulating amoebocytes, lipid droplets and Golgi vesicles in R-cells. Cd also caused dissociation of the fibrillar rough endoplasmic reticulum (RER) and an increase in phagocytotic activity in F- and B-cells. After 21 days, Cd accumulated as crystal deposits on the basal membranes of cells in the haemolymph space and along the microvilli of cells lining the tubular lumen. Elevated Cd concentrations were found in the cytosol, amoebocytes, Golgi vesicles and P/Ca granules in R-cells. Chronic exposure to higher concentrations of Cd (0.5 and 1.0 mg l(-1)) increased crystal deposition, whereas concentrations of Cd, copper and iron decreased in the cell membranes and in amoebocytes and increased in Golgi vesicles. Reduced lipid content, swollen nuclei with vesiculated nucleoli and enhanced activity of RER in R-cells were also noted. Cd was stored in the P/Ca and Ca granules of B-cells. Acute exposure to Cd (2.0 mg l(-1) for 48 h) caused metal granule accumulation along cells lining the tubular lumen and cellular dissociation, with acidosis and necrosis in the cytoplasm and Cd deposits in mitochondria. Cd accumulated in the cells of the digestive gland in a time-, concentration- and cell-type-specific fashion.     

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.     

Phagocytic efficiency and cytotoxic responses of Indian freshwater sponge (Eunapius carteri) cells isolated by density gradient centrifugation and flow cytometry: a morphofunctional analysis

The freshwater sponge Eunapius carteri (Porifera: Demospongiae: Spongillidae), a resident of Indian freshwater ecosystems, has pharmaceutical and ecological potential, but there is inadequate information on its cellular spectrum and cell-mediated immune responses. Microscopical analysis revealed the existence of eight distinct cellular variants, i.e. blast-like cells, choanocytes, small amoebocytes, granular cells, pinacocytes, large amoebocytes, archaeocytes and sclerocytes. The cells were isolated by density gradient centrifugation and flow cytometry and used for a morphofunctional analysis. We investigated the phagocytic efficiency of E. carteri cells under the challenge of yeast particles in vitro and spectrophotometrically quantified the generation of cytotoxic molecules (superoxide anions and nitric oxide) in different isolated cellular fractions. The two cell separating technologies did not yield any significant differences in the major findings on morphology, phagocytic response and generation of superoxide anions and nitric oxide. Archaeocytes, granular cells and large amoebocytes were identified as chief phagocytes with a high phagocytic potential as recorded by light microscopy. Archaeocytes were the principal generators of superoxide anions, whereas nitric oxide was recorded in the fractions rich in archaeocytes and large amoebocytes. The present investigation thus provides useful information regarding cellular variation, cytotoxic status and innate phagocytic response of the cells of E. carteri, a common but less studied sponge of India.     

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.     

Blood proteins in bipolar Priapulida

Summary Molecular weights are provided for 44 blood serum proteins from four species of Priapulida: 21 peptides in Priapulus caudatus and P. tuberculatospinosus, 19 in Priapulopsis bicaudatus, and 24 in P. australis. One (exceptionally two) proteins predominate in the investigated sera. While electrophoretic patterns are variable within species, phenetic trees constructed on the basis of Czekanowski's distances of peptide pattern resemblance match the taxonomic hierarchy of priapulids. This shows that electrophoretic profiles of denatured serum peptides have a phylogenetic basis, in spite of considerable nongenetic diversity which limits phylogenetic interpretations of the present data set. One may tentatively conclude that molecular differentiation of bipolar priapulids is more extensive than morphological differences, at least in the genus Priapulus.