Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.     

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

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.     

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.     

The connective tissue index of <Emphasis Type="Italic">Helix aspersa</Emphasis> as a metal biomarker

The digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island (Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantify the relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, through light microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cu and Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetric density of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein is suggested as a biomarker of Cu exposure in terrestrial mollusks.     

Cuprolinic Blue visualization of cytosolic and membrane-associated glycosaminoglycans in the rat junctional epithelium and gingival epithelia

Summary The gingiva of rat molars was studied at the light microscope level using glutaraldehyde as fixative, Cuprolinic Blue for visualizing polyanionic glycosaminoglycans and the autometallographic technique for enhancing the copper signal of the cationic dye. The polyanions were located inside the epithelial cells in the junctional epithelium, whereas a network located along either the plasma membrane or the intercellular spaces, or both, of the gingival oral epithelium and sulcular oral epithelium was evident with autometallography. In these cases, positive staining was limited to the basal and spinous layers, the granular and keratinized layers being unstained. With the transmission electron microscope, electron-dense aggregates were seen in the gingival lamina propria, in the basement membrane and along the plasma membrane of the keratinocytes of the basal and spinous layers of the gingival and sulcular oral epithelia. In the junctional epithelium, Cuprolinic Blue-positive granules, 25 nm in diameter, were seen in the cytoplasm. Together with some vesicles containing electron-dense material, they may account for the staining process noted after autometallography. When the ultra-thin sections were digested with bovine testicular hyaluronidase, the staining was abolished. This indicates that glycosaminoglycans were primarily responsible for the staining pattern visualized with these methods. In the junctional epithelium, the cytosolic location of the 25 nm granules reflects either transcellular transfer between the plasma membrane and the nucleus or accumulation of glycosaminoglycans in this group of keratinocytes. The glycoconjugates located inside vesicles or vacuoles are related to endocytosis and lysosomal degradation. Interstitial glycosaminoglycans seen in the two types of oral epithelium may play a role in the diffusion of water and nutriments.     

Immunohistochemical localization of vascular endothelial growth factor in the globule leukocyte/mucosal mast cell of the rat respiratory and digestive tracts

 Vascular endothelial growth factor (VEGF) is a potent angiogenic mitogen that also increases vascular permeability. Immunohistochemical localization of VEGF in the respiratory and digestive tracts of healthy adult rats was investigated at light and electron microscopic levels using a specific antibody. The results revealed solitary cells with strong VEGF immunoreactivity scattered in the epithelium of the respiratory tract as well as in the lamina propria and epithelium of the intestine. From ultrastructural features of their large cytoplasmic granules, VEGF-positive cells in the respiratory tract were identified as globule leukocytes (GL). The immunoreactivity was localized exclusively in the cytoplasmic granules of GL. Most of the VEGF-positive cells in the small intestine were located in the lamina propria, whereas those in the large intestine were found more frequently in the epithelium than in the lamina propria. They showed the same morphological features as respiratory tract GL and were identified as mucosal mast cells (MMC). When examined in serial sections, GL/MMC in the respiratory and digestive tracts showed only weak reactivity to anti-histamine antibody. In contrast, connective tissue mast cells (CTMC), which were located in the submucosa of the digestive tract and in the connective tissues of the respiratory tract and other organs, were intensely immunopositive for histamine, whereas they showed no reactivity to anti-VEGF antibody. The specific occurrence of VEGF in GL/MMC suggests that this cell type is involved in paracrine regulation of the permeability of nearby microvessels, and that VEGF immunoreactivity can be used as a histochemical marker to distinguish GL/MMC from CTMC. Accepted: 28 July 1998     

Histophysiology of polysaccharide and lipid reserves in various tissues of Littorina littorea exposed to sublethal concentrations of cadmium

1. Sublethal exposure to cadmium causes glycogen depletion in connective tissues of the mantle, kidney folds, and digestive gland-gonad complex. Glycogen levels are lower at higher environmental concentrations of metal and at longer exposure times.2. Simultaneously with glycogen level reduction in reserve tissues, higher levels of glycogen than in control specimens have been detected in the digestive gland of cadmium exposed winkles. Phosphoglucomutase activity has been detected in kidney, connective tissues, and intestine, but not in digestive tubules. This suggests glycogen mobilisation through digestive tubule epithelia.3. Phosphoglucomutase activity in gills is associated with glycogen level increases in blood vessels and in distal portion of gill lamellae after proximal epithelium disruption.4. Lipid contents of the studied organs are only decreased when glycogen levels are largely reduced. Lipase activity has been demonstrated in digestive tubule, kidney and gill epithelia, but not in connective tissues. It is concluded that lipidic store is intracellular while the polysaccharidic one is organismic.5. Sublethal concentrations of cadmium do not cause impairment of phosphoglucomutase and lipase activities: enzymatic activity is well correlated with reserve consumption, demonstrable activity being lost only after substrate (glycogen or lipid) depletion.     

Fine structure of the midgut of Sinopanorpa tincta (Navás) (Mecoptera: Panorpidae)

Fine structure of the midgut and degeneration of the midgut epithelium of the scorpionfly Sinopanorpa tincta (Navás) adults were investigated using light microscopy and scanning and transmission electron microscopy. The results show that the tubular midgut lacks gastric caeca and is composed of an outer longitudinal and an inner circular muscle layer, a basal lamina, an epithelium and a lumen from the outside to inside. A peritrophic membrane was not found in the lumen. A mass of nodules was observed on the surface of the basal lamina. Three types of cells were recognized in the epithelium: digestive, secretory, and regenerative cells. The digestive cells contain irregular-shaped infoldings in the basal membrane and two types of microvilli in the apical membrane. The secretory cells are characterized by irregular shape and large quantities of secretory granules in the basal cytoplasm. The regenerative cells are triangular in shape and distributed only in the nodules. The epithelial cells are degenerated through programmed cell-death mechanisms (apoptosis and necrosis). The type, function, and degeneration of the epithelial cells of the midgut are briefly discussed.     

Ultrastructure of midgut endocrine cells in the adult mosquito, Aedes aegypti

The ultrastructure of endocrine cells in the midgut of the adult mosquito, Aedes aegypti, resembled that of endocrine cells in the vertebrate gastro-intestinal tract. Midgut endocrine cells, positioned basally in the epithelium as single cells, were cone-shaped and smaller than the columnar digestive cells. The most distinctive characteristic of endocrine cells was numerous round secretory granules along the lateral and basal plasma membranes where contents of the granules were released by exocytosis. Secretory granules in each individual cell were exclusively of one type, either solid or 'haloed', and for all cells observed, the range in granule diameter was 60-120 nm. The cytoplasm varied in density from clear to dark. Lamellar bodies were prominent in the apical and lateral cellular regions and did not exhibit acid phosphatase activity. The basal plasma membrane was smooth adjacent to the basal lamina, whereas in digestive cells the membrane formed a labyrinth. Some endocrine cells reached the midgut lumen and were capped by microvilli; a system of vesicles and tubules extended from beneath the microvilli to the cell body. An estimated 500 endocrine cells were distributed in both the thoracic and abdominal regions of the adult midgut. In one midgut, we classified a sample of endocrine cells according to cytoplasmic density and granule type and size; endocrine cells with certain types of granules had specific distributions within the midgut.     

Histological and electron microscopical observations on the effects of different salinities and heavy metal Ions,on the gills of Jaera nordmanni (Rathke) (Crustacea,Isopoda)

Summary The effects of different salinities and concentrations of copper, mercury and cadmium ions on the gills of Jaera nordmanni are investigated by means of light and electron microscopy. After exposure to 10% and 50% sea water the gill epithelium cells show a marked uniformity in appearance, possessing characteristically large, sub-cuticular spaces which are prominent between microvilli. With exposure to the heavy metal ions a similar sequence of histological and ultrastructural changes occur in all the gill epithelial cells, culminating in cell breakdown. The ultrastructural changes include distended microvilli, dilated endoplasmic reticulum, dissociated ribosomes, diffuse (swollen) cytoplasm, swollen mitochondria and a basal membrane withdrawn from the basal lamina. An increase in the number of haemocytes is also commonly observed in the haemolymph spaces during heavy metal ion exposure. The significance of the morphological changes undergone by the gill epithelial cells after exposure to different salinities and heavy metal ion concentration, are discussed in relation to the physiological functioning of the gill.The author wishes to thank Dr. M.B. Jones and Mr. R.H. Moore for setting up the experiments and to Professor E. Naylor for providing laboratory facilities at the Department of Marine Biology, University of Liverpool, Port Erin, Isle of Man. This work was supported by a Ministry of Defence (Navy) Contract No. AT/2198/010/CDL.     

The fine structure of the female reproductive tract of adult Loa loa

Weber P. 1987. The fine structure of the female reproductive tract of adult Loa loa. International Journal for Parasitology17: 927–934. The wall of the female reproductive tract of Loa loa was studied by electron microscopy. The wall is composed of a monolayered epithelium covered by a basal lamina. The epithelium of the ovary has a moderately developed basal labyrinth, abundant organelles, and a few secretory granules. In the oviduct, the basal lamina intrudes septa-like into the epithelium. Abundant myofilaments are attached to it. Microvilli cover the luminal cell border. The seminal receptacle contains few muscle cells in its basal lamina. It shows a highly developed spongy zone at its luminal surface. The uterine epithelium contains glycogen deposits and lipid droplets. In its anterior parts it shows a highly developed basal labyrinth and an abundance of secretory granules. The vagina has several layers of muscle cells in the basal lamina. Its epithelium contains few organelles, a small number of secretory granules, and is devoid of storage deposits.     

DISTRIBUTION OF METALS IN THE DIGESTIVE GLAND -- GONAD COMPLEX OF THE MARINE GASTROPOD NUCELLA LAPILLUS

The amounts of phosphate, zinc, copper and calcium in gonadmaterial from Nucella lapillus (L.) was higher in males comparedwith females. Water insoluble extracts from the digestive glandwere found to contain granules high in concentrations of phosphate,magnesium, zinc, copper and calcium when compared with watersoluble extracts and both types of extract from gonad tissueof male and female dog whelks. Male N. lapillus contained thehighest amounts of zinc and copper in isolated digestive glandgranules. Intracellular granules containing metal were locatedhistochemically in cells of the digestive gland and visceralhaemocoelic spaces (Received 5 December 1978;     

The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and non-specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa , and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.
In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.
In the basiphil cells of M. edulis , most of the enzymes are localized in a few cytoplasmic granules; non-specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.
In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.     

Some functional and morphological adaptations in the gills of dipnoi

Summary The gill epithelium of the Neoceratodus forsteri contains ce0lls which produce generations of dark vesicles. These vesicles move in waves towards the surface of the cells, break the cellular membranes and pour their content onto the cellular surfaces. This surface coat consists of threads and granules. The breaking of cell membranes by the densely situated vesicles leads to formation of processes, which join frequently to form a surface net: conversely the cytoplasm of the epithelial cells forms large invaginations penetrated by fluid. The reduction of cytoplasm and the mechanical loss of the cytoplasmic processes leads finally to the destruction of the cell.A small number of granulated cells with long processes extending between other cells have also been observed. The border between the gill epithelium and the connective tissue is characterised by the presence of bundles of thin fibers penetrating the epithelium.The above findings are discussed in relation to the differentiation of the epithelial cells, the facilitation of respiration in poorly oxygenated waters, the protection of gill surfaces against abrasion and with regard to the survival of the lung fish in different conditions.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

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.     

A morphological study on gills of the brown shrimp, Penaeus aztecus

The gills of Penaeus aztecus were examined by light and electron microscopy. They are dendrobranchiate, consisting of a central axis with biserially arranged branches that subdivide into bifurcating filaments. A septum divides the lumina of these structures into afferent and efferent channels. Hemolymph from the sternal sinus flows through the afferent channels into the filaments where it is directed into the efferent channels and finally to the pericardial cavity. In addition to these channels, numerous blood vessels permeate the gill. The cuticle covering the gill overlies a thin epithelium which is separated from hemolymph by a basal lamina. The epithelium, which is active in cuticle secretion, has a series of pillar processes that form subcuticular lacunae. The apical membranes of epithelial cells become folded in shrimp exposed to hypo- and hyperosmotic salinities. Granular cells that contain elaborate Golgi apparati and several types of granules are present throughout the gill. Nephrocytes resembling glomerular podocytes line the efferent channels. A large nerve traverses the septum in the axis.     

The roles of haemocytes during degeneration and regeneration of crayfish muscle fibres

Summary Crayfish haemolymph contains three types of haemocytes with cytoplasmic granules: coagulocytes, granulocytes and amoebocytes. Muscle degeneration was induced by either a gross mechanical injury or a mild puncture injury of m. extensor carpopoditi. Granulocytes and amoebocytes were involved in the phagocytosis of disintegrating muscle fibres. Within three weeks after the gross injury the first myotubes were found. The formation of regenerated fibres started before the degenerating material was removed completely. Mild injury resulted in the formation of contraction clots, localized at the ends of a fibre and connected to a persistent external lamina in the form of an empty sheath. The external lamina sheaths were invaded by amoebocytes. They arranged themselves into a superficial layer similar to an epithelium, formed gap junctions and zonulae adherentes, and showed an increase in the number of cytoplasmic microtubules. These transformed haemocytes retained their ability to engulf material of the disintegrating fibre. In about three weeks the number of microtubules in the transformed haemocytes decreased, and newly formed contractile filaments appeared. Satellite cells are present along the normal crayfish muscle fibres. Following their activation in degenerated material, they might conceivably induce the transformation of haemocytes into myogenic cells.     

Effects of external osmolality,calcium and prolactin on growth and differentiation of the epidermal cells of the cichlid teleost Sarotherodon mossambicus

Summary Osmolality and concentrations of divalent cations calcium, and to a lesser extent magnesium of the water are the main environmental factors that determine development and degree of mucification of the skin epithelium of Sarotherodon mossambicus. Epithelial thickness and number of mucocytes in fish exposed to low (freshwater level) concentrations of calcium and magnesium are directly related to the height of the osmotic gradient between water and blood plasma. No such relationship is found in fish exposed to a high (seawater level) concentration of calcium in the water, irrespective of the height of the osmotic gradient.The results strongly indicate that the effects of osmolality and divalent cations are indirect, and mediated by prolactin, since administration of ovine or fish prolactin stimulates growth and multiplication of the cells of the basal layer of the epidermis, and promotes the differentiation of the mucocytes.     

Selective staining and disintegration of intestinal eosinophilic granule cells in Atlantic salmon after intraperitoneal injection of the zinc chelator dithizone

Following intraperitoneal injection of the zinc ion chelator dithizone into Atlantic salmon Salmo salar , staining and morphological changes in intestinal eosinophiic granule cells (EGC) were observed in both cryo-preserved and chemical-preserved tissue sections. In cryo sections rapid and selective vital staining of EGC was observed within 5 min of dithizone injection. Intensely stained red granules appeared as a result of the formation of a complex between dithizone and zinc ions. Stained EGC appeared on both sides of the stratum compactum as well as in the lamina propria . Stained EGC started to disintegrate 5 min after the injection of dithizone, and little of the zinc-dithizone complex was observed 15 h later. The disappearance of the stain coincided with an intense degranulation and disintegration of EGC. Fifteen hours after dithizone treatment regenerating cells were observed in Masson's trichrome stained sections. Four to five days after injection, a fully regenerated continuous EGC layer was observed. Simultaneously with the disintegration of EGC an increase in plasma lysozyme activity occurred. EGC resembles mammalian Paneth cells in their possession of lysozymecontaining granules and their staining by, and response to, dithizone. EGC may represent a central component of zinc metabolism in Atlantic salmon.