Autometallographic localization of protein-bound copper and zinc in the common winkle,Littorina littorea: A light microscopical study

Summary Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrare for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and dithizone procedures exhibited poor sensitivity: there was no positive reaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in sperm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.     

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.     

Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

Gill‐derived glands in species of Astyanax (Teleostei: Characidae)

The presence of a gill‐derived gland is herein reported for the first time in males of species of Astyanax and related genera; they are described through histological cuts and SEM. The gill‐derived glands described for the Characidae, when fully developed, present a similar structure in different species. The main external feature of gill‐derived glands is the fusion of anteriormost gill filaments on the ventral branch of first gill arch. This fusion is caused by squamous stratified epithelial tissue that covers adjacent filaments, forming a series of chambers. In the region where the gill‐derived gland develops, the secondary lamellae of the gill filaments are much reduced or completely atrophied being characterized by the presence of glandular cells forming nests.     

Ecotoxicological impacts of exposure to copper oxide nanoparticles on the gill of the Swan mussel,Anodonta cygnea (Linnaeus, 1758)

This study was conducted to investigate the ecotoxicological effects of exposure to copper oxide nanoparticles (CuO NPs) on the gill of the swan mussel Anodonta cygnea using several approaches including qualitative and quantitative histopathology, ultra-morphology (scanning electron microscopy [SEM]) and measures of clearance rate (CR) and bioaccumulation of CuO NPs. Histological alterations in mussels exposed to 0.25 (T1), 2.5 (T2) and 25.0?µg L^?1 (T3) CuO NPs for 12 days include changes in the length and form of gill lamellae, changes in inter-lamellar spaces, epithelial hyperplasia, atrophy and tissue rupture. Ultra-morphological changes following CuO NP exposure included epithelial hyperplasia and hypertrophy, epithelial lifting, tissue rupture (water channel fusion) and extensive necrosis of the gill surfaces. I_Gill (gill damage severity) index values for both histopathological and ultra-morphological data were significantly (P?0.05) higher in T3. The CR of mussels was significantly (P??1 g^?1 dry weight]) in comparison to controls (CR?=?108?±?47.14 [L min^?1 g^?1 dry weight]). CuO NPs accumulated in exposed mussels at all exposure concentrations until day 4, but there was no further change in accumulation levels by the end of the exposure period. The accumulated content of CuO NPs was significantly (P??1 exposure concentration. Based on these results, significant accumulation of CuO NPs in the gills of swan mussel could affect histological and ultra-structural characteristics of this organ and consequently have deleterious impacts on its filtration activity.     

Ultrastructure and development of the gills in<Emphasis Type="Italic"> Rana dalmatina</Emphasis> (Amphibia,Anura)

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Ortons larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

Cellular responses to increasing Cd concentrations in the freshwater crab,<Emphasis Type="Italic"> Potamonautes warreni</Emphasis>, harbouring microbial gill infestations

We investigated the uptake, transport, storage and defence mechanisms in the freshwater crab, Potamonautes warreni, harbouring microbial gill infestations and exposed to increasing chronic (0.2, 0.5, 1.0 mg l^–1) and acute (2.0 mg l^–1) cadmium (Cd) concentrations under controlled laboratory conditions over a period of 21 days. Transmission electron microscopy and X-ray microanalysis revealed that the microbial gill fauna was eliminated on exposure to 0.2 mg Cd²⁺ l^–1 and that Cd became increasingly adsorbed and incorporated into lamellar crystal deposits and permeated the cuticle of the gills of P. warreni. Degeneration of the apical membrane infoldings and vacuolation of epithelial cells occurred concurrently with pinocytosis, endocytosis and pronounced phagocytotic activity in the epithelia and haemal canal of the gills. Elevated Cd exposures (0.5 or 1.0 mg l^–1) resulted in the swelling and dissociation of mitochondrial outer membranes together with an increase in transport of Cu, Cl and S by haemocytes in the haemal canal to epithelial tissues depleted in these elements. Cd also accumulated in tightly coiled concentric membrane whorls in the haemal canal, whereas the highest concentrations of Cd were found within aggregates of lysosome-like bodies in cuticulin-secreting cells of the gill stem. Chronic exposure to Cd induced increased fatigue and mild uncoordinated motor activity. In contrast, at an acute exposure of 2.0 mg l^–1 over 48 h, P. warreni showed a time-specific rapid loss of motor function, although only mild cellular lesions occurred in the gill tissues. The significance of cellular changes in the gill epithelia and altered motor activity of P. warreni with increased waterborne Cd are discussed as potential biomarker responses in monitoring aquatic pollution.     

Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea

An infestation of young tilapia, Oreochromis mossambicus Peters, by the flagellate Cryptobia branchialiswas observed at the Salton Sea, California, in September, 1997. This is the first report of C. branchialis in a highly saline water-body (43 g l^–1). Ultrastructure of C. branchialis as well as its effect on the gills of tilapia were studied using the scanning and transmission electron microscopy. No direct effect of C. branchialis on the epithelial cells of fish gills was observed. However, alterations of gill general structure, such as deposition of copious mucus on the gill surface, swelling of filaments, reduction of respiratory lamellae and their transformation into short club-shaped structures were found in infected fish. This suggests mortality of young tilapia may arise from decreased gill function in response to Cryptobia infestation.     

斑马鱼鳃的光镜和透射电镜观察

应用光学显微镜和透射电镜对斑马鱼(Danio rerio)鳃的组织结构及鳃丝、鳃小片超微结构进行了观察。结果表明,斑马鱼有4对全鳃,鳃耙呈长锥状,鳃丝呈梳状排列在鳃弓上,鳃小片均匀排列在鳃丝两侧。鳃小片由上皮细胞、柱细胞、内皮细胞和毛细血管网组成,鳃小片基部和血管周围分布有泌氯细胞,胞内有丰富的线粒体和排泄小泡,根据线粒体形态特征和细胞质电子密度可将其分为两个亚型。黏液细胞通常与泌氯细胞对生存在,并且有通外的开口。斑马鱼鳃组织结构与其他硬骨鱼鳃结构相似,其结构和功能有密切的关系。     

Substance P-, neurotensin- and bombesin-like immunoreactivities in the gill epithelium of Ciona intestinalis L.

Summary Substance P-, neurotensin- and bombesin-like immunoreactivities were localised in some gill epithelial cells in the pharynx of Ciona intestinalis L. No immunoreactivity was obtained with antisera to gastrin, glucagon, insulin, pancreatic polypeptide or calcitonin. Some of the epithelial cells of the gills were shown to be argyrophilic with the Grimelius technique.     

Electron microscopical and histochemical studies of differentiation and function of the cephalopod gill (Sepia officinalis L.)

Summary Phase contrast and electron microscopical investigations on the gill ofSepia officinalis L., from the stage shortly before hatching to the adult stage, demonstrate (1) peripheral respiratory epithelial areas and (2) highly folded epithelia rich in mitochondria in the concave recesses of the gill lamellae. Enzyme-histochemical and cytochemical findings, and in particular the GOT proof, suggest by analogy with the chloride cells of the teleosts that the cells of this second type of epithelium are capable of active transport, and that they probably secrete ammonia. Apparently the gill of cephalopods, too, serves both respiratory and excretory functions.This study was supported by the Deutsche Forschungsgemeinschaft     

Sublethal Effect of Copper Toxicity Against Histopathological Changes in the Spiny Lobster, <Emphasis Type="Italic">Panulirus homarus</Emphasis> (Linnaeus, 1758)

The tissue damage induced by various organic pollutants in aquatic animals is well documented, but there is a dearth of information relating to the histological alterations induced by copper in the spiny lobster. In the present study, intermoult juveniles of the spiny lobster Panulirus homarus (average weight 150–200 g) were exposed to two sublethal concentrations of the copper (9.55 and 19.1 μg/l) for a period of 28 days. The muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of the lobsters were then dissected out and processed for light microscopic studies. Exposure to copper was found to result in several alterations in the histoarchitecture of the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of P. homarus. The alterations included disruption and congestion of muscle bundle in muscle tissue; blackened haemocytes; distended lumen and F cell; necrosis of the tubules of the hepatopancreas; disarrangement of circular muscle of the midgut; accumulation of haemocytes in the haemocoelic space; swelling and fusion of lamellae; abnormal gill tips; hyperplastic, necrotic, and blackened secondary gill lamellae of the gills; damaged neurosecretory cell and sensory and motor fibre; necrotic of the thoracic ganglion; dispersedly arranged muscle bands; clumped satellite cells and nucleus of the heart. The results obtained suggest that the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of lobsters exposed to copper were structurally altered. Such alterations could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, digestion of gut and respiration, osmotic and ionic regulations of the gills, which in turn could ultimately affect the survival and growth of P. homarus. Thus, all possible remedial measures should be adopted to prevent the occurrence of copper contamination in the aquatic environment.     

Morphological and functional characterization of a novel Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase-immunoreactive,follicle-like structure on the gill septum of Japanese banded houndshark, <Emphasis Type="Italic">Triakis scyllium</Emphasis>

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na⁺/K⁺-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na⁺/H⁺ exchanger 3 and Ca²⁺ transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.     

Primary cell culture from gill explants of rainbow trout

Primary cultures of gill cells were initiated from gill filament explants of rainbow trout, Oncorhynchus mykiss . The explants were cultured in Leibovitz l -15 medium with 5, 10 or 20% foetal calf serum (FCS) and l -glutamine. The attachment efficiency was serum-dependent though increased FCS concentration did not stimulate further outgrowth of cells. The explants produced cell outgrowth 24 h after attachment as a sheet of cells which exhibited characteristics of gill pavement epithelial cells as indicated by surface microridges revealed by scanning electron micrographs. There was high proliferation for the first 14 days then a stable plateau for 30 days followed by a decline phase from 45 days. Following removal of cells, the explants produced further cell outgrowth which was especially active at the proliferation phase (14 days). Removal of these cells caused the explants to produce a further proliferation of cells reaching confluence in 10–14 days. After the third cell removal cell outgrowth from explants showed migratory activity but did not develop to resemble gill epithelial cells. The use of gill explants to establish primary cultures of fish gill cells has advantages which include longevity of the culture and successive proliferations from explants which could provide a useful tool for the investigation of long-term processes in cellular biology and reduce the number of culture preparations.     

Radiation-induced cytogenetic instability in vivo.

Radiation-induced cytogenetic instability has been well documented in a number of laboratories, and we have hypothesized that such instability is the initiating event in the process leading to radiation-induced cancer. To date most studies of radiation-induced instability have used systems in which cells are rapidly dividing. For this phenomenon to have significance for radiation carcinogenesis, it must be established that instability can be induced in vivo in less rapidly dividing fully differentiated tissues known to be at risk. In the present study, we have examined the kinetics of radiation-induced cytogenetic instability in mammary epithelial cells after irradiation in vivo. Having established that instability could arise in vivo in intact mammary tissue, we subsequently demonstrated a dose-response relationship both in vitro and in vivo and demonstrated a lower frequency of instability after fractionated exposures.     

Gill tissue recovery after copper exposure in Carassius gibelio (Pisces: Cyprinidae)

We investigated the influence of copper in a long-time treatment with concentrations of 0.05 mg L^?1 and 0.1 mg L^?1 to track the histopathological changes in gills of Carassius gibelio, and to find at what extent they will recover after the effect of the copper concentrations stops. Treatment with copper lasted 21 days and the recovery time was of the same duration. The results of histological examination showed degenerative changes (resulting in thinner secondary lamellae and filamentary epithelium), and hyperplastic and hypertrophic changes (proliferation, vasodilatation, aneurysms, epithelial interstitial edema, and fusion) in gills under the influence of two concentrations. The degenerative changes have higher prevalence at low concentrations, while hyperplastic and hypertrophic ones — at high concentrations. After the period of recovery they remained the same, but the extent of expression on the surface of gill filaments changed. The long-time copper intoxication in low concentrations of copper affects gill structure, causing severe changes whose recovery is a slow process that requires a longer period of time.     

Ultrastructure and ion permeability of the two types of epithelial cell arranged alternately in the gill of the fresh water branchiopod Caenestheriella gifuensis (Crustacea)

 The adult freshwater branchiopod, Caenestheriella gifuensis, has, as respiratory organs, fifteen pairs of slender cone-shaped gills composed of a thick epithelium. The silver nitrate/nitric acid technique revealed that the gill epithelium consisted of two kinds of cell, types I and II, which were alternately arranged with irregular interdigitations to form a unique, daisy pattern. Only type I cells were darkly stained by this technique, indicating high permeability of these cells to chloride ions and appearing to be responsible for the ion transport and osmoregulation. Further, electron microscopy disclosed fine structural characteristics of the two distinct types of epithelial cell covered by an extremely thin and soft cuticle layer, suggesting high permeability to gases and ions. The type I epithelial cell was characterized by an abundance of mitochondria, well-developed infoldings of the basal cell membrane exceeding two-thirds of the epithelial thickness, (which produce a magnification of the basolateral surface area of the cell), sparse microvillous projections of the apical border, and complicated interdigitations with the other type of epithelial cell. In the type II epithelial cell, on the other hand, these characteristics were less developed. These results suggest that in addition to their respiratory function, type I epithelial cells are of the ion-transporting type and play an important role in the active absorption of electrolytes to maintain a constant osmotic pressure of the hemolymph in extremely salt-deficient, freshwater environments. The type II epithelial cells may function mostly as respiratory epithelial cells. Accepted: 20 November 1996     

Subcellular accumulation of Cu in the Antarctic bivalve<Emphasis Type="Italic"> Laternula elliptica</Emphasis> from a naturally Cu-elevated bay of King George Island

Subcellular Cu sequestration was examined in the digestive gland, kidney and gill of the Antarctic bivalve Laternula elliptica collected from a Cu-elevated bay in King George Island. Cu was associated with both the soluble cytosolic and insoluble particulate cell fractions in all three organs, but their relative contributions to Cu sequestration varied with tissue type and the total amount of Cu accumulated. Low-molecular-weight (10–13 kDa) metallothionein-like proteins were the major Cu-binding ligands in the cytosol of all three organs. Significant portions of the cytosolic Cu were also bound to proteins with different molecular weights in the kidney and gill. A strong immunological response to a metallothionein (MT) antibody confirmed the presence of MTs in all three organs. Numerous electron-dense granules, which are likely to be metal-rich, were observed in renal epithelial cells by transmission electron microscopy, suggesting that these granules also play a role in Cu sequestration.     

Evaluation of the genotoxicity of cadmium in gill cells of the clam <Emphasis Type="Italic">Corbicula japonica</Emphasis> using the comet assay

Cadmium-induced DNA degradation in gill cells of the clam Corbicula japonica was assessed using the comet assay (single-cell gel electrophoresis). Accumulation of highly toxic cadmium in the gill cells of bivalve is accompanied by damage to the cell genome, which is revealed as DNA migration in the comet assay. The main mechanisms of Cd effects on the integrity of the DNA structure are discussed.