THE INFLUENCE OF DIETARY CALCIUM ON THE TISSUE DISTRIBUTION OF Cu, Zn, Mg & P AND HISTOLOGICAL CHANGES IN THE DIGESTIVE GLAND CELLS OF THE SNAIL ACHATINA FULICABOWDICHM

Dietary calcium had a varying effect on the concentration ofother metals located in the tissues and excreted in the faeces.Faecal phosphate, copper and zinc concentrations decreased withincreased dietary calcium but showed a peak of excretion 10–12weeks after the beginning of the experiment, while the totalamounts of the studied elements lost in the faeces increased.Phosphate values in the digestive gland of snails on calcium-supplementeddiets were lower than metal values found in control snails.Although the digestive gland magnesium concentrations were alwayslower and copper levels higher than the snails on the omnivorousdiet, the trend was for a decrease in tissue magnesium and copperwith increased calcium in the diet. In foot tissue, the trendwas towards a decrease in Mg with decreased dietary calcium.The zinc concentration in the digestive gland and foot of snailson the omnivorous diet was not different from the zinc levelsin snails on the three highest calcium-supplemented diets. Itis suggested that high levels of dietary calcium might act asan environmental stressor characterised by changes in the morphologyand histo-chemistry of the digestive tubules. (Received 14 August 1991; accepted 10 October 1991)     

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.     

Sublethal effects of experimental exposure to mercury in European flat oyster Ostrea edulis: Cell alterations and quantitative analysis of metal

Oysters display a diversity of uptake mechanisms for metallic elements and distribution in the target organs, namely gills and the digestive gland. Various tissues of the flat oyster, Ostrea edulis, were studied following experimental exposure to 0.025 m (5 g l) of mercury, for up to 34 days. All animals survived the treatment. Data indicate Hg accumulation in gill tissue with a maximum concentration of 38.76 g g dry weight after 25 days of exposure. Hg levels were lower in remaining tissues, in which the maximum concentration (18.47 mg g-1 dry weight) was reached after 18 days of exposure. After these times, concentration in both tissues decreased. Results show that oysters can accumulate Hg from the environment, without their survival being affected during the experimental period. Structural alteration of epithelial tissues of gill and digestive gland of flat oyster was comparable with effects described for other metallic elements in bivalve molluscs. Interstitial tissue was disorganized in the digestive gland, and ultrastructural changes in intracellular endomembranes were detected in epithelial cells of the digestive gland after 18 days of treatment. After 25 days, absorptive epithelial cells of gills showed highly dilated, swollen microvilli. These intracellular alterations are parameters of the incipient response to the accumulation of mercury.     

The fine structural localization of acid phosphatase in the gut epithelial cells of the slug,Avion ater (L.)

Summary Acid phosphatase, generally thought of as a lysosomal enzyme and indeed widely employed as a lysosomal marker, has been found associated with the Golgi complex of all cell types from the crop, intestine and digestive gland ofArion ater. Reaction product was also detected within the multivesicular bodies and cytoplasm of columnar cells from the crop and the multivesicular bodies of mucous cells from the intestine. A vacuolar localization was obtained in the digestive cells of the intestine and digestive gland. Secretory protein granules in the calcium cells of the same gland and apical vacuoles in the so-called thin cells also showed a positive reaction.This work was undertaken as part of a slug research project under the direction and co-ordination of Dr. D. K.Roach, supported by A.R.C. Assistance was given by Mr. T. R.Mainwaring in the preparation of tissue for electron microscopy.I would like to thank Professor J.Brough and Professor D.Bellamy for providing facilities and encouragement.     

氟西汀与三氯生复合暴露对麦穗鱼的毒性效应

为探讨药品和个人护理产品(PPCPs)复合污染对水生生态系统的影响,本试验以东北地区土著鱼类麦穗鱼为试验材料,研究了氟西汀(FLX)与三氯生(TCS)复合暴露对其不同器官的毒性效应.经急性(4 h)与慢性(42 d)复合暴露试验后分别检测麦穗鱼Ⅰ相和Ⅱ相解毒酶、神经系统、消化系统及抗氧化系统等受到的影响.结果表明: 在FLX/TCS复合暴露条件下,麦穗鱼脑部乙酰胆碱酯酶活性受到短暂抑制,肝中细胞色素P450活性持续受到抑制,肠中α-葡萄糖苷酶活性在急性暴露后受到诱导但是长期暴露后被抑制,同时长期复合暴露导致肝中脂质过氧化水平升高.氟西汀和三氯生对麦穗鱼的复合暴露可对麦穗鱼多个器官产生急性毒性应激效应,而随着暴露时间的延长,麦穗鱼可产生一定的适应性,但这种适应作用的机制有待进一步研究.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Heat tolerance in Mediterranean land snails: histopathology after exposure to different temperature regimes

The physiological and biochemical adaptation mechanisms of heat-tolerantsnail species are poorly known. We investigated cellular adaptationsto heat stress in four heat-exposed snail populations (two ofXeropicta derbentina, one each of Cernuella virgata and Thebapisana). Snails were exposed to different temperature regimes(25, 33, 38, 40, 43, 45, 48, 50 and 52°C) in the laboratoryfor 8 h. After exposure, immobility rates were determined anda qualitative and semi-quantitative histopathological analysisof the digestive gland (calcium and digestive cells) and mucocytesin the foot was conducted. In addition, the coloration of theshells was densitometrically determined. The shells of T. pisanawere significantly darker than the shells of the other populations.The immobility rates and histopathological examinations showedthat X. derbentina (both populations) was slightly better adaptedto heat than C. virgata, and much better adapted than T. pisana.The observed differences in heat tolerance seemed to be connectedto the ability for rapid and extreme proliferation of calciumcells in the digestive gland. (Received 26 November 2007; accepted 25 July 2008)     

The localization of aluminium in the digestive gland of the terrestrial snail Helix aspersa

The terrestrial snail Helix aspersa was exposed to food containing elevated levels of aluminium for up to 33 days and the digestive gland examined by light and electron microscopy and X-ray microanalysis. Four types of cell are found in the digestive gland, (digestive, excretory, calcium and thin) although aluminium was only found in the excretory cells. The aluminium was localised in the 'yellow' or excretory granules that are a characteristic feature of the excretory cells. Aluminium was only found in the granules of snails fed aluminium but there was no difference in the appearance of granules from control or aluminium-fed snails. The granules were large (up to 20 mum in diameter), irregularly shaped and electron-dense. Sulphur, phosphorus and calcium were detected in granules from all snails. The presence of sulphur may indicate protein residues. The amount of aluminium and phosphorus in the granules increased over the experimental period but the number of granules did not change. Levels of aluminium in the granules decreased when the snails were given control food. The role of the excretory granules in the localisation, detoxification and excretion of aluminium is discussed.     

Effect of phytate-removal and deamidation of soybean proteins on calcium absorption in the in situ rats

Soybean proteins were deamidated by cation-exchange resins after phytate, the inhibitor for calcium absorption from the small intestine, was removed in order to provide the enhancement function of calcium absorption to soybean proteins. About 92% of the phosphorus was removed from the soybean proteins by anion-exchange-resin treatment, indicating that most of the phytate was removed. About 70% of the acid amide was deamidated by cation-exchange-resin treatment, and phytate-removed and deamidated soybean proteins (PrDS) having high calcium binding properties were obtained. PrDS were hydrolyzed by digestive enzymes and their calcium-binding properties and the enhancement function of the calcium absorption from the small intestine of rats were examined. As a result, PrDS retained their high calcium binding properties even after hydrolysis by digestive enzymes. In situ experiments showed that PrDS and their hydrolysates enhanced the calcium absorption from the intestine.     

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;     

Localization and fate of aluminium in the digestive gland of the freshwater snail Lymnaea stagnalis

The digestive gland of the freshwater snail Lymnaea stagnalis, exposed to water containing an elevated concentration of aluminium at neutral pH for up to 30 days, followed by a 20 day recovery period, was examined by light and electron microscopy and X-ray microanalysis. Aluminium was localized in the yellow granules present in the digestive and excretory cells and in the green and small granules present in the digestive cells. More aluminium, silicon, phosphorus and sulphur were present in all three granule types from aluminium exposed snails. The number of yellow and green granules from the digestive gland of aluminium exposed snails showed a progressive increase over the experimental period compared to controls. The number and aluminium content of the granules is likely to reflect the role of the digestive gland as a 'sink' for accumulated aluminium. We propose that intracellular monomeric silica is involved in the detoxification of aqueous aluminium which at neutral pH is largely in the form of an insoluble polyhydroxide. The increased amounts of sulphur and phosphorus in the granules are likely to be part of a broad response to metal loading but probably do not play a significant role in the storage and detoxification of aluminium.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

东方铃蟾消化道组织学的初步研究

采用组织学方法对东方铃蟾的消化道进行了研究。结果表明:肠分为十二指肠、空肠和大肠。消化道管壁由粘膜层、粘膜下层、肌层和浆膜层构成。食道、胃和肠均为单层柱状上皮。胃和十二指肠的粘膜皱褶最丰富。食道腺为复泡状腺,胃腺属于单管状腺,肠的各段无多细胞腺体,但空肠和大肠有丰富的杯状细胞。肌层均为平滑肌,内层环肌较厚,外侧纵肌较薄,其中大肠的外侧纵肌最发达。     

Adrenal catecholamines and related changes during different phases of morphine administration--a histochemical study

(1) Acute morphine treatment of rats reduced the total catecholamine content as well as the noradrenaline content of the adrenal gland. This was accompanied by increased ATPase activity and increased calcium content of the medullary cells. (2) After chronic morphine treatment, the total catecholamine content remained more or less unaltered, but the noradrenaline content was markedly increased together with the noradrenaline-containing areas, in comparison with what was observed in control animals. The ATPase activity of the medullary cells was found to be diminished along with the decreased calcium content. (3) In nalorphine-induced abstinence, a decrease in total catecholamine content together with a reduction of noradrenaline content and of noradrenaline-containing areas were observed. ATPase activity was also found to be increased with increased calcium content.     

Behavior of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration.

We monitored the differential reduction rates and elimination patterns of Escherichia coli and male-specific (F+) bacteriophage during UV depuration for 48 h in oysters (Crassostrea gigas) and mussels (Mytilus edulis) contaminated by short-term (1 to 3 weeks) and long-term (more than 6 months) exposure to sewage in the marine environment. The time taken to reduce levels of E. coli by 90% was 6.5 h or less in all cases. In contrast, the amounts of time needed to reduce levels of F+ bacteriophage by 90% were considerably longer: 47.3 and 41.3 h (after short- and long-term exposures, respectively) in mussels and 54.6 and 60.8 h (after short- and long-term exposures, respectively) in oysters. No differences in the rates of reduction of indicators of viral pollution following exposure of the shellfish to either short- or long-term sewage contamination were observed. Further experiments were conducted with mussels to determine the relative distributions of E. coli and F+ bacteriophage in tissue before and during depuration. Prior to depuration the majority of E. coli organisms (90.1%) and F+ bacteriophage (87.3%) were detected in the digestive tract (i.e., the digestive gland and intestine). E. coli and F+ bacteriophage were reduced in all tissues except the digestive gland to undetectable levels following depuration for 48 h. Within the digestive gland, levels of F+ bacteriophage were reduced to 30% of initial levels, whereas E. coli was reduced to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute and acclimated digestive responses of the cockle Cerastoderma edule (L.) to changes in the food quality and quantity. II. Enzymatic, cellular and tissular responses of the digestive gland

Cockles Cerastoderma edule were fed two different concentrations of two diets with different qualities which were achieved by mixing different proportions of ashed silt particles with cells of the microalgae Tetraselmis suecica. After 3 days (acute response) and 11 days (acclimated response) of exposure to the diets, we analysed the digestive activity of the digestive gland using cyto-histological and enzymatic techniques. We measured (i) the volumetric fraction of digestive and basophilic cells in digestive tubules, (ii) the diverticular radius and the thickness of digestive epithelia, (iii) the stereological parameters characterizing the lysosomal system and, (iv) dry weight, soluble protein content and specific and total amylase, cellulase, laminarinase, and protease activities of the digestive gland. In the conditions of the present study, specific cellulase and laminarinase activities in the digestive gland of cockles were correlated with the volumetric fraction of basophilic cells (r=0.672 and 0.642, respectively), whereas the specific protease was highly correlated (r=0.866) with lysosomal volume density. The implications of these correlations are discussed in relation to the feeding and absorptive parameters reported in the preceding publication. In the acute response, adjustments of the synthesis of constituents of the lysosomal/proteolytic system of the digestive cells seemed to be the only mechanism operating at the digestive level to respond to the changes in food availability. Lysosomal volume density increased with rising ingestion rate of organic matter, however, the occurrence of a limit in this short-term tissular response would account for the recorded trade-off between absorption efficiency and ingestion rate with different food qualities. With regard to acclimation, food quality determined the nature of the response of the digestive gland. With low quality diets, a time-dependent capability of the digestive gland for intensifying lysosomal/proteolytic production explains the increase of food absorption rates that result from higher filtration and ingestion rates. With high quality food, digestive acclimation differed with food particle concentration: with low rations, in spite of constant morphometrical and stereological parameters, the significant changes in the absorptive balance of biochemical components suggests the existence of an increased production of lysosomes that promotes an accelerated turn-over rate of the digestive epithelia. With high food concentrations, this response was coupled with increased activities of cellulase and laminarinase enzymes, probably as a consequence of higher rates of enzyme secretions from basophilic cells.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.     

Comparative histopathological alterations in the digestive gland of marine bivalves exposed to Cu and Cd.

This study compares the histopathological alterations in the digestive gland cells of mussels, Mytilus galloprovincialis and clams, Ruditapes phillipinarum following exposure to copper and cadmium. The results show degenerative processes undergone in the digestive gland ranging from inflammatory responses to extreme vacuolation, particularly in Cd-exposed individuals. Unsaturated neutral lipids tend to accumulate in pathologically enlarged lysosomes of the homogeneous-type or heterogeneous-type depending of the species and of metal. Lipofucsins containing granules were mainly found in Cu-exposed mussels and Cd-exposed clams. No granules were detected in Cd-mussels. The comparison of the methods indicate that paraffin sections are also a suitable material for the localization of lipofucsins.     

Localization of Metals in the Gastropod Littorina saxatilis (Prosobranchia: Littorinoidea) from a Polluted Site

Organ heavy metal levels, and ultrastructural localization were examined in the marine prosobranch mollusc Littorina saxatilis from a metal-contaminated site. Copper and zinc are localized in the digestive gland, stomach and kidney. The digestive gland does not appear to be a significant site of iron accumulation. High levels of copper and zinc in the stomach may indicate significant uptake from the food. Relatively high heavy metal levels in several organs were reflected in the presence of a variety of metal-containing aggregates (granules) within the tissues. In common with other invertebrates two broad classes of granule were present. The first appears to be proteinaceous, with a high sulphur content. This type contained copper and iron and was found in the stomach, kidney, pore cells, and rectum. The second type was inorganic and mineralized, containing phosphorus. They contained calcium, zinc and manganese, and were observed in the kidney and digestive gland. Differences in the number of metals were observed between the various granule types, possibly reflecting specific pathways involved in metal sequestration.