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.     

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.     

Biomarkers of oxidative stress in Biomphalaria alexandrina snails for assessing the effects of certain inorganic fertilisers

Freshwater snails are often used to monitor aquatic pollution. The present study aims to use Biomphalaria alexandrina snails as bio-indicators for three types of NPK fertilisers (high nitrogen, high phosphorus and balanced). Data showed that high phosphorus and high nitrogen fertilisers have a negative impact on the growth rate of juvenile snails. Such observations were supported by determining antioxidant parameters in the digestive gland. Results indicated a significant increase in the catalase activity of snails that were exposed to ½ LC₅₀ concentrations of balanced and high phosphorus fertilisers. Meanwhile, the glutathione S-transferase, glutathione peroxidase and reduced glutathione parameters showed varying results representing an increase or decrease in the activities of all treated groups after both the first and second weeks, according to the type of fertiliser the snails were exposed to.     

Paxillus involutus — Pinus sylvestris Mycorrhizae from Heavily Polluted Forest.

The electron-opaque granules localized in vacuoles of Paxillus involutus hyphae associated with Pinus sylvestris mycorrhizae, collected from heavily polluted sites were analysed by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) connected with the electron microscope (TEM 902, Zeiss). On the basis of the elemental composition two kinds of granules were distinguished. The first, similar in appearance to polyphosphate granules, described already for several fungi, was characterized by high contents of phosphorus accompanied by sulphur, calcium and often aluminium. More common, however, was the second sort of granules containing more nitrogen, sulphur and cadmium, while the amount of phosphorus was much lower. The data reveal accumulation of cadmium inside fungal vacuoles and suggest the possibility of detoxification of heavy metal by the symbiotic fungus.     

The potential role of spherocrystals in the detoxification of essential trace metals following exposure to Cu and Zn in the fighting conch <Emphasis Type="Italic">Strombus</Emphasis> (<Emphasis Type="Italic">Lobatus</Emphasis>) <Emphasis Type="Italic">pugilis</Emphasis>

Crypt cells—one of the three cell types composing Strombidae digestive tubules—are characterized by the presence of numerous metal-containing phosphate granules termed spherocrystals. We explored the bioaccumulation and detoxification of metals in Strombidae by exposing wild fighting conch Strombus pugilis for 9 days to waterborne CuSO₄ and ZnSO₄. The total amount of Cu and Zn was determined in the digestive gland and in the rest of the body by Inductively Coupled Plasma (ICP) analyses. The digestive gland spherocrystal metal content was investigated based on the semi-quantitative energy dispersive X-ray (EDX) elemental analysis. ICP analyses of unexposed individuals revealed that 87.0?±?5.9% of the Zn is contained in the digestive gland, where its concentration is 36 times higher than in the rest of the body. Regarding Cu, 25.8?±?16.4% of the metal was located in the digestive gland of the control individuals, increasing to 61.5?±?16.4% in exposed individuals. Both Cu and Zn concentrations in the digestive gland increased after exposures, pointing to a potential role of this organ in the detoxification of these metals. EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn in unexposed individuals. No difference was found in the relative proportion of Zn in spherocrystals of exposed versus control individuals. Contrastingly, copper was never detected in the spherocrystals from controls and Zn-exposed individuals, but the relative proportion of Cu in spherocrystals of Cu-exposed individuals varied from 0.3 to 5.7%. Our results show the direct role of spherocrystals in Cu detoxification.     

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.     

Histochemical changes of carbohydrate and protein contents in the digestive gland cells of the land snail Monacha cartusiana following starvation

The present study was designed to investigate histochemically the detection of carbohydrate and protein in the normally feeding snails and after 15 and 30 days of starvation. Generally, abundant carbohydrate and protein materials were detected in the component cells of the digestive gland of normally feeding snails. The results of this investigation revealed a pronounced decline of carbohydrates in the digestive gland cells of Monacha cartusiana snails after starvation. Severe decline in carbohydrate content was observed especially after 30 days of starvation. Moreover, protein inclusions have exhibited a week stainability in the digestive gland cells of these snails as a consequence of starvation.     

The cytology,histochemistry, and ultrastructure of the cell types found in the digestive gland of the slug,Agriolimax reticulatus (Müller)

Summary Four cell types have been identified in the digestive glands from light and electron microscope studies. The possible functions of each cell type are discussed.Thin cells are undifferentiated. Calcium cells contain spherules of calcium salts which have a characteristic ultrastructure. Different protein granules are found apically. Digestive cells are present as two distinct forms. One form is believed to be absorbing food material and digesting it intracellularly, and the other form is a secreting cell. Both forms contain green and yellow granules and histochemistry shows these granules to be distinct. Protein granules also occur apically.Excretory cells are distinguished by having a large central vacuole containing excretory granules. Histochemistry shows these granules, like the yellow granules of digestive cells, to be composed mainly of lipofuscin.It is suggested that digestive cells form excretory cells.     

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.     

Ultrastructural localization of esterase and acid phosphatase in digestive gland cells of fed and starvedCepaea nemoralis (L.) (Mollusca: Helicidae)

Summary The ultrastructural localizations of thiolacetic acid esterase, indoxyl acetate esterase and acid -glycerophosphatase have been studied in the digestive gland cells of fed and starvedCepaea nemoralis. In fed snails the major localization of all three enzymes was in the green granule vacuoles of digestive cells. In addition, the cytoplasm of calcium cells and the Golgi apparatus and GERL (?) of all cell types were acid phosphatase positive. Many digestive cells of starved snails showed a similar enzyme distribution to that found in fed snails but other digestive cells showed a very high cytoplasmic activity of all three enzymes. It is suggested that these cells are in the process of autolysis. New light is also thrown on the process by which food is transported from the digestive gland lumen to the phagosomes of digestive cells.     

Physiology and lipid metabolism of Littorina saxatilis infected with trematodes

Physiological and biochemical alterations in Littorina saxatilis infected with larval trematodes were investigated and compared with the metabolism of non-parasitized snails. Oxygen consumption rates of infected snails differed from those of non-infected controls in medium sized individuals (30 to 130 mg) but not in very large infected individuals (> 200 mg). Small snails (0.5 to 8.5 mg) were seldom infected by parasites, and this size-class consisted only of non-infected specimens. The specific oxygen consumption rate of infected snails was not dependent on their mass and remained constant over the size ranges investigated. Alterations in the snail metabolism appeared to be connected to injuries to digestive gland tissues caused by the parasites. The glycogen concentration and fatty acids of neutral lipids and phospholipids in the digestive gland were determined. Infected snails differed from uninfected snails in the complete absence of glycogen in digestive gland and had proportionally higher quantities of eicosenoic (20:1) acid in the total phospholipids. It remains unclear whether infection by trematodes activates enzymes in the snail's digestive gland to synthesize eicosenoic (20:1) acid, or whether the sporocysts themselves possess these enzymes. The role of phospholipid fatty acids in the regulation and maintenance of the parasite's metabolism is briefly considered. Biochemical alterations observed in the fatty acid composition may have an adaptive significance, by helping to stabilize the host-parasite system.     

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu²⁺, Cd²⁺ or Zn²⁺ (40 μg/l/animal) were investigated.2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed.3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules.4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects.5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.     

ULTRASTRUCTURAL LOCALIZATION OF COMPLEX CARBOHYDRATES AND PHOSPHATASES IN DIGESTIVE GLAND CELLS OF FEEDING AND HIBERNATING SNAILS, HELIX LUCORUM (GASTROPODA: HELICIDAE)

The digestive gland of normally-fed snails Helix lucorum, aswell as that of snails which had hibernated for 4 months wereexamined by the use of cytochemical techniques for detectionof acid and alkaline phos-phatase, as well as of periodate-reactive(PA-TCH-SP technique), sulfated (HID-TCH-SP technique) and carboxylatedcarbohydrates (LID-TCH-SP technique). The cytochemical resultssupport the hypothesis of intracellular digestion via lysosomalactivity of material taken up by endocytotic processes by thecells of the digestive gland. Four months hibernation did notaffect the intracellular distribution of polysac-charides andphosphatases in the cells of the digestive gland of H. lucorumcompared to that in the control snails. In addition, hibernationaffected the percentage of the calcium cells which significantlyincreased compared to the non-hibernating snails, whiie thepercentages of the digestive and excretory cells remained almoststable. However, the periodate-reactive sulfated and carboxylatedpolysaccharides of the digestive gland cells decreased in thehibernated snails compared to the controls. The results suggestthat the cytochemistry of periodate-reactive, sulfated and carboxylatedpolysaccharides used in the present study could, also, be appliedto the study of lysosomal activities. (Received 1 October 1991; accepted 4 December 1991)     

Green cocoons in silkworm Bombyx mori resulting from the quercetin 5-O-glucosyltransferase of UGT86, is an evolved response to dietary toxins

The glycosylation of UDP-glucosyltransferases (UGTs) is of great importance in the control and elimination of both endogenous and exogenous toxins. Bm-UGT10286 (UGT86) is the sole provider of UGT activity against the 5-O position of quercetin and directly influences the formation of green pigment in the Bombyx cocoon. To evaluate whether cocoon coloration evolved for mimetic purposes, we concentrated on the expression pattern of Ugt86 and the activities of the enzyme substrates. The expression of Ugt86 was not only detected in the cocoon absorbing and accumulating tissues such as the digestive tube and silk glands, but also in quantity in the detoxification tissues of the malpighian tubes and fat body, as well as in the gonads. As in the green cocoon strains, Ugt86 was clearly expressed in the yellow and white cocoon strains. In vitro, the fusion protein of UGT86 showed quercetin metabolic activity. Nevertheless, Ugt86 expression of 5th instar larvae was not up-regulated in the silk gland by exogenous quercetin. However, it was significantly up-regulated in the digestive tube and gonads (P < 0.05). A similar result was observed in experiments where larvae were exposed to rutin, an insect resistance inducer and growth inhibitor typically found in plants, and to 20-hydroxylecdysone (20E), an insect endocrine and plant source hormone. On the contrary, up-regulated Ugt86 expression was almost nil in larvae exposed to juvenile hormone III (P > 0.05). The results of HPLC revealed that a new substance was formed by mixing 20E with the recombinant UGT86 protein in vitro, indicating that the effect of Ugt86 on 20E was similar to that on exogenous quercetin derived from plant food, and that the effect probably initiated the detoxification reaction against rutin. The conclusion is that the reaction of Ugt86 on the silkworm cocoon pigment quercetin is not the result of active mimetic ecogenesis, but derives from the detoxification of UGTs.     

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.     

幼龄皱纹盘鲍唾液腺和消化腺的超微结构与组织化学

以透射电镜观察和组织化学方法研究了45日龄皱纹盘鲍的唾液腺和消化腺。唾液腺由粘液细胞和纤毛细胞组成,粘液细胞含发达的粗面内质网和大量的粘原颗粒,分泌中性和酸性混合粘多糖。消化腺由消化细胞和嗜碱性细胞组成,消化细胞呈现活跃的内吞和细胞内消化,并具蛋白酶和非特异性酯酶活性。嗜碱性细胞含发达的粗面内质网和大量含铁的折光小体,折光小体的电子密度较低。     

Schistosoma mansoni: effect of infection on reproduction and gonadal growth in Biomphalaria glabrata

Sexually mature Biomphalaria glabrata were exposed to 12 miracidia of Schistosoma mansoni, and egg production of snails was monitored over a period of 5 weeks. During the study period, exposed snails grew at approximately the same rate as unexposed controls. Castration, as measured by a reduction in the mean number of eggs laid per snail occurred between 14 and 21 days postexposure (PE). The reduction in fecundity in infected snails coincided with the migration and establishment of daughter sporocysts in the digestive gland and gonad. Enumeration of individual oocytes in longitudinal sections of the ovotestis revealed that uninfected snails contained significantly more oocytes per section than infected snails at 27, 31, and 40 days PE. In addition, the mean area of gonadal sections of control snails increased over the 40-day experimental period, whereas there was no such increase in gonadal area of infected snails. These data suggest that there is an inhibition in gonadal growth in infected snails. When oocyte data were expressed in terms of mean gonadal area, the mean number of oocytes per mm2 of gonad of uninfected and infected snails did not differ significantly over the study period, except at Day 14 PE, when infected snails contained a significantly greater number of oocytes per mm2 of gonad than did uninfected controls. It is hypothesized that daughter sporocysts of S. mansoni are primarily responsible for the inhibition of host reproductive activity, and may be mediating their effects through mechanisms involved in the regulation of gonadal growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR studies on adenylates and other phosphorus metabolites in the schistosome vector Biomphalaria glabrata

31P nuclear magnetic resonance spectroscopy was applied successfully to whole intact and de-shelled snails and to the isolated digestive gland-gonad tissue complex as well as other tissues of Biomphalaria glabrata. Several phosphorus metabolites, including ATP and ADP, were observed. The mean ATP/ADP ratio calculated for the tissue complex was 3.1 and the ATP concentration was 0.73 nmoles/mg tissue fresh weight. Assignments for AMP, sugar phosphates, and inorganic phosphate peaks were tentatively made. A major phosphorus component was identified as a phosphonate and this metabolite was also present in egg masses and the albumin gland. Phosphoarginine was not observed in the tissue complex but was present in whole animals. Infection by Schistosoma mansoni resulted in marked alteration in the relative levels of phosphorus metabolites in the digestive gland-gonad complex during the course of infection. The decrease in phosphonate was particularly notable. The relative level of a metabolite occurring at -1.1 ppm was also decreased but its identity remained unknown. The ATP/ADP ratio was not affected by infection, but an increase in the relative level of inorganic phosphate suggested a possible decrease in phosphorylation potential.     

Parasite development and visceral pathology in Galba truncatula co-infected with Fasciola hepatica and Paramphistomum daubneyi

Histological investigations in Galba truncatula naturally or experimentally co-infected with Fasciola hepatica and Paramphistomum daubneyi were carried out to study parasite development and the responses of the digestive gland and kidney of snails, as larval forms of these digeneans often use these two sites for their growth within the snail's body. The number of live rediae per snail ranged from 2.4 to 4.2 for the dominating parasite (it developed in the digestive gland) and was less than 2.0 for the other species. When the dominating species was F. hepatica, most snails harboured cercariae-containing rediae; if this parasite was P. daubneyi, procercariae-containing rediae with or without free procercariae were observed in most snails. In contrast, most rediae of the other species were immature. The pathology caused by the dominating species in the digestive gland was greater than that recorded in the kidney, where the other parasite was generally located. The most frequent tissue lesions in the digestive gland were generalized epithelial necrosis and epithelial reconstitution. In the kidney, multifocal epithelial necrosis was frequently observed, particularly when P. daubneyi was the dominating species. The frequencies of lesions in the digestive gland agreed with percentages reported by our team in other snails mono-infected with F. hepatica or P. daubneyi. In contrast, multifocal necrosis in the kidney was clearly greater in the present study and this finding might be explained by assuming that a sufficient number of free larvae within the snail would be necessary for the development of epithelial necrosis in the whole kidney.