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)     

HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803)

The crypt cells lining the Aplysia punctata digestive tubulescomprise of three types of cell; calcium, excretory, and thincells. The calcium cells play a role in osmoregulation, mineral storage,exocrine secretion, iron detoxification, and excretion processes.They possess well- developed microvilli and a basal labyrinth,suggesting a role in absorption. The Golgi apparatus is involvedin the production of two main components of calcium spherules;the fibrillar material and mineralized granules. Golgi complex,rough endoplasmic reticulum (RER), ribosomes, and altered mitochondriaare involved in the formation of calcium spherules. Secretoryactivity is indicated by the formation of dense granules containingiron and calcium salts. Lipofuscin pigment has been found inlarge concretions which may arise from cytoplasmic areas surrounded byendoplasmic reticulum, RER and Golgi tubules. There are threestages of excretory cells, called early, mature, and post-excretorycells. This study traces the development of granulofibrillarvacuoles up to the formation of the lipofuscin concretions andshows that excretory cells are in fact degenerating calciumcells. The fine structure of thin cells suggests that they areyoung calcium cells. (Received 29 December 1997; accepted 15 November 1998)     

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.     

THE FINE STRUCTURE AND FUNCTION OF THE ANAL GLAND OF THE MURICID NUCELLA LAPILLUS (NEOGASTROPODA) (AND A COMPARISON WITH THAT OF THE TROCHID GIBBULA CINERARIA)

The fine structure of the anal gland of Nucella lapillus isdescribed, and compared with that of Gibbula cineraria. It isconcluded that the glands are not analogous in function andare unlikely to be homologous. The anal gland of Gibbula isa small rectal diverticu-lum near the anal papilla, lined bymucus-secreting cells. The evidence suggests that the largeanal gland of Nucella extracts macromolecules and cations fromthe blood, metabolizes or sequesters them in lyso-somes andultimately expels the resultant residual bodies by apocrinesecretion. It has large reserves of lipid and glycogen, andthe abundance of melanin indicates that it is a major site oftyrosine degradation. The invariable presence of bacteria inthe lumen, and of pits in the epithelium to house them, impliesa symbiotic relationship in which the bacteria metabolize anddegrade the cell debris, some of which is resorbed. The lossof the anal gland in bucci-nids and nassariids is accompaniedby great reduction in size and importance of the rectal sinus,and increase in complexity of the kidney. (Received 31 December 1991; accepted 23 January 1992)     

INFLUENCE OF THE CLAY TYPES ILLITE AND MONTMORILLONITE ON THE UPTAKE OF65 Zn BY SCENEDESMUS OBLIQUUS.

SUMMARY

The stimulatory effect of the clays illite and montmorillonite on zinc uptake by Scenedesmus obliquus is discussed. The competitive effects of hydrogen, phosphate, magnesium and copper ions on zinc uptake are illustrated. This competition is reduced in the presence of illite and montmorillonite and zinc uptake is stimulated. Ferrous ions have no effect on zinc uptake, while calcium ions antagonize zinc uptake at high concentrations. The latter effect was remarkable in the presence of montmorillonite.     

FURTHER OBSERVATIONS ON THE GROWTH OF THE COMMON DOG-WHELK, NUCELLA LAPILLUS (L.), IN THE LABORATORY

Juvenile Nueella lapillus, reared in aquaria from adults collectedat Hele in North Devon, did not change shape during the firsttwo years of life. In an earlier paper, Crothers (1977b)foundthat juveniles, reared under identical conditions from adultscollected at Porlock Weir in West Somerset, grew allometricallyduring their second year. The different pattern of growth isyet another feature, to add to differences in size and shape,of the unusual populations of Nucella found in the Severn Estuary. (Received 11 June 1979;     

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.     

EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

Rat liver mitochondria allowed to accumulate maximal amounts of Ca⁺⁺ and HPO₄⁼ ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca⁺⁺ and HPO₄⁼ from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca⁺⁺-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca⁺⁺-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca⁺⁺-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca⁺⁺ and phosphate from the mitochondria into the medium.     

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.     

PHENOTYPIC CHANGES IN THE PROGENY OF NUCELLA LAPILLUS (GASTROPODA) TRANSPLANTED FROM AN EXPOSED SHORE TO SHELTERED INLETS

Nucella lapillus adults were transplanted from the exposed northCornish coast (Bude) to two sheltered inlets in south Devon(Dart Estuary and Plymouth Sound), sites where the native populationshad ceased breeding as a result of sterilisation by tributyltin(TBT) pollution. The transplants produced progeny, many of whichsurvived to maturity and beyond (6 + years). The shell formof Bude individuals is typical of animals exposed to severewave-action, i.e. of light construction with a large apertureto accommodate a broad foot allowing for strong adhesion. However,in contrast, the shell form of the progeny differed markedlyfrom that of their parents; they had the characters associatedwith development under sheltered conditions and subject to severecrab predation, i.e. a robust structure and small aperture.Statistical analysis of the shell characters of the two generationsindicated highly significant differences. It is concluded theseobservations provide evidence of the remarkable plasticity inthe shell form of N lapillus in that both ‘exposed morph’and ‘sheltered morph’ can be produced by the samegene pool when subject to the actions of the appropriate selectionpressures. (Received 25 September 1992; accepted 14 October 1992)     

THE COMPOSITION OF PHOSPHATE GRANULES IN THE DIGESTIVE GLANDS OF MARINE PROSOBRANCH GASTROPODS: VARIATION IN RELATION TO TAXONOMY

The composition of some 1150 phosphate granules in the digestiveglands of over 40 species of marine prosobranch gastropods hasbeen surveyed using a simple preparation technique and semi-quantitativeSEM x-ray microanalysis. Spectral peaks for Mg, K, Ca, Mn, Feand Zn were compared to that of P. Four major types of phosphategranule can be recognised, each generally characteristic ofa taxonomic grouping: high Mg in archaeogastropods and littorinids,multiple metal in higher mesogastropods, and, in neogastropods,Mg-Ca in muricoideans and high Zn in buccinoideans. At leastone Conus species (C. ventricosus) has high-Mg granules. Somecauses of variation in granule composition are discussed: speculatively,it is suggested a palaeoenvironmental influence seems possible. (Received 5 November 1997; accepted 6 January 1998)     

EFFECTS OF WOUND HEALING ON ZINC DISTRIBUTION AND ALKALINE PHOSPHATASE ACTIVITY OF HELIX ASPERSA (GASTROPODA: PULMONATA)

Helix aspersa were loaded with ⁶⁵zinc and after 2 and 5 daysstarvation there was no significant reduction in the zinc concentrationof the digestive gland and shell. Wounding of the optic tentacle,foot tissue and partial shell removal of H. aspersa resultedin deposition of ⁶⁵zinc in the wound area. Alkaline phosphataseshowed no increase in concentration at the sites of wounding. (Received 15 October 1985;     

DISTRIBUTION OF DIGESTIVE TUBULES AND FINE STRUCTURE OF DIGESTIVE CELLS OF APLYSIA PUNCTATA (CUVIER, 1803)

The distribution of digestive tubules of Aplysia punctatahas been studied in animals under experimental feeding conditions.Histological analysis of the digestive gland has revealed twotypes of tubules, called tubules A and B. Tubules of type Awere composed of basiphilic cells (calcium, excretory and thincells) and tubules of type B were lined by large digestive cellsand basiphilic cells. The latter occur in small groups, usuallyin the corners of the tubules. Type A tubules are involved inion metabolism and show a diphasic cycle (absorptive and reconstitutive)according to the height and the stage of calcium cells. TypeB tubules are involved in digestive processes and display atetraphasic cycle (holding, absorption, fragmentative and reconstitutive)depending upon the height and the stage of the digestive cells.The tetraphasic cycle was compared with the four categoriesof tubules in bivalves. It is proposed that digestive processesmay be continuous in digestive cells of A. punctata. (Received 16 November 1999; accepted 1 October 2000)     

SEXUAL DIMORPHISM OF NUCELLA LAPILLUS (GASTROPODA: MURICIDAE) IN NORTH WALES, UK

Multivariate statistical methods were employed to examine sexualdimorphism in size and shape of Nucella lapillus collected from 16sheltered sites along coasts of Anglesey and the Lleyn Peninsula, NorthWales, UK. Females were significantly larger than males in overallsize; among 12 relative measures of shell shape, two ratios (shellwidth/shell length and aperture length/shell length) were significantlydifferent between males and females, but these differences usuallydecreased with increasing age (shell length). The observed hypoallometricdimorphism could be a result of selection on increased femalefecundity, which may be positively correlated with shell sizein N. lapillus as in other gastropod species. (Received 22 November 1999; accepted 10 April 2000)     

The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouse

The distribution of metals within the hepatopancreas of Oniscus asellus (Crustacea, Isopoda) from two uncontaminated sites, and two sites contaminated with zinc, cadmium and lead, has been studied by atomic absorption spectrophotometry, light microscopy, transmission and scanning electron microscopy and X-ray microanalysis. The hepatopancreas contains two types of intracellular granule. The first type, in the S cells, are spherical granules which contain copper, sulphur and calcium. In woodlice from contaminated sites, these ‘copper’ granules, also contain zinc, cadmium and lead. The second type, in the B cells, are flocculent deposits which contain iron. In woodlice from contaminated sites, these ‘iron’ granules also contain zinc and lead. Moribund woodlice from contaminated sites have large numbers of ‘copper’ and ‘iron’ granules in the hepatopancreas and a fine deposit of zinc and lead on the membranes of the cells. There are numerous microorganisms in close association with the microvillous border of the hepatopancreas of woodlice from all four sites. Within the microorganisms of Oniscus asellus from contaminated sites, there are deposits of material which contain zinc, lead, calcium and phosphorus ‘Copper’ and ‘iron’ granules could have evolved as storage sites for essential metals to be utilized when demand from the body exceeds uptake from the food. Woodlice in contaminated sites may be able to ‘detoxify’ potentially harmful amounts of essential and non-essential metals by storing them in a relatively insoluble form within these granules.     

MORPHOLOGICAL AND CHEMICAL CAMOUFLAGE OF THE MEDITERRANEAN NUDIBRANCH DISCODORIS INDECORA ON THE SPONGES IRCINIA VARIABILIS AND IRCINIA FASCICULATA

The nudibranch Discodoris indecora shows a perfect camouflageon its prey, the dictyoceratid sponge Ircinia variabilis. Shapeand colour of the nudibranch are remarkably similar to thatof the sponge which is widespread in the shallow waters of theMediterranean Sea. The metabolite pattern of I. variabilis isdominated by unusual molecules, the sesterter-penoids palinurinand variabilin, containing 25 carbons and displaying at theends a ß-substituted furan ring and a tetronic acid.However, a chemical polymorphism has been recently describedfor I. variabilis. Some specimens contain either palinurin orvariabilin whereas others possess a mixture of both sestertepenoids.During the study of Mediterranean sponges, a linear sesterterpenoidrelated to palinurin and variabilin was found in Ircinia fasciculata,that from a taxonomic point of view is closely relatedto Irciniavariabilis. Discodoris indecora shows a typical defence behaviourwhen it is disturbed, secreting a copious white slime that containslarge amounts of palinurin and variabilin. Most likely the animalis able to transfer the sponge metabolites from the digestiveglands to mantle glands. Electron micrographs of the nudibranchmantle showed the presence of unicellular glands characterizedby a cylindrical vacuole with a big nucleus on the basis. Theglands were concentrated in the most conspicuous tubercles ofthe dorsal mantle. Probably, the sponge metabolites are storedin these glands. Comparative chemical analysis of mucous secretions, dorsal tubercles,mantle, foot, digestive glands and hepatopancreas of D. indecoraconfirmed this hypothesis. Palinurin and fasciculation were,of course, present in the digestive glands but they were alsofound in the mucus and in the mantle sections enriched by theabove described unicellular glands. The defensive role of palinurinand variabilin, strongly suggested by their anatomical location,was supported by recording the induction of feeding-deterrencein the fresh-water fish Carassius auratus and in the marinefish Chromis chromis and Sparus auratus. (Received 15 March 1996; accepted 27 January 1997)     

Kinetics of metals in molluscan faecal pellets and mineralized granules,incubated in marine sediments

Marine snails excrete metals via the gut. The sediment-feeding, tower shell Cerithium vulgatum, from a polluted environment, accumulates Cr and Ni to 3500 ppm dry weight in the faecal pellets. Pellets from C. vulgatum and the grazing, top shell Monodonta mutabilis were incubated in surface and deeper anoxic layers of clean and metal-polluted sediment. Pellets retained the original load of metals and in some cases gained Ti, Cr, Mn, Fe and Ni. Effects were modified by sediment properties. Pellets of C. vulgatum are durable, membrane-bound structures and they reduce metal bioavailability to food chains by compartmentalization. Intracellular, phosphate granules bind metals in digestive glands of snails and they are excreted via the gut and faecal pellets. These granules were extracted from digestive glands of both species of snail and incubated in the same sediments. Magnesium phosphate granules from M. mutabilis dissolved but calcium/metal phosphate granules from C. vulgatum remained; they had differentially retained or lost Mn, Fe, Co, Ni and Zn according to the type of sediment.     

L-Tyrosine carboxy-lyase of barley roots

_L-Tyrosine carboxy-lyase (E.C. 4. 1. 1. 25) was isolated fromroots of germinating barley (Hordeum vulgare). The enzyme requirespyridoxal phosphate for maximum activity. The optimum pH foractivity is about 7.0. The enzyme is inhibited by p-chloromercuribenzoateand hydroxylamine at 10^–3 M. Enzyme activity is foundin extracts from young roots, especially from those in earlystages of development, but not in extracts from shoots of thesame plant. Localization and changes in the amounts of _L-tyrosinecarboxy-lyase and aromatic amines in developing barley seedlingswere measured. Participation of carboxy-lyase in the formationof aromatic amines in barley roots is suggested. (Received July 17, 1970; )     

CALCIUM PHOSPHATE GRANULES IN THE HEPATOPANCREAS OF THE BLUE CRAB CALLINECTES SAPIDUS

The hepatopancreas of the adult male blue crab Callinectes sapidus in intermolt was found to contain substantial amounts of calcium, magnesium, and inorganic phosphorus, averaging about 260, 20, and 250 µg-atoms per g wet tissue, respectively, accounting for over 10% of the tissue dry weight. Electron microscopy of the intact tissue showed three qualitatively different granular structures having electron densities suggestive of high mineral content. After fractionation of the tissue using centrifugal techniques, almost 95% of the total mineral was found to reside in a heavy, nonmitochondrial particulate fraction(s). The bulk of the low-speed pellet consisted of relatively dense, roughly spherical granules 1–5 µm in diameter, which could be considerably purified by repeated suspension in water and low-speed sedimentation. In the electron microscope the isolated granules appeared basically similar to one of the three characteristic types of electron-dense granules seen in the intact tissue. Although the freshly isolated granules lost approximately 50% of their wet weight when dried at 105°C, only 10% more was lost upon dry ashing at 450°C, suggesting a fairly low content of organic material. Chemical analysis revealed calcium, magnesium, and inorganic phosphate at 5.7, 2.1, and 4.4 µg-atoms per mg dried granules, respectively, accounting for 69% of the dry weight of the fraction. By specific enzymatic assays, the freshly isolated granules were found to contain ATP, ADP, and AMP at levels of 0.13, 0.03, and 0.01 µmol/mg, or 8% of their total dry weight. The remainder of the total phosphorus contributed an additional 3%, whereas carbonate, citrate, oxalate, and protein each constituted no more than 1%. The mineral granules of the crab hepatopancreas appear to function as storage forms of calcium and phosphate during the intermolt period. This tissue appears promising as a model for study of the cellular events associated with biological calcification, since conventional biochemical techniques can be employed. Furthermore, the major mineralized component of the tissue can be obtained in large amounts for direct study by a simple fractionation procedure.     

Magnesium,calcium, copper,and zinc in meals

Meals of 12 diets were prepared from conventional foods with precautions against contamination by metallic elements because of epidemiologic associations between ischemic heart disease and the metabolism of magnesium, calcium, copper, and zinc. Magnesium, calcium, copper, and zinc were measured by atomic absorption spectrometry with satisfactory accuracy and precision. The mean daily amount of copper in the diets was less than the apparent adult requirement. Mean amounts of magnesium and zinc were close to apparent requirements; however, adults consuming amounts less than one standard deviation below these means may be depleting body stores and be at risk of pathology. Magnesium and copper were highly correlated (r=0.849,P=0.0001) in meals. This and other significant correlations probably will prevent the relationships of these elements to ischemic heart disease from being elucidated by epidemiology. Metabolic experiments will be necessary to differentiate among several hypotheses.