Outer Mantle Epithelium Research Articles

A calcification-related calmodulin-like protein in the oyster Crassostrea gigas mediates the enhanced calcium deposition induced by CO2 exposure

Calcium transportation and homeostasis are essential for marine bivalves to maintain basic metabolism and build their shells. Calmodulin-like proteins (CaLPs) are important calcium sensors and buffers and can respond to ocean acidification (OA) in marine calcifiers. However, no further study of their physiological function in calcium metabolism under elevated CO2 has been performed. Here, we identified a novel CaLP (designated CgCaLP) in the Pacific oyster Crassostrea gigas and demonstrated its participation in the calcification process: the mRNA expression level of CgCaLP peaked at the trochophore larval stage and remained high at stages when shells were shaped; the mRNA and protein of CgCaLP were more highly expressed in mantle tissue than in other tissues. Under elevated CO2 levels, the protein expression level of CgCaLP in hemocytes increased, while in contrast, significantly decreased protein levels were detected in gill and mantle tissues. Shell dissolution caused the imbalance of calcium in hemocytes and decreased calcium absorption and transportation demand in gill and mantle tissues, inducing the molecular function allocation of CgCaLP under CO2 exposure. Despite the decreased protein level in mantle tissue, CgCaLP was found to translocate to outer mantle epithelium (OME) cells where condensed calcium-rich deposits (CRDs) were detected. We further demonstrated that CgCaLP mRNA and protein expression levels could respond to seawater Ca2+ availability, suggesting that the calcium deposition capacity of oysters might be enhanced to fight against shell dissolution problems and that CgCaLP might serve as an essential participator of the process. In summary, CgCaLP might enhance calcium deposition under CO2 exposure and thus play a significant and flexible molecular function involved in a compensation strategy of oysters to fight against the acidified ocean.

Dilution of Seawater Affects the Ca2 + Transport in the Outer Mantle Epithelium of Crassostrea gigas.

Varying salinities of coastal waters are likely to affect the physiology and ion transport capabilities of calcifying marine organisms such as bivalves. To investigate the physiological effect of decreased environmental salinity in bivalves, adult oysters (Crassostrea gigas) were exposed for 14 days to 50% seawater (14) and the effects on mantle ion transport, electrophysiology and the expression of Ca2+ transporters and channels relative to animals maintained in full strength sea water (28) was evaluated. Exposure of oysters to a salinity of 14 decreased the active mantle transepithelial ion transport and specifically affected Ca2+ transfer. Gene expression of the Na+/K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase was decreased whereas the expression of the T-type voltage-gated Ca channel and the Na+/Ca2+-exchanger increased compared to animals maintained in full SW. The results indicate that decreased environmental salinities will most likely affect not only osmoregulation but also bivalve biomineralization and shell formation.

Terebratulide brachiopod shell biomineralization by mantle epithelial cells

To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles.At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, in cross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference.FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of mineralization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

In situ Measurements of pH, CA2+, and Dic Dynamics within the Extrapallial Fluid of the Ocean Quahog Arctica islandica

This study investigated to what extent the extrapallial fluid (EPF) of the marine bivalve Arctica islandica (Linneaus, 1767) is involved in shell formation. With in situ pH microscopy, pH gradients were identified between inner shell surface and outer mantle epithelium (OME). pH at the OMEvaried rapidly between neutral and values above 9, suggesting active H+ pumping. Microsensor measurements showed also remarkable short-term dynamics in pH and Ca2+ concentrations, again suggesting active ion pumping. Further focus was on pH, Ca2+, and dissolved inorganic carbon dynamics within the EPF to determine whether calcium carbonate precipitation is possible within the EPF. The data show that the bulk of the inner EPF rarely reaches calcium carbonate saturation and, thus, cannot be the site of shell formation. At the OME surface, however, pH levels of up to 9.5 were observed, corresponding to a 30-fold carbonate supersaturation. Thus, ion pumping by the OME can drive calcification when the OME is just a few mm distant from the inner shell surface, as it is the case in the outer EPF.

Calcite fibre formation in modern brachiopod shells

The fibrous calcite layer of modern brachiopod shells is a hybrid composite material and forms a substantial part of the hard tissue. We investigated how cells of the outer mantle epithelium (OME) secrete calcite material and generate the characteristic fibre morphology and composite microstructure of the shell. We employed AFM, FE-SEM, and TEM imaging of embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze substituted samples. Calcite fibres are secreted by outer mantle epithelium (OME) cells. Biometric analysis of TEM micrographs indicates that about 50% of these cells are attached via hemidesmosomes to an extracellular organic membrane present at the proximal, convex surface of the fibres. At these sites, mineral secretion is not active. Instead, ion transport from OME cells to developing fibres occurs at regions of closest contact between cells and fibres, however only at sites where the extracellular membrane at the proximal fibre surface is not developed yet. Fibre formation requires the cooperation of several adjacent OME cells. It is a spatially and temporally changing process comprising of detachment of OME cells from the extracellular organic membrane, mineral secretion at detachment sites, termination of secretion with formation of the extracellular organic membrane, and attachment of cells via hemidesmosomes to this membrane.

Calcium transfer across the outer mantle epithelium in the Pacific oyster, Crassostrea gigas.

Calcium transport is essential for bivalves to be able to build and maintain their shells. Ionized calcium (Ca2+) is taken up from the environment and eventually transported through the outer mantle epithelium (OME) to the shell growth area. However, the mechanisms behind this process are poorly understood. The objective of the present study was to characterize the Ca2+ transfer performed by the OME of the Pacific oyster, Crassostrea gigas, as well as to develop an Ussing chamber technique for the functional assessment of transport activities in epithelia of marine bivalves. Kinetic studies revealed that the Ca2+ transfer across the OME consists of one saturable and one linear component, of which the saturable component fits best to Michaelis-Menten kinetics and is characterized by a Km of 6.2 mM and a Vmax of 3.3 nM min-1 The transcellular transfer of Ca2+ accounts for approximately 60% of the total Ca2+ transfer across the OME of C. gigas at environmental Ca2+ concentrations. The use of the pharmacological inhibitors: verapamil, ouabain and caloxin 1a1 revealed that voltage-gated Ca2+-channels, plasma-membrane Ca2+-ATPase and Na+/Ca2+-exchanger all participate in the transcellular Ca2+ transfer across the OME and a model for this Ca2+ transfer is presented and discussed.

Connecting pattern to process: Growth of spiral shell sculpture in the gastropod Nucella ostrina (Muricidae: Ocenebrinae).

Shell morphology is a well-suited and underused system to examine the development of novel forms. The three-dimensional structure produced (the shell) is separate from the largely two-dimensional tissue that secretes it (the mantle), allowing us to disentangle the pattern from the process. Despite knowing a great deal about the mechanics of shell secretion (process), and the variety of shell shapes that exist (pattern), no effort has been made to understand how the mantle changes to produce different shell shapes. We investigated this question in the dimorphic snail Nucella ostrina, which exhibits both smooth and ribbed shells to determine how ribs are formed by the mantle. Rib thickenings are produced only in the outer calcitic shell layer and secreted by the distal Outer Mantle Epithelium (OME) with increased acid phosphatase activity. The evenly thick inner aragonitic layers are secreted by the proximal OME which expresses acid phosphatase. Here we show that locally thicker ribs in N. ostrina are produced by changing the dimensions of the distal OME: elongation in the direction of growth and increased cell height. This should increase the amount of shell material secreted, producing locally thicker shell (ribs). Preliminary evidence suggests this mechanism may be widespread in gastropods.

Congruence and conflict: case studies of morphotaxonomy versus rDNA gene tree phylogeny among articulate brachiopods (Brachiopoda: Rhynchonelliformea), with description of a new genus

We present five case studies among articulate (rhynchonelliform) brachiopods, i.e. of Rhynchonellida, Cancellothyridoidea, Terebratuloidea, Dyscolioidea, Laqueoidea, and various terebratulids with modified long-loops, in an attempt to illustrate and better understand congruence and conflict between morpho-classification and rDNA-based molecular clade structure, having been prompted to address these issues by difficulties encountered when describing the newly collected brachiopod, Ebiscothyris bellonensis gen. et sp. nov. The five studies reveal dramatic conflict in the Rhynchonellida and Terebratuloidea/Dyscolioidea, good congruence in the Cancellothyridoidea and Laqueoidea, and fair congruence (albeit with weak phylogenetic signal) in the long-looped terebratulids. We suggest that the leading cause of the observed conflict lies in the use of inadequately specific morphological characters and morpho-classification. Phylogenetic systematic (cladistic) analyses of Rhynchonellida also conflict markedly with the rDNA gene tree, leading us to recognize that such analyses are not only conceptually circular (using morphological characters to assess a morphological classification) but also to propose that they are biased by the act of classification that necessarily precedes the identification of putatively homologous characters; when the prior classification does not reflect evolutionary history, phylogenetic analysis will do likewise. In addition, we propose that the brachiopod community has overlooked the significance of two sources of morphological homoplasy affecting brachiopod systematics: (1) the loss of co-adapted genomic complexes caused by mass extinctions at the end of the Permian; and (2) the pervasive consequences of developmental integration and constraint resulting from the integrated roles of the outer mantle epithelium in shell deposition and growth that underly the determination of form and the shell-based classification. © 2015 The Linnean Society of London

The outer mantle epithelium of Haliotis tuberculata (Gastropoda Haliotidae): an ultrastructural and histochemical study using lectins

AbstractThe mantle of molluscs has been the subject of many studies as it is the organ that forms the shell. Microscopic studies in particular focus on the outer mantle epithelium, but few studies address this epithelium in a histochemical way. In this study, the outer mantle epithelium in adult specimens of Haliotis tuberculata is studied, that is, in specimens involved in maintaining and repairing the shell rather than in generating it. The epithelial cells are studied by scanning (SEM) and transmission electron microscopy (TEM), and by histochemical techniques, including the use of lectins for their biochemical characterization. The epithelium is composed of pigmented epidermal cells with small microvilli and junctional complexes. It furthermore contains a few ciliated cells, as well as two types of secretory cells which differ in the ultrastructural appearance of their secretory granules and their glycoconjugate content. Histochemical study shows secretory cells containing sulphated glycoconjugates such as glycosaminoglycans or mucins rich in N‐acetylgalactosamine and N‐glycoproteins rich in fucose. Furthermore, the apical regions of the epidermal cells are positive for lectins that label fucose, mannose and N‐acetylglucosamine. The role of epithelial cells in the synthesis of structural components of the shell is discussed.

2,4-Dichlorophenoxyacetic acid alters intracellular pH and ion transport in the outer mantle epithelium of the bivalve Anodonta cygnea

Bivalve molluscs, due to their sedentary mode of life and filter-feeding behavior, are very susceptible to pollutant bioaccumulation and used as sentinel organisms in the assessment of environment pollution. Herein we aimed to determine the in vivo, ex vivo and in vitro effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, in Anodonta cygnea shell growth mechanisms. For that, we evaluated the effect of 2,4-D (100μM) exposure on the transepithelial short-circuit current (Isc), potential (Vt) and conductance (Gt), as well as on OME ion transport systems and intracellular pH (pHi). In vivo exposure to 2,4-D caused an increase of 50% on the Isc generated by OME and ex vivo addition of that compound to the apical side of OME also induced an Isc increase. Furthermore, 2,4-D was able to cause a pHi increase in isolated cells of OME. Noteworthy, when 2,4-D was added following the exposure to specific inhibitors of several membrane transporters identified as responsible for pHi maintenance in these cells, no significant effect was observed on pHi except when the V-type ATPase inhibitor was used, indicating an overlap with the effect of 2,4-D. Thus, we concluded that 2,4-D is able of enhancing the activity of the V-ATPases present on the OME of A. cygnea and that this effect seems to be due to a direct stimulation of those H+ transporters present on the apical portion of the membrane of OME cells, which are vital for shell maintenance and growth. This study allows us to better understand the molecular mechanisms behind 2,4-D toxicity and its deleterious effect in aquatic ecosystems, with particular emphasis on those involved in shell formation of bivalves.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75μM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75μM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na+/H+ exchangers, Na+–K+ pump, Na+-driven and Na+-independent HCO3−/Cl− transporters and Na+/HCO3− co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4′-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3−/Cl− co-transporters present on the basolateral membrane.

A New Species of Gymnophallid (Digenea) and an Amended Diagnosis of the GenusGymnophalloidesFujita, 1925

Morphological and molecular evidence suggest that specimens formerly described as Lacunovermis sp. from Nacella (Patinigera) spp. (Patellogastropoda: Patellidae) belong to a new species of Gymnophalloides Fujita, 1925 . Based on the new information, they are identified as Gymnophalloides nacellae n. sp. The new species differs from Gymnophalloides tokiensis, Gymnophalloides seoi , and Gymnophalloides heardi mainly through the presence of a group of papillae located on the ventral surface between oral and ventral suckers. A detailed morphological study revealed the lack of pars prostatica, a character previously reported in G. seoi , which is why it was formerly placed in the Gymnophallinae. Molecular information proved that G. nacellae is close to G. seoi , being nestled together with Parvatrema representatives. This molecular information, along with the absence of pars prostatica, allows these 2 genera to be placed in Parvatrematinae. An amended diagnosis of Gymnophalloides is provided. Histological sections of mantle epithelium of the limpet show metacercariae attached by their oral and ventral suckers in a similar manner to G. seoi in its host, the oyster Crassostrea gigas . Tissue reaction includes cells of outer mantle epithelium being stretched by sucker attachment, hemocyte infiltration of connective tissue between mantle epitheliums, and abnormal calcareous deposition on the inner surface of the shell.

Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization

BackgroundThe shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.ResultsWe report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.ConclusionsThis EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

Needles and pins: acicular crystalline periostracal calcification in venerid bivalves (Bivalvia: Veneridae)

A scanning electron microscope study of the periostracum of 50 species of venerid bivalves revealed that periostracal calcification in the form of aragonitic needles and shorter pins is widespread within the family. Together with organic and sediment coatings that are found in some species, these needles form an integral part of the functional shell. Visible as a white ‘crust’ on the outside of shells, long slender needles (up to 400 mm long and 1 mm wide) without adherent material are seen in species of Tivela and Lioconcha and in Gomphina undulosa. Other venerids including Pitar species, Mysia undata and Compsomyax subdiaphana have short pins, capped with a fibrous organic matrix and significant coatings of sediment. Callocardia hungerfordi and Clementia papyracea have very thick sediment coatings underlain by short pins, while Gafrarium and Circe species have short pins with a thin, robust, organic coating and little particulate material. Finally, there are species, including Venus verrucosa, Chione elevata and Mercenaria mercenaria, where minute, ,1 mm long pins also underpin a thin organic coating. Details of formation were studied in Tivela lamyi and Lioconcha ornata, where the needles are elongate hexagonal crystals of aragonite enveloped by an organic sheath, which grow at their proximal ends from within the periostracum, connected to the outer mantle epithelium via narrow channels. Growth of needles ceases following the onset of shell calcification. The distribution of the periostracal structures was examined in relation to a published molecular phylogeny that recognized two major clades within the family. Larger needles and pins are confined to the clade that includes subfamilies Pitarinae, Gouldinae, Meretricinae and Petricolinae, while submicron-sized pins are found only in the Venerinae and Chioninae of the second clade. Calcified periostracal structures appear to be absent in Tapetinae and Dosiniinae.

A Mathematical Model of the Proton Balance in the Outer Mantle Epithelium of Anodonta cygnea L.

In the freshwater mollusc Anodonta cygnea and other unionids, the mantle plays an important role in the regulation of the movements of ions between the shell and the extrapaleal fluid. In this report, a mathematical model that attempts to describe the cell metabolic mechanisms underlying the operation of the outer mantle epithelium as a source of protons is presented. We encoded the information gathered by studying the epithelium in vitro, which includes the electrophysiology of the preparation, measurements of basic rates of transport of protons and base, the effect of metabolic and transport inhibitors on its electrical behavior and the dynamic measurements of pHi. The model was conceived so that the short-circuit current (Isc) and fluxes of Na+, K+ and Cl(-); intracellular volume; electrical potential; and ionic concentrations can be computed as a function of time. Furthermore, the analytical descriptions of all ionic fluxes involved are such that the effect of transport inhibitors can be simulated. In all the simulations performed, it was possible to reproduce the experimental results obtained with specific inhibitors of transport systems on the Isc and on pHi. In some cases, it was necessary to make alterations to one or more parameters of the reference condition. For each simulation carried out, the analysis of the results was consistent. The model is an analytical tool that can be used to show the internal coherence of the qualitative model previously proposed and to plan further experiments.

Shell matrices of Recent rhynchonelliform brachiopods: microstructures and glycosylation studies

ABSTRACTLike most metazoan biomineralisations, the brachiopod shell is the end product of a biologically controlled calcification process. The main agent of the control is the extracellular matrix, which is secreted by the outer mantle epithelium. This matrix mediates the calcification process by allowing crystal nucleation and elongation in specific orientations and finally, by stopping crystal growth. The proteinaceous moiety of brachiopod shell matrices has been extensively studied. Less known are the post-translational modifications that occur in these matrices, in particular glycosylations. In this comparison of five species of Recent articulated brachiopods, the ratio of soluble to insoluble organic matrix varies between the species. Polydisperse macromolecular materials occur in each of these species with discrete proteins of 50 kDa in Notosaria nigricans, Calloria inconspicua and Neothyris lenticularis, 37 kDa in Terebratulina retusa and Gryphus vitreus and 20–25 kDa in N. nigricans. Protein mixtures from all five species respond differently to anionic stains (Stains-All and Alcian Blue). PAS staining results in a positive smear in C. inconspicua and T. retusa and highlights low molecular weight glycoproteins in C. inconspicua. The polysaccharide composition of the soluble matrix of T. retusa is different from the others due to high proportions of arabinose and low proportions of fucose. In all cases, polysaccharide composition of the insoluble matrix is dominated by glucose and glucosamine. Insoluble matrices have more glucose and xylose and less galactosamine and glucosamine than the corresponding soluble matrix. Relatively high amounts of glucosamine may suggest the presence of chitin in the shell matrix of rhynchonelliform brachiopods.

CGRP REGULATES THE ACTIVITY OF MANTLE CELLS AND HEMOCYTES IN ABALONE PRIMARY CELL CULTURES (HALIOTIS TUBERCULATA)

Mollusc shell formation requires calcium (Ca ) and bicarbonate (CO3 −) ions, transported through the mantle via the hemolymph, and the secretion of an organic matrix that interacts with the mineral ions to form either the aragonite or calcite polymorph of calcium carbonate (CaCO3). The biomineralization process takes place in the extrapallial space, between inner shell and outer mantle epithelium, and is believed to be under the control of calcium-binding proteins and hormonal factors such as calcitonin-related molecules (CGRP-like peptides). Epithelial cells from the mantle are responsible from the secretion of matrix molecules whereas hemocytes are involved in ion transportation during shell formation and regeneration. To understand the biochemical and cellular events implied in the biomineralization process, we developed primary cell cultures from the nacreous gastropod Haliotis tuberculata. Mantle cells as well as hemocyte cell cultures were successfully maintained in vitro and evaluated for their viability and proliferation using semiautomated assays. The effect of calcitonin-related molecules (h-CGRP) on cellular activity and proliferation was evaluated in those primary cell cultures. The results indicate that human-CGRP modulates the activity of both mantle cells and hemocytes and increases carbonic anhydrase activity in vitro. These results are consistent with those obtained in vivo, supporting the hypothesis that CGRP-like molecules control the activity of target cells involved in the biomineralization process.

Studies on a PMCA-like protein in the outer mantle epithelium of Anodonta cygnea: insights on calcium transcellular dynamics

Early studies on the outer mantle epithelium (OME) cells of the freshwater bivalve Anodonta cygnea (Linnaeus, 1758) revealed high ionic calcium concentrations by electrophysiological methods and subsequently a high tendency to reach an intracellular toxic condition. This toxicity could be neutralized by specific mechanisms in the cytosol of OME cells of A. cygnea. The present immunocytochemistry studies of OME cells by light and transmission electron microscopy (TEM) clearly showed a positive reaction of an antibody directed against the human plasma membrane Ca(2+)-ATPase 1 (PMCA-1) in the cytoplasm of OME cells. Also, western blot analysis of different fractions of OME cells with anti human PMCA-1 and C28R2 antibodies confirmed the presence of a PMCA-like protein with an unusual topographical localization and a molecular weight of only 70-80 kDa. These results lead us to speculate that this PMCA-like protein is distributed either in the plasma membrane or in the entire cytosol, where it eventually regulates intracellular calcium levels. Interestingly, the antibody reactions showed seasonal variations, being highest in OME samples prepared during summer when A. cygnea live under natural acidosis and absent in samples taken in winter conditions, which is in accordance with the seasonal variation of shell calcification rates. During winter, PMCA-1 antibody reaction was also detected in OME cells of animals kept only under experimentally induced acidosis conditions. Therefore, we assume that a functional role for this PMCA-like protein in the intracellular calcium regulation of OME cells during the mineralization of the shells of A. cygnea can be speculated.

Light Induces an Increase in the pH of and a Decrease in the Ammonia Concentration in the Extrapallial Fluid of the Giant ClamTridacna squamosa

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.

H +-ATPase of crude homogenate of the outer mantle epithelium of Anodonta cygnea

The outer mantle epithelium of the freshwater bivalve, Anodonta cygnea, is responsible for the mineralisation of the shell. Under short circuit conditions, it generates a current that is due to the operation of an electrogenic proton pump located in the apical barrier of that epithelium. Bafilomycin A 1 and Concanamycin A inhibited the short circuit current. The IC 50 and maximum inhibition dose were 0.17 and 0.5 μM for Bafilomycin A 1, and 0.7 and 5 μM for Concanamycin A. The present work was undertaken to further characterise V-type ATPase of the outer mantle cells. The V-ATPase enzymatic activity of crude homogenate, measured as the amount of inorganic phosphorous released, due to ATP hydrolysis, in the presence of Na 2SO 3 (200 mM) was found to be 4.6±1.1 μmol Pi/mg protein/h, at 27 °C, pH 7.0–7.4 and ATP 4.5–6.0 μM. Bafilomycin A 1 and Concanamycin A inhibit the V-ATPase activity with an IC 50 of 14 and 8 nmol mg −1, respectively. Dicyclohexylcarbodiimide (DCCD; 100 mM) and NaNO 3 (100 μM) inhibited the V-type ATPase in what it seems a non-specific manner and NEM (100 mM) was unable to do it. Bafilomycin A 1 (10 μM) and Concanamycin A (10 μM), inhibited 50–60% of the total activity.