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PubMed1999

A new matrix protein family related to the nacreous layer formation of Pinctada fucata.

We have isolated a new matrix protein family (N16) which is specific to the nacreous layer of the Japanese pearl oyster, Pinctada fucata, and have cloned and characterized the cDNAs coding for the components. Analysis of the deduced amino acid sequence revealed that N16 showed no definitive homology with other proteins. The in vitro studies of the crystallization clarified that N16 induced aragonite crystals when fixed on the substrate but inhibited crystal formation without it. The aragonite crystals showed platy morphology different from those formed inorganically, and long intervals of incubation resulted in crystalline layers highly similar to the nacreous layer.

Amino Acid Sequence↗
PubMed1997

Accumulation of copper, nickel, lead and zinc by snail, Lunella coronatus and pearl oyster, Pinctada radiata from the Kuwait coast before and after the Gulf War oil spill.

This study investigated the contribution of the 1991 oil spill to heavy metal contamination in the marine environment of the Gulf in Kuwait by analyzing moleskin organisms (gastropod snail, Lunella coronatus and bivalve Pearl oyster, Pinctada radiata) for their heavy metal contents before and after the spill. Concentrations of copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn) were determined in the soft tissue of both snail and oyster samples from three coastal stations during 1990 and 1994. In the 1990 samples, the metal concentrations in snail and oysters from different sampling stations were between 0.35 and 0.67; 0.11 and 2.29 micrograms/g for Cu, 1.50 and 4.50; 0.47 and 1.33 micrograms/g for Ni, 0.16 and 2.98; 0.44 and 0.69 microgram/g for Pb and 19.94 and 54.79; 247.20 and 1204.40 micrograms/g for Zn, respectively. In the 1994 samples, the metal concentrations were between 11.24 and 55.00; 28.90 and 168.43 micrograms/g for Cu, 15.33 and 16.96; 0.08 and 1.54 micrograms/g for Ni, 0.37 and 0.57 microgram/g; 0.07 and 0.44 microgram/g for Pb and 28.86 and 486.61; 17.75 and 575.00 micrograms/g for Zn respectively. The 1994 samples have significantly higher mean concentrations of Cu, Ni and Zn than the 1990 samples, except Pb in the 1994 samples, which showed a slightly lower mean concentration. The difference in patterns of metal occurrence and the significant increase in the Cu, Ni, and Zn concentrations in the 1994 snail and oyster samples were due to a contribution from the 1991 Gulf War oil spill.

Animals↗
PubMed2003

Biomineralisation markers during a phase of active growth in Pinctada margaritifera.

To search for the biochemical parameters involved in calcium and carbonate transport during crystal formation and biomineralisation in nacreous molluscs, the carbonic anhydrase activity, the levels of calciotropic hormones in hemolymph and in tissues and the circulating concentration of calcium were measured in pearl oysters (Pinctada margaritifera) during a phase of active growth. Activity of carbonic anhydrase in gill tissue increased linearly with age of the animals, while no age variation in activity was noted for the mantle. The circulating level of total calcium increased during the growth of the animals. Calciotropic hormones were radioimmunoassayed in gill, mantle and hemolymph. Only a calcitonin-gene related peptide (CGRP) could be detected and its concentration decreased as a function of growth, both in hemolymph and mantle. No variation in CGRP concentration with age was observed in gill tissue. Our data demonstrate that carbonic anhydrase and a molecule biologically and immunologically related to CGRP are involved during growth of the animals. In addition, this study shows the presence of three main calcium compartments, gill, hemolymph and mantle, involved in the biomineralisation process.

Animals↗
PubMed2002

Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts.

In vivo and in vitro studies provide strong evidence of the osteogenic activity of nacre obtained from Pinctada maxima. The in vitro studies indicate that diffusible factors from nacre are involved in cell stimulation. The water-soluble matrix (WSM) was extracted from nacre by a non-decalcifying process, and four fractions (SE(1)-SE(4)) were separated by SE-HPLC. Those fractions were tested in vitro on MRC5 fibroblasts. Alkaline phosphatase (ALP) activity was measured as a marker of osteoblastic differentiation. The anti-apoptotic protein Bcl-2 was also immunodetected in cultured osteoblasts from rat calvaria. WSM and fraction SE(4) increased ALP activity. BMP-2 had the same effect on the cells as WSM and SE(4). WSM greatly increased the amount of Bcl-2 in the cytoplasm and nucleus of osteoblasts. These in vitro studies support our initial hypothesis that nacre organic matrix (WSM) of a bivalve mollusk contains signal-molecules that can stimulate the osteogenic pathway in mammalian cells that are targets for bone induction.

Alkaline Phosphatase↗
PubMed2003

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata).

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.

Amino Acid Sequence↗
PubMed2003

A novel matrix protein participating in the nacre framework formation of pearl oyster, Pinctada fucata.

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.

Amino Acid Sequence↗
PubMed1985

Detection of two lectins in haemolymph from the oyster Pinctada maxima.

Two lectins were detected in haemolymph from the oyster Pinctada maxima, a macromolecular (approximately 8 X 10(6) dalton) N-acetyl-galactosamine-binding lectin and a lower molecular weight (approximately 4 X 10(5) dalton) alpha-galactose-binding lectin. The macro-molecular lectin agglutinated human type A erythrocytes to a greater extent than human type B or O erythrocytes and was the major lectin detected in the haemolymph. The 4 X 10(5) dalton lectin agglutinated human type A, B and O erythrocytes to the same extent.

Acetylgalactosamine↗
PubMed2004

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata).

The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.

Amino Acid Sequence↗
PubMed2003

Large discrepancies in differentiation of allozymes, nuclear and mitochondrial DNA loci in recently founded Pacific populations of the pearl oyster Pinctada margaritifera.

This study presents a comparative analysis of population structure applied to the pearl oyster (Pinctada margaritifera) from the Central Pacific islands using three classes of molecular markers: two mitochondrial genes (mtDNA), five anonymous nuclear loci (anDNA), and eight polymorphic allozymes. Very low levels of haplotype diversity and nucleotidic divergence detected for mtDNA validate the hypothesis of a recent (re)colonization of Polynesian lagoons after their exondation during the last glaciations. Some nuclear loci, however, showed highly significant FST values, indicating a reduced amount of larval exchange between archipelagos at present. A large interlocus variance of FST was nevertheless observed. We discuss whether this pattern is inherent to the stochasticity of the drift process since recolonization, or if it could result from balancing selection acting on certain loci. This study illustrates once more the need to combine the analysis of several kinds of loci when unrelated phenomena are likely to leave their footprints on genetic structure.

Animals↗
PubMed2003

Evidence for male-biased effective sex ratio and recent step-by-step colonization in the bivalve Pinctada mazatlanica.

This paper presents a comparison of the geographical distribution of genetic variability at mitochondrial and nuclear loci among pearl oyster populations from the tropical American Pacific coast (Pinctada mazatlanica). Surprisingly, both mitochondrial and nuclear gene variability decreased regularly from north to south of the studied area, which, altogether with a significant correlation between genetic and geographical distances for mtDNA, suggests a recent colonization or re-colonization of the southern areas. However, the loss of diversity between north and south was much more important for mitochondrial than for nuclear DNA, and this did not translate into measurable fixation index at nuclear loci (theta = 0.03, n.s.), contrary to the mitochondrial data (theta = 0.18*). Smaller effective size of mtDNA accentuated by a strong male-biased effective sex ratio and step-by-step colonization from northern areas can explain this discrepancy among natural populations of this protandric species.

Animals↗
PubMed2001

Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture (Haliotis tuberculata).

In mollusks, the mantle is responsible for the secretion of an organic matrix that mineralizes to form the shell. A model of mantle cell culture has been established from the nacreous gastropod Haliotis tuberculata. First, viability of cells, quantified by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) reduction assay, was monitored in order to determine a cell density and a time-culturing period in order to investigate biomineralization processes in vitro. During the first 11 days of culture, an increase of MTT response demonstrated an activation of cultured cells mitochondrial activity as confirmed by the total protein content assay. The effect of a water-soluble extract from the organic matrix of Pinctada maxima (WSM) was tested on this cell culture system for 11 days-period exposure. WSM reduced the global viability of mantle cells in a dose-dependent way which corresponded to a cell death. Alkaline phosphatase activity normalized to total protein content increased in the presence of WSM. This increase may be due to an activation of cells and a selection of one (or a few) cell type(s). Further investigations will help us to determine this selectivity issue.

Animal Structures↗
PubMed2003

Microtopography and antifouling properties of the shell surface of the bivalve molluscs Mytilus galloprovincialis and Pinctada imbricata.

Biofouling rapidly covers most submerged surfaces in the marine environment. However, some marine organisms remain clean despite strong fouling pressure. Potential physical inhibitors of fouling were investigated by comparing the thickness, cover, and microtopographic structure of the periostracum of two bivalve molluscs, the blue mussel, Mytilus galloprovincialis, and the pearl oyster, Pinctada imbricata. The cover and thickness of the periostracum were measured on four size classes of each species using histological and microscopic techniques. The periostracum of M. galloprovincialis was significantly thicker than that of P. imbricata and did not differ significantly between size classes. In contrast, the periostracum of P. imbricata decreased significantly with increasing size in both thickness and cover. The microtopography of the shell surface of both species was measured using atomic force microscopy (AFM) and scanning electron microscopy (SEM), which revealed a homogeneous ridged surface for M. galloprovincialis with a uniform distance of 1-2 microns between ridges with a mean depth of 1.5 microns. P. imbricata had a heterogeneous surface structure without a repeating structural pattern. To compare the potential antifouling properties of the shell surface the four size classes of both species were tested in fourteen-week field exposure trials. M. galloprovincialis was rarely fouled over the trial period with less than 10% of M. galloprovincialis shell across all size classes being fouled. In contrast, P. imbricata had significantly higher levels of fouling. Both the proportion of P. imbricata shells fouled and the density of fouling organisms were positively correlated with the age of the shell and the amount of intact periostracum. The relationship between the shell surface microtopography and the intensity of fouling is discussed.

Animal Structures↗
PubMed2004

Spatio-temporal variation in the genetic composition of wild populations of pearl oyster (Pinctada margaritifera cumingii) in French Polynesia following 10 years of juvenile translocation.

Abstract The genetic impact of the cultural practice of spat collection and translocation between genetically distinct stocks of black-lipped pearl oyster, Pinctada margaritifera cumingii, was studied by comparing samples collected in the 1980s and 2000s from seven atolls in French Polynesia. An amova revealed homogenization of the previously genetically distinct wild stocks of Tuamotu-Gambier and Society archipelagos (the indices of genetic differentiation among archipelagos and among populations within archipelagos, respectively, Phi(CT) and Phi(ST), decreased from 0.032* and 0.025*, respectively, to 0.006(NS) and 0.007(NS)). These results suggest high success of spontaneous reproduction in farms, probably due to the very high density of cultivated pearl oysters, and underline the importance of genetic monitoring of future hatchery produced stocks.

Analysis of Variance↗
PubMed2005

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata.

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.

Amino Acid Sequence↗
PubMed2004

Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii).

We examined the effect of tributyltin (TBT) on reproduction of the pearl oyster (Pinctada fucata martensii). In a maternal exposure test, five female pearl oysters were exposed to TBT at measured concentrations of 0 (control), 0.092, or 0.191 microg/L at 25 degreesC for one week, and the embryo developmental success (the ratio of normal D-larvae to all larvae) was measured. The embryo developmental success was significantly decreased in the 0.191-microg/L treatment group (65.5%) compared to that in the control group (82.5%; p = 0.031). Concentrations of TBT in the ovary reached 0.088 microg/g in the 0.191-microg/L treatment group. In a waterborne exposure test, inseminated eggs were exposed to TBT at measured concentrations of 0 (control), 0.020, 0.045, 0.091, 0.192, or 0.374 microg/L for 24 h. The embryo developmental success also was significantly decreased in the 0.192-microg/L treatment group (78.3%; p = 0.020) and no development at all was observed in the 0.374-microg/L treatment group compared with that in the control group (95.4%). These results clearly demonstrate that TBT accumulating in the bodies of bivalves has the potential to inhibit reproduction.

Animals↗
PubMed2005

The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata.

Signals and organic matrix proteins secreted from the mantle are critical for the development of shells in molluscs. Nacrein, which is composed of a carbonic anhydrase domain and a Gly-X-Asn repeat domain, is one of the organic matrix proteins that accumulates in shells. In situ hybridization revealed that nacrein was expressed in the outer epithelial cells of the mantle of the pearl oyster Pinctada fucata. The recombinant nacrein protein inhibited the precipitation of calcium carbonate from a saturated solution containing CaCl2 and NaHCO3, indicating that it can act as a negative regulator for calcification in the shells of molluscs. Because deletion of the Gly-X-Asn repeat domain of nacrein had a significant effect on the ability of nacrein to inhibit the precipitation of calcium carbonate, it is conceivable that the repeat domain has a primary role in the inhibitory function of nacrein in shell formation. Together these studies suggest that nacrein functions as a negative regulator in calcification in the extrapallial space between the shell and the mantle by inhibiting the precipitation of CaCO3.

Animal Structures↗
PubMed2000

Clinical trial results on the use of a recombinant feline interferon-omega to protect Japanese pearl oysters Pinctada fucata martensii from akoya-virus infection.

Japanese pearl oysters (akoya oyster: Pinctada fucata martensii) are currently suffering mass mortalities from akoya-virus infection. In the present study, akoya oysters were injected with an anti-viral, recombinant feline interferon-omega) (rFeIFN-omega) in an attempt to confer resistance to this virus. In infectivity experiments, oysters were twice injected with rFeIFN-omega at 1 mega unit kg(-1) of the meat weight. They were challenged with a single inoculation of cultured akoya-virus and held for 20 to 30 d at 25 degrees C. Control oysters received only the viral challenge without rFeIFN-omega administration. In prophylactic experiments, oysters that were given the akoya-virus on Days 1 to 5 after rFeIFN-omega administration showed lower mortalities. Furthermore, the survivors had fewer muscular lesions resulting from the virus infection than the controls. In treatment experiments, the virus was inoculated on Days 1 to 3 before rFeIFN-omega administration. None of the treated oysters died within a 30 d experimental period. Survivors displayed repaired lesions with fibrous tissues that were produced by enhanced agranulocytes in the body musculature. Moreover, rFeIFN-omega) was not toxic to akoya oysters. Thus, rFeIFN-omega administration is efficacious in preventing mortality of akoya oysters with akoya-virus infection.

Animals↗