Pinctada Fucata Research Articles

Laser imaging polarimetry of nacre.

Nacre is a complex biomaterial made of aragonite-tablet bricks and organic mortar that is considerably resilient against breakage. Nacre has been studied with a wide range of laboratory techniques, leading to understanding key fundamentals and informing the creation of bio-inspired materials. In this article, we present an optical polarimetric technique to investigate nacre, taking advantage of the translucence and birefringence of its microcomponents. We focus our study on 3 classes of mollusks that have nacreous shells: bivalve (Pinctada fucata), gastropod (Haliotis asinina and Haliotis rufescens) and cephalopod (Nautilus pompilius). We sent polarized light from a laser through thin samples of nacre and did imaging polarimetry of the transmitted light. We observed clear distinctions between the structures of bivalve and gastropod, due to the spatial variation of their birefringence. The patterns for cephalopod were more similar to bivalve than gastropod. Bleaching of the samples disrupted the transmitted light. Subsequent refilling of the bivalve and gastropod nacre samples with oil produced optical patterns similar to those of unbleached samples. In cephalopod samples, we found that bleaching produced irreversible changes in the optical pattern.

Molecular and functional analysis of PmCHST1b in nacre formation of Pinctada fucata martensii.

Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of a CHST1 gene identified from the pearl oyster Pinctada fucata martensii (PmCHST1b) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5' PSB motif and 3' PB motif) which interacted with 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC (P<0.05), and sulfate polymer content in the extrapallial fluid significantly reduced (P<0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation.

Seasonal and positional variations in the rate of nacre coating in Indian pearl oyster Pinctada fucata (Gould, 1850)

Recently technology has been perfected for the production of quality image pearls (mabe pearls) in India which fetches a good price ranging from $20-50 per pearl. This study focused on variations in the rate of nacre coating during different seasons when implanted with image pearl nucleus in different positions of the pearl oyster shell. The implantation was done at three positions at P1, P2 and P3 in both shells and the experiments were repeated in three seasons namely pre monsoon, monsoon and post monsoon. Among the positions, the position P2 which is the wide marginal mantle region gave the maximum nacre coating rate. Hydrological parameters such as atmospheric and water temperature, salinity, pH, turbidity, ammonia, nitrite, nitrate, phosphate, silicate, dissolved oxygen, primary productivity and chlorophyll a were also recorded during the study. Rate of nacre coating was found to be maximum (6.79 ± 2.07 μ/ day) during post monsoon months corresponding to maximum productivity (Net Primary Production of 204.40 ± 45.86 mgC/m3/day and Gross Primary Production of 361.40 ± 96.92 mgC/m3/day) and chlorophyll concentration (2.79 ± 0.13 mg/m³). The rate of coating was minimum (2.19 ± 0.17 μ/day) during pre monsoon season. The influence of hydrological parameters and positions of the implanted nuclei on the nacre coating were statistically analysed and discussed in the present paper.

Alv Protein Plays Opposite Roles in the Transition of Amorphous Calcium Carbonate to Calcite and Aragonite during Shell Formation

Amorphous calcium carbonate (ACC) is an important precursor in biominerals such as shells, coral, foraminiferans, and urchin spine. However, the mechanism underlying the transition from ACC to stable biosynthetic crystals is still poorly understood. Herein, we identified a matrix protein referred to as Alv in Pinctada fucata, which has dramatically opposite functions during the different transition processes from ACC to stable crystals—calcite and aragonite in shell formation. The functions of Alv were studied by RNA interference, binding of recombinant Alv (rAlv) to chitin, calcite and aragonite assay, ACC transition, in vitro crystallization, calcium carbonate precipitation, and near-UV CD spectra. We found that rAlv could promote nucleation during ACC crystallization, stimulate the transition from ACC to calcite, but suppress transition from ACC to aragonite. It is concluded that Alv is involved in the transition of ACC, and plays a crucial role in the formation of shells. As far as we know, Alv is one...

Sex-changing patterns of Akoya pearl oyster (Pinctada fucata)

BackgroundPearl production by transplantation in Akoya pearl oyster (Pinctada fucata) is a biotechnology developed in Japan that skillfully utilizes the pearl-forming ability of oysters. In this method, cultured pearls are formed from a pearl nucleus and a small piece of mantle transplanted into the gonads of recipient pearl oysters. In this study, we hypothesized that the sex of the recipient pearl oyster might affect the quality of pearl produced. While some previous studies have examined the sex of Akoya pearl oyster, detailed information is lacking.ResultsTo investigate sex in Akoya pearl oyster, we collected small gonadal fragments from 1-year-old pearl oysters by biopsy. Using the collected gonad fragment, the sex of the oysters was determined by microscopic observation, and the remaining samples were stored for gene expression analyses. All oysters were labeled to distinguish each individual for serial samplings every four months over the 2-year study period. At the start of experiment, nearly all of the pearl oysters were male, but the male:female ratio ofmale decreased over the course of the experiment. Interestingly, the number of males increased after spring, during the breeding season. This suggests that, in pearl oyster, sex is affected by season. Expression analysis of sex-related genes (Dmrt2, Vtg, Zp) indicated that all genes were expressed in all individuals and all periods.ConclusionsThese results suggest that Akoya pearl oysters are hermaphroditic, and that females appear as necessary, such as during the breeding season. These findings could contribute to higher efficiency and quality of pearl cultivation.

Molecular cloning and characteristics analysis of Pmtgfbr1 from Pinctada fucata martensii

Pinctada fucata martensii is cultured for pearl production. Growth improvement has received considerable research interest. Transforming growth factor β type Ⅰ receptor (TβR-I), which is involved in signals transmission of transforming growth factor beta (TGF-β), participates in cell proliferation and growth. In this study, we characterized a Tgfbr1 gene which encoded TβR-I from P. fucata martensii (Pmtgfbr1). Pmtgfbr1 cDNA contains an open reading frame of 1569 bp and encodes a polypeptide of 522 amino acids (aa). Pmtgfbr1 possesses a typical TβR-I structure (extracellular receptor ligand domain, transmembrane domain, and cytoplasmic tyrosine kinase catalytic domain). Pmtgfbr1 is expressed in all the studied tissues and exhibited the highest expression level in the adductor muscle. Moreover, Pmtgfbr1 exhibited the lower expression level in the larger group (L) than that in the smaller group (S) and is negatively correlated with growth traits (P < 0.01). Our results indicated that Pmtgfbr1 is a candidate functional gene associated with growth traits.

Molecular cloning and characterization of a putative mitogen-activated protein kinase (Erk1/2) gene: Involvement in mantle immunity of Pinctada fucata

Extracellular signal-regulated kinases (ERKs) are conserved and related with protein-serine/threonine kinases that participate in the regulation of multiple biological processes, such as cell survival, cell differentiation, proliferation, metabolism, and inflammation. However, little is known about the roles of this kinase in the pearl oyster. In this study, we cloned and identified an ERK homolog from Pinctada fucata (PfErk). Furthermore, we have unraveled its expressional kinetics after lipopolysaccharide (LPS) and polyinosinic-epolycytidylic acid (poly I:C) immune challenge. Pferk harbored a 5′ untranslated region (UTR) of 12 bp, a coding sequence of 1074 bp, and a 3′ UTR of 882 bp. The putative peptide comprised a predicted molecular mass of 41.19 kDa, with a theoretical pI of 6.15. Sequence analysis showed that it possesses one STK catalytic domain and a conserved His-Arg-Asp (HRD) domain. In addition, a canonical Thr-Glu-Tyr (TEY) dual phosphorylation motif and an ATRW substrate binding site were also identified in the coding protein. Homology assessment of PfErk showed high similarity to Homo sapiens ERK. Phylogenetic analysis supported a close evolutionary relationship with molluscan orthologs. The expression patterns of Pferk were observed in seven different tissues of pearl oyster, with highest expression in the mantle and lowest expression in the digestive gland. Pferk mRNA expression levels were detected at developmental stages, with the highest expression in D-shaped larvae, followed by the 32-cell stage. The mRNA expression of Pferk was upregulated significantly in P. fucata mantle primary cells and mantle tissue after LPS and poly (I:C) treatment, and PfErk phosphorylation levels were activated by LPS and poly (I:C) challenges. Overall, our results suggested that PfErk may play important roles in pearl oyster innate immunity, and provided a new understanding of mantle immunity in the pearl oyster.

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in Pinctada fucata.

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.

Identification, Characterization, and Evaluation of Polymorphic Microsatellite Markers for Parentage Assignment in Pinctada fucata

AbstractParentage analysis is a useful tool for establishing pedigree relationships in selective breeding. In this study, 31 microsatellite markers were tested, and 13 loci were subsequently used to identify parenthood in Pinctada fucata. According to simulation analysis, the power of nine loci to exclude false parents was 99.94%, and that of 10 loci was 99.95%. Moreover, using 10 loci and known parental and filial information, the cumulative assignment success rate to one true parental pair was as high as 99%. Of the 120 progeny tested, 93.3% were exclusively assigned to their parental pairs in mixed pedigrees. These results suggested that these microsatellite markers can be used for rapid and effective parentage assignment and contribute to the selective breeding of P. fucata.

Relationship between primary structure or spatial conformation and functional activity of antioxidant peptides from Pinctada fucata

Antioxidant peptides from Pinctada fucata are ubiquitous in nature and can be utilized as ingredients in cosmetics or functional foods to positively regulate oxidative species in the human body against oxidation. Here, mutated peptides and a homologous series of peptides were designed and prepared, based on the original peptide sequence of an antioxidant peptide from Pinctada fucata meat (PFMAP), to better understand the structural relationships, including primary structure, spatial conformation and activity. The results showed that the antioxidant activity factors in order of importance were spatial conformation, amino acid sequence, amino acid position, amino acid composition, peptide chain length and degree of side-chain amino acid glycosylation. Moreover, this protocol had incomparable advantages in purity, speed and cost of preparing polypeptides, relative to enzymatic hydrolysis or chemosynthesis methods. This study also supplies test data information and a theoretical basis for the higher-value application of polypeptides from marine sources.

DNA Techniques Applied to the Identification of Pinctada Fucata Pearls from Uwajima, Ehime Prefecture, Japan

DNA Techniques Applied to the Identification of Pinctada Fucata Pearls from Uwajima, Ehime Prefecture, Japan

Newly isolated bacterium Tenacibaculum sp. strain Pbs-1 from diseased pearl oysters is associated with black-spot shell disease

In recent years, black-spot shell disease has appeared on the shells of Akoya pearl oysters, Pinctada fucata, causing serious problems in cultured pearl production. This disease was believed to be caused by boring of Polydora ciliata into calcareous substrate, and a control measure against P. ciliata was developed. As a result, boring activity of P. ciliate decreased, but black-spot shell disease remains a serious problem. We detected the genus Tenacibaculum specifically from the shells of diseased oysters using PCR-DGGE, and isolated Tenacibaculum sp. strain Pbs-1 from the diseased oyster shells. Analyses of 16S rRNA gene sequence homology and its biochemical and morphological characteristics suggested that strain Pbs-1 was a new species of Tenacibaculum. We conducted four infectivity experiments by different infection methods: smear, injection, immersion, or a combination of wound and immersion. The highest infectivity (100%) in the shell was observed in the test group by the smear method, which was accompanied by severe clinical signs that included a focal blackish discoloration of the shell. The results of the infectivity experiments indicated that the newly isolated Tenacibaculum sp. strain Pbs-1 is one of the putative causative agents of black-spot shell disease in Akoya pearl oysters.

Adaptive response of pearl oyster Pinctada fucata martensii to low water temperature stress

The pearl oyster Pinctada fucata martensii is a warm-water shellfish that is sensitive to cold environments. To investigate its potential adaptation to low-temperature stress, the selected line (SL) and based population (BP) were sampled to undergo transcriptome sequence. Results of transcriptome analysis showed 572 significant differentially expressed genes. The typical HSP70 and HSP40 exhibited the polar expression model in the two groups. Meanwhile, the related genes that involved in energy release mediated by oxidative phosphorylation and the biosynthesis of unsaturated fatty acid were increased in the SL. The apparent enrichment of different expressed genes in amino acid metabolism indicated that the small molecule system with amino acids was one of the main regulator for low-temperature stress. The different expressions of immune-related and lysosome protein encoding genes also reflected the variation of immunity in the two groups and indicated that it could affect the adaptation ability in different temperature. In addition, the similar trends of different expression of typical genes between two groups were obtained by using RNA-seq and qRT-PCR. These results suggested that multi-system adjustments are involved in the processes of low water temperature stress in pearl oyster, providing insights into the response systems of shellfish to acclimatise with ambient environment change.

Selective Synergism Created by Interactive Nacre Framework-Associated Proteins Possessing EGF and vWA Motifs: Implications for Mollusk Shell Formation.

In the nacre layer of the Pinctada fucata oyster shell there exists a multimember proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally defined combinatorial model consisting of two recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain (vWA)) and r-n16.3 (containing an EGF-like domain), whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca2+-mediated protein-protein interactions (∼4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca2+ there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp/Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization.

Growth and survival of host pearl oyster Pinctada fucata martensii (Dunker, 1880) treated by different biofouling-clean methods in China

Pearl oyster Pinctada fucata martensii is an economically important species farmed to produce round nucleated pearl in China. However, pearl oyster shells are commonly exposed to biofouling, which is manually cleaned. This study experimentally evaluates the effects of biofouling cleaning by Siganus fuscescens (Houttuyn, 1782) foraging on growth performance and pearl traits of host pearl oyster P. fucata martensii. Three groups designated as EG1, EG2 and EG3 were set in the experiment. In EG1, biofouling was manually cleaned every 2 weeks and oysters were cultured in natural sea conditions. In EG2, biofouling was cleaned by foraging behavior of S. fuscescens every 2 weeks and oysters were cultured in natural sea conditions. In EG3, oysters were farmed in the net cage where S. fuscescens were stored. The survival rate of EG2 was higher than those of EG1 and EG3 and the daily growth rates of length and weight of shell and the thickness and weight of pearl of EG3 were significantly larger than those of EG1 and EG2. The survival rate, daily growth rates of the length and weight of shell, and thickness and weight of pearl were not significantly different between EG1 and EG2. This study suggested S. fuscescens foraging could evidently improve survival of host pearl oyster, which is promising to improve pearl yield in pearl oyster farms in China.

Deleterious Effects of Harmful Dinoflagellates and Raphidophytes on Egg Viability and Spermatozoa Swimming Velocity in the Japanese Pearl OysterPinctada fucata martensii

Several species of harmful algal blooms (HAB) exert inimical effects on bivalve molluscs, with mass mortalities and pathologies reported worldwide. Information on the effects of HAB on the reproduction of bivalve molluscs, however, remains very limited. The Japanese pearl oyster Pinctada fucata martensii is an important economic species cultured in Japan. In previous studies, harmful dinoflagellates and raphidophytes were shown to affect embryos and larvae of Japanese pearl oyster. In the present study, quantitative deleterious effects of Alexandrium catenella, Alexandrium affine, Heterosigma akashiwo, and Chattonella marina on egg viability and spermatozoa swimming velocity are reported. All four HAB species significantly reduced the swimming velocity of spermatozoa. Egg viability was affected only after exposure to A. catenella and A. affine. An additional cytotoxicity test using Vero cells showed that bioactive exudates are associated with the deleterious effects and these effects are more prominent during the death phase of the algal culture. The results of the present study warrant further research on the effects of HAB species on gamete quality of bivalves, further characterization of bioactive compounds secreted by Alexadnrium spp., and highlight the role of physical activation of the toxic mechanism of harmful raphidophytes suggested in a previous study and confirmed in the present study.

Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization

In bivalves, the mantle tissue secretes organic matrix and inorganic ions into the extrapallial space (EPS) to form the shells. In addition, more and more evidences indicate the participation of hemocytes in shell mineralization, but no direct evidence has been reported that verifies the presence of hemocytes in the EPS, and their exact roles in biomineralization remain uncertain. Here, we identified hemocytes from the EPS of Pinctada fucata. Numerous components involved in cellular and humoral immunity were identified by proteome analysis, together with several proteins involved in calcium metabolism. The hemocytes exerted active phagocytosis and significantly upregulated the expression of immune genes after immune stimulation. A group of granulocytes were found to contain numerous calcium-rich vesicles and crystals, which serve as a calcium pool. During shell regeneration, some genes involved in calcium metabolism are upregulated. Strikingly, most of the shell matrix proteins were absent in the hemocytes, suggesting that they might not be solely responsible for directing the growth of the shell. Taken together, our results provided comprehensive information about the function of hemocytes in immunity and shell formation.

Functional Expression, Purification and Identification of Modified Antioxidant Peptides from Pinctada fucata Muscle

The aim of this study was to establish a system for the functional expression, purification and identification of new subtypes of modified antioxidant peptides from Pinctada fucata muscle (named MMP). After molecular modification, MMP had a primary coil spatial conformation that resembled an α-helix and improved the functional activity of MMP. The MMP DNA sequence was modified and codon optimized based on the amino acid sequence of the natural antioxidant peptide (named MP), which was isolated and purified from Pinctada fucata muscle by enzymatic hydrolysis. Antioxidant activity assay results showed that the IC50 values for DPPH-radical scavenging activity, superoxide-radical scavenging activity and hydroxyl-radical scavenging activity of MMP were 0.056, 0.310 and 0.126 mg/ml, respectively, which were higher than the antioxidant activities of enzymatically digested or chemically synthesized MMP (p < 0.05). In addition, this protocol exhibited great advantage in purity, speed, cost, and degree of automation for preparing MMP compared with enzymatic hydrolysis or chemical synthesis. Thus, the proposed method can be used to obtain MMP with high antioxidant activity and at a high yield. In addition, this method provided test data and a theoretical basis for high-value application of polypeptides from marine sources.

Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin

Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5′untranslated region (UTR) of 11 bp, a 3′UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.

Fam20C participates in the shell formation in the pearl oyster, Pinctada fucata

Kinase-family with sequence similarity 20, member C (Fam20C) is a protein kinase, which can phosphorylate biomineralization related proteins in vertebrate animals. However, the function of Fam20C in invertebrate animals especially the role in biomineralization is still unknown. Herein, we cloned the cDNA of fam20C from the pearl oyster, Pinctada fucata. It is showed that the expression of fam20C in the mantle edge was much higher than other tissues. In situ hybridization showed that fam20C was expressed mostly in the outer epithelial cells of the middle fold, indicating it may play important roles in the shell formation. Besides, fam20C expression increased greatly in the D-shape stage of pearl oyster development, when the shell was first formed. During the shell repair process, the expression level of fam20C increased 1.5 times at 6 h after shell notching. Knockdown of fam20C in vivo by RNA interference resulted in abnormally stacking of calcium carbonate crystals at the edges of nacre tablets, showing direct evidence that fam20C participates in the shell formation. This study provides an insight into the role of kinase protein in the shell formation in mollusk and broaden our understanding of biomineralization mechanism.