Japanese Pearl Oyster Pinctada Fucata Research Articles

Effect of different microalgal diets on the growth performance of Japanese pearl oyster Pinctada fucata martensii broodstock

Aquaculture ResearchVolume 51, Issue 11 p. 4809-4813 SHORT COMMUNICATION Effect of different microalgal diets on the growth performance of Japanese pearl oyster Pinctada fucata martensii broodstock Satoshi Yamamoto, Corresponding Author Satoshi Yamamoto y-sato4@bio.mie-u.ac.jp orcid.org/0000-0003-3429-5938 Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, Japan Correspondence Satoshi Yamamoto, Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan. Email: y-sato4@bio.mie-u.ac.jpSearch for more papers by this authorTsutomu Tsutsui, Tsutomu Tsutsui Faculty of Marine Bioresources, Mie Maritime High School, Shima, JapanSearch for more papers by this authorTomoyuki Saguchi, Tomoyuki Saguchi Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this authorNaoaki Tsutsui, Naoaki Tsutsui Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this authorTakao Yoshimatsu, Takao Yoshimatsu Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this author Satoshi Yamamoto, Corresponding Author Satoshi Yamamoto y-sato4@bio.mie-u.ac.jp orcid.org/0000-0003-3429-5938 Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, Japan Correspondence Satoshi Yamamoto, Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan. Email: y-sato4@bio.mie-u.ac.jpSearch for more papers by this authorTsutomu Tsutsui, Tsutomu Tsutsui Faculty of Marine Bioresources, Mie Maritime High School, Shima, JapanSearch for more papers by this authorTomoyuki Saguchi, Tomoyuki Saguchi Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this authorNaoaki Tsutsui, Naoaki Tsutsui Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this authorTakao Yoshimatsu, Takao Yoshimatsu Laboratory of Shallow Sea Aquaculture, Graduate School of Bioresources, Mie University, Tsu, JapanSearch for more papers by this author First published: 17 July 2020 https://doi.org/10.1111/are.14785Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume51, Issue11November 2020Pages 4809-4813 RelatedInformation

Troponin T from the Japanese Pearl Oyster <i>Pinctada fucata</i>: Molecular Cloning, Tissue Distribution, Gene Structure, and Interaction Analysis with Tropomyosin

Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.

Pearl formation in the Japanese pearl oyster ( Pinctada fucata ) by CaCO 3 polymorphs: Pearl quality‐specific biomineralization processes and their similarity to shell regeneration

Pearl oyster shell consists of two layers: a calcite prismatic layer (outer layer) and an aragonite nacreous layer (inner layer). Calcite and aragonite are CaCO3 polymorphs, and their formations are controlled by shell-forming tissue called mantle. Pearl sacs originating in the mantle form cultured pearls. Therefore, it has been widely accepted that pearl and shell are produced by the same process. However, this idea has been called into question by some recent mineralogical studies indicating microstructural and crystal-polymorphic diversity in pearls. The pearl biomineralization process is still not fully understood in detail. Thus, in this study, we focused on the diversity of CaCO3 polymorphism of non-nacreous structures (NNSs) underlying the nacreous layer in pearl and regenerated shell, to reveal the biomineralization process of the Japanese pearl oyster (Pinctada fucata). Using Meigen's stain and scanning electron microscope-energy dispersive X-ray (SEM-EDX), NNSs polymorphs in valuable and valueless pearls, in addition to regenerated shell, were compared. Aragonite was exclusively observed in the NNSs of valuable pearls, whereas calcite was dominant in those of valueless pearls. The same analysis of NNSs of regenerated shells was carried out. As in valueless pearls, almost all regenerated shell NNSs consisted of calcite, but one NNS was composed of aragonite. Accordingly, it seems that pearls are formed by the same biomineralization process as shell regeneration rather than shell formation.

Tropomyosin Isoform Expression in the Adductor Muscle of the Japanese Pearl Oyster, <i>Pinctada fucata</i>

We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.

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.

Ca<sup>2+</sup>-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, <i>Pinctada fucata</i>

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational change in Pifuc-TnC.

Lethal effects of ichthyotoxic raphidophytes, Chattonella marina, C. antiqua, and Heterosigma akashiwo, on post-embryonic stages of the Japanese pearl oyster, Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5×102cellsml−1), C. antiqua (103cellsml−1), and H. akashiwo (5×103cellsml−1) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5×103cellsml−1 and C. marina at 8×103cellsml−1. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 105cellsml−1 more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8×103cellsml−1, 24h), C. marina (8×103cellsml−1, 24h), and H. akashiwo (104cellsml−1, 24h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (103cellsml−1, 24h),C. marina (8×103cellsml−1, 24h), and H. akashiwo (5×104cellsml−1, 24h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5×102–8×103cellsml−1, 48–86h) and C. antiqua (103–8×103cellsml−1, 72–86h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.

Pif97, a von Willebrand and Peritrophin Biomineralization Protein, Organizes Mineral Nanoparticles and Creates Intracrystalline Nanochambers.

The formation of the mollusk nacre layer involves the assembly and organization of mineral nanoparticles into fracture-toughened mesoscale-sized aragonite tablets that possess intracrystalline nanoporosities. At least one nacre protein family, known as the framework proteome, is strategically located as part of a macromolecular coating around each nacre tablet and is believed to participate in tablet formation. Here, we report new studies of a recombinant form (rPif97) of a unique Japanese pearl oyster (Pinctada fucata) nacre framework biomineralization protein, Pif97. This unique protein possesses both a von Willlebrand factor type A domain (vWA, F23-Y161) and a Peritrophin A chitin-binding domain (PAC, E234-D298). rPif97 self-associates or aggregates to form amorphous protein phases that organize both amorphous and single-crystal calcium carbonate nanoparticles in vitro. Further, in the presence of nucleating calcite crystals, rPif97 protein phases deposit onto these crystals and become occluded over time, forming nanochambers within the crystal interior. The formation of these mineral-modifying amorphous protein phases is linked to the presence of intrinsic disorder and amyloid-like cross-β-strand aggregation-prone regions, and three-dimensional modeling indicates that both the vWA and PAC domains are accessible for intermolecular interactions. Thus, the vWA- and PAC-containing Pif97 protein exhibits key functionalities that would allow its participation in mollusk nacre layer tablet assembly and porosity formation.

Evaluation of genetic characteristics of wild and cultured populations of the Japanese pearl oyster Pinctada fucata martensii by using AFLP markers

Genetic characteristics of four wild (Mie, Fukui, Shimane and Nagasaki) and five cultured populations (selectively bred for 12 years with an origin of Ehime population) of the Japanese pearl oyster, Pinctada fucata martensii, were evaluated by using AFLP markers. Six primer pairs generated 1019 loci in total, among which 45.2–55.1 % was polymorphic among populations. Although there was no significant difference in gene diversity between wild (0.170–0.174) and cultured (0.158–0.173) populations, genetic relatedness in cultured populations (0.316–0.450) was about three times higher than that in wild populations (0.110–0.165). In addition, genetic differentiation was about twenty times larger in cultured populations (Nei’s distance: 0.0111) than in wild populations (Nei’s distance: 0.0005). These results mean that selective breeding can cause marked inbreeding as well as large genetic differentiation among cultured populations in a short period. On the other hand, it was suggested that genetic homogenization in the wild, probably due to a large-scale transport of cultured oysters, had progressed in the sea around Japan. In conclusion, it is necessary to prevent inbreeding by the reconsideration of the style of selective breeding in cultured populations, while the escape of gametes or spats of cultured strains in the wild should be avoided for the preservation of genetic characteristics in native populations of P. f. martensii.

Correlation between Nacre-deposition Ability and Shell-closing Strength in Japanese Pearl Oyster Pinctada fucata

Correlation between Nacre-deposition Ability and Shell-closing Strength in Japanese Pearl Oyster Pinctada fucata

Differential inimical effects of Alexandrium spp. and Karenia spp. on cleavage, hatching, and two larval stages of Japanese pearl oyster Pinctada fucata martensii

The effects of four harmful and potentially harmful dinoflagellates, Alexandrium affine, Alexandrium catenella, Karenia mikimotoi and Karenia papilionacea, on the early-life development of Japanese pearl oyster, Pinctada fucata martensii, were assessed. Density- and time-dependent, mild to severe effects on cleavage, hatching, D-larvae, and pre-settling larvae of pearl oysters were found. The non-PST-producer A. affine was highly toxic to both cleavage and hatching with potent lytic activity at a density of 2.5×102cellsml−1. The PST-producer A. catenella also affected cleavage and reduced hatching, but at 2-fold higher density with lytic activity only at the highest density tested (2×103cellsml−1). Cleavage was affected by K. mikimotoi and K. papilionacea at 1.5×104cellsml−1, but hatching was only affected by K. mikimotoi. Mortalities in both larval stages were not observed. D-larvae were not affected by K. papilionacea, but their activity decreased following exposure to A. affine, A. catenella, and mainly K. mikimotoi. Pre-settling larvae were more sensitive to all four harmful algae than were D-larvae. The dinoflagellate A. catenella had the highest effect on the activity of pre-settling larvae (10cellsml−1), followed by K. mikimotoi (5×102cellsml−1), A. affine (5×102cellsml−1), and K. papilionacea (1.5×104cellsml−1). The results of this study suggest that complex mechanisms, including paralytic shellfish toxins (PST), brevetoxins (PbTx) and a variety of lytic and membrane-disruptive toxins and/or other metabolites, could have been involved in such inimical stage-specific effects. It also highlight the threat posed by harmful algae on the recruitment of Japanese pearl oysters and potentially other bivalve species, and show that they could exert subchronic to chronic effects at realistically low cell densities, and impact bivalve populations through altered reproductive and recruitment processes, ultimately hampering both wild populations and aquaculture industries.

Nacre Protein Sequence Compartmentalizes Mineral Polymorphs in Solution

The Japanese pearl oyster (Pinctada fucata) n16 framework matrix protein is an integral part of the growth and formation of the mollusk shell biomineralization mechanism. It is a required component of the extracellular matrix with a dual mineralization role, as an anchor component to synchronize the assembly of the beta-chitin and N-series, Pif-series protein extracellular matrix for aragonite formation and as a regulator of aragonite formation itself. However, the mechanism by which this protein controls aragonite formation is not understood. Here, we investigate the mineralization potential and kinetics of the 30 AA N-terminal portion of the n16 protein, n16N. This sequence has been demonstrated to form either vaterite or aragonite depending upon conditions. Using in situ potentiometric titration methods, we find that n16N is indeed responsible for the self-assembly characteristics found in vivo and in vitro but is not involved with active Ca2+ binding or mineral nucleation processes. Upon the basis of time- and peptide concentration-dependent sampling of mineral deposits that form in solution, we find that n16N is responsible for controlling where mineralization occurs in bulk solution. This protein sequence acts as a molecular spacer that organizes the mineralization space and promotes the formation of mineral constituents that contain ACC, vaterite, and aragonite. Without the concerted action of the n16N assemblage, unregulated calcite formation occurs exclusively. Thus, the n16 protein provides the regulation needed to have the characteristic polymorph, crystalline orientations, and related mechanical properties associated to the microstructure of mollusk shells.

A Pearl Protein Self-Assembles To Form Protein Complexes That Amplify Mineralization

The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada fucata mantle gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess an EF-hand-like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered "imitator" EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF-hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF-hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-β-strand sequences within the putative PFMG1 pseudo-EF-hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously sized protein oligomers and films in vitro, and this process is enhanced by Ca(2+), which promotes the formation of aggregation-prone extended β-strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing invasive threats to the mantle organ.

Evolution and functional analysis of the Pif97 gene of the Pacific oyster Crassostrea gigas

Abstract Mollusc shell matrix proteins (SMPs) are important functional components embedded in the shell and play a role in shell formation. A SMP (Pif177) was identified previously from the nacreous layer of the Japanese pearl oyster Pinctada fucata, and its cleavage products (named pfPif97 and pfPif80 proteins) were found to bind to the chitin framework and induce aragonite crystal formation and orient the c axis. In this study, a homologue of pfPif177 was cloned from the mantle of the Pacific oyster Crassostrea gigas, containing the homologue of pfPif97 only and not pfPif80. This finding hints at the large divergence in gene structure between the two species. This homologue (cgPif97) shares characteristics with pfPif97, and suggests that the biological functions of these two proteins may be similar. The expression pattern of cgPif97 in different tissues and development stages indicates that it may play an important role in shell formation of the adult oyster. The morphology of the inner shell surface was affected by injected siRNA of cgPif97 and the calcite laths of the shell became thinner and narrower when the siRNA dose increased, suggesting that the cgPif97 gene plays an important role in calcite shell formation in C. gigas. In conclusion, we found evidence that the Pif177 gene evolved very fast but still retains a similar function among species.

Development of an artificial fertilization method for the Japanese pearl oyster Pinctada fucata martensii by using a microplate

Development of an artificial fertilization method for the Japanese pearl oyster Pinctada fucata martensii by using a microplate

Effects of cooling rate on post-thaw motility and fertility of Japanese pearl oyster Pinctada fucata martensii spermatozoa

The post-thaw motility and fertility of Japanese pearl oyster sperm show large variances, even among sperm samples obtained from the same individuals. This study aimed to clarify the factors that cause such differences. Spermatozoa were diluted 50 times with diluent comprising 10 % methanol, 18 % fetal bovine serum, and 72 % seawater, and dispensed into 0.25 ml straws. A total of 59 straws were cooled, one by one, at 11 different heights from the surface of liquid nitrogen (LN) to −50 °C, and then immediately immersed in LN. After thawing the straws, the relationships between the cooling rate and the post-thaw motility and post-thaw fertility of the spermatozoa were examined. Both the post-thaw motility and the post-thaw fertility showed a sharp peak when the straws were cooled at around −20 °C/min. There was a strong correlation between post-thaw motility and fertility (P < 0.001). There was a large difference in the cooling rates and the post-thaw motilities and fertilities of the spermatozoa, even between straws cooled at the same height. These results indicate that the optimum range for the cooling rate of oyster spermatozoa is quite narrow, and the method of cooling straws at a fixed distance from the LN surface is unsuitable for the cryopreservation of Japanese pearl oyster spermatozoa.

Polymorph Crystal Selection by n16, an Intrinsically Disordered Nacre Framework Protein

Framework proteins are a subclass of mollusk shell nacre-associated polypeptides that form supramolecular assemblies with β-chitin and other matrix proteins. These macromolecular assemblies manage the energetics of aragonite polymorph nucleation, and thus, there is keen interest in understanding the molecular characteristics of framework proteins. Here, we report the mineralization activity, oligomerization, and structural features of a recombinant framework nacre protein n16, isoform 3 (r-n16.3, Japanese pearl oyster Pinctada fucata). We find that r-n16.3 assembles in mineralization solutions to form spheroidal-fibril and mineralized thin film assemblies, in addition to spherical vaterite mineral deposits and aragonite single crystal deposits that possess unusual texture and layered morphologies. The oligomerization of r-n16.3 is spontaneous over the pH range 5–8.5, and protein particle sizes are observed to increase in radii when Ca(II) is present. Bioinformatics studies reveal that the r-n16.3 molecule is intrinsically disordered (random coil) and possesses residual α helix and β sheet structure. Experimentally, we confirmed that the secondary structure of apo-r-n16.3 assemblies is largely disordered (50% random coil, 20% β strand, 8% α helix). However, in the presence of high Ca(II) concentrations, we observe IDP disorder-to-order transformations that increase β turn structure and decrease random coil, α helix, and β strand contents. We conclude that r-n16.3 is an intrinsically disordered oligomeric nacre framework protein that nucleates vaterite and single crystal aragonite in vitro and possesses target-specific IDP disorder-to-order transformation capabilities in response to Ca(II).

Identification and Characterisation of a Calcium Carbonate‐Binding Protein, Blue Mussel Shell Protein (BMSP), from the Nacreous Layer

The nacreous layer of molluscan shells consists of a highly organised, layered structure comprising calcium carbonate aragonite crystals, each surrounded by an organic matrix. In the Japanese pearl oyster Pinctada fucata, the Pif protein from the nacreous layer functions in aragonite binding, and plays a key role in nacre formation. Here, we investigated whether the blue mussel Mytilus galloprovincialis also has a protein with similar functions in the nacreous layer. By using a calcium carbonate-binding assay, we identified the novel protein blue mussel shell protein (BMSP) 100 that can bind calcium carbonate crystals of both aragonite and calcite. When the entire sequence of a cDNA encoding BMSP 100 was determined, it was found that BMSP is a preproprotein consisting of a signal peptide and two proteins, BMSP 120 and BMSP 100. BMSP 120 contains four von Willebrand factor A (VWA) domains and one chitin-binding domain, thus suggesting that it has a role in maintaining structure within the matrix. Immunohistochemical analysis revealed that BMSP 100 is present throughout the nacreous layer with dense localisation in the myostracum. Posttranslational modification analysis indicated that BMSP 100 is phosphorylated and glycosylated. These results suggest that there is a common molecular mechanism between P. fucata and M. galloprovincialis that underlies the nacreous layer formation.

Silk Fibroin Hydrogels Coupled with the n16N−β-Chitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization

Previous results have shown that the nacre specific peptide, n16N, from the Japanese pearl oyster Pinctada fucata has a binding affinity for β-chitin. As a result, the n16N-chitin assembly is able to selectivity nucleate aragonite. Here, we have added silk fibroin hydrogels to the in vitro assay to more fully represent the in vivo matrix. Crystallization, with a silk fibroin hydrogel and n16N on β-chitin, results in metastable vaterite and amorphous calcium carbonate, which form as flat deposits with hemispherical centers. Acidic peptide controls (p-Asp/p-Glu) were also tested in the silk-chitin assay and result in flat calcite that grows into the β-chitin substrate. Fluorescence imaging of that matrix, made with labeled n16N, shows that n16N binds to β-chitin in the presence of silk gel. These results demonstrate that the addition of a silk hydrogel to the n16N−β-chitin assembly changes the microenvironment for mineralization. This work contributes to our understanding of the roles of individual nacre ma...

Can the Quality of Pearls from the Japanese Pearl Oyster (Pinctada fucata) be Explained by the Gene Expression Patterns of the Major Shell Matrix Proteins in the Pearl Sac?

For pearl culture, the pearl oyster is forced open and a nucleus is implanted into the gonad with a mantle graft. The outer mantle epithelial cells of the implanted mantle graft elongate and surrounding the nucleus a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in the regulation of pearl formation. Recently, seven shell matrix proteins were identified from the pearl oyster Pinctada fucata. However, there is a paucity of information on the function of these proteins and their gene expression patterns. Our study aims to elucidate the relationship between pearl type, quality, and gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the pearl sac based on real-time PCR analysis. After culturing for about 2 months, the pearl sac tissues were collected from 22 individuals: 12 with high quality (HP), nine with low quality (LP), and one with organic (ORG) pearl formation. The surface of each of the 12 HP pearls was composed only of a nacreous layer; in contrast, that of the nine LP pearls was composed of nacreous and prismatic layers. The six target gene expressions were detected in all individuals. However, delta threshold cycle (ΔC(T)) for msi31 was significantly higher in the HP than in the LP individuals (Mann-Whitney's U test, p=0.02). This means that the relative expression level of msi31, which constitutes the framework of the prismatic layer, was higher in the LP than in the HP individuals.