Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata.

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Structural and functional analyses of calcium ion response factors in the mantle of Pinctada fucata.

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.

A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Structural and functional analyses of a TIMP and MMP in the ligament of Pinctada fucata.

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods. PfTIMP consists of 143 amino acid residues and has a molecular weight of 13,580.4. To investigate the activity of PfTIMP, inhibition of matrix metalloproteinase (MMP) activity was measured. PfTIMP strongly inhibited human MMP13 and MMP9. Eight MMP homologs were identified from a P. fucata genomic database by BLAST search. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double stranded (ds) RNA targeting MMP54089 was injected into living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.

Cryopreservation of sperm from seven-band grouper, Epinephelus septemfasciatus.

In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me2SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6±8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min⁻¹); however, the post-thaw motility of sperm cooled to -30 °C was significantly lower than that of other cooled temperatures (-40 to -70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me2SO with 95% FBS and cooled to at least -40 °C before immersion in liquid nitrogen.