Comparison of expression patterns of shell matrix protein genes in the mantle tissues between high- and low-quality pearl-producing recipients of the pearl oyster, Pinctada fucata

The production of a cultured pearl is the result of a complex interplay between the donor and recipient oysters. However, there is a paucity of information on the relationship between donor and recipient oyster gene expression patterns and pearl quality. Shell matrix proteins affect not only the formation of the shell, but also that of the pearls. We compared the gene expression patterns of five shell matrix proteins (msi60, nacrein, msi31, prismalin-14, and aspein) in the mantle edge (ME), which forms the prismatic layer, and the mantle center (MC), which forms the nacreous layer, between high- (HP) and low quality pearl- (LP) producing recipient oysters. After culturing for about two months, ME and MC tissues were collected from nine recipient oysters: four with HP, five with LP. In the ME, the average threshold cycle (ΔC(T)) for aspein was higher in HP than in LP (t-test, p = 0.03). Additionally, in the MC, the average ΔC(T) for msi60 was lower in HP than in LP (p = 0.06). This means the relative expression level of msi60 in the mantle of HP was higher than that of LP, and expression level of aspein in the mantle of HP was lower than that of LP. Pearl quality was closely related to the expression patterns of shell matrix protein genes of recipient oysters.     

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in <Emphasis Type="Italic">Pinctada fucata</Emphasis>

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.     

Temperature and Food Influence Shell Growth and Mantle Gene Expression of Shell Matrix Proteins in the Pearl Oyster Pinctada margaritifera

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.     

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

Cultured Pearl Surface Quality Profiling by the Shell Matrix Protein Gene Expression in the Biomineralised Pearl Sac Tissue of <Emphasis Type="Italic">Pinctada margaritifera</Emphasis>

Nucleated pearls are produced by molluscs of the Pinctada genus through the biomineralisation activity of the pearl sac tissue within the recipient oyster. The pearl sac originates from graft tissue taken from the donor oyster mantle and its functioning is crucial in determining key factors that impact pearl quality surface characteristics. The specific role of related gene regulation during gem biogenesis was unknown, so we analysed the expression profiles of eight genes encoding nacreous (PIF, MSI60, PERL1) or prismatic (SHEM5, PRISM, ASP, SHEM9) shell matrix proteins or both (CALC1) in the pearl sac (N?=?211) of Pinctada margaritifera during pearl biogenesis. The pearls and pearl sacs analysed were from a uniform experimental graft with sequential harvests at 3, 6 and 9 months post-grafting. Quality traits of the corresponding pearls were recorded: surface defects, surface deposits and overall quality grade. Results showed that (1) the first 3 months of culture seem crucial for pearl quality surface determination and (2) all the genes (SHEM5, PRISM, ASP, SHEM9) encoding proteins related to calcite layer formation were over-expressed in the pearl sacs that produced low pearl surface quality. Multivariate regression tree building clearly identified three genes implicated in pearl surface quality, SHEM9, ASP and PIF. SHEM9 and ASP were clearly implicated in low pearl quality, whereas PIF was implicated in high quality. Results could be used as biomarkers for genetic improvement of P. margaritifera pearl quality and constitute a novel perspective to understanding the molecular mechanism of pearl formation.     

Gene Expression Patterns in the Outer Mantle Epithelial Cells Associated with Pearl Sac Formation

For pearl culture, nucleus and mantle grafts are implanted into the gonad of the host oyster. The epithelial cells of the implanted mantle graft elongate and surround the nucleus, and a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in pearl formation. We studied the gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the epithelial cells associated with pearl sac formation. There were differences in the expression patterns of the six genes in the epithelial cells, and the relative expression levels for msi60 and aspein differed between the mantle graft and pearl sac (48 days after implantation). Therefore, the gene expression patterns of the epithelial cells were genetically undetermined, and changed between before and after pearl sac formation. The gene expression patterns of the epithelial cells of the pearl sac may be regulated by the host oysters.     

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.     

Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.     

Can the Quality of Pearls from the Japanese Pearl Oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>) 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.