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The triangle-sail mussel (Hyriopsis cumingii) is the most important freshwater pearl-bearing mussel in China. Nucleated pearls from the mantle of H. cumingii are emerging pearl cultivation products. Compared to traditional non-nucleated and nucleated pearls from visceral mass, nucleated pearls from the mantle show significant cultivation advantages, such as high pearl quality and short nurturing cycles. These nucleated pearls are widely known as "freshwater Akoya" in the pearl market because they resemble Japanese saltwater Akoya pearls. However, techniques for cultivating nucleated pearls from the mantle of H. cumingii are still immature and lack a targeted quality evaluation system. Therefore, this study was conducted to identify growth traits related to the quality of nucleated pearls from the mantle of H. cumingii. Two-year-old H. cumingii were implanted with 10 nuclei, 5 mm each in size per mussel. These host mussels were cultured for 19 months to form commercial pearls. Correlation and path analyses of the key quality traits of pearls (pearl number/pearl formation percentage, maximum diameter, minimum diameter, roundness, nacre thickness) and the growth traits of host mussels (shell length, shell width, shell height, body weight, shell weight, soft tissue weight) and nuclei (maximum diameter, minimum diameter, roundness) were performed. The results showed that the pearl formation rate varied from 50% to 100%, with 43.33% of mussels exhibiting a pearl formation rate of 70% and only 3.33% of mussels exhibiting a pearl formation rate of 50% or 100%. The average pearl formation rate was (70.00±10.82)%. The variation in pearl size (minimum diameter) was 4.72‒9.19 mm with an average of (6.91±0.67) mm. The variation in pearl roundness (diameter variation) was 0‒45.60% with an average of (6.74±7.78)%. The highest percentage of pearls were round, followed by near-round pearls, and then by positive round pearls at 21.90%. The nacre thickness was thin in 1.43% of pearls and extra thick in 98.57%. Pearl quantity and roundness were not correlated with all quantitative traits of pearls, host mussels, or nuclei (P > 0.05). Pearl size and nacre thickness were highly significantly and positively correlated with shell width, body weight, and shell weight (P < 0.01) and were significantly and positively correlated with shell height and soft tissue weight of the host mussels (P < 0.05). Among pearl quantitative traits, path analysis of pearl size and nacre thickness showed (highly) significant correlations with host mussels and nuclei. Stepwise regression analysis was performed with the pearl minimum diameter (y1) and nacre thickness (y2) as dependent variables and the shell length (x1), shell width (x2), shell height (x3), body weight (x4), shell weight (x5), and soft tissue weight (x6) of host mussels, along with the maximum diameter (x7), minimum diameter (x8), and roundness (x9) of the nuclei, as independent variables. The regression equations were y1 = 3.464 + 0.078x2 − 0.055x9 (R2 = 0.75; P < 0.01) and y2 = −1.243 + 0.077x2 − 0.055x9 (R2 = 0.75; P < 0.01). The direct effect of shell width on pearl size was the highest (0.69). The direct effect of nucleus roundness on pearl size was −0.23. The indirect effect of shell width on pearl size through nucleus roundness (0.05) was significantly greater than that of nucleus roundness on shell width (−0.02). The direct and indirect effects of shell width and nucleus roundness on nacre thickness were similar to those on pearl size. The direct determinant coefficient of shell width on pearl size (0.50) was significantly higher than that of nucleus roundness (0.08) and that of shell width via nucleus roundness (−0.03). The direct and indirect determinant coefficients of nucleus thickness by shell width and nucleus roundness in pearl-bearing mussels showed the same trend. These findings suggest that the shell width of host mussels is a key trait for controlling pearl size and pearl nacre thickness in nucleated pearls from the mantle of H. cumingii. These results will contribute to the development of China´s pearl industry.

BackgroundShell and pearl formation in bivalves is a sophisticated biomineralization process that encompasses immunological and mineralization aspects, particularly during shell repair and the initial stages of pearl cultivation when a nucleus is inserted. Here, we describe a novel C-type lectin, HcLec1, isolated and characterized from the freshwater pearl mussel Hyriopsis cumingii Lea. MethodsImmune challenge, RNA interference (RNAi) experiments, ELISA, and antibacterial assays were employed to investigate the role of HcLec1 in innate immunity. We also established shell damage repair and pearl nucleus insertion models to examine the impact of HcLec1 on the biomineralization process in Hyriopsis cumingii Lea. In vitro calcium carbonate crystallization assays were conducted to explore the direct role of HcLec1 in calcium carbonate crystal formation.ResultsThe HcLec1 gene sequence is a full-length cDNA of 1552 bp, encoding 240 amino acids. HcLec1 comprises an N-terminal signal peptide and a carbohydrate-recognition domain (CRD), with QPD (Gln-Pro-Asp) and MND (Met-Asn-Asp) motifs for polysaccharide binding. Tissue expression analysis showed that HcLec1 is predominantly expressed in the gill tissue of Hyriopsis cumingii Lea under normal conditions, and its expression is significantly elevated in both gill and pearl sac tissues following nucleus insertion for pearl cultivation (P < 0.05). After immune stimulation with Aeromonas hydrophila and lipopolysaccharides (LPS), HcLec1 expression levels significantly increased in both cases (P < 0.01), indicating a role in bivalve innate immunity. RNA interference (RNAi)-mediated knockdown of HcLec1 led to a significant decrease in the expression levels of immune-related genes (WAP, α2m, and Lyso) and mineralization-related genes (CA, CHS, Nacrein, and Pif) (P < 0.05). In animal models for shell damage and nucleus insertion in pearl cultivation, HcLec1 showed a consistent expression pattern, with an initial significant decrease followed by a marked increase, peaking at day 14 (P < 0.05). This suggests a role for HcLec1 in pearl formation and shell repair. The recombinant HcLec1 protein demonstrated binding affinity to LPS and PGN, a robust ability to agglutinate Escherichia coli, Staphylococcus aureus, Aeromonas veronii, and Aeromonas hydrophila, and significantly inhibited bacterial growth (P < 0.05). Moreover, rHcLec1 promoted calcite crystal formation in saturated calcium carbonate solutions and altered crystal morphology.DiscussionThe HcLec1 gene plays a pivotal role in both innate immunity and biomineralization in the triangle sail mussel. This study enhances our understanding of the functional diversity of C-type lectins and provides a foundation for future studies on shell repair and pearl growth.

Hyriopsis cumingii is a species that naturally inhabits substrates rich in various metal elements. Iron, essential for several biological processes, significantly influences the growth and development of bivalves. This study established nine full-sibling families and conducted an 11-month cultivation experiment involving five different levels of iron content in substrates (1.75 %, 3.85 %, 4.75 %, 5.95 %, and 6.85 %) to investigate the impact of varying iron concentrations on the growth and inner shell color of H. cumingii. The findings revealed that H. cumingii demonstrated significantly enhanced growth under the Iron 3 condition (4.75 %), while individuals cultivated under Iron 4 (5.95 %) exhibited a deeper purple pigmentation in their inner shell. Heritability estimates for growth and inner shell color traits ranged from 0.07 to 0.25 across the experimental population. The anal angle radial rib length (AARRL) displayed stronger genetic and phenotypic correlations with body weight (BW) and shell width (SW) compared to shell height (SH) and total shell height (TSH). Notably, significant gene-environment interaction effects (G × E) were notably observed for the color exhibited by the inner shell and growth-related traits, especially between Iron 1 and Iron 3, as well as between Iron 3 and Iron 5. The comprehensive estimation of breeding values showed that Family 7 exhibited superior performance across all traits under different conditions, positioning it as a promising candidate for genetic improvement. This research underscores the considerable influence of iron concentrations in substrates on the growth and shell coloration of H. cumingii, offering valuable insights for developing selective breeding strategies.

Hyriopsis cumingii is one of the most popular freshwater mussels for the pearl culture industry in China. Three elite strains, i.e., Shenzi No. 1 (purple inner shell, SZ1), Shenzhe No. 3 (white inner shell and rapid growth, SZ3), and Golden strain (golden inner shell, GS), exhibit advantageous characteristics in terms of pearl coloration and biomineralization, which result in a significantly higher pearl quality. To explore the genetic basis of phenotypic variation, the three populations were subjected to whole-genome resequencing. A total of 38,781,748 high quality single nucleotide polymorphisms (SNPs) were identified across the genomes of the three strains. The three populations exhibited high levels of genetic diversity of individuals within each population, as expressed by high heterozygosity (Ho: 0.3099–0.3170, He: 0.2969). The GS population appeared to have experienced a greater selective pressure than SZ1 and SZ3, as denoted by its higher linkage disequilibrium (LD) level. By combining the pairwise comparison of the fixation index (Fst) and nucleotide diversity ratio (θπ), we identified four critical biomineralization-related genes (CaM, CHST, CHSY, and COL6A3) and three color-related genes (TRY, FER, and BCO1) that may serve as selective signatures for the breeding of H. cumingii. In summary, the identified functional genes as well as their corresponding SNP loci provided a genetic basis for selecting traits related to inner shell color and biomineralization and may accelerate the progress of marker-assisted selection in H. cumingii.

The color of crayfish (Procambarus clarkii) shells not only affects their value, but also has a significant impact on individual size. Astaxanthin is a common carotenoid that has been found in many studies to have different functions, including regulating body color formation. In this study, the levels of different carotenoids were detected using high performance liquid chromatography (HPLC) and found a significant correlation between astaxanthin content and shell color. An astaxanthin gene (PcASTA) was identified from P. clarkii, and it was expressed in tissues of crayfish with dark-red shells and green shells. The highest expression levels of PcASTA were found in the hepatopancreas, followed by the muscle. The expression of PcASTA in all tissues of red shell crayfish was higher than that of green shell crayfish, indicating that PcASTA may be involved in shell color formation in crayfish. Moreover, high water temperature and feeding β-carotene can lead to an increase in tissue expression of PcASTA and astaxanthin content in crayfish shells. The findings indicate that PcASTA may affect shell color in P. clarkii by being involved in astaxanthin synthesis and transformation.

Biomineralization is a widespread phenomenon in marine mollusks and is responsible for the production of shells and pearls. However, the regulatory mechanisms governing the adaptive immune responses in the mollusk mantle and visceral mass during mineralization remain unclear. In this work, we examined the mantle and visceral mass immune responses of Hyriopsis cumingii during pearl culture using high-throughput sequencing techniques. A mantle transcriptome database was established using transcriptome sequencing technology and reference to the major databases. Digital gene expression profiling was used to identify the differentially expressed genes of mantle and visceral mass at different insertion periods. Moreover, quantitative real-time PCR was used to verify the expression of five immune-related genes. Transcriptome sequencing results showed 257,457 unigenes were identified. Digital gene expression profiles showed 1389, 3572, 1888, and 2613 differentially expressed genes (DEGs) in the mantle and visceral mass at 5, 20, 50, and 90 d after insertion, respectively, with the highest number at 20 d and the lowest at 5 d after insertion (q < 0.05). A cluster analysis of the DEGs showed similar clustering and expression features in the mantle to the control group, and at 5, 50 and 90 d, after mantle insertion. The DEGs in the visceral mass showed similar clustering and expression features to the control group and at 5, 20 and 50 d after insertion. We also screened 22 immune-related DEGs in the mantle and visceral mass during the same pearl culture period, including serine/threonine-protein kinase NLK, C-type lectin, and galectin. The greatest number of DEGs was found 90 d after insertion. Compared with the mantle, more immune-related DEGs were down-regulated than up-regulated in the visceral mass during pearl culture, indicating that the immune regulatory mechanisms in the visceral mass and the mantle differ during pearl culture, and that the visceral mass is liable to higher infection and mortality rates. Quantitative real-time PCR results showed that the expression of five immune-related genes was consistent with DGE results. Our findings will further knowledge of the immune systems that are present in the mantle and visceral mass during pearl culture.

Abstract Background Protein kinase C (PKC) is a multifunctional serine and PKC can phosphorylate serine residues in the cytoplasmic domain of tyrosinase, thereby regulating the activity of tyrosinase. Activated PKC is bound to the melanosome membrane, and unactivated PKC is free in the cytoplasm of melanocytes. In this study, we study the role of PKC gene in the melanin synthesis pathway and its effect on the color of the nacre of H. cumingii. Results In this study, a HcPKC gene in H. cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcPKC was expressed in both purple and white mussels, and the level of mRNA expression was higher in the purple mussels than in white mussels. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcPKC may be involved in nacre formation. After SNP association with inner shell color related traits, according to the principle that 0.25

Abstract Background Monochasma savatieri Franch. ex Maxim is a medicinally valuable herb. However, the collection and protection of the wild germplasm resources of M. savatieri are still insufficient, and their genetic diversity and population structure have been poorly studied. Results We collected and examined 46 M. savatieri individuals from Fujian, Hunan, Jiangxi, and Zhejiang provinces for genetic diversity and population structure, using 33 newly developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. Applying these markers, we detected a total of 208 alleles, with an average of 6.303 alleles per locus. The polymorphic information content varied from 0.138 to 0.884 (average: 0.668), indicating a high level of polymorphism. At the population level, there was a low degree of genetic diversity among populations (I = 0.535, He = 0.342), with Zhejiang individuals showing the highest genetic diversity among the four populations (Fst = 0.497), which indicated little gene flow within the M. savatieri populations (Nm = 0.253). Mantel test analysis revealed a significant positive correlation between geographical and genetic distance among populations (R 2 = 0.3304, p

Mussel cell culture is a challenging problem and serum serves a crucial biological role in cell culture as an autologous supply and an immunizing agent. In this study, the biology (calcium ions, total protein, pH, and osmotic pressure) of fetal bovine serum (FBS) and Hyriopsis cumingii serum (HCS) was investigated, and the development of Hyriopsis cumingii (H. cumingii) mantle cells in HCS and FBS systems was examined. The results showed that total protein, calcium ions, and osmotic pressure varied significantly (p

Uroporphyrinogen III synthetase (UROS) is an important enzyme involved in the structural formation of porphyrin, which is responsible for color intensity. A UROS homolog was isolated and characterized from the pearl mussel, Hyriopsis cumingii (HcUros). The HcUros cDNA was 858 bp in length with an open reading frame that encoded a 285 amino acid protein, which contained a conserved HemD domain. HcUros exhibited widespread tissue distribution, displaying particular enrichment in the mantle and haemolymph of purple mussels, compared to white mussels. In situ hybridization of HcUros in mantle tissue demonstrated that it was specifically expressed in the dorsal epithelial cells of the mantle pallia. Additionally, HcUros expression was upregulated following implantation of the pearl sac to produce pearls. The data suggest that HcUros contributes to shell pigmentation, and nacre formation and coloring in H. cumingii.

Cyclin dependent kinase 6 (CDK6) is a serine/threonine kinase that plays important roles in cell cycle progression and differentiation. In this study, full-length cDNA of Hc-CDK6 was obtained from freshwater pearl mussels (Hyriopsis cumingii, Hc) with 3´,5´ rapid-amplification of cDNA ends (RACE). The Hc-CDK6 expression profiles were analyzed with quantitative real-time PCR and in situ hybridization. The function of the Hc-CDK6 gene was studied with both RNA interference (RNAi) and overexpression in H. cumingii. Hc-CDK6 was found to encode 331 amino acids and to have a CDK4/6-like serine/threonine kinase catalytic structural domain. In terms of the amino acid sequence, the protein Hc-CDK6 was most closely related to its homolog in Crassostrea gigas, with a similarity of 75.23%. Hc-CDK6 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonads), and the highest expression was observed in the gonads (P

Color is an important trait of pearls which significantly affects their economic value. Golden seawater pearls with excellent luster are becoming popular among consumers, but golden freshwater pearls are rare. Hyriopsis cumingii is the most important freshwater mussel for pearl production in China and a selective breeding program has recently been developed to improve the yellowness and luster of the inner shell of H. cumingii for better production of golden freshwater pearls. As part of this breeding program, it has been necessary to evaluate the genetic parameters of the target traits and possible genotype-by-environment interactions. In this study, we investigated the effects of the environment on the genetic parameters of the inner shell color and luster of F1 golden strain H. cumingii, reared at two sites (Chongming and Wuyi). Inner shell color parameters and inner shell luster were recorded in 570 19-month-old individuals. Microsatellite-based DNA parentage analysis was used to assign the mussels to 19 maternal half-sib families, including 41 full-sib families. The results showed that the estimated heritability of the inner shell color parameters L*, a*, b* and dE were low, 0.13 ± 0.04, 0.14 ± 0.04, 0.12 ± 0.03 and 0.14 ± 0.05, respectively. The heritability of inner shell luster was medium (0.28 ± 0.07). The correlations between the genetic and phenotypic parameters for inner shell color and inner shell luster were insignificant at less than 0.8. Genetic correlations between the inner shell color parameters and inner shell luster in Chongming and Wuyi ranged from −0.07 ± 0.28 to −0.37 ± 0.24 and showed significant genotype-by-environment interaction. This study demonstrated that inner shell color parameters and inner shell luster could be improved by selective breeding. Environmental influences on genotype for inner shell color and inner shell luster should be taken into account in future selective breeding programs in golden strain H. cumingii.

In the original publication [...]

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

Insulin-like growth factor 1 (IGF1) plays an important regulatory role in the regulation of growth, differentiation, and anabolism in a variety of cells. In this study, the full-length cDNA of the IGF1 gene was cloned from Hyriopsis cumingii, named HcIGF1. The expression level of HcIGF1 in six tissues (adductor muscle, foot, hepatopancreas, gill, mantle, and gonad) was determined. In addition, the localization of HcIGF1 in the mantle was analyzed by in situ hybridization, and finally the function of HcIGF1 was explored by RNA interference and prokaryotic expression. The results showed that the amino acid sequence contained a typical IIGF structural domain. The phylogenetic tree showed that HcIGF1 clustered with other marine bivalve sequences. Quantitative real-time PCR and in situ hybridization analysis showed that HcIGF1 was expressed in all tissues. The highest expression was in the foot and the lowest was in the mantle. In the mantle tissue, the hybridization signal was mainly concentrated in the outer mantle. After RNA interference, the expression of IGF1 was found to be significantly decreased (p < 0.05), and its related genes IGF1R, AKT1, and cyclin D2 were downregulated, while MAPK1 were upregulated. The recombinant HcIGF1 protein was purified and its growth-promoting effect was investigated. The results showed that the recombinant HcIGF1 protein could significantly promote the proliferative activity of the mantle cells of mussels, with the best proliferative effect at 12.5 μg/mL. The results of this study provide a new method to solve the problem of weak proliferation of shellfish cells in vitro and lay the foundation for further understanding of the growth regulation mechanism of H. cumingii, as well as a better understanding of the physiological function of IGF1 in mollusks.

Freshwater pearl mussels and marine pearl oysters are major producers of cultured pearls. With the development of pearl farming, pearl bivalve mollusks have attracted significant research attention over the past 25 years. To provide an overview of this research, we conducted a bibliometric analysis of publications from the Web of Science Core Collection database from 1995 to 2020. A network map generated by VOSviewer software was used to evaluate studies of pearl bivalve mollusks in terms of author, country, organization, journal, and keywords. A total of 2,288 publications were obtained, showing an annual increase in the number of papers. Researchers based in China were a major contributor to the field and there was active cooperation among authors. Research focused on mussel and oyster developmental biology, growth, culture mode, molecular biology, and ecological conservation. In recent years, hot topics, such as growth performance and traits, pearl formation and biomineralization, and immune response, have been widely discussed. Genomics, transcriptomics, proteomics, and metabonomics analyses were commonly used to analyze the regulatory mechanism of coding and non-coding genes. The interaction between the environment and farming activities showed the importance of sustainable development. Interdisciplinary research could solve some of the issues facing the pearl bivalve mollusk farming. In conclusion, our findings could serve as another way to understand research trends in pearl bivalve mollusks and contribute to future studies.

Creb (Cyclic AMP response element binding protein) is a nuclear regulatory factor that regulates transcription through autophosphorylation. In melanocytes, cAMP's corresponding elements bind to the Creb protein to autophosphorylation and activate MITF (Microphthalmia-associated transcription factor). MITF stimulates Tyrosine(tyr) to induce melanocytes to differentiate into eumelanin and pheomelanin. In this study, a HcCreb gene in Hyriopsis cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcCreb was expressed in both purple and white mussels, and there was a significant difference in expression between adductor muscle (p

Shell biomineralization is a process in which inorganic minerals assemble under accuracy control of macromolecule, mainly the matrix proteins. A shell-notching assay was used in Hyriopsis cumingii and CaCO3 deposition during shell regeneration and the expression pattern of five shell matrix protein encoding genes, HcTyp-1, hic31, hic52, silkmapin and Hcperlucin were analyzed. Macro- and microscopic observation revealed that shell regeneration can be broadly separated into three stages. (1) The formative period of the periostracum (from 0th day to 6thday), the pellicle formed and covered the gap creating a sealed space for mineralized layer origin. (2) The period of prismatic layer mineralization (from 14 to 21 days), the periostracum continued to grow, and aragonite seeds originated from the organic membrane accumulated and grew laterally to cover the whole periostracum. (3) The growth period of the nacreous layer (from 29 to 39 days), the initial nacreous layer formed, and regular aragonite crystals deposited, nucleated and eventually grew to form a distinctive brick wall structure. Furthermore, the periodic high-expression of five shell matrix protein genes during periostracum formation and prismatic or nacreous layer mineralization suggested they had a dominant role in the programmed reparative process. These observations contribute to understanding the molecular mechanism of shell formation. Keywords: Matrix protein, Shell, Biomineralization, Hyriopsis cumingii

Microphthalmia-associated transcription factor (MITF) is an essential transactivator in melanin synthesis. To characterize the role of MITF in the pearl mussel Hyriopsis cumingii, the MITF homolog of H. cumingii was isolated. The full-length HcMitf cDNA consisted of a 1332-bp with an open reading frame that encode for a 443 amino acid protein that contain a conserved basic helix-loop-helix zipper domain. The HcMitf was found to widespread tissue distribution but expression was higher in purple mussels than in white mussels, mostly in mantle, liver, kidney, gill, and foot with the exception of the adductor mussel. HcMitf and its downstream gene tyrosinase (HcTyr) were highly expressed at the nacre deposition stage after implantation of mantle tissue to produce pearls. Using RNA interference, the expression of HcMitf was reduced by 78% (P

The Carassius auratus complex in natural populations includes diploid triploid and polyploidy individuals. Diploid individuals belong to the species Carassius auratus whereas triploid and polyploidy individuals are from the subspecies Carassius auratus gibelio. Triploid individuals are all female and reproduce clonally by gynogenesis. Therefore the Carassius auratus complex is an ideal system for studying evolution of unisexual reproduction. Identification of triploid individuals and clonal lines is the first step towards understanding of the evolution of unisexual clonal lines. We examined the ability of 10 microsatellites in identifying triploid individuals in 94 individuals from Japan and China. In 40 confirmed triploid individuals and eight confirmed diploid individuals, all triploid and diploid individuals can be identified by genotyping 10 microsatellite, and four triploid clonal lines were identified. Using the 10 microsatellites we genotyped 46 adult individuals (40 females and six males) from a natural population in China and found that all six males were diploid whereas the majority of females (36 of 40) were triploid and three triploid clonal lines were detected. In 18 diploid individuals from China, all individuals showed different genotypes, suggesting there is no diploid clonal line in diploid crucian carp. A phylogenetic analysis of 94 individuals from China and Japan showed that triploid individuals and clonal lines have originated recurrently.

The triangle sail mussel Hyriopsis cumingii (Lea) is the most important mussel species used for commercial freshwater pearl production in China. Mussel color is an important indicator of pearl quality. To identify genes involved in the nacre coloring, we conducted RNA-seq and obtained 541,268 sequences (298 bp average size) and 440,034 sequences (293 bp average size) in secreting purple and white nacre libraries (P- and W-libraries), respectively. The 981,302 Expressed Sequence Tags (ESTs) were assembled into 47,812 contigs and 289,386 singletons. In BLASTP searches of the deduced protein, 22,495 were proteins with functional annotations. Thirty-three genes involved in pearl or shell formation were identified. Digital expression analysis identified a total of 358 differentially expressed genes, and 137 genes in the P-library and 221 genes in the W-library showed significantly higher expression. Furthermore, a set of SSR motifs and SNPs between the two samples was identified from the ESTs, which provided the markers for genetic linkage, QTL analysis and future breeding. These EST sequences provided valuable information to further understand the molecular mechanisms involved in the formation, color determination and evolution of the pearl or shell.

Iron is one of the most important minor elements in the shells of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell growth. A novel ferritin subunit (Fth1) cDNA from the freshwater pearl mussel (Hyriopsis cumingii) was isolated and characterized. The complete cDNA contained 822 bp, with an open reading frame (ORF) of 525 bp, a 153 bp 5' untranslated region (UTR) and a 144 bp 3' UTR. The complete genomic DNA was 4125 bp, containing four exons and three introns. The ORF encoded a protein of 174 amino acids without a signal sequence. The deduced ferritin contained a highly conserved motif for the ferroxidase center comprising seven residues of a typical vertebrate heavy-chain ferritin. It contained one conserved iron associated residue (Try27) and iron-binding region signature 1 residues. The mRNA contained a 27 bp iron-responsive element with a typical stem-loop structure in the 5'-UTR position. Copy number variants (CNVs) of Fth1 in two populations (PY and JH) were detected using quantitative real-time PCR. Associations between CNVs and growth were also analyzed. The results showed that the copy number of the ferritin gene of in the diploid genome ranged from two to 12 in PY, and from two to six in JH. The copy number variation in PY was higher than that in JH. In terms of shell length, mussels with four copies of the ferritin gene grew faster than those with three copies (P

In triangle sail mussel (Hyriopsis cumingii), shell biomineralization is a complicated process that involves multiple gene products. Shell matrix proteins are involved in the formation of the organic framework and play an important role in the regulation of calcium carbonate deposition. In this study, A new shell matrix protein gene Hc-transgelin was identified in H. cumingii. The full-length cDNA of Hc-transgelin was 1200 bp, including a 501 bp open reading frame, which encoded 166 amino acids. Hc-transgelin is rich in lysine, it accounts for 11.40% of the protein. The predicted transgelin protein contained a conserved calmodulin homologous domain. A tissuespecific expression assay indicated that Hc-transgelin exhibited significantly highest expression in the mantle. Furthermore, Hc-transgelin in situ hybridization detected positive signals at the edge of the mantle outer fold, where nacre and prismatic layer biomineralization occur. An RNA interference assay showed that the shape of aragonite flakes in nacre changed and their growth was inhibited, and cracks appeared in the prismatic layer organic sheath when the expression of Hc-transgelin was suppressed. In a shell repair assay, a higher expression of Hc-transgelin appeared from day 12 to day 25 when the nacre accumulated quickly. These findings indicate that Hc-transgelin may be involved in the formation of aragonite flakes in the nacre and play a role in the formation of the organic sheath in the prismatic layer of the shell. This study provides new insights into the role of the Hctransgelin gene and also contributes to the molecular understanding of mollusk shell formation. [ABSTRACT FROM AUTHOR]