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Circular RNAs (circRNAs) play key roles in several biological processes in animals and their regulatory mechanism in body color formation or pigmentation in fish remains unclear. Here, circRNAs from black and red individuals of Plectropomus leopardus were identified to clarify the mechanism of circRNAs and the competing endogenous RNA (ceRNA) network (circRNA-microRNA (miRNA)-messenger RNA (mRNA)) in body color formation. We detected a total of 1,424 novel circRNAs. Expression analysis of circRNAs in black vs. red P. leopardus revealed 24 differentially expressed circRNAs (DECs), and 11 and 13 of these DECs were up-regulated and down-regulated in red individuals relative to black individuals, respectively (P1). We identified a total of 19 significant miRNA-circRNA-mRNA ceRNA networks through the analysis of DECs, differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs). Pathway enrichment analyses of the DEGs involved in the ceRNA network revealed that they were mainly involved in melanin metabolism and immune response. Our findings showed the possibility of the regulatory functions of circRNAs and the corresponding ceRNA network in the body color formation process and will aid the breeding selection process of P. leopardus.

Long non-coding RNAs (lncRNAs) play a multifaceted role in transcriptional regulation, and the potential molecular regulatory mechanisms of lncRNAs and lncRNA–miRNA–mRNA networks in body color formation are of great significance for its selective breeding. Therefore, lncRNAs and lncRNA-miRNA-mRNA ceRNA network of red- and black-colored Plectropomus leopardus were identified and analyzed. Sequencing analyses identified 167 differentially expressed lncRNAs (DELs) between red- and black-colored P. leopardus, including 89 upregulated and 78 downregulated DELs in the red-colored group (false discovery rate (FDR) < 0.05 and |log2FC| > 1). Differentially expressed miRNA (DEM), genes (DEG), and DEL analyses found 605 and 125 negatively co-expressed miRNA–mRNA pairs and lncRNA–miRNA pairs, respectively. Further correlation analysis with Spearman’s correlation coefficient >0.9 as the threshold identified 3,721 lncRNA–mRNA pairs. Then, a competitive endogenous RNA (ceRNA) network of 325 pairs (p < 0.05) was obtained. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of network DEGs showed that melanin metabolic process, lipid metabolism, and immune-related pathway were enriched. The ceRNA network provided interactions among lncRNAs, miRNAs, and mRNAs and extended the molecular foundation of body color formation.

Salinity is an important abiotic stress that has significant effects on the physiology and metabolism of marine fish. The greater amberjack (Seriola dumerili) is a large, fast-growing species with high commercial value in global aquaculture. To describe the molecular response of the greater amberjack liver to different salinity stresses, RNA-seq analysis was performed to identify the important genes and signaling pathways activated in response to salt stress. Greater amberjack juveniles were reared under different salinity stresses (20, 30, and 40 ppt) for 30 days to evaluate their tolerance, adaptability, and molecular responses. A total of 657 (426 up-regulated and 231 down-regulated) and 65 (17 up-regulated and 48 down-regulated) differentially expressed genes (DEGs) were identified in the group at 30 vs. 20 ppt and 30 vs. 40 ppt salinity, respectively. qPCR and transcriptomic analysis showed that salinity stress affected the expression of genes involved in lipid metabolism (pld2, pla2g7, acacb, and acsl4a), vitamin metabolism (cyp24a1 and cyp2r1), ion transporters (slc4a1a, slc4a4b, rhag, and rh50), and signal transduction (itpkcb, fgf19, and fgfr4). KEGG pathway enrichment analysis showed that the DEGs were primarily involved in metabolism, ribosome biogenesis in eukaryotes, and insulin signaling pathway. The identified candidate genes involved in metabolism pathways, ion transporters, and signal transduction, provide a basis for further study of the molecular mechanisms involved in salinity adaptation and transcriptional plasticity in the livers of marine fish.

During pearl culture, the excess immune responses may induce nucleus rejection and death of pearl oysters after transplantation. To better understand the immune response and pearl formation, lipidomic analysis was applied to investigate changes in the serum lipid profile of pearl oyster Pinctada fucata martensii following transplantation. In total, 296 lipid species were identified by absolute quantitation. During wound healing, the content of TG and DG initially increased and then decreased after 3 days of transplantation with no significant differences, while the level of C22:6 decreased significantly on days 1 and 3. In the early stages of transplantation, sphingosine was upregulated, whereas PC and PUFAs were downregulated in transplanted pearl oyster. PI was upregulated during pearl sac development stages. GP and LC-PUFA levels were upregulated during pearl formation stage. In order to identify enriched metabolic pathways, pathway enrichment analysis was conducted. Five metabolic pathways were found significantly enriched, namely glycosylphosphatidylinositol-anchor biosynthesis, glycerophospholipid metabolism, alpha-linolenic acid metabolism, linoleic acid metabolism and arachidonic acid metabolism. Herein, results suggested that the lipids involved in immune response, pearl sac maturation, and pearl formation in the host pearl oyster after transplantation, which might lead to an improvement in the survival rate and pearl quality of transplanted pearl oyster.

The receptors of serotonin, also known as 5-hydroxytryptamine receptor (5-HTR) can mediate regulatory metamorphosis processes in a variety of mollusks. Studying the mechanisms of metamorphosis of the pearl oyster is significant to elucidate breeding, resource recovery and marine pearl production. In this study, two 5-HTR genes from Pinctada fucata martensii (Pm5-HTR2 and Pm5-HTR4) were cloned. A total of 1623 bp open reading frame was identified in Pm5-HTR2, and a 1185 bp open reading frame was detected in Pm5-HTR4; these open reading frames encoded a 540-residue polypeptide and a 394-residue polypeptide, respectively. We also conducted a domain analysis, which indicated that Pm5-HTR2 and Pm5-HTR4 contained a seven-transmembrane domain and revealed that the receptors had high similarity to Crassostrea gigas 5-HTR2 (54.62%) and 5-HTR4 (66.23%). Sequence analysis demonstrated conserved advanced structure and motifs (the DRY/ERY and NPXXY motifs). The expression pattern analysis revealed high expression levels of Pm5-HTR2 and Pm5-HTR4 during the developmental stages. ISH analysis showed that Pm5-HTR2 was primarily expressed in the FE, B, T, EU, and EL stages and Pm5-HTR4 was mainly expressed in the FE, B, T, D, EU, and EL stages. These results suggest that 5-HTRs may play key roles in P. f. martensii larval metamorphosis.

Body color is an essential ecological phenotypic trait determining the ability of aquatic animals to adapt to or survive in a treacherous environment. However, researches regarding its underlying molecular mechanism in leopard coral groupers has been limited. Therefore, mRNAs, metabolites, and miRNAs of different-colored leopard coral grouper (Plectropomus leopardus) were sequenced to investigate the body color formation mechanism. The transcriptome analysis identified 1236 genes as differentially expressed genes (DEGs), of which 579 were up-regulated and 657 were down-regulated in the red-colored group compared with the brown-colored group (FDR < 0.01 and |log2FC| > 1). Metabolome analysis revealed 439 and 84 SDMs in POS and NEG of liquid chromatography-mass spectrometry, respectively (VIP > 1 and P < 0.05). The miRNA analysis exhibited 149 (120 up-regulated and 29 down-regulated) differentially expressed miRNAs (DEMs) in the analysis of the brown-colored vs. red-colored groups (P < 0.05 and |log2FC| > 1), including miR-144, miR-185, miR-192, miR-2188, miR-6316, novel-m0015, and novel-m0040. Integrated analysis of multiple omics exhibited that carotenoid metabolism, immune response, lipid metabolism, and amino acid metabolism were involved in the body color formation and adaptation to the environment. Therefore, the present multiomics research prepared a foundation for bright body color formation and promoted the process of preferred body-color breeding.

An 8-week growth trial was conducted to assess the effects of dietary vitamin C (VC) on the growth performance, digestive enzyme activity, antioxidant ability, immunity, and resistance to Vibrio harveyi infection of coral trout (Plectropomus leopardus, initial weight 17.42 ± 0.06 g). Four kinds of iso-nitrogenous and iso-lipidic experimental feeds were formulated with covering VC levels of 0 (C0), 50 (C1), 100 (C2), and 200 (C3) mg/kg, respectively. Supplementation of an appropriate amount of VC in diet (a) increased specific growth rate (SGR) and weight gain rate (WGR); (b) increased amylase, lipase, and trypsin activities in the liver; (c) enhanced glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and reduced malonic dialdehyde (MDA) level in serum and liver; (d) increased lysozyme (LZ) and acid phosphatase (ACP) activities in serum and liver, as well as complement (C3 and C4) and immunoglobulin M (IgM) contents in the liver; (e) upregulated the relative expression of copper-zinc superoxide dismutase (SOD-1), manganese superoxide dismutase (SOD-2), CAT, GSH-Px1a, ACP6, AKP, LZ-c, IgM, C3, and C4-b in the liver; (f) improved the survival rate of V. harveyi challenge. In summary, an appropriate amount of VC could improve growth, digestive ability, antioxidant capacity, immunity, and the ability to resist the challenge of V. harveyi in coral trout. In order to ensure that coral trout has good growth performance, digestive and antioxidant capacity, it is recommended that the levels of VC in the feed is between 255.25 and 273.84 mg/kg. The immunity of coral trout is stronger when the VC level in the diet is between 251.95 and 314.03 mg/kg.

The greater amberjack (Seriola dumerili) is a gonochoristic fish with no sexual dimorphism in appearance, making sex identification difficult. Piwi-interacting RNAs (piRNAs) function in transposon silencing and gametogenesis and are involved in various physiological processes, including sex development and differentiation. Exosomal piRNAs can be indicators for the determination of sex and physiological status. In this study, four piRNAs were differentially expressed in both serum exosomes and gonads between male and female greater amberjack. Three piRNAs (piR-dre-32793, piR-dre-5797, and piR-dre-73318) were significantly up-regulated and piR-dre-332 was significantly down-regulated in serum exosomes and gonads of male fish, compared to female fish, consistent with the serum exosomal results. According to the relative expression of four marker piRNAs derived from the serum exosomes of greater amberjack, the highest relative expression of piR-dre-32793, piR-dre-5797, and piR-dre-73318 in seven female fish and that of piR-dre-332 in seven male fish can be used as the standard for sex determination. The method of sex identification can ascertain the sex of greater amberjack by blood collection from the living body, without sacrificing fish. The four piRNAs did not show sex-inclined expression in the hypothalamus, pituitary, heart, liver, intestine, and muscle tissue. A piRNA–target interaction network involving 32 piRNA-mRNA pairs was generated. Sex-related target genes were enriched in sex-related pathways, including oocyte meiosis, transforming growth factor-beta signaling pathway, progesterone-mediated oocyte maturation, and gonadotropin releasing hormone signaling pathway. These results provide a basis for sex determination in greater amberjack and improve our understanding of the mechanisms underlying sex development and differentiation in the species.

Pinctada fucata martensii produces high-quality pearls. However, excess immune and inflammatory response after grafting operation will lead to nucleus rejection, pearl sac formation failure, and death of the host pearl oyster. Polyunsaturated fatty acids (PUFAs) are critically important for its regulation function in inflammation and their synthesis process was limited by the related enzymes including fatty acid desaturase (FAD). In this study, FAD gene in the pearl oyster was characterized in P. f. martensii (PmΔ5FAD). We established that the full-length sequence of PmΔ5FAD contained a 1302-bp open reading frame that encoded a sequence of 433 amino acids. PmΔ5FAD domain analysis revealed the presence of a distinctive Cyt-b5 domain and a FA-desaturase domain that were highly similar to the Δ5FAD protein sequences identified in Haliotis discus hannai (69.25%) and Mimachlamys nobilis (62.96%). PmΔ5FAD expressed in all detected tissues. Functional analysis of PmΔ5FAD in recombinant Saccharomyces cerevisiae yeast showed the existence of Δ5-desaturation activity, which towards PUFA substrates and it efficiently desaturated exogenous PUFA C20:3n-6 to C20:4n-6 (ARA) with a desaturation conversion rate of 19.49%. Furthermore, PmΔ5FAD showed a significantly higher expression level on day 1 after the grafting operation, which may upregulate ARA synthesis in response to inflammatory and immune. These results provide important information concerning the function of FADs during pearl oyster PUFA biosynthesis and grafting operation.

Body color is an important economic and ecological trait in aquatic animals, which influence their economic values and determine the animal ability to survive in the marine environment. Red-colored Plectropomus leopardus is in high demand owing to its bright color and potential value for artificial breeding. High-throughput transcriptome sequencing and liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches were used to identify transcript and metabolic differences between black-colored and red-colored P. leopardus. Compared with the black-colored group, 218 and 181 differentially expressed genes were up- and down- regulated, respectively, in the red-colored group. 425 and 56 significantly different metabolites were identified in LC-MS positive and LC-MS negative ion models, respectively, between two colored groups. Based on the integrative analysis, the red-colored group exhibited greater carotenoid uptake, transport, and accumulation activity potential than those in the black-colored group and may consume more arachidonic acid for body color formation. The black-colored group showed greater melanin synthesis activity compared with the red-colored group. These results substantially improve the understanding of molecular and metabolic mechanisms underlying body color formation in P. leopardus and the potential of body color for the environment adaptation.

Abstract Background Marine bivalves undergo complex development processes, such as shell morphology conversion and changes of anatomy and life habits. In this study, the transcriptomes of pearl oyster Pinctada fucata martensii and Pacific oyster Crassostrea gigas at different development stages were analyzed to determine the key molecular events related to shell formation, settlement and metamorphosis. Result According to the shell matrix proteome, biomineralization-related genes exhibited a consensus expression model with the critical stages of shell formation. Differential expression analysis of P. f. martensii, revealed the negative regulation and feedback of extracellular matrixs as well as growth factor pathways involved in shell formation of larvae, similar to that in C. gigas. Furthermore, neuroendocrine pathways in hormone receptors, neurotransmitters and neuropeptide receptors were involved in shell formation, settlement and metamorphosis. Conclusion Our research demonstrated the main clusters of regulation elements related to shell formation, settlement and metamorphosis. The regulation of shell formation and metamorphosis could be coupled forming the neuroendocrine-biomineralization crosstalk in metamorphosis. These findings could provide new insights into the regulation in bivalve development.

Glycosaminoglycan (GAG), an important component of proteoglycan (PG), was biosynthesized with the initiation by peptide O-xylosyltransferase. O-xylosyltransferase activity was presented as a marker for increased PG synthesis. In the present study, a novel O-xylosyltransferase (OXT) gene was identified from Pinctada fucata martensii (PmOXT). The PmOXT-deduced protein sequence carried a typical water-soluble carbohydrate domain, branch domain, and xylosyltransferase domain. Homologous analysis of PmOXT presented the conserved DXD motif and catalytic structure characteristics. Phylogenetic tree analysis showed the traditional taxonomy and PmOXT clustered with Crassostrea gigas. PmOXT was expressed in all the detected tissues and developmental stages. PmOXT had a significantly higher expression level in the shell formation associated tissues and developmental stages. PmOXT expressed significantly decreased in the central zone of the mantle and marginal zone of the mantle after RNA interference; additionally, OXT activity and GAG content in the extrapallial fluid were significantly reduced compared with the control. Furthermore, the crystal tablets of prismatic layer displayed obvious holes and disordered crystals with obviously rough surface and irregular crystal tablets were observed in the nacre after RNAi. Results suggested that PmOXT affected the shell formation by influencing the formation of GAGs in the process of addition to the core proteins.

The protein inhibitor of activated STAT (PIAS) functions in diverse aspects, including immune response, cell apoptosis, cell differentiation, and proliferation. In the present study, the PIAS in the pearl oyster Pinctada fucata martensii was characterized. The sequence features of PmPIAS were similar to that of other PIAS sequences with PIAS typical domains, including SAP, Pro-Ile-Asn-Ile-Thr (PINIT), RLD domain, AD, and S/T-rich region. Homologous analysis showed that PmPIAS protein sequence showed the conserved primary structure compared with other species. Ribbon representation of PIAS protein sequences also showed a conserved structure among species, and the PINIT domain and RLD domain showed the conserved structure compared with the sequence of Homo sapiens. The expression pattern of PmPIAS in different tissues showed significant high expression in the gonad. PmPIAS also exhibited a significantly higher expression in the 1 and 2 days after cold tolerance stress (17°C) and showed its potential in the cold tolerance. The SNP analysis of the exon region of PmPIAS obtained 18 SNPs, and among them, 11 SNPs showed significance among different genotypes and alleles between cold tolerance selection line and base stock, which showed their potential in the breeding for cold tolerance traits.

Larval development, especially settlement and metamorphosis, has a vital influence on commercial culture during the life cycle of marine invertebrates. Sipunculus nudus, a peanut worm, is an important economic aquaculture species. However, information about the mechanism of its larval settlement and metamorphosis is still limited. Transcriptomic analysis was performed to determine the DEGs between the pelagospheric larva and creeping larva of S. nudus. A total of 96,455 unique unigenes were obtained and 46,355 were annotated. Transcriptome analysis revealed 19,352 DEGs, including 9,923 that were upregulated and 9,429 downregulated after settlement and metamorphosis. KEGG pathway enrichment analyses revealed that these DEGs were mainly involved in the Notch signaling pathway, TNF signaling pathway, protein digestion and absorption, thyroid hormone signaling pathway, and ECM–receptor interaction, and so on. Furthermore, the changes in genes related to cytoskeleton and cell adhesion indicated that protein degradation and apoptosis during metamorphosis mediate the loss of larval cilia and ECM remodeling functions in S. nudus metamorphosis. Changes in genes related to signal transduction and nervous system suggested the cessation of larval swimming, which is involved in settlement and metamorphosis. Changes in genes related to ingestion and digestion indicated that dietary shift occurred with metamorphic transition. Changes in some genes related to stress response and immunity indicated that conditioning induced the immune response of peanut worm. The results improve understanding of the physiological traits controlling S. nudus metamorphosis and provide a solid basis for further study.

In this study, we developed microsatellite markers, analyzed their distribution and frequency and verified polymorphism based on the transcriptome data of pearl oyster Pinctada maxima. Totally, 58,121 potential EST-SSRs from 145,877 sequences were identified. We randomly selected 100 of these primer pairs for further validation in the stock, 76 primer pairs were successfully amplified and 25 primer pairs were found to be polymorphic. The number of alleles at each locus ranged from 2 to 5, with an average allele number of 2.96. The observed heterozygosity ranged from 0.185 to 0.931 with an average of 0.656. The expected heterozygosity ranged from 0.337 to 0.754 with an average of 0.552. The polymorphic information content ranged from 0.263 to 0.690 with an average of 0.462. These markers will provide an effective tool for genetic diversity, genetic map construction and molecular-assisted breeding in the species of P. maxima. Keywords: Pinctada maxima, SSR markers, Polymorphism, Genetic diversity

Background: Molluscs can accumulate carotenoids in their body tissues by predominantly feeding on aquatic plant sources. Carotenoid transport and absorption are determined by the regulation of various proteins such as Scavenger receptor class B(SR-BI). We report the identification and characterisation of pearl oyster Pinctada fuctada martensii SR-BI (PmSR-BI). The correlation between total carotenoid content (TCC) and gene expression was also estimated. Results: The full-length cDNA of PmSR-BI was 1828 bp, including an open-reading frame encoding of 1518 bp with a pI value of 5.83. PmSR-BI protein contains a hydrophobic CD36 domain and four centrally clustered cysteine residues for the arrangement of disulphide bridges. The deduced amino acid sequence had an identity of 30% to 60% with the SR-B of other organisms. Reverse transcription polymerase chain reaction analysis showed that mRNA transcripts were expressed in multiple tissues of adult pearl oyster. A higher expression of PmSR-BI gene was observed in the hepatopancreas than in the adductor muscle, gill and mantle. The TCC and gene expression of PmSR-BI were significantly correlated (P

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Fibroblast growth factor 18 (FGF18) plays an important functional role in skeletal growth and development. The FGF18 gene was characterized in pearl oyster Pinctada fucata martensii (PmFGF18) with the full-length sequence containing an open reading frame of 714 bp encoding 237 amino acids. The domain analysis of PmFGF18 showed a distinctive FGF domain, with a high similarity to FGF18 protein sequences from Crassostrea gigas (43.35%) and C. virginica (37.43%). PmFGF18 expression was revealed in all analyzed tissues with a significantly higher expression level in the fast-growing group than the slow-growing group. The analysis of PmFGF18 polymorphism demonstrated 33 SNPs (single nucleotide polymorphisms) in the CDS and promoter region of PmFGF18 sequence. Association analysis revealed 19 SNPs (2 SNPs from CDS and 17 SNPs from the promoter region) associating significantly with growth traits. Among the associated SNPs, one SNP g.50918198 A > C was verified in the other breeding line. Therefore, PmFGF18 can be utilized as a candidate gene for growth, and its related SNPs could be used in selective breeding of P. f. martensii for the improvement of growth traits.

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

Natural disasters and environmental pollution are the main problems in traditional offshore cultivation. While culturing pearl oysters through industrial farming can avoid these problems, food availability in this case is limited. This study compares the metabolomics responses of pearl oysters, Pinctada fucata martensii, fed a formulated diet indoors with those of oysters cultured with natural diet outdoors by using a gas chromatography time-of-flight mass spectrometry (GC-TOF/MS)-based metabolomics approach. The animals were divided into two groups as follows: the experimental group (EG) was fed a formulated diet indoors and the control group (CG) was cultured with natural diet outdoors. After 45 days of feeding, the survival rate of EG was significantly higher than that of CG. The absolute growth rate (AGR) of the total weight of EG did not significantly differ from that of CG, but the AGRs of the shell length, shell height, and shell width of CG were significantly higher than those of EG. EG showed significantly higher amylase activities than CG, and the hexokinase and glucose-6-phosphate isomerase concentrations of the former were significantly lower than those of the latter. Metabolomics revealed 125 metabolites via mass spectrum matching with a spectral similarity value > 700 in the hepatopancreas, and 48 metabolites were considered to be significantly different between groups (VIP > 1 and P < 0.05). Pathway analysis results indicated that these significantly different metabolites were involved in 34 pathways. Further integrated key metabolic pathway analysis showed that, compared with CG, EG had lower capabilities for cysteine and methionine metabolism, sulfur metabolism, and starch and sucrose metabolism. This study demonstrated that the formulated diet could be an excellent substitute for natural diet; however, its nutrients were insufficient. Effective strategies should be developed to enhance the utilization of formulated diets.

Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many aspects of the biomineralization process and its evolutionary origin remain unknown. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms that underlie its production of shells and pearls are not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins, chondroitin sulfotransferases, and regulatory elements. Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre suggests that elements of chitin- and collagen-based matrices have deep roots and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems. [ABSTRACT FROM AUTHOR]