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Biomineralization‐controlled exo‐/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified "pearl" involved in invading defence. Notably, exo‐/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole‐genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization‐controlled growth and ectopic biomineralization. The insulin‐like growth factor (IGF) endocrine signal interacted with ancient single‐copy transcription factors to form the regulatory network. Moreover, the "cross‐phylum" regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo‐/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo‐/endoskeleton animals and ectopic biomineralization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Postoperative care is a critical step of pearl culture that ultimately determines culture success. To determine the effect of dietary vitamin D3 (VD3) levels on immunity and antioxidant capacity of pearl oyster Pinctada fucata martensii during postoperative care and explore the mechanisms behind this phenomenon, five isonitrogenous and isolipidic experimental diets were formulated by adding different levels of dietary VD3 (0, 500, 1000, 3000, and 10000 IU/kg), and the diets were fed to five experimental groups (EG1, EG2, EG3, EG4, and EG5) in turn and cultured indoors. The control group (CG) was cultured in the natural sea. Pearl oysters that were 1.5 years old were subjected to nucleus insertion. After culturing for 30 days, EG3 exhibited significantly higher survival rates than those in CG and EG5 (P < 0.05). Moreover, EG3 exhibited the highest activities of alkaline phosphatase, acid phosphatase, catalase, superoxide dismutase, and lysozyme. However, EG5 achieved the highest activities of glutathione peroxidase. Metabolomics-based profiling of pearl oysters fed with high levels of dietary VD3 (EG5) and optimum levels of dietary VD3 (EG3) revealed 76 significantly differential metabolites (SDMs) (VIP > 1 and P < 0.05). Pathway analysis indicated that SDMs were involved in 21 pathways. Furthermore, integrated key metabolic pathway analysis suggested that pearl oysters in EG5 regulated the pentose phosphate pathway, glutathione metabolism, sphingolipid metabolism, and arachidonic acid metabolism in response to stress generated from excessive VD3. These findings had significant implications on strengthening the future development and application of VD3 in aquaculture of pearl oyster P. f. martensii. • This metabolomics study is the first to assess the effects of high dietary levels of VD3 on pearl oyster. • Suitable doses of VD3 increased the immunity and antioxidant capacity of pearl oysters. • Pearl oysters regulated metabolism in response to stress generated from excessive VD3. [ABSTRACT FROM AUTHOR]

Abstract Similar to other marine bivalves, Pinctada fucata martensii presents unsynchronized growth, which is one of the problems farmers currently face. However, the underlying mechanisms have not been studied. In the present study, pearl oyster P. f. martensii from cultured stocks were selected to produce a progeny stock. At 180 days, the stock was sorted by size, and fast- and slow-growing individuals were separately sampled. Then, metabolomic and transcriptomic approaches were applied to assess the metabolic and transcript changes between the fast- and slow-growing P. f. martensii groups and understand the mechanism underlying their unsynchronized growth. In the metabolomics assay, 30 metabolites were considered significantly different metabolites (SDMs) between the fast- and slow-growing groups and pathway analysis indicated that these SDMs were involved in 20 pathways, including glutathione metabolism; sulfur metabolism; valine, leucine, and isoleucine biosynthesis; and tryptophan metabolism. The transcriptome analysis of different growth groups showed 168 differentially expressed genes (DEGs) and pathway enrichment analysis indicated that DEGs were involved in extracellular matrix-receptor interaction, pentose phosphate pathway, aromatic compound degradation. Integrated transcriptome and metabolome analyses showed that fast-growing individuals exhibited higher biomineralization activity than the slow-growing group, which consumed more energy than the fast-growing group in response to environmental stress. Fast-growing group also exhibited higher digestion, anabolic ability, and osmotic regulation ability than the slow-growing group. This study is the first work involving the integrated metabolomic and transcriptomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying unsynchronized bivalve growth. Graphical abstract Unlabelled Image Highlights • Metabolomics and transcriptomic were applied to analyze the bivalve unsynchronized growth. • Fast-growing group hold high biomineralization activity. • Slow-growing group consumed more energy in response to environment stress. • Fast-growing group exhibited high digestion and anabolic ability. • Fast-growing group possessed high osmotic regulation ability. [ABSTRACT FROM AUTHOR]

Heterosis improves growth and survival of shellfish species. Although breeders have widely exploited heterosis, its underlying molecular mechanisms remain unclear. In this study, a 2 × 2 complete diallel cross was facilitated between two full‐sib families to produce two inbred families (A and D) and their reciprocal hybrid families (B and C) of pearl oyster Pinctada fucata martensii. Growth traits of the four families were compared at the adult stages. Transcriptome analysis was conducted on the four families using an Illumina sequencing platform. The results revealed that the growth traits of the four families significantly varied (P < 0.05). The mid‐parent heterosis values of shell length, shell height, shell width, shell weight, and total weight were 12.9%, 14.9%, 18.2%, 17.2%, and 33.2%, respectively. The B‐ and C‐inbred (A and D) triads had 79 and 68 differentially expressed genes (DEGs), respectively, which were dominantly nonadditive, including overdominance, underdominance, and low‐parent dominance. Gene ontology term analysis showed that the DEGs in the B‐ and C‐inbred triads were enriched for metabolic process, cellular process cell part, binding, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs in the B‐ and C‐inbred triads were involved in focal adhesion, the P13K‐Akt signaling pathway, the mRNA surveillance pathway, and the focal adhesion pathway. The reliability of the sequencing data was confirmed by real‐time polymerase chain reaction analysis of six growth‐related genes. The findings of this study provide new insights into heterosis for growth traits and the design of genetic breeding programs for this species. Two inbred families (A and D) and their reciprocal hybrid families (B and C) were developed with breeders from two full‐sib families. The mid‐parent heterosis values of growth traits at adult stages ranged from 12.9% to 33.2%. The differentially expressed genes in the B‐ and C‐inbred triads were 79 and 68, respectively, which were dominantly nonadditive, including overdominance, underdominance, and low‐parent dominance. [ABSTRACT FROM AUTHOR]

Marine pearl production is directly influenced by the growth speed of Pinctada fucata martensii. However, the slow growth rate of this organism remains the main challenge in aquaculture production. Epidermal growth factor receptor (EGFR), an important receptor of tyrosine kinases in animals, plays versatile functions in development, growth and tissue regeneration. In this study, we described the characteristic and function of an EGFR gene identified from P. f. martensii (PmEGFR). PmEGFR possesses a typical EGFR structure and is expressed in all studied tissues, with the highest expression level in adductor muscle. PmEGFR expression level is significantly higher in the fast-growing group than that in the slow-growing one. Correlation analysis represents that shell height and shell weight show positive correlation with PmEGFR expression (p < 0.05), and total weight and tissue weight exhibit positive correlation with it (p < 0.01). This study indicates that PmEGFR is a valuable functional gene associated with growth traits. [ABSTRACT FROM AUTHOR]

Analyses of the transcriptome and metabolome were conducted to clarify alterations of key genes and metabolites in pearl oysters following exposure to short-term hypoxic treatment. We totally detected 209 DEGs between the control and hypoxia groups. Enrichment analysis indicated the enrichment of GO terms including "oxidation-reduction process", "ECM organization", "chaperone cofactor-dependent protein refolding", and "ECM-receptor interaction" KEGG pathway by the DEGs. In addition, between the two groups, a total of 28 SDMs were identified, which were implicated in 13 metabolic pathways, such as "phenylalanine metabolism", "D-amino acid metabolism", and "aminoacyl-tRNA biosynthesis". Results suggest that pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. Also, pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity, modulating immune and biomineralization activities, maintaining protein homeostasis, and reorganizing the cytoskeleton. The results of our study help unveil the mechanisms by which pearl oysters respond adaptively to short-term hypoxia. • Pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. • Pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity. • Pearl oysters might adapt to short-term hypoxic stress by modulating immune activities. • Pearl oysters might adapt to short-term hypoxic treatment by maintaining protein homeostasis. • Pearl oysters might adapt to short-term hypoxic treatment by reorganizing the cytoskeleton. [ABSTRACT FROM AUTHOR]

Abstract Land-based culturing can avoid the effects of environmental pollution and natural disasters, thus ensuring food safety for shellfish. However, food availability, in this case, is limited. To achieve the optimum balance of dietary carbohydrates and proteins and explore the mechanisms behind the phenomenon, we formulated five isoenergetic and isolipidic diets (C30P40, C35P35, C40P30, C45P25, and C50P20) with different levels of carbohydrates (C) and proteins (P). There were five experimental groups (C30P40, C35P35, C40P30, C45P25, and C50P20) and two control groups (CG1 and CG2). CG1 was fed with mixed powders of yeast and Chlorella sp., and CG2 was cultured in natural sea. After 60-day feeding, the highest rates of survival and absolute growth appeared in C45P25. C45P25 exhibited significantly higher activities of amylase, protease, alkaline phosphatase, acid phosphatase, superoxide dismutase, catalase, glutathione peroxidase, and phenoloxidase and significantly lower malondialdehyde content than C30P40, C35P35, C40P30, C50P20, and CG1. No significant differences were observed between C45P25 and CG2. Furthermore, the total antioxidant capacity of the pearl oysters in C45P25 was significantly higher than that in C30P40, C35P35, C40P30, and C50P20. On the basis of these results, the optimal balance of proteins and carbohydrates for pearl oysters was the C45P25 diet. Metabolomics-based profiling of the pearl oysters fed with high-carbohydrate/low-protein diet (C45P25) and low-carbohydrate/high-protein diet (C30P40) revealed 80 significantly different metabolites (VIP > 1 and P < 0.05). Furthermore, integrated key metabolic pathway analysis showed that C45P25 regulated starch and sucrose metabolism, alanine, aspartate and glutamate metabolism and glycine, serine and threonine metabolism to meet the energy demand and increase the glucogenic amino acid, thereby promoting protein synthesis and reducing fatty acid β-oxidation in comparison with C30P40. This finding helps elucidate the underlying mechanisms leading to the high-carbohydrate/low-protein diet characteristic of the optimal dietary carbohydrate and protein levels of P. f. martensii. Graphical abstract Unlabelled Image Highlights • The study is the first to explore the optimum balance of dietary carbohydrates and proteins of pearl oyster. • Dietary carbohydrates were used as the main energy source for pearl oyster. • C45P25 enhanced starch and sucrose metabolism to meet the energy demand. • C45P25 regulated glycine, serine and threonine metabolism to reduce β-oxidation. • C45P25 modified alanine, aspartate and glutamate metabolism to promote protein synthesis. [ABSTRACT FROM AUTHOR]

Natural disasters and environmental pollution are the main problems for traditional offshore culture. Pearl oyster culture through industrial farming can prevent these difficulties, but food availability is limited. In this study, we formulated two diets, P3 and P5, with yeast powder and corn gluten, respectively, as major protein sources. Two experimental groups (EG1 and EG2) were separately fed with P3 and P5 diets indoors. After 45 days of feeding, GC–TOF/MS-based metabolomics of the hepatopancreas was performed to assess the effect of different formulated diets on pearl oyster ( Pinctada fucata martensii ) combined with growth performance. EG1 displayed significantly faster absolute growth rate and higher survival rate than EG2. In the metabolomic assay, 125 metabolites were identified by mass spectrum matching with a spectral similarity value (SV) > 700, and 35 were considered significantly different metabolites (SDMs) (VIP > 1 and P < 0.05). Pathway analysis indicated that the SDMs were involved in 34 pathways, including seven influential pathways (pathway impact value > 0.1). The impact values of phenylalanine, tyrosine, and tryptophan biosynthesis, ascorbate and aldarate metabolism, phenylalanine metabolism, inositol phosphate metabolism, tyrosine metabolism, sulfur metabolism, and cysteine and methionine metabolism were 0.500, 0.500, 0.308, 0.219, 0.207, 0.167, and 0.161, respectively. Further integrated key metabolic pathway analysis showed that EG2 exhibited lower capability for inositol phosphate metabolism, ascorbate and aldarate metabolism, phenylalanine metabolism, and tyrosine metabolism. This study suggested that yeast powder is a better protein source of formulated diet for pearl oysters than corn gluten. [ABSTRACT FROM AUTHOR]

microRNAs are a class of non-coding RNAs with post-transcriptional regulatory functions in eukaryotes. In our previous study, miR-184-3p was identified in the hemocyte transcriptome of Pinctada fucata martensii (Pm-miR-184-3p), and its expression was shown to be up-regulated following transplantation surgery; however, its role in regulating transplantation immunity has not yet been clarified. Here, the role of Pm-miR-184-3p in regulating the immune response of P. f. martensii was studied. The expression of Pm-miR-184-3p increased following the stimulation of pathogen-associated molecular patterns, and Pm-miR-184-3p overexpression increased the activity of antioxidant-related enzymes, such as superoxide dismutase and catalase. Transcriptome analysis obtained 1096 differentially expressed genes (DEGs) after overexpression of Pm-miR-184-3p, and these DEGs were significantly enriched in conserved pathways such as the Cell cycle pathway and NF-kappa B signaling pathway, as well as GO terms including base excision repair, cell cycle, and DNA replication, suggesting that Pm-miR-184-3p could enhance the inflammation process. Target prediction and dual luciferase analysis revealed that pro-inflammatory related genes Pm-TLR3 and Pm-FN were the potential target of Pm-miR-184-3p. We speculate that Pm-miR-184-3p may utilize negative regulation of target genes to delay the activation of corresponding immune pathways, potentially preventing excessive inflammatory responses and achieving a delicate balance within the organism. Overall, Pm-miR-184-3p play a key role in regulating cellular responses to transplantation. Our findings provide new insights into the immune response of P. f. martensii to transplantation. • Expression of Pm-miR-184-3p was increased after PAMPs stimulation. • Overexpression of Pm-miR-184-3p enhanced the activities of antioxidant enzymes. • Overexpression of Pm-miR-184-3p activated cell cycle related-genes. • Pm-TLR3 and Pm-FN are potential targets of Pm-miR-184-3p. [ABSTRACT FROM AUTHOR]

The globular C1q domain-containing (C1qDC) protein can recognize a variety of ligands, such as pathogen-associated molecular patterns, and plays an important role in the innate immune response. Our previous studies showed that a novel globular C1q domain-containing protein (PmC1qDC-1) is involved in the damage repair process of pearl oyster shells. However, the function of PmC1qDC-1 in pearl oyster innate immunity remains unknown. In the present study, the high-level structural analysis showed that PmC1qDC-1 was a spherical structure composed of 10 strands and was similar to the AiC1qDC-2 of bay scallop (Argopecten irradians). In situ hybridization indicated that PmC1qDC-1 had strong fluorescence signal in gills. Furthermore, the mRNA expression of PmC1qDC-1 was highly induced at 6–48 h in gill after lipopolysaccharide, peptidoglycan and polyinosinic–polycytidylic acid stimulation. Additionally, we obtained the recombinant protein of PmC1qDC-1 (rPmC1qDC-1) and found that rPmC1qDC-1 had antibacterial activity against Gram-negative (i.e., Pseudomonas aeruginosa , Vibrio parahaemolyticus , Escherichia coli , and Aeromonas hydrophila) and Gram-positive (i.e., Staphylococcus aureus and Bacillus subtilis) bacteria. These results indicated that PmC1qDC- 1 might play an important role in the immune response against bacteria and viruses. This study provides clues for further studying the immune defense of Pinctada fucata martensii against pathogens and exploring the evolution of the classic pathway of complement system. • PmC1qDC- 1 has a strong fluorescence signal in gills. • PmC1qDC-1 can be rapidly induced by PAMP stimulation. • Obtaining the recombinant protein of PmC1qDC-1. • The recombinant PmC1qDC-1 protein exhibits antibacterial activity. [ABSTRACT FROM AUTHOR]

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus which hold the potential function in the shell formation as shell matrix proteins. In this study, a C1qDC protein was identified and characterized in pearl oyster (Pinctada fucata martensii) (PmC1qDC) to explore its function in nacre formation. The PmC1qDC-deduced protein sequence carried a typical globular C1q (gC1q) domain that possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1qDC family members and shared high homology with other gC1q domains. Homologous analysis of PmC1qDC presented it contained conserved secondary structure and Phe135, Phe155, Tyr166, Phe173, Tyr181, Phe183, and Phe256 amino acid residues. Expression pattern analysis showed that PmC1qDC expressed in all the detected tissues and exhibited a significantly higher expression level in nacre formation-associated tissues. After the shell notching, the expression level of PmC1qDC showed significantly up-regulation after 12 h in the central zone of mantle (MC). PmC1qDC expression significantly decreased in the MC after RNA interference (RNAi). Furthermore, disordered crystals with evident rough surface and irregular crystal tablets were observed in the nacre after RNAi. Results suggested that PmC1qDC affects the shell nacre formation, which is significant to improve the pearl production of pearl oyster. • PmC1qDC expressed significantly higher in nacre formation associated tissues. • PmC1qDC was involved in shell regeneration after injury. • PmC1qDC affected the shell nacre formation. [ABSTRACT FROM AUTHOR]

The pearl oyster, Pinctada fucata martensii , produces high-quality pearls. During pearl production, excess immune and inflammatory response after transplantation will lead to nucleus rejection, pearl sac formation failure, and death of the host pearl oyster. The hemocyte transcriptome and fatty acid (FA) contents in the adductor muscle before and after transplantation were analyzed to investigate the response of pearl oyster P. f. martensii to allograft-induced stress from lipid metabolism. The hemocyte transcriptome analysis detected 193 lipid metabolism-related genes, such as the elongation of very long-chain FA protein 5, acyl-CoA 6-desaturase, cytochrome P450, phospholipase A2, glycerol-3-phosphate O-acyltransferase, and prostaglandin-H2 d -isomerase. Pathway enrichment analyses revealed that these genes were mainly involved in the "biosynthesis of unsaturated FAs," "FA biosynthesis," "ARA metabolism," and "glycerolipid metabolism." An analysis of FA contents in the adductor muscle indicated no significant difference in the contents of lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, arachidic acid, α-linolenic acid, eicosadienoic acid, docosadienoic acid, and 11,14,17-eicosatrienoic acid. However, ARA, DHA, and EPA in the adductor muscle after transplantation were significantly greater than those processed without grafting surgery. These results suggest that pearl oysters require more polyunsaturated FAs (PUFAs) to regulate their inflammatory and immune response after transplantation. However, their ability to biosynthesize unsaturated FAs is limited. Given these results, the addition of PUFA-containing diets or selection of a line with strong ability to biosynthesize unsaturated FAs may be valuable for pearl oyster recovery after transplantation. • This study is the first to assess the response of pearl oysters to allograft-induced stress from lipid metabolism. • Pearl oysters require PUFA to regulate their inflammation and immune response after transplantation. • Pearl oysters have a limited ability to biosynthesize unsaturated fatty acids. [ABSTRACT FROM AUTHOR]

Abstract Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in immune responses. In this study, we identified 57 IL-17 genes from the genomes of six marine invertebrates, including Pinctada fucata martensii , Crassostrea gigas , Lottia gigantea , Capitella teleta , Mizuhopecten yessoensis , and Mytilus galloprovincialis. Phylogenetic analysis showed that all invertebrate IL-17 genes were clustered into one group, implying that invertebrate IL-17 evolved from one common ancestral gene. From the extron–intron analysis, we found many intronless IL-17 genes in mollusks, which may be caused by retroposition. Tissue and development transcriptomic analysis showed that the expression of PmIL-17 was tissue and developmental stage-specific. Moreover, we cloned the full length of the IL-17-2 gene from P. f. martensii (PmIL-17-2) and explored its function in the immune response. The full-length cDNA of PmIL-17-2 is 719 bp, containing an open reading frame of 564 bp, a 5′ -untranslated region (UTR) of 31 bp, and a 3′ -UTR of 124 bp with a 30 bp poly (A) tail. PmIL-17-2 had a strong response to lipopolysaccharide (LPS), indicating that the PmIL-17-2 participates in innate immune responses. In situ hybridization of hemocytes showed that PmIL-17-2 was mainly produced by granulosa cells, and the number of the stained granulosa increased after LPS stimulation. These results lay the foundation for the research of IL-17 family in marine invertebrates. Highlights • A total of 57 IL-17 genes were identified from 6 marine invertebrates. • IL-17 expression in pearl oyster was tissue and developmental stage specific. • IL-17s in invertebrates originate from the same ancestral gene. • PmIL-17-2 is involved in the innate immune response after LPS stimulation. [ABSTRACT FROM AUTHOR]

Abstract β -carotene-15,15-dioxygenase is an enzyme involved in carotenoid metabolism to catalyze oxidative cleavage of β -carotene at its central double bond to two molecules of retinal in intestinal cells of vertebrate. In this study, we cloned and characterized β -carotene-15,15-dioxygenase in pearl oyster Pinctada fucata martensii (PmβCDOX). The full length of PmβCDOX gene was 1802 bp, including 1554 bp of the open reading frame (ORF) that encoded 517 amino acids, a 5′UTR of 134 bp and a 3′ UTR of 114 bp. PmβCDOX was expressed at various tissues with highest level in hepatopancreas. Eighteen and fifteen single nucleotide polymorphisms (SNPs) were separately obtained in the exon and promoter of PmβCDOX. Eight SNPs (six SNPs in the exon and two SNPs in the promoter region) were significantly associated to total carotenoid content (TCC) (P <.05). The eight SNPs of significantly associated TCC were divided three haploblocks. Haplotypes CCTT had larger TCC than other haplotypes. The present results suggest that PmβCDOX is involved in carotenoid metabolism in pearl oyster. Our study will be helpful for development gene marker in selective breeding programs for TCC trait of the species. Highlights • We cloned the full-length and promoter of βCDOX in Pinctada fucata martensii. • Eighteen and fifteen single nucleotide polymorphisms (SNPs) were separately obtained in the exon and promoter of PmβCDOX. • Haplotypes CCTT had larger TCC (total carotenoid content) than other haplotypes. [ABSTRACT FROM AUTHOR]

Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of a CHST1 gene identified from the pearl oyster Pinctada fucata martensii ( PmCHST1b ) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5′ PSB motif and 3′ PB motif) which interacted with 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC ( P <0.05), and sulfate polymer content in the extrapallial fluid significantly reduced ( P <0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation. [ABSTRACT FROM AUTHOR]

Novel microRNA miR-63 (novel-miR-63) from pearl oyster Pinctada fucata martensii (Pm-novel-miR-63) is a species-specific miRNA. Our previous research has shown that the expression of Pm-novel-miR-63 was significantly downregulated at 24 h after nucleus transplantation. In this study, we analyzed the function and regulatory role of Pm-novel-miR-63 in the immune response of pearl oysters. The results showed that Pm-novel-miR-63 expression increased after the stimulation of pathogen associated molecular patterns at 6–12 h, and the activity of immune and antioxidant enzymes in the serum decreased after Pm-novel-miR-63 overexpression. Transcriptome analysis revealed that Pm-novel-miR-63 participated in regulating transplantation immunity through the Notch and mRNA surveillance signaling pathways. Target prediction and dual luciferase analysis revealed that Pm-GDP-FucTP, Pm-CysLTR2, and Pm-RLR were the target genes of Pm-novel-miR-63. These results suggested that Pm-novel-miR-63 participated in regulating the immune response in pearl oysters and can serve as a new interference target to reasonably control excessive immune rejection in pearl culture. • Expression of Pm-novel-miR-63 was increased after PAMPs stimulation. • The activity of immune and antioxidant enzymes was decreased after overexpression of Pm-novel-miR-63. • Pm-novel-miR-63 participated in regulating transplantation immunity via Notch and mRNA surveillance signaling pathways. • Pm-GDP-FucTP, Pm-CysLTR2 and Pm-RLR were the potential targets of Pm-novel-miR-63. [ABSTRACT FROM AUTHOR]

Pearl oyster Pinctada fucata martensii is an economically important species farmed to produce round nucleated pearl in China. However, pearl oyster shells are commonly exposed to biofouling, which is manually cleaned. This study experimentally evaluates the effects of biofouling cleaning by Siganus fuscescens (Houttuyn, 1782) foraging on growth performance and pearl traits of host pearl oyster P . fucata martensii . Three groups designated as EG1, EG2 and EG3 were set in the experiment. In EG1, biofouling was manually cleaned every 2 weeks and oysters were cultured in natural sea conditions. In EG2, biofouling was cleaned by foraging behavior of S . fuscescens every 2 weeks and oysters were cultured in natural sea conditions. In EG3, oysters were farmed in the net cage where S . fuscescens were stored. The survival rate of EG2 was higher than those of EG1 and EG3 and the daily growth rates of length and weight of shell and the thickness and weight of pearl of EG3 were significantly larger than those of EG1 and EG2. The survival rate, daily growth rates of the length and weight of shell, and thickness and weight of pearl were not significantly different between EG1 and EG2. This study suggested S . fuscescens foraging could evidently improve survival of host pearl oyster, which is promising to improve pearl yield in pearl oyster farms in China. [ABSTRACT FROM AUTHOR]

Abstract The pearl oyster Pinctada fucata martensii is a warm-water shellfish that is sensitive to cold environments. To investigate its potential adaptation to low-temperature stress, the selected line (SL) and based population (BP) were sampled to undergo transcriptome sequence. Results of transcriptome analysis showed 572 significant differentially expressed genes. The typical HSP70 and HSP40 exhibited the polar expression model in the two groups. Meanwhile, the related genes that involved in energy release mediated by oxidative phosphorylation and the biosynthesis of unsaturated fatty acid were increased in the SL. The apparent enrichment of different expressed genes in amino acid metabolism indicated that the small molecule system with amino acids was one of the main regulator for low-temperature stress. The different expressions of immune-related and lysosome protein encoding genes also reflected the variation of immunity in the two groups and indicated that it could affect the adaptation ability in different temperature. In addition, the similar trends of different expression of typical genes between two groups were obtained by using RNA-seq and qRT-PCR. These results suggested that multi-system adjustments are involved in the processes of low water temperature stress in pearl oyster, providing insights into the response systems of shellfish to acclimatise with ambient environment change. Highlights • HSP70 and HSP40 exhibited the polar expression model in the two populations. • Biosynthesis of unsaturated fatty acid and energy release was increased in the SL. • The small molecular system with amino acids might play a key role in cold stress. • Lysosome and immune function could influence the low temperature resistance. [ABSTRACT FROM AUTHOR]

Effective immune regulation after transplantation during pearl production is crucial for the cultivation of high-quality pearls. MicroRNAs (miRNAs) play an important role in a variety of physiological processes. To understand the regulatory rules of miRNAs after transplantation in Pinctada funcata martensii , we constructed 13 miRNA transcriptomes, including the control group (Con), allograft (Al), and xenograft (Xe) transplantation at six time points (6, 12, and 24 h and 3, 6, and 12 days), in which the xenografted mantle tissue was from Pinctada maxima. We identified 159 differentially expressed miRNAs (DEMs) and found that these DEMs showed high expression at 12 h, 24 h, and 3 days after transplantation. A total of 130 DEMs, such as Let-7, were present in the Al and Xe groups; miR-34 and 16 other DEMs were specifically present in the Al group; miR-216b and 13 other DEMs were specifically present in the Xe group. Compared with the Con group, the target genes of DEMs in the Al group were significantly enriched in protein complex, cytoskeleton, and macromolecular complex, and the Xe group was significantly enriched in ribonucleoside metabolic process, nucleoside binding, and cell division. Compared with the Al group, the target genes in the Xe group were significantly enriched in response to DNA damage stimulation. Overall, multiple pathways associated with cellular activity were enriched in higher numbers of genes in the Xe group than in the Al group. These findings enriched the information on immune regulatory mechanisms at the expression level of miRNAs in P. f. martensii after transplantation. • Thirteen miRNA transcriptomes were constructed. • Obtaining 159 differentially expressed miRNAs after transplantation. • Specific miRNAs involved in the immune response of allografts and xenografts were obtained. • Xenografts were found to induce stronger immune responses than allografts. [ABSTRACT FROM AUTHOR]

In this study, we formulated five diets, namely, P1, P2, P3, P4 and P5, with Chlorella sp. powder, Spirulina platensis powder, yeast powder, soybean meal and corn gluten, respectively, as major protein sources. A feeding experiment was designed to evaluate the effects of formulated diets on the growth performance, immunity and antioxidant and biomineralization capacity of juvenile pearl oyster ( Pinctada fucata martensii ). In the experiments, the five groups were separately fed with P1, P2, P3, P4 and P5 diets. After 45 days of feeding, pearl oysters fed on P1, P2, P3 and P4 diets showed significantly higher absolute growth rate and protease and amylase activities than those fed on P5 diet (P < 0.05). Moreover, pearl oysters fed on P1, P2, P3 and P4 diets exhibited significantly higher activities of alkaline phosphatase (AKP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (P < 0.05). Significantly higher expression levels of SOD, GPx, CAT, heat shock protein (HSP) 70, HSP90, nacrein, pif177 and pearlin mRNA were observed in pearl oysters fed on P1, P2, P3 and P4 diets relative to those fed on P5 (P < 0.05). Results suggested the suitability of Chlorella sp. powder, S. platensis powder, yeast powder and soybean meal as protein sources for development of formulated diets for pearl oyster P. f. martensii . [ABSTRACT FROM AUTHOR]

• Shell injury induced the production of serum ecdysone to regulate shell repair. • The ecdysone signal was transmitted by PmEcR ecdysone receptor in pearl oyster. • Ecdysone induced the expression of biomineralization related genes. • PmEcR and PmRXR was located in the nuclei and cytoplasm. • PmEcR and PmRXR did not form dimers which is different from other results. Ecdysone exists in arthropods, Mollusca and other invertebrates and plays vital roles in exoskeleton formation of Ecdysozoa. However, little is known about its functions in bivalve species. Herein, we identified ecdysone from the serum of pearl oyster Pinctada fucata martensii and obtained the coding sequence of ecdysone receptor (PmEcR) and homologue of its heterodimer protein retinoid X receptor (PmRXR). The deduced amino acid sequences of PmEcR and PmRXR contained a DNA-binding and ligand-binding domain and were very similar to the orthologs of other species. Moreover, PmEcR and PmRXR were located in the nuclei and cytoplasm of HEK-293T cells. PmEcR and PmRXR were highly expressed in early embryos and biomineralized mantle tissue. Moreover, the serum concentration of ecdysone significantly increased at 2, 4, 6, and 8 h post-shell notching. The expression of PmEcR in the mantle tissue was significantly induced at the corresponding time points, while that of PmRXR was significantly induced at 6 h. Ecdysone stimulation remarkably induced the expression of growth factors (BMP2 and BMP7), transcription factors (PmRunt and AP-1), and shell matrix protein genes (chitinase, lysine-rich matrix protein (KRMP), TYR2, and PmCOLVI), which indicated that ecdysone signaling plays important roles in shell repair. However, yeast two-hybrid assay and bimolecular fluorescence complementation showed that PmEcR and PmRXR did not form dimers, suggesting the different molecular interactions of EcR in bivalves. These findings provide insights into the function of ecdysone and its regulation pathway in bivalve species. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) are a class of ligand-gated ion channels that participate in signal transduction and are reported to play an important role in the immunomodulation of vertebrates and invertebrates. Previous studies have shown that the nAChRs in mollusks have undergone large-scale expansion after tandem repeats and retrotransposition, with the most expansion observed in bivalves. This study characterized the sequence of a tandem repeat nAChR unique to several bivalve mollusks and investigated its functions in Pinctada fucata martensii. Firstly, phylogenetic analysis revealed that the tandem arrays of nAChRs existed before bivalve differentiation and m ost tandem-replicated nAChR genes have a conserved genomic structure and domain combination. In present study, five tandemly duplicated nAChR genes were cloned from P. f. martensii and designated as PmnAChR-1 to PmnAChR-5. qRT-PCR analysis revealed that five PmnAChRs were specifically expressed in adult gills. In addition, after PAMP stimulation, the expression of PmnAChRs in hemocytes of P. f. martensii were strongly induced but exhibited different responses to different stimuli. PmnAChR-1 , PmnAChR-4 , and PmnAChR-5 exhibited strong and wide responsiveness to lipopolysaccharide (LPS) stimulation but had no response to peptidoglycan (PGN) stimulation. PmnAChR-2 expression was notably upregulated at 6 h after PGN challenge but had no response to LPS stimulation. Polyinosinic-polycytidylic acid challenge upregulated nearly all PmnAChRs , except for PmnAChR-5. Furthermore, Pm-miR-873-3p, Pm-miR-4577, Pm-miR-103a-3p, and Pm-miR-6753-3p were identified as the regulatory miRNA of PmnAChR-1 , PmnAChR-3 , PmnAChR-4 , and PmnAChR-5 , respectively. These findings suggested that these tandem arrays of nAChRs are unique to bivalves, and the tandem duplication of nAChR genes may be involved in the immune regulation process after pathogen stimulation. [Display omitted] • A tandem array of nAChRs unique to bivalves was identified. • The tandem array of nAChRs exists before bivalves differentiation. • PmnAChR genes expression were high in gill. • The expression of the PmnAChR genes was significantly up-regulated under PAMPs challenge. • Four regulatory miRNAs of PmnAChRs were identified. [ABSTRACT FROM AUTHOR]

Larval metamorphosis, the planktonic larvae transited into benthic juveniles, is a short important phase for mollusks. However, information about the mechanism of its larval settlement and metamorphosis of Pinctada fucata martensii is still limited. In this study, metabolomic and transcriptomic approaches were applied to assess the metabolic and transcript changes among the different larval stage groups of P. f. martensii to understand the mechanism. In the metabolomics assay, 71 pathways of significantly different metabolites (SDM), including phenylalanine, tyrosine, and tryptophan biosynthesis; ubiquinone and other terpenoid–quinone biosynthesis; phenylalanine metabolism and d -glutamine and D-glutamate metabolism, were identified between the eyed larvae and the spat. Fifty-seven pathways of SDMs, including d -glutamine and D-glutamate metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; arachidonic acid metabolism; ubiquinone and other terpenoid–quinone biosynthesis and steroid hormone biosynthesis, were identified between the spat and the juvenile. The transcriptome analysis showed that 4561 and 915 differentially expressed genes (DEG) were identified between the eyed larvae and the spat stages and between the spat and the juvenile stages, respectively. The pathway enrichment analysis in DEGs associated with the neurotransmitter showed that cholinergic, glutamatergic, GABAergic, and serotonergic synapse pathways were significantly enriched between the eyed larvae and the spat stages. This study is the first work involving the integrated metabolomic and transcriptomic analyses to identify the key pathways and understand the molecular and the metabolic mechanisms underlying the larval metamorphosis of P. f. martensii. • Metabolomics and transcriptomic were applied to analyze the larval metamorphosis of P. f. martensii. • The upregulating levels of 5-HT and N -methyl-D-aspartic acid, and their receptors promote the larval metamorphosis. • The levels of dopamine and dopamine receptors downregulated in spat stage. • Multiple neurotransmitter receptors regulate the larval metamorphosis. [ABSTRACT FROM AUTHOR]

The cholinergic anti-inflammatory pathway has been identified as a reflex monitoring system that contributes to the physiological and pathological regulation of cytokines. Nicotinic acetylcholine receptor (nAChR) plays an important role in immune regulation as a key molecule in neuronal communication. In this work, we investigated the characteristics and functions of a novel nAC hR β gene identified from the pearl oyster Pinctada fucata martensii (PmnAC hR-β). PmnAC hR-β displays structural similarities to nAChR molecules described in mammals, including a typical neurotransmitter-gated ion-channel ligand binding domain (LBD) and transmembrane (TM) domain. The result of phylogenetic analysis speculated that nAChR-β in Mollusca, Chordata and Arthropoda were separated into three branches. The LBD of PmnAChR-β was highly conserved, but its TM was variable. PmnAChR-β was highly expressed in eggs and fertilized eggs and had the most abundant mRNA expression in the gills of pearl oyster. The expression of PmnAChR-β mRNA was dramatically upregulated 12 h after lipopolysaccharide stimulation. Furthermore, PmnAChR-β was highly expressed at 12 h and 6–18 d after transplantation in hemocytes. Pm-miR-516b-5p was identified as the regulatory microRNA of PmnAChR-β. These results indicated that PmnAChR-β may be an important component of the cholinergic anti-inflammatory pathway and participates in the immune regulation process of pearl oysters. Unlabelled Image • A novel nAChR-β gene was cloned from pearl oysters. • Tissue and developmental stage specificity expression of PmnAChR-β. • PmnAChR-β was involved in the immunoregulatory of pearl oysters after LPS stimulation and transplantation. • The regulatory miRNA of PmnAChR-β was identified. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with post-transcriptional regulatory activity in various biological processes. Pearl oyster Pinctada fucata martensii is one of the main species cultured for marine pearl production in China and Japan. In this study, we constructed two small RNA libraries of mantle central (MC) and mantle edge (ME) from P. f. martensii and obtained 24,175,537 and 21,593,898 clean reads, respectively. A total of 258 miRNAs of P. f. martensii (Pm-miRNA) were identified, and 93 differentially expressed miRNAs (DEMs) including 49 known Pm-miRNAs and 44 novel Pm-miRNAs were obtained from the MC and ME. The target transcripts of these DEMs were obviously enriched in neuroactive ligand-receptor interaction pathway, and others. After over-expression of Pm-miR-124 and Pm-miR-9a-5p in the MC by mimic injection into the muscle of P. f. martensii , nacre exhibited a disorderly growth as detected by scanning electron microscopy. Pm-nicotinic acetylcholine receptor alpha subunit, Pm-neuropeptide Y and Pm-chitin synthase were investigated as the targets of Pm-miR-124; and Pm-tumor necrosis factor receptor associated factor 2 and Pm-chitin synthase were investigated as the targets of Pm-miR-9a-5p. These predicted target transcripts were down-regulated after the over-expression of Pm-miR-124 and Pm-miR-9a-5p in MC. This study comprehensively analyzed the miRNAs in mantle tissues to enhance our understanding of the regulatory mechanism underlying shell formation. Unlabelled Image [ABSTRACT FROM AUTHOR]

Thioredoxin-like protein 5 (Trxlp-5) is a thioredoxin isoform associated with cellular redox homeostasis through the activity of thiol-disulfide reductase. In our study, Trxlp-5 was identified and characterized in Pinctada fucata martensii. The expression of PmTrxlp-5 was detected in response to polyinosinic: polycytidylic acid (poly I:C) and lipopolysaccharides (LPS) stimulation. The differences in PmTrxlp-5 expression were evaluated between the black coloured selected line and the control stock after grafting operation. The open reading frame (ORF) consisted of 1167bp encoding a 388 amino acid, 5′-UTR of 41bp and a 3′-UTR of 846bp. PmTrxlp-5 exhibited a conserved WCXXC functional motif similar to thioredoxins from other species. Tissue analysis showcased the highest relative mRNA expressions of PmTrxlp-5 in the haemocytes. Interestingly, after the grafting operation, mRNA expression of PmTrxlp-5 in the haemocytes was differentially expressed post grafting with a peak 6 h after grafting suggesting the high involvement of the gene in immune response in the early stage after grafting. The black coloured selected line group (BS) had significantly higher expression than the control group (CG) at 24 h, 6 d and 30 d after grafting operation. PmTrxlp-5 also showed a wave-like pattern in mRNA expression after bacterial endotoxin LPS and viral mimic poly I:C. These results suggested that PmTrxlp-5 plays a vital function in cellular redox homeostasis and immune response against grafting operation and pathogenic infections and can be used as a gene marker for selective breeding programs. • The full length sequence of PmTrxlp-5 was characterized in P. fucata martensii. • PmTrxlp-5 was expressed highly in the black coloured selected line of P. fucata martensii. • PmTrxlp-5 may function in immune response against grafting operation and pathogenic infections. [ABSTRACT FROM AUTHOR]

Odd-skipped related 1 (OSR1) from Pinctada fucata martensii (PmOSR1) was cloned in this study. PmOSR1 contained an open reading frame measuring 951 bp that encoded a polypeptide of 316 residues. Domain analysis also revealed that PmOSR1 had a typical ZnF_C 2 H 2 domain, indicating high identity with OSR1 protein sequence from Crassostrea gigas (55.25%) and Mizuhopecten yessoensis (65.48%). The expression pattern analysis of PmOSR1 revealed its wide expression in the tissues and developmental stages. PmOSR1 also presented a higher expression in the fast-growing group and hybrid families than in the slow-growing group and inbred families. The polymorphism analysis of PmOSR1 revealed 45 single nucleotide polymorphisms (SNPs) in the genomic sequence, and association analysis presented four SNPs significantly associated with growth traits (namely, shell length, width, height, and shell weight; P < 0.05). Three SNPs out of the four SNPs were shaped as a haploblock, and haplotype AGT showed significantly higher growth traits than that of haplotype AACAA. Therefore, PmOSR1 could be used as a growth candidate gene for the selective breeding of P. f. martensii. • Odd-skipped related 1 from Pinctada fucata martensii (PmOSR1) possesses the conserved domain of OSR1. • PmOSR1 holds the potential to influence growth of P. f. martensii. • Four SNPs from PmOSR1 genomic sequence were significantly associated with growth traits. • Haplotype AGT showed significantly higher growth traits than that of haplotype AACAA. [ABSTRACT FROM AUTHOR]

Chitin participates in shell formation as the main component of an organic framework. Chitin-binding protein contains domains that can bind to chitin specifically. In this study, a novel chitin-binding protein from Pinctada fucata martensii (PmCBP) with poly (chitin-binding domain) was cloned, which contains a 5′-untranslated region (UTR) of 114 bp and 3′UTR of 116 bp, and encodes a putative protein of 2044 amino acids. The predicted PmCBP protein was structurally typical of the CBP family with 20 ChtBD2 domains. Phylogenetic and linear relation analyses showed that the ChtBD2 domain has a highly conserved structure among the three species of P. f. martensii , Crassostrea gigas, and Mizuhopecten yessoensis. qRT-PCR and in-situ hybridization analysis revealed that PmCBP was most abundant in the mantle pallium whose expression level was significantly correlated with the growth traits. After RNAi, PmCBP expression was significantly inhibited in the mantle pallium (P < 0.05) and the microstructure of nacreous layers showed a disordered growth in the experiment group. These results indicated that PmCBP may be involved in nacreous layer formation through participation in the process of binding chitin in pearl oyster P. f. martensii. PmCBP expression was down-regulated in the mantle pallium after RNAi (p <.05; Fig. A), and the microstructure of nacreous layers showed a disordered growth in the experiment group (Fig. B). The inner surface microstructure of the shell nacreous layer in the control group was normal under SEM with a stair-like growth pattern. These results indicate that PmCBP may involved in the formation of the shell nacreous layer. Fig. A show relative expressions in MP after RNAi of PmCBP. Mean values with "*" are significantly different (P <.05). Fig. B show microstructure of shells nacreous layer after RNAi. Unlabelled Image • We cloned a novel chitin binding protein from Pinctada fucata martensii (PmCBP) with Poly (chitin binding domain), the complete sequence was 6365 bp, and encodes a putative protein of 2044 amino acids. • Phylogenetic and linear relation analyses showed that the ChtBD2 domain has a highly conserved structure among the three species of P. f. martensii , C. gigas and M. yessoensis. • QRT-PCR and ISH analysis revealed that PmCBP was most abundant in the mantle pallium whose expression level mantle pallium was significantly correlated with the shell length and the shell weight. • RNAi results indicate that the poly (chitin binding domain) could be a molecule involved in exoskeleton formation. [ABSTRACT FROM AUTHOR]