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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]

The polyamine putrescine (Put) is a ubiquitous small cationic amine. It plays an essential role in controlling the innate immune response. However, little is known about its function in mollusks. In this study, the Put content was observed to increase in the serum of pearl oyster Pinctada fucata martensii after 6 and 24 h of lipopolysaccharide (LPS) stimulation. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased, and nitric oxide synthase was downregulated in the Put group (i.e., combined treatment with Put and LPS) compared with that in the LPS group (i.e., combined treatment with phosphate-buffered saline and LPS). Furthermore, activities of alkaline phosphatase and acid phosphatase were inhibited after 6 h of LPS stimulation. The expression levels of the nuclear factor kappa B, IκB kinase, Janus kinase, and signal transducer and activator of transcription proteins genes were all significantly suppressed at 12 and 24 h in the Put group. Pseudomonas aeruginosa and Bacillus subtilis grew better after being incubated with the serum from the Put group than that from the LPS group. Additionally, the Put treatment remarkably inhibited the autophagy of hemocytes mediated by the AMP-activated protein kinase-mammalian target of rapamycin-Beclin-1 pathway. This study demonstrated that Put can effectively inhibit the inflammatory response induced by LPS in pearl oysters. These results provide useful information for further exploration of the immunoregulatory functions of polyamines in bivalves and contribute to the development of immunosuppressive agents. [ABSTRACT FROM AUTHOR]

Pearl oysters are found in the marine ecosystem. Due to their frequent exposure to microbial pathogens and environmental perturbations, they have developed a multifaceted innate immune system with differentially expressed immune‐related genes in response to a range of stress via vast coordinated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, encapsulation and phagocytosis of the circulating haemocytes. The species are farmed to produce pearls. With the increasing adoption of aquaculture techniques in pearl production, there is the need to understand their culture environment and their immune response mechanisms. Multi‐omic studies have documented responses to nuclei insertion operation, diseases and various environmental conditions in Pinctada fucata martensii (Dunker, 1880), Pinctada maxima (Jameson, 1901), Pinctada margaritifera and Pteria penguin (Roding, 1798). Among the various stressors, nuclei insertion operation has been identified as a prevailing factor modifying gene expression. Other factors include life stage, air exposure, temperature, salinity, metals and organic contaminants. This manuscript details the response mechanism of these pearl oysters to the various stressors. [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]

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. [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]

Heterodimeric PEBP2/CBFs are key regulators in diverse biological processes, such as haematopoietic stem-cell generation, bone formation and cancers. In this work, we cloned runt-like transcriptional factor (designated as PmRunt) and CBF β (designated as PmCBF) gene, which comprise the heterodimeric transcriptional factor in Pinctada martensii. PmRunt was identified with an open reading frame that encodes 545 amino acids and has typical Runt domain. Phylogenetic analysis results speculated that runt-like transcriptional factors (RDs) in vertebrates and invertebrates are separated into two branches. In molluscs, PmRunt and other RDs are clustered in one of these branches. Direct interaction between PmRunt and PmCBF was evidenced by yeast two-hybrid assay results. Gene repression by RNA interference decreased the expression level of PmRunt, and subsequent observation of the inner surface of the nacre by scanning electron microscopy demonstrated disordered growth. The luciferase activities of reporters that contain promoter regions of Collagen VI-like (PmColVI) and PmNacrein were enhanced by PmRunt. Meanwhile, Pm-miR-183 apparently inhibited the relative luciferase activity of reporters containing the 3′-UTR of PmRunt. The expression level of PmRunt was repressed after Pm-miR-183 was overexpressed in the mantle tissue. Therefore, we proposed that PmRunt could be targeted by Pm-miR-183 and regulate the transcription of PmColVI and PmNacrein by increasing their transcriptional activity, thereby governing nacre formation. [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]

The effects of three artificial diets (S1, S2 and S3) on survival, growth and biochemical composition of one-year-old pearl oyster Pinctada martensii were investigated. Six experimental groups (EG1, EG2, EG3, EG4, EG5 and EG6) and one control group (CG) were set up. EG1, EG2 and EG3 were solely fed on S1, S2 and S3, respectively. EG4, EG5 and EG6 were fed on mixed diets, as follows: S1 and Platymonas subcordiformis; S2 and P. subcordiformis; and S3 and P. subcordiformis, respectively. CG was fed on only P. subcordiformis. All groups were continuously fed for 60 days. Survival, growth and biochemical composition of soft tissues were compared across the groups. Results showed that survival rate, the absolute growth rate (AGR) and relative growth rate (RGR) of shell length did not differ significantly across the groups ( p > 0.05). The AGR and RGR of total weight differed significantly among the groups ( p < 0.05). AGR and RGR of shell length and total weight were the highest in EG5 and the lowest in EG1. Gross fat content showed insignificant differences among the groups ( p > 0.05). However, gross protein content and ash content showed significant differences across the groups ( p < 0.05). The gross protein of the groups solely fed on artificial diets was lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. The contents of other amino acids, total amino acids (TAAs) and essential amino acids (EAAs) showed significant differences across the groups ( p < 0.05). TAA, EAA and delicious amino acids of the groups fed solely on artificial diets were lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. Results indicated that the artificial diet (S2) can serve as substitutes of microalgal diets for P. martensii. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain. [Display omitted] • Cross-kingdom miRNA transport from microalgae to bivalves by exosomes was detected. • Functions of circulating exosomal miRNAs in pearl oyster were examined. • Microalgae-derived miRNAs have multiple roles in pearl oyster. [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]

Abstract: Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5′ untranslated region (UTR) was 11bp and the 3′UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X1–2-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii. [Copyright &y& Elsevier]

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]

Long non-coding RNAs (lncRNAs) play regulatory roles in various biological processes, including exoskeleton formation and immune response. The exoskeleton-based mantle–shell defense system is an important defense mechanism in shellfish. In this study, we found a novel lncRNA, herein formally named, LncMSEN2 , from the pearl oyster Pinctada fucuta martensii , and its sequence was validated via polymerase chain reaction (PCR). LncMSEN2 was highly expressed in mantle tissues, especially in the central region (P < 0.05), and was also expressed in the pearl sac as detected by quantitative real-time PCR. In situ hybridization experiments revealed that LncMSEN2 had a strong positive signal in the inner and outer epidermal cells of the mantle pallial and central regions. RNA interference experiments showed that interference of LncMSEN2 expression with dsRNA in mantle tissues led to an abnormal crystal structure of the nacre. In addition, LncMSEN2 expression significantly increased 6 h after lipopolysaccharide stimulation in mantle tissues (P < 0.05). These results indicated that LncMSEN2 may be a novel regulator of the mantle–shell defense system of pearl oyster. • LncMSEN2 , a mantle-specific LncRNA was identified from pearl oyster. • LncMSEN2 was located in epidermal cells of mantle tissue and involved in nacre formation. • LncMSEN2 respond to LPS stimulation in pearl oyster. [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]

HSP40, also called DnaJ, functions as a molecular chaperone by binding to Hsp70 and plays critical roles in the growth, development, and response to heat stress. However, this gene family is rarely reported in pearl oyster. In this study, 31 putative HSP40 genes from Pinctada fucata martensii (PmHSP40) were identified through bioinformatics methods and classified into three groups according to the presence of the complete three domains (J, G/F zinc finger domain, and cysteine rich domain). Further analysis showed that the PmHSP40 genes are highly diverse in sequence, domain structure, and tissue and development expression profile, implying diversified functions. In addition, one highly induced PmHSP40 in low-temperature (PmHSP40LT) was cloned, and its function in temperature response was explored. PmHSP40LT has a full length of 1741 bp, containing 1059 bp ORF, 152 bp 5′UTR, and a 507 bp 3′UTR, and encodes 352 amino acids. PmHSP40LT expression was significantly induced at low (17 °C) and high temperature (32 °C) at 6 h, 1 d, and 3 d relative to the control group. Thus, PmHSP40LT possibly participates in response to high and low temperatures in pearl oyster. In conclusion, all these results provide a comprehensive basis for the further analysis of PmHSP40 function in pearl oysters. • Putative HSP40 genes were identified from Pinctada fucata martensii. • PmHSP40 genes are highly diverse in sequence, domain structure. • PmHSP40 genes are diverse in temporal and spatial expression profile. • One highly induced PmHSP40 in low-temperature (PmHSP40LT) was coned. • PmHSP40LT was significantly induced at low temperature and high temperature. [ABSTRACT FROM AUTHOR]

The frequency at which organisms are exposed to hypoxic conditions in aquatic environments is increasing due to coastal eutrophication and global warming. To reveal the effects of long-term hypoxic stress on metabolic changes of pearl oyster, commonly known as Pinctada (Pinctada fucata martensii), the present study performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites following 25 days hypoxia challenge. Transcriptome analysis detected 1108 differentially expressed genes (DEGs) between the control group and the hypoxia group. The gene ontology (GO) analysis of DEGs revealed that they are significantly enriched in functions such as "microtubule−based process", "histone (H3–K4, H3–K27, and H4–K20) trimethylation", "histone H4 acetylation", "kinesin complex", and "ATPase activity", and KEGG pathway functions, such as "DNA replication", "Apoptosis", and "MAPK signaling pathways". Metabolome analysis identified 68 significantly different metabolites from all identified metabolites, and associated with 25 metabolic pathways between the control and hypoxia groups. These pathways included aminoacyl−tRNA biosynthesis, arginine and proline metabolism, and phenylalanine metabolism. Our integrated analysis suggested that pearl oysters were subject to oxidative stress, apoptosis, immune inhibition, and neuronal excitability reduction under long-term hypoxic conditions. We also found a remarkable depression in a variety of biological functions under long-term hypoxia, including metabolic rates, biomineralization activities, and the repression of reorganization of the cytoskeleton and cell metabolism. These findings provide a basis for elucidating the mechanisms used by marine bivalves to cope with long-term hypoxic stress. • Pearl oysters were subject to oxidative stress, apoptosis, and immune inhibition under long-term hypoxic conditions. • Pearl oysters altered their neuronal activity under long-term hypoxic conditions. • Pearl oysters depressed biomineralization process under long-term hypoxia. • Pearl oysters declined the repression of reorganization of the cytoskeleton under long-term hypoxia. [ABSTRACT FROM AUTHOR]

Pre-grafting conditioning is a crucial procedure before transplant surgery during pearl production. To investigate the molecular response of the pearl oyster Pinctada fucata martensii to conditioning, we constructed two hemocyte transcriptomes from pearl oysters with and without conditioning. A total of 134,222,686 raw reads were generated and assembled using the reference genome of the pearl oyster. Transcriptome analysis revealed 3,074 differentially expressed genes (DEGs). Gene ontology and pathway enrichment analyses revealed that these DEGs were mainly associated with "microtubule-based process", "regulation of actin cytoskeleton", and "cell cycle". All related genes were over-expressed in pearl oysters after conditioning. Some nucleotide-binding oligomerization domain-like receptors (NLR), toll-like receptor, myd88, proinflammatory cytokine interleukin-17 (IL-17), and apoptosis-related genes were highly expressed in pearl oysters after conditioning, indicating that conditioning induced the immune response of pearl oysters. "Fatty acid biosynthesis" (FA biosynthesis) was included in the enriched terms, and all eight FA synthase genes in this pathway were highly induced after conditioning. Four tandemly duplicated arginine kinase genes (PmAK) were found in the genome of P. f. martensii , gene structure and sequence analysis indicated PmAK genes were more diverse compared with that from human and zebra fish. The four tandemly duplicated PmAKs were highly up-regulated after conditioning. These findings will help to elucidate the responding molecular events after conditioning and explain the high pearl oyster survival rate with conditioning after transplantation, thereby providing useful information in perfecting the conditioning method to improve pearl oyster survival rate after transplantation. • Immune response was induced after pre-grafting conditioning. • Fatty acid biosynthesis were involved in the response to conditioning. • Four tandemly duplicated AK were found in the genome of P. f. martensii. • AK was functional in the energy production after conditioning. [ABSTRACT FROM AUTHOR]

The immune response after allograft or xenograft transplantation in the pearl oyster is a major factor that cause its nucleus rejection and death. To determine the mechanism underlying the immune response after allograft and xenograft transplantations in the pearl oyster Pinctada fucata martensii , we constructed two sets of transcriptomes of hemocytes at different times (6 and 12 h; 1, 3, 6, 12, and 30 d) after allograft and xenograft transplantations, in which the xenografted mantle tissue was from Pinctada maxima. The transcriptomic analysis reveals many genes are involved in the immune response to transplantation, such as transient receptor potential cation channel (TRP), calmodulin (CaM), DNA replication-related genes, and sugar and lipid metabolism-related genes. The expression of these identified genes was higher in the host pearl oyster transplanted with xenograft than that by allograft. The histological analysis of the pearl sac also confirmed that many hemocytes were still gathered around the transplanted nucleus, and no pearl sac was formed in the host pearl oysters at 30 d after xenograft transplantation. The genomic analysis indicated that pearl oysters evolved many copies of genes, such as TRP, CaM, and GST, to sense and cope with the immune response after transplantation. "Ribosome" and "Cytosolic DNA-sensing pathway" were specifically induced in the xenograft group, whereas "Notch signaling pathway" specifically responded to the allograft transplantation. These results can improve our understanding of the mechanism underlying the immune response of pearl oysters after allograft and xenograft transplantations. • Fifteen transcriptomes of the hemocytes from pearl oyster were constructed. • Obtained many pathways and genes involved in transplantation response. • Find out the difference in molecular response between allograft and xenograft. • Pearl oysters evolved many copies of genes involved in transplantation response. [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]

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]

Similar to other marine bivalves, Pinctada maxima shows unsynchronized growth, but the underlying metabolic mechanisms have not been studied. In this study, gold-lipped pearl oyster P. maxima from cultured stocks were selected to produce progeny stock. At 180 days, the stock was sorted by size, and fast-growing individuals and slow-growing individuals were separately sampled. Then, a metabolomic approach based on gas chromatography–mass spectroscopy was applied to assess the metabolite changes between the fast-growing and slow-growing groups of P. maxima and to understand the mechanism of their unsynchronized growth. In the metabolomics assay, among the 896 peaks isolated, 111 metabolites were revealed a spectral similarity value of >700 by using mass spectrum matching, and 48 were considered as significantly different metabolites (SDMs; VIP > 1 and P < 0.1) between the fast-growing and slow-growing groups. Results revealed that pearl oyster P. maxima changes metabolic status with different growth performance. Pathway analysis indicated that these SDMs were involved in 11 pathways. Further integrated key metabolic pathway analysis showed that pearl oysters possessed different capabilities in valine, leucine, and isoleucine biosynthesis, glycine, serine, and threonine metabolism, pyrimidine metabolism, cysteine and methionine metabolism, and glutathione metabolism between fast-growing and slow-growing groups. This study is the first metabolomics study to identify the key pathways and crucial metabolites so as to understand the metabolic mechanism of unsynchronized growth of bivalves. [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]

Metamorphosis of pearl oyster Pinctada maxima pediveliger is affected by physical and chemical characteristics of the collectors. In the present study, we conducted four experiments to evaluate the effects of collector characteristics on the settlement and growth of hatchery-reared pearl oyster. In the first experiment, black, red, yellow, and white plastic sheets were used as collectors. Settlement of P. maxima larvae in black and red plastic sheets was significantly higher than that in yellow and white plastic sheets ( P < 0.05). Mean shell length was not significantly different among the four colored sheets ( P > 0.05). In the second experiment, pediveliger larvae were settled onto palm rope and polypropylene rope collectors. The number of spat in the palm rope collector was significantly higher than that of spat in the polypropylene rope collector ( P < 0.05). Mean shell length was not significantly different between palm rope and polypropylene rope collectors ( P > 0.05). In the third experiment, settlement and subsequent growth were compared between plastic sheets with and without biofilm. The number of spat in the plastic sheets with biofilm was significantly higher than that in the plastic sheets without biofilm ( P < 0.05). Mean shell length after settlement were not significantly affected ( P > 0.05). In the fourth experiment, pediveliger larvae were stored in tanks with (experimental group) and without collectors (control group). The settlement rate of larvae on the wall was not significantly different between the two groups( P > 0.05). The settlement rates of the larvae on the wall and collectors in the experimental group were significantly larger than those on the wall in the control group( P < 0.05). Mean shell length was not significantly different between the groups ( P > 0.05). Our results suggest that appropriate selection of collector types, color, and conditioning can improve the settlement of P . maxima larvae in hatcheries. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (LncRNAs) are abundant in the genome of higher forms of eukaryotes and implicated in regulating the diversity of biological processes partly because they host microRNAs (miRNAs), which are repressors of target gene expression. In vertebrates, miR-133 regulates the differentiation and proliferation of cardiac and skeletal muscles. Pinctada martensii miR-133 ( pm-miR-133 ) was identified in our previous research through Solexa deep sequencing. In the present study, the precise sequence of mature pm-miR-133 was validated through miR-RACE. Stem loop qRT-PCR analysis demonstrated that mature pm-miR-133 was constitutively expressed in the adductor muscle, gonad, hepatopancreas, mantle, foot, and gill of P. martensii . Among these tissues, the adductor muscle exhibited the highest pm-miR-133 expression. Target analysis indicated that pm-RhoA was the potential regulatory target of pm-miR-133 . Bioinformatics analyses revealed that a potential LncRNA (designated as Lnc133 ) with a mature pm-miR-133 could generate a hairpin structure that was highly homologous to that of Lottia gigantea . Lnc133 was also highly expressed in the adductor muscle, gill, hepatopancreas, and gonad. Phylogenetic analysis further showed that the miR-133s derived from chordate and achordate were separated into two classes. Therefore, Lnc133 hosting pm-miR-133 could be involved in regulating the cell proliferation of adductor muscles by targeting pm-RhoA . [ABSTRACT FROM AUTHOR]

Sperdimine (Spd) and spermine (Spm) play an important role in control of innate immune responses in vertebrates and invertebrates. However, little is known about their functions in mollusks. In this study, we identified two Spd synthase and two Spm synthase genes from the genome of the pearl oyster Pinctada fucata martensii. Spd content gradually reduced in serum after lipopolysaccharide (LPS) stimulation, whereas Spm content increased. In addition, Spd/Spm treatment can significantly up-regulate the activities of superoxide dismutase, catalase, and glutathione peroxidase, and inhibit the activity of inducible nitric oxide synthase. We also found that the content of TNF-α was significantly inhibited in the Spm group, while it was significantly induced at 48 h in the Spd group. IL-17 content was down-regulated at 6, 24 and 48 h in the Spm group, while IL-17 showed no significant change in the Spd group. The expression levels of NF-κB, IKK, STAT, and JAK genes were significantly suppressed at 6 h and induced at 48 h after Spm treatment. However, the expression levels of NF-κB, IKK, and JAK were remarkably upregulated at 12, 24 and 6 h after Spd treatment. STAT gene was suppressed at 6 h and then induced at 12 h in the Spd group. Finally, compared with that in the control group, the growth of Pseudomonas aeruginosa was promoted after being incubated with serum from the Spm group at 6 h but inhibited after being incubated with serum from the Spd group at 12 and 24 h. Collectively, these results indicate that Spm and Spd will be disordered after LPS stimulation, which may be important immune regulatory molecules in pearl oysters. These results provide references for in-depth study of the functions of polyamines in mollusks. • The pearl oyster genome contains two spermidine synthase and two spermine synthase genes. • Lipopolysaccharide (LPS) stimulation reduces spermidine (Spd) level and increases spermine (Spm) content. • Spd or Spm treatment improves the antioxidant capacity of pearl oysters. • Spm treatment reduces the inflammatory response induced by LPS, whereas Spd promotes inflammation. • NF-κB and JAK-STAT signaling pathways are the key pathways for Spm or Spd to regulate the immune response of pearl oysters. [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]

Excessive immune response of pearl oyster after transplantation is the main factor that causes its nucleus rejection and death. In this study, to clarify how the cholinergic system regulates the immune response after transplantation, ACh and mecamylamine hydrochloride (nAChR inhibitor) were used to activate or block the cholinergic system of pearl oysters before transplantation. Transcriptomes of hemocytes were constructed at different times (6, 12, 24, and 48 h) after transplantation. Transcriptome analysis showed that the Kyoto Encyclopedia of Genes and Genomes pathway related to "inositol phosphate metabolism" pathway and Gene Ontology (GO) terms "calcium-ion binding" were significantly enriched in the differential expression genes of the two comparisons, and calmodulin genes were upregulated at 12–24 h after activation of the cholinergic system. Further experiments showed that ACh treatment significantly induced intracellular Ca2+ concentration. GO terms related to "antioxidant activity" and "superoxide dismutase activity" were significantly enriched in the two comparisons. The result of enzyme activity showed that the activities of superoxide dismutase and catalase in serum were significantly upregulated after activation of the cholinergic system but downregulated after blockade of this system. Furthermore, the cholinergic system maybe participate in regulating the "DNA replication", "cell cycle", "mismatch repair", and "apoptosis" of the hemocytes after transplantation. These results provide new insights into the molecular mechanism of the cholinergic system in regulating the immunity of pearl oyster transplantation and facilitate the development of immunosuppressive agents to improve the effectiveness of cultured pearls. • This study is the first to assess the function of the cholinergic system in pearl oyster transplantation immunity. • ACh treatment induced the Ca2+ concentration and calmodulin expression in the hemocytes of pearl oysters. • Obtained many pathways and genes involved in cholinergic system regulation of transplant immunity. • The cholinergic system maybe regulate the proliferation and apoptosis of the hemocytes after transplantation. [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]

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]