The role of Pm-miR-184-3p in regulating the immune response in the pearl oyster Pinctada fucata martensii.

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.

Characterization of transcription factor activator pretein-1 (AP-1) and its association with cold tolerance in Pinctada fucata martensii.

AP-1 is an important transcription factor for cell proliferation/differentiation and animal immunity/development; however, its role in research in shellfish is poorly understood. Here, the cDNA of AP-1 gene from Pinctada fucata martensii was characterized. Its expression was detected in all six examined tissues, and a high level was observed in the gill and hepatopancreas. Analysis of the developmental transcriptomes showed that the PmAP-1 gene expression levels were high during D-stage larval and spat stages. The gene also exhibited a significantly high expression under cold tolerance stress. SNP analysis of the exon region and 5' flanking region of PmAP-1 revealed 19 SNPs of which 8 showed significant differences between cold tolerance selection line and base stock. Furthermore, three haplotypes generated by the SNPs of PmAP-1 were significantly associated with cold tolerance, respectively.These results suggest that the PmAP-1 gene plays an important role in the response of P. f. martensii to low temperature stress. These SNPs and haplotypes of PmAP-1 may be related to the cold tolerance of P. f. martensii, and could be candidate markers potentially for further selective breeding.

LncMSEN1, a mantle-specific LncRNA participating in nacre formation and response to polyI:C stimulation in pearl oyster Pinctada fucata martensii.

Long noncoding RNA (LncRNA) regulates various life processes, including biomineralization and innate immune response through complex mechanisms. In this research, we identified a LncRNA named LncMSEN1 from pearl oyster Pinctada fucata martensii. LncMSEN1 sequence was validated by PCR, and its expression was high in mantle tissues according to qRT-PCR. LncMSEN1 was co-located with the nacre matrix protein N-U8 and fibrinogen domain-containing protein. And LncMSEN1 and N-U8 expression levels in the mantle were positively correlated. RNA interference was used to detect its effect on nacre formation in shells. Results showed that the decreased LncMSEN1 expression in mantle can cause the disordered growth of crystals on the inner surface of nacre in the shells, as well as the decrease expression of N-U8. In addition, the LncMSEN1 expression level significantly increased at 24 h after polyI:C stimulation in the mantle (P < 0.05). These findings suggested the involvement of LncMSEN1 in the formation of nacre in shells and related to innate immune response in pearl oyster, which provided additional insights into the roles of LncRNAs in pearl oysters.

Effect of vitamin D3 on immunity and antioxidant capacity of pearl oyster Pinctada fucata martensii after transplantation: Insights from LC-MS-based metabolomics analysis.

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.

HSP40 gene family in pearl oyster Pinctada fucata martensii: Genome-Wide identification and function analysis.

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.

Comprehensive transcriptome analysis reveal key molecular events in the pearl oyster after pre-grafting conditioning.

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.

Genome and transcriptome analyses providing insight into the immune response of pearl oysters after allograft and xenograft transplantations.

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.

Evolution and function analysis of interleukin-17 gene from Pinctada fucata martensii.

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.

Adaptive response of pearl oyster Pinctada fucata martensii to low water temperature stress.

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.

Identification of EGFR in pearl oyster (Pinctada fucata martensii) and correlation analysis of its expression and growth traits.

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.

Effects of protein sources on growth, immunity and antioxidant capacity of juvenile pearl oyster Pinctada fucata martensii.

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.

Identification of a novel miR-146a from Pinctada martensii involved in the regulation of the inflammatory response.

Increasing evidence demonstrated that microRNAs (miRNAs) play critical roles in innate immunity in vertebrates and invertebrates. MiR-146a/b is reported as a key regulator of the immune response through mediating Toll-like receptor and cytokine signalling. In this study, a novel miR-146a was identified and characterised from Pinctada martensii (designated as pm-miR-146a), and its roles in modulating the inflammatory response after LPS stimulation were also investigated. Pm-miR-146a ubiquitously expressed in all examined tissues, with the highest level in the mantle and lowest expression in the haemolymph. Pm-miR-146a increased at 24 h after lipoplysaccharide injection, in union with up-regulated NF-κB (P < 0.05). The over-expression of pm-miR-146a in vivo could significantly inhibit the expression of macrophage migration inhibitory factor (MIF), the potential target gene predicted by miRanda, while enforcing pm-miR-146a involved in the down-regulation of NF-κB. Thus, we propose that pm-miR-146a plays a role of negative feedback regulation to the NF-κB signal by repressing the expression of the pro-inflammatory cytokine MIF. These findings revealed that miR-146a represents a critical role in inflammatory response and offers new evidence for miRNAs in the innate immunity of molluscs.

MicroRNA, Pm-miR-2305, Participates in Nacre Formation by Targeting Pearlin in Pearl Oyster Pinctada martensii.

MicroRNAs (miRNAs) are noncoding RNA molecules that function as negative regulators of target genes. In our previous research, 258 pm-miRNAs were identified in Pinctada martensii by Solexa deep sequencing. Pm-miR-2305 was one of the identified pm-miRNAs with a potential function in biomineralization. In the present study, the precursor of pm-miR-2305 was predicted with 96 bp, containing a characteristic hairpin structure. Stem-loop qRT-PCR analysis indicated that pm-miR-2305 was constitutively expressed in all the tissues (adductor muscle, gill, mantle, hepatopancreas, foot, and gonad) of P. martensii and was highly expressed in the foot. After the over-expression of pm-miR-2305 in the mantle by mimics injection into the muscle of P. martensii, nacre demonstrated disorderly growth, as detected by scanning electron microscopy. Dual luciferase reporter gene assay indicated that pm-miR-2305 mimics could significantly inhibit the luciferase activity of the reporter containing the 3'UTR of the pearlin gene. Western blot analysis demonstrated that the protein expression of pearlin was down-regulated in the mantle tissue after the over-expression of pm-miR-2305. Therefore, our data showed that pm-miR-2305 participated in nacre formation by targeting pearlin in P. martensii.

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation.

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5' untranslated region (UTR) of 51 bp, a 3' UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.

Molecular characterization of the BMP7 gene and its potential role in shell formation in Pinctada martensii.

Bone morphogenetic protein 7 (BMP7), also called osteogenetic protein-1, can induce bone formation. In this study, the obtained full-length cDNA of BMP7 from Pinctada martensii (Pm-BMP7) was 2972 bp, including a 5'-untranslated region (UTR) of 294 bp, an open reading fragment of 1290 bp encoding a 429 amino acid polypeptide and a 3'-UTR of 1388 bp. The deduced protein sequence of Pm-BMP7 contained a signal peptide, a pro-domain and a mature peptide. The mature peptide consisted of 135 amino acids and included a transforming growth factor ß family domain with six shared cysteine residues. The protein sequence of Pm-BMP7 showed 66% identity with that from Crassostrea gigas. Two unigenes encoding Pm-BMPRI (Pm-BMP receptor I) and Pm-BMPRII were obtained from the transcriptome database of P. martensii. Tissue expression analysis demonstrated Pm-BMP7 and Pm-BMPRI were highly expressed in the mantle (shell formation related-tissue), while Pm-BMPRII was highly expressed in the foot. After inhibiting Pm-BMP7 expression using RNA interference (RNAi) technology, Pm-BMP7 mRNA was significantly down-regulated (p < 0.05) in the mantle pallium (nacre formation related-tissue) and the mantle edge (prismatic layer formation related-tissue). The microstructure, observed using a scanning electron microscope, indicated a disordered growth status in the nacre and obvious holes in the prismatic layer in the dsRNA-Pm-BMP7 injected-group. These results suggest that Pm-BMP7 plays a crucial role in the nacre and prismatic layer formation process of the shell.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

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.

The first high-quality chromosome-level genome of the Sipuncula Sipunculus nudus using HiFi and Hi-C data.

Sipuncula is a class of exocoelomic unsegmented animals whose evolutionary relationships are unresolved. The peanut worm Sipunculus nudus is a globally distributed, economically important species belonging to the class Sipuncula. Herein, we present the first high-quality chromosome-level assembly of S. nudus based on HiFi reads and high-resolution chromosome conformation capture (Hi-C) data. The assembled genome was 1,427 Mb, with a contig N50 length of 29.46 Mb and scaffold N50 length of 80.87 Mb. Approximately 97.91% of the genome sequence was anchored to 17 chromosomes. A BUSCO assessment showed that 97.7% of the expectedly conserved genes were present in the genome assembly. The genome was composed of 47.91% repetitive sequences, and 28,749 protein-coding genes were predicted. A phylogenetic tree demonstrated that Sipuncula belongs to Annelida and diverged from the common ancestor of Polychaeta. The high-quality chromosome-level genome of S. nudus will serve as a valuable reference for studies of the genetic diversity and evolution of Lophotrochozoa.

Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation.

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 797 bp long, containing an open reading fragment (ORF) of 537 bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4 kDa and theoretical isoelectric point of 5.97. The 5' untranslated region (UTR) was 11 bp and the 3'UTR was 249 with 18 bp 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-X(1-2)-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.

Lipidomic insights into the immune response and pearl formation in transplanted pearl oyster Pinctada fucata martensii.

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

Integrated GC-MS- and LC-MS-Based Untargeted Metabolomics Studies of the Effect of Vitamin D3 on Pearl Production Traits in Pearl Oyster Pinctada fucata martensii.

Pearl oyster Pinctada fucata martensii is widely recognized for biomineralization and has been cultured for high-quality marine pearl production. To ascertain how dietary vitamin D3 (VD3) levels affect the features of pearl production by P. f. martensii and discover the mechanisms regulating this occurrence, five experimental diets with variable levels of VD3 were used with inclusion levels of 0, 500, 1,000, 3,000, and 10,000 IU/kg. The distinct inclusion levels were distributed into five experimental groups (EG1, EG2, EG3, EG4, and EG5). All the experimental groups were reared indoors except the control group (CG) reared at the sea. Pearl oysters, one year and a half old, were used in the grafting operation to culture pearls. During the growing period that lasted 137 days, EG3 had the highest survival rate, retention rate, and high-quality pearl rate. A similar trend was found for EG3 and CG with significantly higher pearl thickness and nacre deposition rates than other groups, but no significant differences were observed between them. A metabolomics profiling using GC-MS and LC-MS of pearl oysters fed with low quantities of dietary VD3 and optimal levels of dietary VD3 revealed 135 statistically differential metabolites (SDMs) (VIP > 1 and p < 0.05). Pathway analysis indicated that SDMs were involved in 32 pathways, such as phenylalanine metabolism, histidine metabolism, glycerophospholipid metabolism, alanine aspartate and glutamate metabolism, arginine and proline metabolism, glycerolipid metabolism, amino sugar and nucleotide sugar metabolism, and tyrosine metabolism. These results provide a theoretical foundation for understanding the impacts of VD3 on pearl production traits in pearl oyster and reinforce forthcoming prospects and application of VD3 in pearl oyster in aquaculture rearing conditions.