Comparative transcriptomics identifies genes underlying growth performance of the Pacific black-lipped pearl oyster Pinctada margaritifera.

BACKGROUND: In bivalves, the rate at which organisms grow is a major functional trait underlying many aspects of their commercial production. Growth is a highly polygenic trait, which is typically regulated by many genes with small to moderate effects. Due to its complexity, growth variability in such shellfish remains poorly understood. In this study, we aimed to investigate differential gene expression among spat of the pearl oyster Pinctada margaritifera with distinct growth phenotypes. RESULTS: We selected two groups of P. margaritifera spat belonging to the same F2 cohort based on their growth performance at 5.5 months old. Transcriptome profile analysis identified a total of 394 differentially expressed genes between these Fast-growing (F) and Slow-growing (S) phenotypes. According to functional enrichment analysis, S oysters overexpressed genes associated with stress-pathways and regulation of innate immune responses. In contrast, F oysters up-regulated genes associated with cytoskeleton activity, cell proliferation, and apoptosis. Analysis of genome polymorphism identified 16 single nucleotide polymorphisms (SNPs) significantly associated with the growth phenotypes. SNP effect categorization revealed one SNP identified for high effect and annotated for a stop codon gained mutation. Interestingly, this SNP is located within a gene annotated for scavenger receptor class F member 1 (SRF1), which is known to modulate apoptosis. Our analyses also revealed that all F oysters showed up-regulation for this gene and were homozygous for the stop-codon mutation. Conversely, S oysters had a heterozygous genotype and a reduced expression of this gene. CONCLUSIONS: Altogether, our findings suggest that differences in growth among the same oyster cohort may be explained by contrasted metabolic allocation between regulatory pathways for growth and the immune system. This study provides a valuable contribution towards our understanding of the molecular components associated with growth performance in the pearl oyster P. margaritifera and bivalves in general.

PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome.

BACKGROUND: The pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms. RESULTS: Herein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment. CONCLUSIONS: Our results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.

Identification of a long non-coding RNA (LncMSEN2) from pearl oyster and its potential roles in exoskeleton formation and LPS stimulation.

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.

Transcriptional regulation of the matrix protein Shematrin-2 during shell formation in pearl oyster.

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata, has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer-binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata, improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level.

Gene expression profiles at different stages for formation of pearl sac and pearl in the pearl oyster Pinctada fucata.

BACKGROUND: The most critical step in the pearl formation during aquaculture is issued to the proliferation and differentiation of outer epithelial cells of mantle graft into pearl sac. This pearl sac secretes various matrix proteins to produce pearls by a complex physiological process which has not been well-understood yet. Here, we aimed to unravel the genes involved in the development of pearl sac and pearl, and the sequential expression patterns of different shell matrix proteins secreted from the pearl sac during pearl formation by pearl oyster Pinctada fucata using high-throughput transcriptome profiling. RESULTS: Principal component analysis (PCA) showed clearly different gene expression profiles between earlier (before 1 week) and later stages (1 week to 3 months) of grafting. Immune-related genes were highly expressed between 0 h - 24 h (donor dependent) and 48 h - 1 w (host dependent), and in the course of wound healing process pearl sac was developed by two weeks of graft transplantation. Moreover, for the first time, we identified some stem cell marker genes including ABCG2, SOX2, MEF2A, HES1, MET, NRP1, ESR1, STAT6, PAX2, FZD1 and PROM1 that were expressed differentially during the formation of pearl sac. The expression profiling of 192 biomineralization-related genes demonstrated that most of the shell matrix proteins (SMPs) involved in prismatic layer formation were first up-regulated and then gradually down-regulated indicating their involvement in the development of pearl sac and the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated at 2 weeks after the maturation of pearl sac. Nacrein, MSI7 and shematrin involved in both layer formation were highly expressed during 0 h - 24 h, down-regulated up to 1 week and then up-regulated again after accomplishment of pearl sac formation. CONCLUSIONS: Using an RNA-seq approach we unraveled the expression pattern of the key genes involved in the development of pearl sac and pearl as a result of host immune response after grafting. These findings provide valuable information in understanding the molecular mechanism of pearl formation and immune response in P. fucata.

Developmental characteristics of pearl oyster Pinctada fucata martensii: insight into key molecular events related to shell formation, settlement and metamorphosis.

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.

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.

Identification and analysis of an MKK4 homologue in response to the nucleus grafting operation and antigens in the pearl oyster, Pinctada fucata.

The mitogen-activated protein kinase kinase 4 (MKK4) is a key component of the c-Jun N-terminal kinase (JNK) signaling pathway and regulates multiple cellular activities. However, little is known about the roles of this kinase in pearl oyster. In this study, we identified an MKK4 homologue in Pinctada fucata by using a transcriptome database. Sequence analysis and protein structure prediction showed that PfMKK4 is highly conserved to MKK4 from other vertebrate and invertebrate species. Phylogenetic analysis revealed that PfMKK4 has the closest relationship with that from Crassostrea gigas. QPCR was used to investigate expression profiles in different healthy adult tissues and developmental stages of P. fucata. We found that PfMKK4 was ubiquitously expressed in all tissues and developmental stages examined except for in D-shaped larvae. Gene expression analysis suggested that PfMKK4 is involved in the response to the nucleus insertion operation. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [poly(I:C)] stimulation in vivo reduced PfMKK4 mRNA expression at 6 h, 48 h and 48 h, 72 h, respectively. LPS and poly(I:C) induced PfMKK4 phosphorylation in a primary mantle cell culture. These results contribute to better understanding of the potential role played by PfMKK4 in protecting the pearl oyster from injury caused by grafting or disease.

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.

Irregularity of the Linear Growth of the Margaritifera margaritifera (Bivalvia: Margaritiferidae) Population of the Nemina River, Karelia.

The dependence of shell growth in length and height during ontogeny has been studied in the pearl mussel Margaritifera margaritifera inhabiting the Nemina River (basin of Lake Onega, Karelia). It has been shown that the population is heterogenous based on the height-to-length ratio. It has been found that during ontogeny M. margaritifera from the studied population undergoes a constant change in the relative growth of the shell leading to either lengthening or rounding of the shell.

Microarray: a global analysis of biomineralization-related gene expression profiles during larval development in the pearl oyster, Pinctada fucata.

BACKGROUND: The molluscan Pinctada fucata is an important pearl-culturing organism to study biomineralization mechanisms. Several biomineralization-related genes play important roles regulating shell formation, but most previous work has focused only on their functions in adult oysters. Few studies have investigated biomineralization during larval development, when the shell is initially constructed and formed until the juvenile stage in dissoconch shells. Here, we report, for the first time, a global gene analysis during larval development of P. fucata based on a microarray and reveal the relationships between biomineralization-related genes and the shell formation process. RESULTS: Based on the P. fucata mantle transcriptome, 58,940 probes (60 nt), representing 58,623 transcripts, were synthesized. The gene expression profiles of the fertilized egg, trochophore, D-shaped, and umbonal stage larvae, as well as juveniles were analyzed by microarray performance. The expression patterns of the biomineralization-related genes changed corresponding to their regulatory function during shell formation. Matrix proteins chitin synthase and PFMG2 were highly expressed at the D-shaped stage, whereas PFMG6, PFMG8 and PfN23 were significantly up-regulated at the umbonal stage, indicating different roles regulating the formation of either periostracum, Prodissoconch I or Prodissoconch II shells. However, the majority of matrix proteins were expressed at high levels at the juvenile stage, and the shells comprised both an aragonitic nacreous layer and a calcitic prismatic layer as adults. We also identified five new genes that were significantly up-regulated in juveniles. These genes were expressed particularly in the mantle and coded for secreted proteins with tandem-arranged repeat units, as most matrix proteins. RNAi knockdown resulted in disrupted nacreous and prismatic shell layers, indicating their potential roles in shell formation. CONCLUSIONS: Our results add a global perspective on larval expression patterns of P. fucata genes and propose a mechanism of how biomineralization-related genes regulate the larval shell formation process. These results increase knowledge about biomineralization-related genes and highlight new aspects of shell formation mechanisms.

Cloning and gene expression of signal transducers and activators of transcription (STAT) homologue provide new insights into the immune response and nucleus graft of the pearl oyster Pinctada fucata.

The signal transducers and activators of the transcription (STAT) family play an important role in regulatory and cellular functions by regulating the expression of a variety of genes, including cytokines and growth factors. In the present study, a Pinctada fucata STAT protein, termed PfSTAT, was described. The deduced amino acid sequence of PfSTAT contains the conserved STAT_bind domain and the SH2 domain, and the additional Bin/Amphiphysin/Rvs (BAR) domain, but does not have STAT_alpha and STAT_int domains. Multiple sequence alignments revealed that PfSTAT showed relatively low identity with vertebrate and other invertebrate STATs, and phylogenetic analysis indicated that the evolution of STAT may have been more complex and ancient. Gene expression analysis revealed that PfSTAT is involved in the immune response to polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus insertion operation. This study contributes to a better understanding of PfSTAT in protecting the pearl oyster from disease or injury caused by grafting.

Molecular characterization and analysis of a putative 5-HT receptor involved in reproduction process of the pearl oyster Pinctada fucata.

5-HT (5-hydroxytryptamine; serotonin) has been linked to a variety of biological roles including gonad maturation and sequential spawning in bivalve molluscs. To gain a better understanding of the effects of 5-HT on developmental regulation in the pearl oyster Pinctada fucata, the isolation, cloning, and expression of the 5-HT receptor was investigated in this study. A full-length cDNA (2541 bp) encoding a putative 5-HT receptor (5-HTpf) of 471 amino acids was isolated from the ovary of the pearl oyster. It shared 71% and 51% homology, respectively, with the Crassostrea gigas 5-HT receptor and the Aplysia californica 5-HT1ap. The 5-HTpf sequence possessed the typical characteristics of seven transmembrane domains and a long third inner loop. Phylogenetic analysis also indicated that 5-HTpf was classified into the 5-HT1 subtype together with other invertebrate 5-HT1 receptors. Quantitative RT-PCR showed that 5-HTpf is widely expressed in all tissues tested, is involved in the gametogenesis cycle, embryonic and larval development stages, and expression is induced by E2 in ovarian tissues. These results suggest that 5-HTpf is involved in the reproductive process, specifically in the induction of oocyte maturation and spawning of P. fucata.

Efficacy of calcein and Coomassie Blue dyeing of shell growing-edges and micro growth-bands: ageing juvenile of Pinctada mazatlanica (Pterioida: Pteriidae).

Age validation is the first step to determine shellfish species age determination. This information is vital for different inferential models used in marine ecosystem management activities. In spite that various validation techniques are used for marking carbon calcium structures, the calcein marking technique for oysters had never been used for age validation in Pinctada mazatlanica. Thus the objectives of this study included: the evaluation of calcein to mark a shell growing-edge, and the efficacy of Coomassie Blue staining on posterior shell growth, to produce visible micro growth-bands that would enable age validation of juvenile mother-of-pearl oysters. Oysters were collected and cultivated at The Perlas del Cortez S. de R. L. MI. pearl-farming operation, in Pichilingue, La Paz Bay, Baja California Sur, Mexico; a total of 36 oysters (shell height 11.5-36.4 mm) were injected with calcein (0.125 g/L), and another 50 oysters (shell height 14.8-42.7 mm) were submersed in calcein (0.4 and 0.7 g/L). Shell slices of calcein-marked oysters were posteriourly stained with Coomassie Blue R-25 for micro growth-band recognition. Our results showed that Calcein marking only worked by submersion and produced a concise bright lime-green florescent band along the growing-edge with clear boundaries for both concentrations. However, marks resulted better at the lower calcein concentration (0.4 g/L) with more "perfect" and "good" marks on the growing-edge (p = 0.0012). Commassie Blue staining technique was successful, and allowed to conclude that one micro growth-band was laid down per day, similar to other oyster species. Mean 15-d increment of shell growth height was slightly greater at the lower calcein concentration (= 0.735 mm) than at the higher one (= 0.577 mm) (not significant difference, p = 0.198). Calcein marking of shell growing-edges and Commassie Blue staining of posterior shell growth, as a method for age validation is recommended for shellfish shell growth-band counts. This will allow back-dating for estimation of very precise colonization dates, both spatially and temporally in future work.

Chemical toxicity of leachates from synthetic and natural-based spat collectors on the embryo-larval development of the pearl oyster, Pinctada margaritifera.

In French Polynesia, the pearl farming industry relies entirely on collecting natural spat using a shade-mesh collector, which is reported to contribute to both plastic pollution and the release of toxic chemicals. With the aim of identifying more environment-friendly collectors, this study investigates the chemical toxicity of shade-mesh (SM) and alternative materials, including reusable plates (P), a newly developed biomaterial (BioM) and Coconut coir geotextile (Coco), on the embryo-larval development of Pinctada margaritifera. Embryos were exposed during 48 h to four concentrations (0, 0.1, 10 and 100 g L-1) of leachates produced from materials. Chemical screening of raw materials and leachates was performed to assess potential relationships with the toxicity observed on D-larvae development. Compared to the other tested materials, results demonstrated lower levels of chemical pollutants in BioM and no toxic effects of its leachates at 10 g L-1. No toxicity was observed at the lowest tested concentration (0.1 g L-1). These findings offer valuable insights for promoting safer spat collector alternatives such as BioM and contribute to the sustainable development of pearl farming.

Comprehensive analysis of microRNAs in the mantle central and mantle edge provide insights into shell formation in pearl oyster Pinctada fucata martensii.

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.

Novel nicotinic acetylcholine receptor involved in immune regulation in pearl oyster (Pinctada fucata martensii).

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 nAChR ß gene identified from the pearl oyster Pinctada fucata martensii (PmnAChR-ß). PmnAChR-ß 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-ß in Mollusca, Chordata and Arthropoda were separated into three branches. The LBD of PmnAChR-ß was highly conserved, but its TM was variable. PmnAChR-ß 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-ß mRNA was dramatically upregulated 12 h after lipopolysaccharide stimulation. Furthermore, PmnAChR-ß 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-ß. These results indicated that PmnAChR-ß may be an important component of the cholinergic anti-inflammatory pathway and participates in the immune regulation process of pearl oysters.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

SEM observation of early shell formation and expression of biomineralization-related genes during larval development in the pearl oyster Pinctada fucata.

Shell formation of Pinctada fucata in larval development stages plays a crucial role in their survival. Scanning electron microscopy (SEM) was used to observe the morphological changes during larval development. We found that the early shell forms soon after enlargement of the blastopore at the anterior end of the trochophore stage and the complete shell forms in the spats stage, required for metamorphosis of P. fucata. Based on our transcriptome data of trochophore, D-shaped, umbonal, eyespots and spats stages, including the whole process of shell formation, 93 differentially expressed biomineralization-related genes were identified, of which 25 genes were unique to P. fucata, 30 were identical to genes in pacific oyster, and the remaining genes were annotated to other species. Two-dimensional and three-dimensional principal components analysis (PCA) showed that different developmental stages were significantly different, with the early two stages exhibiting a larger difference compared with the next stages. The 93 genes were sorted into 20 trends with three trends being significantly enriched: an initial increase and then a decrease, a monotonic decrease, and a monotonic increase. Gene expression patterns changed with regulatory function during shell formation. Almost all the biomineralization-related genes were up-regulated in the D-shaped stage, but only five genes were up-regulated in that stage but down-regulated in the remaining stages. There were also 11 genes up-regulated in the last three stages, and a total of 24 genes showed high expression level during the last four stages. The 55 genes selected for shell incision experiment sorted into five trends and most genes presented differences in expression between 24 h and other time points. Considering all these results, there is a correlation with the morphological change and the expression of biomineralization-related genes during larval developmental stages, especially of differently expressed genes.

The role of a new insulin-like peptide in the pearl oyster Pinctada fucata martensii.

Pinctada fucata martensii, is an economically important marine bivalve species cultured for seawater pearls. At present, we know little about the molecular mechanisms of the insulin signalling pathway in this oyster. Herein, we cloned and analysed an insulin-like peptide (PfILP) and its signalling pathway-related genes. We detected their expression levels in different tissues and developmental stages. Recombinant PfILP protein was produced and found to significantly increase primary mantle cell activity and induce the expression of the proliferating cell nuclear antigen (PCNA) gene. PfILP could also regulate the 293T cell cycle by stimulating the S phase and inhibiting the G1 and G2 phases. Recombinant PfILP protein induced the expression of its signalling pathway-related genes in mantle cells. In vitro co-immunoprecipitation analysis showed that PfILP interacts with PfIRR. PfILP activated expression of the pfIRR protein, and also activated the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways by stimulating phosphorylation of MAPK and AKT. Further analysis showed that PfILP up-regulated glycogen synthesis-related genes glycogen synthase kinase-3 beta (GSK-3ß), protein phosphatase 1 (PP1) and glucokinase (GK) at the mRNA level, as well as the expression of the PP1 protein, and phosphorylation of GSK-3ß. These results confirmed the presence of a conserved insulin-like signalling pathway in pearl oyster that is involved in cell activity, glycogen metabolism, and other physiological processes.