Characterization and expression analysis of tandemly duplicated nicotinic acetylcholine receptors in pearl oysters after stimulation of pathogen-related molecular patterns.

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.

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.

Tissue-specific metabolic responses of the pearl oyster Pinctada martensii exposed to benzo[a]pyrene.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is well known for its teratogenic, mutagenic and carcinogenic effects. In this study, we applied metabolomics to investigate the tissue-specific metabolic responses of the Pinctada martensii digestive glands and gills after a short-duration exposure to BaP (1 µg/L and 10 µg/L). After 72 h of exposure to BaP, the majority of metabolite changes were related to osmolytes, energy metabolites, and amino acids. BaP (1 µg/L) accelerated energy deterioration and decreased osmotic regulation, while BaP (10 µg/L) disturbed energy metabolism and increased osmotic stress in the digestive glands. Both BaP doses disturbed osmotic regulation and energy metabolism in the gills. BaP also induced neurotoxicity in both tissues. These findings demonstrated that BaP exhibited tissue-specific metabolic responses in P. martensii. The difference in these metabolite responses between the digestive glands and gills might prove to be suitable biomarkers for indicating exposure to specific marine pollutants.

A Sperm Spawn-Inducing Pheromone in the Silver Lip Pearl Oyster (Pinctada maxima).

Pheromones are considered to play an important role in broadcast spawning in aquatic animals, facilitating synchronous release of gametes. In oysters, the sperm has been implicated as a carrier for the spawn-inducing pheromone (SIP). In hatchery conditions, male pearl oysters (Pinctata maxima) can be stimulated to spawn through a variety of approaches (e.g. rapid temperature change), while females can only be induced to spawn through exposure to conspecific sperm, thus limiting development of targeted pairing, required for genetic research and management. The capacity for commercial production and improvement of genetic lines of pearl oysters could be greatly improved with access to a SIP. In this study, we prepared and sequenced crude and semi-purified P. maxima sperm extracts that were used in bioassays to localise the female SIP. We report that the P. maxima SIP is proteinaceous and extrinsically associated with the sperm membrane. Bioactivity from pooled RP-HPLC fractions, but not individual fractions, suggests that the SIP is multi-component. We conclude that crude sperm preparations, as described in this study, can be used as a sperm-free inducer of female P. maxima spawning, which enables for a more efficient approach to genetic breeding.

An integrated metabolomic and proteomic study of toxic effects of Benzo[a]pyrene on gills of the pearl oyster Pinctada martensii.

Benzo[a]pyrene (BaP) is one of the most important polycyclic aromatic hydrocarbons (PAHs), which are widely present in the marine environment. Because of its teratogenic, mutagenic, and carcinogenic effects on various organisms, the toxicity of BaP is of great concern. In this study, we focused on the toxic effects of BaP (1 µg/L and 10 µg/L) on gills of the pearl oyster Pinctada martensii using combined metabolomic and proteomic approaches. At the metabolome level, the high concentration of BaP mainly caused abnormal energy metabolism, osmotic regulation and immune response marked by significantly altered metabolites in gills. At the proteome level, both concentrations of BaP mainly induced signal transduction, transcription regulation, cell growth, stress response, and energy metabolism. Overall, the research demonstrated that the combination of proteomic and metabolomic approaches could provide a significant way to elucidate toxic effects of BaP on P. martensii.

A novel toll-like receptor from the pearl oyster Pinctada fucata martensii is induced in response to stress.

Graft rejection due to immune incompatibility is a common occurrence in pearl culture, which often cause death to the host oyster. To improve cultured pearl production, host mortality and bead rejection rates must be reduced. Toll-like receptor 4 (TLR4) plays an important role in innate immunity, and may be related to allograft rejection. Here, we cloned the TLR4 cDNA from the pearl oyster Pinctada fucata martensii (PmTLR4). PmTLR4 cDNA was 3138bp, including a 2625bp open reading frame encoding 874 amino acids. The predicted PmTLR4 protein was structurally typical of the TLR family. PmTLR4 had relatively high sequence similarity and identity to the TLR4 of the Cyclina sinensis (48.1% and 27.6%, respectively). Multiple alignment of TLR4 sequences across species indicated that the Toll/interleukin-1 (IL-1) receptor domain was conserved among species. PmTLR4 mRNA was expressed in all tissues tested, with the most abundant mRNA expression in hepatopancreas and gill in P. fucata martensii. After being stressed by either lipopolysaccharide (LPS) exposure or the nucleus insertion operation, PmTLR4 mRNA expression increased significantly in the hemocytes as compared to controls. Peak level of PmTLR4 mRNA was observed 6h after the LPS injection, and 2d after the nucleus insertion operation. These data suggest that PmTLR4 may play a vital role in the induction of innate immunity and is therefore associated with allograft immunity in the pearl oyster P. fucata martensii.

Response of the pearl oyster Pinctada margaritifera to cadmium and chromium: Identification of molecular biomarkers.

This study was designed to identify in the pearl oyster Pinctada margaritifera, used as a bio-accumulator, molecular biomarkers for the presence of heavy metals in the lagoon environment. Pearl oysters were exposed to 2 concentrations (1 and 10µgL-1) of cadmium (Cd) and chromium (Cr) compared to a control. Twelve target genes encoding proteins potentially involved in the response to heavy metal contamination with antioxidant, detoxification or apoptosis activities were selected. P. margaritifera accumulated Cd but not Cr, and mortality was related to the amount of Cd accumulated in tissues. In response to Cd-Cr contamination, metallothionein (MT) was significantly up-regulated by Cd-Cr at both concentrations, while 7 others (SOD, CAT, GPX, GSTO, GSTM, CASP, MDR) were down-regulated. Based on the development of these molecular tools, we propose that the pearl oyster, P. margaritifera, could be used as a sentinel species for heavy metal contamination in the lagoons of tropical ecosystems.

Effects of pyrene exposure on immune response and oxidative stress in the pearl oyster, Pinctada martensii.

Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly observed in aquatic ecosystems, which originates primarily from the incomplete combustion of fossil fuels and the use of petroleum compounds. Pyrene can cause the immune disturbance and oxidative stress, result in immunotoxicity, DNA damage, reduce reproduction significantly, and induce behavioral changes. Marine bivalves are commonly used as bioindicators for marine pollution, and hemolymph is a metabolite transfer medium for PAH pollutant. However, the vital immune indicator responses of pearl oyster Pinctada martensii hemolymph exposed to pyrene is still unclear. Thus, the immunotoxic responses of pyrene on the hemolymph of the Pinctada martensii were investigated in this study. After exposure to pyrene for 7 days, the total number of hemocytes (THC), cell membrane stability (CMS), phagocytic activity (PA) and total glutathione (GSHT) all decreased significantly. Pyrene also caused a significant increase in lipid peroxidation (LPO). Median effective concentrations (EC50) of pyrene on THC (4.5 µg L-1) and LPO (5.2 µg L-1) were lower than those for CMS (13.8 µg L-1), PA (12.1 µg L-1) and GSHT (7.2 µg L-1), which indicates that THC and LPO were more sensitive. Additionally, a clear dose-effect relationship indicated that pyrene stimulated a marked immune response, as well as oxidative stress in P. martensii, which demonstrates the subtle effects of pyrene exposure on marine invertebrates and the potential associated risk.

Proteomic and metabolomic analysis on the toxicological effects of Benzo[a]pyrene in pearl oyster Pinctada martensii.

Benzo[a]pyrene (BaP) is one of the typical toxic polycyclic aromatic hydrocarbons (PAHs) that are widely present in marine environment. BaP has diverse toxic effects, including teratogenic, carcinogenic, mutagenic effects and so on, in various organisms. In this work, we focused on the differential proteomic and metabolomic responses in the digestive gland of pearl oyster Pinctada martensii exposed to two doses of BaP (1 and 10µg/L). Metabolic responses revealed that the high dose of BaP (10µg/L) mainly caused disturbances in osmotic regulation and energy metabolism in the digestive gland. Proteomic responses indicated that both doses of BaP induced disturbances in energy metabolism, cytoskeleton, cell injury, oxidative stress and signal transduction based on the differential proteomic biomarkers. Overall, these results demonstrated a number of potential biomarkers that were characterized by an integrated proteomic and metabolomic approach and provided a useful insight into the toxicological effects on pearl oyster P. martensii.

Gene cloning and expression analysis of AhR and CYP4 from Pinctada martensii after exposed to pyrene.

Pyrene, a typical polycyclic aromatic hydrocarbon, is a common pollutant in the marine environment. Polycyclic aromatic hydrocarbons initiate cellular detoxification in an exposed organism via the activation of the aryl hydrocarbon receptor (AhR). Subsequent metabolism of these xenobiotics is mainly by the cytochrome P450 enzymes of the phase I detoxification system. Full-length complementary DNA sequences from the pearl oyster Pinctada martensii (pm) encoding AhR and cytochrome P4 were cloned. The P. martensii AhR complementary DNA sequence constitutes an open reading frame that encodes for 848 amino acids. Sequence analysis indicated PmAhR showed high similarity with its homologues of other bivalve species. The cytochrome P(CYP)4 complementary DNA sequence of P. martensii constitutes an open reading frame that encodes for 489 amino acids. Quantitative real-time analysis detected both PmAhR and PmCYP4 messenger RNA expressions in the mantle, gill, hepatapancreas and adductor muscle of P. martensii exposed to pyrene. The highest transcript-band intensities of PmAhR and PmCYP4 were observed in the gill. Temporal expression of PmAhR and PmCYP4 messenger RNAs induction was observed in gills and increased between 3 and 5 days post exposure; then returned to control level. These results suggest that messenger RNAs of PmAhR and PmCYP4 in pearl oysters might be useful parameters for monitoring marine environment pyrene pollution.

Molecular characterization and expression analysis of lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) from pearl oyster Pinctada fucata.

The lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) plays an important function in the innate immune response of invertebrates as a pattern recognition receptor (PRR). Herein, we described the isolation and characterization of pearl oyster Pinctada fucata LGBP (designated as poLGBP). The poLGBP cDNA was 2,075 bp long and consisted of a 5'-untranslated region (UTR) of 18 bp, a 3'-UTR of 299 bp with one cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1,758 bp encoding a polypeptide of 585 amino acids with an estimated molecular mass of 65.1 kDa and a theoretical isoelectric point of 5.80. Homology analysis of the deduced amino acid sequence of the poLGBP with other known LGBP sequences by MatGAT software revealed that the poLGBP shared 26.3-56.7% identity and 40.5-70.9% similarity to the other known LGBP sequences. SMART and alignment analysis revealed that the poLGBP possessed a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site, a ß-1,3-linkage of polysaccharide, a glycine-rich region, a threonine-rich region and two N-glycosylation sites. In healthy pearl oyster, the poLGBP mRNA was specifically expressed in digestive gland, and not detected in gill, adductor muscle, gonad, intestine, mantle and hemocytes. However, after bacteria stimulation, the expression of the poLGBP mRNA was significantly up-regulated in digestive gland and also weakly detected in haemocytes, gonad and intestine. After LPS stimulation, the poLGBP mRNA expression was significantly up-regulated at 8 and 12 h in digestive gland, and the expression level was 10.7-fold higher than the PBS group at 12 h. After bacteria stimulation, the expression level of the poLGBP mRNA was also significantly up-regulated in digestive gland and was 12.9-fold higher than the PBS group at 8 h. However, during the experiment, the poLGBP mRNA expression was not detected in gill after LPS or bacteria stimulation. The tissue-specific expression and the expression up-regulation after LPS or bacteria stimulation in digestive gland suggested that the poLGBP was an inducible acute-phase protein and might play an important function in digestion as digestive enzyme and pattern recognition receptor.

In vitro regulation of CaCO(3) crystal polymorphism by the highly acidic molluscan shell protein Aspein.

Biominerals, especially molluscan shells, generally contain unusually acidic proteins. These proteins are believed to function in crystal nucleation and inhibition. We previously identified an unusually acidic protein Aspein from the pearl oyster Pinctada fucata. Here we show that Aspein can control the CaCO(3) polymorph (calcite/aragonite) in vitro. While aragonite is preferentially formed in Mg(2+) -rich solutions imitating the extrapallial fluids of marine molluscs, Aspein exclusively induced calcite precipitation. Our results suggest that Aspein is involved in the specific calcite formation in the prismatic layer. Experiments using truncated Aspein demonstrated that the aspartic acid rich domain is crucial for the calcite precipitation.

Metal accumulation and enzyme activities in gills and digestive gland of pearl oyster (Pinctada fucata) exposed to copper.

The effects of exposure to copper under laboratory-controlled conditions were investigated in the pearl oyster, Pinctada fucata. Metal accumulation and the activity of five enzymes were measured: two immune defense involved enzymes [acid phosphatase (AcPase) and phenoloxidase (PO)], two antioxidant enzymes [superoxide dismutase (SOD) and Se-dependent glutathione peroxidase (Se-GPx)] and one metal-sensitive enzyme [alkaline phosphatase (ALP)]. Analyses were carried out in gills and digestive gland of oysters exposed to 0.05 microM and 0.5 microM copper, respectively, at 12, 24, 48 and 72 h of exposure. The digestive gland of P. fucata was the main copper accumulation organ when oysters were exposed to low concentrations, whereas gills became the target organ in oysters exposed to high concentrations. The adaptation and recovery of the oysters were observed in our study. Levels of the copper accumulation and the sensitivity to copper were the main, if not, part of the reasons for the various responses of the selected enzymes. Se-GPx may potentially be used as biomarkers in biotesting of marine heavy metal pollutions. The enzymatic responses were compared with those of other studies and the possible reasons were discussed.

CGRP stimulates gill carbonic anhydrase activity in molluscs via a common CT/CGRP receptor.

The physiological significance of calcitonin gene-related peptide (CGRP) during biomineralization was investigated by assessing the effect of human CGRP on the carbonic anhydrase activity in gill membranes of the pearl oyster, Pinctada margaritifera. Salmon CT and human CGRP were able to induce a 150% increase of the basal activity. No additive effect was observed suggesting that both activities are mediated by the same receptor. The CGRP-stimulated effect was specific as demonstrated by the inhibition produced by the CGRP antagonist, hCGRP8-37. So, CGRP by its specific action on gill carbonic anhydrase controls the calcification process, an ancient role both in invertebrates and non-mammalian vertebrates.