Pearl Oyster Pinctada Martensii Research Articles

Proteins extracted from pearl oyster (Pinctada martensii) with efficient accelerated wound healing in vitro through promoting cell proliferation, migration, and collagen formation

Ethnopharmacological relevancePearl oyster (Pinctada martensii) is used in Chinese traditional medicine use in photoprotective, anti-inflammatory, and wound treatment.Aim of the study: This study explored whether the mucus protein of Pearl oyster (protein of Pinctada martensii, PMP) affects human skin fibroblast (HSF) proliferation, migration, collagen-related gene expression related to collagen formation, and in vivo healing effects. Materials and methodsThe PMP component was analyzed by LC-MS/MS. The cell viability was evaluated using a CCK-8 kit. The expression genes were measured by reverse transcription polymerase chain reaction. A full-thickness excisional wounding model in Sprague-Dawley (SD) rats was used to test the repairing effect of PMP in vivo, and Hematoxylin-Eosin (H&E) and Masson's Trichrome staining were applied to evaluate skin structure. ResultsThe components of PMP were identified using LC-MS/MS proteomics, and a total of 3023 proteins were detected. The results of PMP-treated HSF showed that PMP effectively promoted cell proliferation by 1.6-fold and cell migration by 1.5-fold at a concentration of 1 mg/mL. Additionally, PMP treatment up-regulated the expression levels of collagen-related genes COL1A1, COL3A1, and MMP-1 in fibroblasts. Furthermore, PMP was applied in the therapy of full-thickness excisional wounds in rats. The results demonstrated that PMP significantly accelerated wound healing time, resulted in the recovery of dermal and epithelial thickness, and stimulated collagen regeneration. The regenerated skin closely resembled the structure of normal skin. ConclusionsThese findings provide solid evidence supporting the potential of PMP as a promising candidate for the treatment of skin wounds.

Study on the Effect of Mass Selection and Hybridization on Growth Performance of Chinese Pearl Oyster Pinctada martensii

The pearl oyster Pinctada martensii is an important species for sea pearl production in China. To explore the influence of the combination of mass selection and hybridization on growth performance of P. martensii, we established four selected groups and four control groups (each with two within-family crosses and two reciprocal hybrid crosses) using 1-year-old offspring of two families (Family A and Family B) from Beihai, Guangxi Province, China. Generally, the growth of the selected group was greater than that of the control group for both within-family crosses and reciprocal hybrid crosses. Shell length and width were affected by genotype, environmental factors, and the interaction between the two on Days 210 and 360. The shell widths of the four reciprocal hybrid crosses all showed heterosis on Day 360. The four within-family crosses showed a certain degree of inbreeding depression during the growth period. On Day 360, the three genetic parameters for shell width of the selected group of ♀ Family B and ♂ Family A were the largest, with values of 0.70, 1.17, and 0.06 for realized heritability, standard response to selection, and current genetic gain, respectively. Overall, the growth performance of ♀ Family B × ♂ Family A in the selected group was the best. Therefore, the combination of mass selection and hybridization could be an effective way to improve the growth performance of P. martensii.

Comparative study on the functional properties of the pearl oyster (Pinctada martensii) protein isolates and its electrostatic complexes with three hydrophilic polysaccharides

ABSTRACT Pinctada martensii protein isolates (PPIs) and polysaccharide complexes of different mixing ratios (10:1, 10:2, 10:3) were prepared at the optimal pH. The characteristics of PPIs and complexes were compared by determination of solubility, emulsifying properties, surface hydrophobicity, thermal properties, rheological behavior, and fourier transform infrared (FT-IR) spectroscopy. Compared with PPIs, the solubility of complexes changed to different degrees, and 10:3 PPI-pectin complex had the highest solubility (10.02 ± 0.68%). The emulsion properties of 10:2 and 10:3 PPI-alginate complexes were significantly higher than produced from other systems. The conjugation of PPI and pectin evidently reduced surface hydrophobicity of PPI. Denaturation temperatures of complexes were generally higher than that of PPIs. The gel network of PPI and complexes was formed within 70–80°C at pH 7.0 and G′ values of complexes were lower than that of PPIs. FT-IR spectroscopy showed O-H stretching of water absorption, C-C and C-O stretching and C-H bending of colloidal polysaccharides were improved in complexes.

Occurrence and potential health risks assessment of polycyclic aromatic hydrocarbons (PAHs) in different tissues of bivalves from Hainan Island, China

The levels of 16 PAHs were determined in the adductor, gills, gonads, hepatopancreas and mantles of the pearl oyster (Pinctada martensii) and the mussel (Perna viridis) collected from coasts of Li'an and Xincun Bays. The levels of ΣPAHs ranged from 597.1 to 2332 ng g−1 d w in the various tissues of bivalves. The pyrolytic source played an important role in the local coastal environment. Significantly higher levels of M-PAHs and H-PAHs were observed in Pinctada martensii than in Perna viridis. The ΣPAHs at different tissues showed the following order from high to low: mantles > hepatopancreas > gonads > gills > adductor. When levels of individual PAHs in the five bivalve tissues have been compared with each other, high correlations have been found (r2 = 0.793–0.975). A general trend was observed that log transformed BSAFs declined with increase of Kow values. The estimated amount of ΣPAHs via ingestion of oyster and mussel varied from 1.35 × 10−2-1.70 × 10−1 and 2.15 × 10−2-1.91 × 10−1 μg kg−1 body weight day−1, respectively. The THQs and CRs calculated for regular consumption of raw bivalves were in the acceptable ranges and may not pose health risk concerns. But for certain population with higher consumption rate for PAHs contaminated bivalves, cautions should be taken for their higher cancer risk.

Deep transcriptome profiling sheds light on key players in nucleus implantation induced immune response in the pearl oyster Pinctada martensii

Complete mitochondrial genome (mitogenome) provides important information for better understanding of gene rearrangement, molecular evolution and phylogenetic analysis. Here we determined the complete mitogenome sequence of Chiromantes eulimene (Brachyura: Sesarmidae) for the first time. The total length is 15,894 bp and includes 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, as well as a putative control region. The genome composition is highly A + T biased (75.5%), and exhibits a negative AT-skew (−0.017) and GC-skew (−0.206). All of the 13 PCGs are initiated by the start codon ATN, with an exception (GTG) in ND1. The typical stop codon (TAA or TAG) is detected in ten PCGs, whereas the remaining three PCGs (COI, COII and Cyt b) terminate by an incomplete T. The gene order in C. eulimene mitogenome was rearranged compared with that of the ancestor of Decapoda. The gene order of F-ND5-H changed to H-F-ND5. Like other sesarmid crabs, the I-Q-M gene cluster in the pancrustacean ground pattern became Q-I-M order in C. eulimene genome. Tandem duplication-random loss model and slipped-strand mispairing mechanism are determined as most likely to explain the observed gene rearrangements. Phylogenetic analysis places all Sesarmidae species into one group. Almost all families except Xanthidae, Gecarcinidae and Homolidae form a monophyletic clade and the polyphyly of Eriphioidea, Ocypodoidea and Grapsoidea is well supported. These results will help to better understand the gene rearrangements and evolutionary position of C. eulimene and lay a foundation for further phylogenetic studies of Brachyura.

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.

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.

Deep transcriptome profiling sheds light on key players in nucleus implantation induced immune response in the pearl oyster Pinctada martensii

Immunological rejection of the pearl oysters following nucleus implantation is a major issue limiting the successful rate of cultured pearls. To date, the molecular mechanism of immune tolerance during pearl formation in the pearl oysters is still largely unknown. Through the RNA sequencing platform and comparative transcriptomic analysis, we investigated the chronic gene expression changes at seven time points (0, 5, 10, 15, 20, 30, 60 days post implantation or dpi) over a period of 60 days following nucleus implantation in the pearl oyster Pinctada martensii. A total of 81,390 unique transcripts (or unigenes) with a combined length of 96.8 million bp and a N50 value of 2227 bp were obtained. When compared with sequences in the nr, nt, Swiss-Prot, KEGG, COG and GO databases, 36,380 unigenes can find homologous genes. Pairwise comparison of gene expression among all the samples showed that the largest number (or 6846) of differentially expressed genes was observed at 10 dpi. The number then decreased to below 5000 at 15, 20 and 30 dpi and increased again to 6679 at 60 dpi. PCA analysis further showed that the seven time points can be roughly divided into four groups. Comparative transcriptomic analysis between the four groups identified a variety of genes showing differential expression at different time points, including many immune-related genes such as those encoding for toll-like receptor, lectin, scavenger receptor, and peroxidase. In addition, GO and KEGG enrichment analysis revealed that these differentially expressed genes were mainly associated with metabolism, ribosome function, immune response, signaling transduction, and cytoskeleton organization. Notably, two KEGG pathways, namely “cell adhesion molecules” and “primary immunodeficiency” were significantly enriched during the whole process. This finding indicates that genes in these pathways are likely to play critical roles in the immune tolerance of the pearl oysters. To conclude, the data obtained contribute to a better understanding of the molecular mechanisms of nucleus implantation induced immune response in the pearl oysters, and will facilitate the development of effective measures to improve the performance of pearl culture.

Dynamic responses of antioxidant enzymes in pearl oyster Pinctada martensii exposed to di(2-ethylhexyl) phthalate (DEHP)

Di(2-ethylhexyl) phthalate (DEHP) is recognized as one of the most ubiquitous contaminants in marine environments and causes adverse effects on the health of marine organisms. The purpose of this study was to investigate the toxic effects of DEHP on the pearl oyster Pinctada martensii. The Pinctada martensii was exposed to 0.0, 0.5, 2, 8, or 32mgL−1 DEHP for 7 and 10days using parameters of antioxidant. Antioxidant indicators included levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), peroxidase (POD), and total antioxidant capacity (T-AOC) in the gills and hepatopancreas of Pinctada martensii for 7 and 10days. Besides, we used the lowest observed effect concentration (LOEC) of five enzyme activities in different tissues of Pinctada martensii for 7 and 10days to compare sensitivity. The results showed that the gills were more sensitive than the hepatopancreas of Pinctada martensii and that GSH activity in the gills and CAT activity in the hepatopancreas might be suitable biomarkers after 7days of DEHP exposure. After 10days of DEHP exposure, the GSH activity and CAT activity in the gills and SOD activity in the hepatopancreas could be regarded as biomarkers. Compared to the LOEC, GSH activity in the gills and CAT activity in the hepatopancreas after 7days of DEHP exposure were more sensitive than any other biomarkers. In addition, after 10days of DEHP exposure, GSH activity in the gills and hepatopancreas were much more sensitive than other activities. In conclusion, GSH activity demonstrated its potential to be used as a biomarker for the monitoring of DEHP pollution in the marine environment.

PmRunt regulated by Pm-miR-183 participates in nacre formation possibly through promoting the expression of collagen VI-like and Nacrein in pearl oyster Pinctada martensii.

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.

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.

Pm-miR-133 hosting in one potential lncRNA regulates RhoA expression in pearl oyster Pinctada martensii

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.

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.

Molecular cloning and expression analysis of heat shock protein 20 (HSP20) from the pearl oyster Pinctada martensii.

Small heat shock proteins (HSPs) are molecular chaperones with ATP-independent properties. They are involved in a variety of physiological and stress processes. In this study, the full-length HSP 20 (HSP20) from Pinctada martensii, designated as PmHSP20, was obtained from hemocytes using rapid amplification of cDNA ends technology. The PmHSP20 cDNA was 952 bp in length, containing an open reading frame of 534 bp that encoded 177-amino acid residues, with an isoelectric point of 5.86 and molecular weight of 20.24 kDa. The sequence of this deduced polypeptide contained typical structure and function domains conserved in the HSP20 family, providing evidence that PmHSP20 belongs to the HSP20 family. The PmHSP20 mRNA expression levels were detected in various tissues of P. martensii and in hemocytes after challenges with the bacteria Vibrio harveyi and lipopolysaccharide (LPS) using quantitative real-time polymerase chain reaction amplification. The results indicated that PmHSP20 is constitutively expressed in all tissues tested and might be involved in the immune response. The upregulation of PmHSP20 after V. harveyi and LPS challenge suggests that PmHSP20 plays an important role in anti-bacterial immunity. Studies on PmHSP20 are a valuable resource to further explore the immune system in pearl oysters and might enhance our knowledge of molluscan innate immunity.

Growth performance and biochemical composition of juvenile pearl oyster Pinctada martensii fed on artificial diets

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). 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.

Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster.

Characterization of 25 single nucleotide polymorphism markers in the pearl oyster Pinctada martensii (Dunker)

Twenty-five single nucleotide polymorphism (SNP) markers were developed and characterized from the expressed sequence tags of the pearl oyster Pinctada martensii in this study. The SNP genotyping of the wild population and an artificially bred population was analyzed using unlabeled probe high resolution melting. All the loci contained two alleles, and the minor allele frequency ranged from 0.117 to 0.494. The observed and expected heterozygosities ranged from 0.234 to 0.767 and from 0.207 to 0.726 respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium, and significant linkage dis-equilibrate was detected in two pairs of markers. Two loci deviated significantly from the Mendelian ratios (P < 0.05), suggesting that these two sites may be subject to selection pressure. Markers developed in this study can be used for population evaluation, parentage analysis and genetic improvement in P. martensii.

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225mgL(-1)) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated β-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115°C) and sodium chloride concentration (from 2.5 to 20.0%, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent.

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.

Highly efficient identification of thousands of microsatellite loci in the pearl oyster (Pinctada martensii) from RNA-Seq

High-throughput RNA-Seq affords a cost and time effective means of obtaining large numbers of genetic markers for aquatic genomics. Here, we present thousands of novel microsatellite loci developed for the pearl oyster, Pinctada martensii from the Illumina HiSeq™ 2000 library of the pearl sac. Free user-friendly bioinformatics tools were employed to screen for microsatellite loci and design appropriate primers in 102,762 unigenes with 7216 microsatellite loci identified in total, 4862 of which had flanking sequences suitable for polymerase chain reaction primer design. The 50 randomly chosen primer pairs were tested in two populations of pearl oyster (base population (POP1) and selected population (POP2), with 30 individuals of each population). All the primer pairs were amplified successfully in two populations. All loci were polymorphic in POP1, while there were 3 loci showing monomorphism in POP2. In POP1 and POP2, observed heterozygosity from 0.033 to 1.000 and 0.000 to 1.000, 19 and 16 microsatellite loci deviated significantly from Hardy–Weinberg expectations including a Bonferroni correction (P < 0.001). Thirteen loci were highly informative content (PIC ≥ 0.5) in both populations. These identified loci will be useful for potential application for evolutionary, population genetic and chromosome linkage mapping research on pearl oyster.