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.

Diversification of von Willebrand Factor A and Chitin-Binding Domains in Pif/BMSPs Among Mollusks.

Pif is a shell matrix protein (SMP) identified in the nacreous layer of Pinctada fucata (Pfu) comprised two proteins, Pif97 and Pif 80. Pif97 contains a von Willebrand factor A (VWA) and chitin-binding domains, whereas Pif80 can bind calcium carbonate crystals. The VWA domain is conserved in the SMPs of various mollusk species; however, their phylogenetic relationship remains obscure. Furthermore, although the VWA domain participates in protein-protein interactions, its role in shell formation has not been established. Accordingly, in the current study, we investigate the phylogenetic relationship between PfuPif and other VWA domain-containing proteins in major mollusk species. The shell-related proteins containing VWA domains formed a large clade (the Pif/BMSP family) and were classified into eight subfamilies with unique sequential features, expression patterns, and taxa diversity. Furthermore, a pull-down assay using recombinant proteins containing the VWA domain of PfuPif 97 revealed that the VWA domain interacts with five nacreous layer-related SMPs of P. fucata, including Pif 80 and nacrein. Collectively, these results suggest that the VWA domain is important in the formation of organic complexes and participates in shell mineralisation.

Substantial gene expression shifts during larval transitions in the pearl oyster Pinctada margaritifera.

Early development stages in marine bivalve are critical periods where larvae transition from pelagic free-life to sessile mature individuals. The successive metamorphosis requires the expression of key genes, the functions of which might be under high selective pressure, hence understanding larval development represents key knowledge for both fundamental and applied research. Phenotypic larvae development is well known, but the underlying molecular mechanisms such as associated gene expression dynamic and molecular cross-talks remains poorly described for several nonmodel species, such as P. margaritifera. We designed a whole transcriptome RNA-sequencing analysis to describe such gene expression dynamics following four larval developmental stages:  d-shape, Veliger, Umbo and Eye-spot. Larval gene expression and annotated functions drastically diverge. Metabolic function (gene expression related to lipid, amino acid and carbohydrate use) is highly upregulated in the first development stages, with increasing demand from  d-shape to umbo. Morphogenesis and larval transition are partly ordered by Thyroid hormones and Wnt signaling. While larvae shells show some similar characteristic to adult shells, the cause of initialization of biomineralization differ from the one found in adults. The present study provides a global overview of Pinctada margaritifera larval stages transitioning through gene expression dynamics, molecular mechanisms and ontogeny of biomineralization, immune system, and sensory perception processes.

Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

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.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

Pearl Farming Micro-Nanoplastics Affect Oyster Physiology and Pearl Quality.

Pearl farming is crucial for the economy of French Polynesia. However, rearing structures contribute significantly to plastic waste, and the widespread contamination of pearl farming lagoons by microplastics has raised concerns about risks to the pearl industry. This study aimed to evaluate the effects of micro-nanoplastics (MNPs, 0.4-200 µm) on the pearl oyster (Pinctada margaritifera) over a 5-month pearl production cycle by closely mimicking ecological scenarios. MNPs were produced from weathered plastic pearl farming gear and tested at environmentally relevant concentrations (0.025 and 1 µg L-1) to decipher biological and functional responses through integrative approaches. The significant findings highlighted the impacts of MNPs on oyster physiology and pearl quality, even at remarkably low concentrations. Exposure to MNPs induced changes in energy metabolism, predominantly driven by reduced assimilation efficiency of microalgae, leading to an alteration in gene expression patterns. A distinct gene expression module exhibited a strong correlation with physiological parameters affected by MNP conditions, identifying key genes as potential environmental indicators of nutritional-MNP stress in cultured oysters. The alteration in pearl biomineralization, evidenced by thinner aragonite crystals and the presence of abnormal biomineral concretions, known as keshi pearls, raises concerns about the potential long-term impact on the Polynesian pearl industry.

A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19.

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10-7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC50) values of 398.1 µM and 462.4 µM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections.

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii).

With the advancement of nanotechnology and the growing utilization of nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments, posing potential ecotoxicological risks to aquatic organisms. In this study, the toxicological effects of TiO2 nanoparticles were investigated on the intestinal health of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a 14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period. Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle increased the susceptibility of pearl oysters to potential pathogenic bacteria infections. Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial communities in the gut of pearl oysters. Notable changes included a decrease in the relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria, which could potentially impact the immune function of pearl oysters. The abundance of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting that TiO2 nanoparticles exposure may affect the digestive and energy metabolic functions of pearl oysters. Furthermore, the short-term recovery of seven days did not fully restore these levels to normal. These findings provide crucial insights and serve as an important reference for understanding the toxic effects of TiO2 nanoparticles on bivalves.

A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

Comparison of in situ black-lipped oyster spat collection and larval dispersal modelling results in semi-closed pearl-farming lagoons of the Tuamotu Archipelago.

Spat collection of the pearl oyster Pinctada margaritifera in atoll lagoons of French Polynesia is the fundamental sustain of black pearl farming. Spat collection has always yielded variable results in space and time, but obvious signs of steady decreases, even collapses, have emerged in several lagoons. Spat collection materializes the ecological connectivity pathways between wild spawning populations and the location of artificial larval settlement substrates. To assess if oyster larval dispersal modelling could capture such pathways, we compared four six-week long spat collector deployment periods with dispersal simulations in two different lagoons. Spat collectors displayed wide spatial and temporal variations. Numerical modelling and field experiments were generally not in agreement. Although both methods have limitations, they can still approximate each other. But the accuracy of model simulations cannot be ascertained with spat collection data only. Using a SWOT (Strength-Weakness-Opportunities-Threats) analysis, we emphasize the complementarity of both approaches for management decisions.

The physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii).

To evaluate the physiological responses to titanium dioxide nanoparticles exposure in pearl oysters (Pinctada fucata martensii), pearl oysters were exposed for 14 days to different levels (0.05, 0.5, and 5 mg/L) of nano-TiO2 suspensions, while a control group did not undergo any nano-TiO2 treatment. And then recovery experiments were performed for 7 days without nano-TiO2 exposure. At days 1, 3, 7, 14, 17, and 21, hepatopancreatic tissue samples were collected and used to examine the activities of protease, amylase, lipase, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), lysozyme (LYS), alkaline phosphatase (AKP), and acid phosphatase (ACP). The microstructure of the nacreous layer in shell was also analyzed by scanning electron microscopy. Results showed that pearl oysters exposed to 5 mg/L of TiO2 nanoparticles had significantly lower protease, amylase, and lipase activities and significantly higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did even after 7-day recovery (P-values <0.05). Pearl oysters exposed to 0.5 mg/L or 0.05 mg/L of TiO2 nanoparticles had lower protease, amylase, and lipase activities and higher CAT, SOD, GPx, LYS, ACP, and AKP activities than control pearl oysters did during the exposure period. After 7-day recovery, no significant differences in protease, lipase, SOD, GPx, CAT, ACP, AKP, or LYS activities were observed between pearl oysters exposed to 0.05 mg/L of TiO2 nanoparticles and control pearl oysters (P-values >0.05). In the period from day 7 to day 14, indistinct and irregular nacreous layer crystal structure in shell was observed. This study demonstrates that TiO2 nanoparticles exposure influences the levels of digestion, immune function, oxidative stress, and biomineralization in pearl oysters, which can be partially and weakly alleviated by short-term recovery. These findings contribute to understanding the mechanisms of action of TiO2 nanoparticles in bivalves. However, studies should evaluate whether a longer recovery period can restore to their normal levels in the future.

Hazardous impacts of heavy metal pollution on biometric and biochemical composition of pearl oyster Pinctada radiata from five sites along Alexandria coast, with reference to its potential health risk assessment.

This study investigated the effect of heavy metals on the pearl oyster Pinctada radiata from 5 sites along the coast of Alexandria, with focus on its ecological health and potential risks to human consumption. Pollution results showed that Abu-Qir had the highest Cu and Cd values. Montaza and Eastern Harbor had the highest Fe and Pb values, respectively. Statistically, differences in metal concentrations among study sites were significant (p < 0.05). Non-carcinogenic risk (TTHQ) of tested metals and carcinogenic ones of Cd and Pb showed "high risk" on human health by consuming pearl oysters. Morphometric measurements and condition indices were studied to assess growth patterns and health in relation to heavy metals exposure. Key findings showed detectable declines in size and condition index in Eastern Harbor, whereas Abu-Qir recorded the highest values. This condition index performance presented Abu-Qir, Mammora, and Miami as ideal locations for spat collection and oyster rearing, potentially enhancing Egyptian pearl farming. Average values of spatial proximate contents of pearl oyster showed that it was rich in proteins (33.07-58.52%) with low fat content (1.39-1.87%) and carbohydrates (9.72-17.63%). Biochemical composition of pearl oyster demonstrated its high nutritional value which supported its promotion as a functional food for human consumption. The calorie content of pearl oyster was less than 2 Kcal, making this species an alternative source of healthy food to reduce obesity. Regression analysis indicated that Cu, Cd, and Pb had significant effect on 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, calories, vitamins, and pigment content of the collected oysters.

cAMP-Mediated CREM-MITF-TYR Axis Regulates Melanin Synthesis in Pacific Oysters.

Colorful shells in bivalves are mostly caused by the presence of biological pigments, among which melanin is a key component in the formation of shell colours. Cyclic adenosine monophosphate (cAMP) is an important messenger in the regulation of pigmentation in some species. However, the role of cAMP in bivalve melanogenesis has not yet been reported. In this study, we performed in vitro and in vivo experiments to determine the role of cAMP in regulating melanogenesis in Pacific oysters. Besides, the function of cAMP-responsive element modulator (CREM) and the interactions between CREM and melanogenic genes were investigated. Our results showed that a high level of cAMP promotes the expression of melanogenic genes in Pacific oysters. CREM controls the expression of the MITF gene under cAMP regulation. In addition, CREM can regulate melanogenic gene expression, tyrosine metabolism, and melanin synthesis. These results indicate that cAMP plays an important role in the regulation of melanogenesis in Pacific oysters. CREM is a key transcription factor in the oyster melanin synthesis pathway, which plays a crucial role in oyster melanin synthesis through a cAMP-mediated CREM-MITF-TYR axis.

Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors.

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, 4 µg/µL of myoglobin, 10 µg/µL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

Background: Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq™ 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac. Results: A total of 3168 microsatellite loci were identified in 39,078 unigenes, and 1977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters. Conclusions: The present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.

Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.

Molecular cloning and characterisation of scavenger receptor class B in pearl oyster Pinctada fuctada martensii

Background: Molluscs can accumulate carotenoids in their body tissues by predominantly feeding on aquatic plant sources. Carotenoid transport and absorption are determined by the regulation of various proteins such as Scavenger receptor class B(SR-BI). We report the identification and characterisation of pearl oyster Pinctada fuctada martensii SR-BI (PmSR-BI). The correlation between total carotenoid content (TCC) and gene expression was also estimated. Results: The full-length cDNA of PmSR-BI was 1828 bp, including an open-reading frame encoding of 1518 bp with a pI value of 5.83. PmSR-BI protein contains a hydrophobic CD36 domain and four centrally clustered cysteine residues for the arrangement of disulphide bridges. The deduced amino acid sequence had an identity of 30% to 60% with the SR-B of other organisms. Reverse transcription polymerase chain reaction analysis showed that mRNA transcripts were expressed in multiple tissues of adult pearl oyster. A higher expression of PmSR-BI gene was observed in the hepatopancreas than in the adductor muscle, gill and mantle. The TCC and gene expression of PmSR-BI were significantly correlated (P b 0.05), with a correlation coefficient of 0.978. Conclusions: The results suggested that PmSR-BI is involved in the absorption of carotenoids in the pearl oyster P. fuctada martensii.

Histoquímica de la glándula digestiva en la ostra perla Pinctada imbricata (Pterioida: Pteriidae) durante su ciclo gametogénico, Venezuela / Histochemistry of the digestive gland of the pearl oyster Pinctada imbricata (Pterioida: Pteriidae) during the gametogenic cycle, Venezuela

ResumenLas técnicas histoquímicas hoy en día permiten seleccionar áreas de tejido y generar información confiable sobre la distribución de reservas energéticas en los moluscos bivalvos durante su ciclo de vida. Mensualmente se examinaron las gónadas y la glándula digestiva (GD) de 15 individuos recolectados entre abril 2012 y febrero 2013 por técnicas histológicas e histoquímicas de microscopia de luz, para relacionar el ciclo gametogénico y la disponibilidad de reservas energéticas con los parámetros ambientales. En el ciclo gametogénico, la proporción mensual de organismos maduros fue mayor en los machos entre agosto (40 %) y noviembre (53 %), mientras que las hembras tienden a presentar un ciclo más corto y sincronizado de liberación de gametos (septiembre 67 % y octubre 60 %). Los períodos intensos de desoves coinciden en ambos sexos (octubre-enero). Entre abril-agosto 2012 y enero-febrero 2013, se observan los valores más altos del IGl (índice de glúcido), mientras que en septiembre disminuyen y alcanzaron valores mínimos entre octubre y diciembre. El IL (índice de lípidos) presentó valores máximos en abril-2012 y febrero-2013, con un valor intermedio en agosto. Los resultados indican que las reservas de la GD presentan un patrón de movilización en relación inversa con la proliferación de los gametos de ambos sexos, vinculado directamente con la disponibilidad de nutrientes como la clorofila a y el seston orgánico.

AbstractHistochemical techniques today allow you to select areas of tissue and generate reliable information on the distribution of energy reserves in bivalve molluscs during their life cycle. The main objective of this study was to describe and relate the gametogenic cycle with the availability of energy reserves and the environmental parameters. For this, we sampled and examined the gonads and digestive glands (DG) of 15 individuals collected monthly during April 2012 and February 2013. We processed and analyzed the samples by standard histological and histochemical light microscopy techniques. Our results showed that for the gametogenic cycle, the monthly proportion of mature organisms was higher for males, between August (40 %) and November (53 %), while the females tend to have a shorter synchronized cycle and release of gametes in September (67 %) and October (60 %). The intense spawning periods in both sexes was the same (October to January). Between the periods April-August 2012 and January-February 2013, we observed the highest values of IGl and glucide index (instead, a decrease was observed in September, reaching minimum values during the period October-December). Besides, the maximum values of IL, lipid index, were observed in April-2012 and February-2013, with an intermediate value in August-2013. The results indicated that the reserves of the GD have a pattern of mobilization inversely related to the proliferation of gametes in both sexes; this was directly linked to the availability of nutrients such as chlorophyll a and the organic seston. Rev. Biol. Trop. 64 (2): 849-858. Epub 2016 June 01.