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Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

ABSTRACT: In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.

SUBMITTER: Joubert C

PROVIDER: S-EPMC4133174 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

Joubert Caroline C Linard Clémentine C Le Moullac Gilles G Soyez Claude C Saulnier Denis D Teaniniuraitemoana Vaihiti V Ky Chin Long CL Gueguen Yannick Y

PloS one 20140814 8

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was asse ...[more]

PMID: 25121605

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Project description:BackgroundColor polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.ResultsOut of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.ConclusionsOur results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.

Project description:The genomics of economically important marine bivalves is studied to provide better understanding of the molecular mechanisms underlying their different reproductive strategies. The recently available gonad transcriptome of the black-lip pearl oyster Pinctada margaritifera is a novel and powerful resource to study these mechanisms in marine mollusks displaying hermaphroditic features. In this study, RNAseq quantification data of the P. margaritifera gonad transcriptome were analyzed to identify candidate genes in histologically-characterized gonad samples to provide molecular signatures of the female and male sexual pathway in this pearl oyster. Based on the RNAseq data set, stringent expression analysis identified 1,937 contigs that were differentially expressed between the gonad histological categories. From the hierarchical clustering analysis, a new reproduction model is proposed, based on a dual histo-molecular analytical approach. Nine candidate genes were identified as markers of the sexual pathway: 7 for the female pathway and 2 for the male one. Their mRNA levels were assayed by real-time PCR on a new set of gonadic samples. A clustering method revealed four principal expression patterns based on the relative gene expression ratio. A multivariate regression tree realized on these new samples and validated on the previously analyzed RNAseq samples showed that the sexual pathway of P. margaritifera can be predicted by a 3-gene-pair expression ratio model of 4 different genes: pmarg-43476, pmarg-foxl2, pmarg-54338 and pmarg-fem1-like. This 3-gene-pair expression ratio model strongly suggests only the implication of pmarg-foxl2 and pmarg-fem1-like in the sex inversion of P. margaritifera. This work provides the first histo-molecular model of P. margaritifera reproduction and a gene expression signature of its sexual pathway discriminating the male and female pathways. These represent useful tools for understanding and studying sex inversion, sex differentiation and sex determinism in this species and other related species for aquaculture purposes such as genetic selection programs.

Project description:BACKGROUND: The shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell. RESULTS: We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time. CONCLUSIONS: This EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

Project description:BACKGROUND:Black pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis. RESULTS:The gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera. CONCLUSIONS:The study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.

Dataset Information

Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

Publications

Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

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