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To explore the molecular mechanism of triploidy effect in the pearl oyster Pinctada fucata, two RNA-seq libraries were constructed from the mantle tissue of diploids and triploids by Roche-454 massive parallel pyrosequencing. The identification of differential expressed genes (DEGs) between diploid and triploid may reveal the molecular mechanism of triploidy effect. In this study, 230 down-regulated and 259 up-regulated DEGs were obtained by comparison between diploid and triploid libraries. The gene ontology and KEGG pathway analysis revealed more functional activation in triploids and it may due to the duplicated gene expression in transcriptional level during whole genome duplication (WGD). To confirm the sequencing data, a set of 11 up-regulated genes related to growth and development control and regulation were analyzed by RT-qPCR in independent experiment. According to the validation and annotation of these genes, it is hypothesized that the set of up-regulated expressed genes had the correlated expression pattern involved in shell building or other interactive probable functions during triploidization. The up- regulation of growth-related genes may support the classic hypotheses of 'energy redistribution' from early research. The results provide valuable resources to understand the molecular mechanism of triploidy effect in both shell building and producing high-quality seawater pearls., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Climate change and ocean acidification are likely to have a profound effect on marine molluscs, which are of great ecological and economic importance. One process particularly sensitive to climate change is the formation of biominerals in mollusc shells. Fundamental research is broadening our understanding of the biomineralization process, as well as providing more informed predictions on the effects of climate change on marine molluscs. Such studies are important in their own right, but their value also extends to applied sciences. Biominerals, organic/inorganic hybrid materials with many remarkable physical and chemical properties, have been studied for decades, and the possibilities for future improved use of such materials for society are widely recognised. This article highlights the potential use of our understanding of the shell biomineralization process in novel bio-inspired and biomimetic applications. It also highlights the potential for the valorisation of shells produced as a by-product of the aquaculture industry. Studying shells and the formation of biominerals will inspire novel functional hybrid materials. It may also provide sustainable, ecologically- and economically-viable solutions to some of the problems created by current human resource exploitation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to (45)Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The mantle is an organ common to all molluscs and is at the forefront of the biomineralisation process. The present study used the Mediterranean mussel (Mytilus galloprovincialis) as a model species to investigate the structural and functional role of the mantle in shell formation. The transcriptomes of three regions of the mantle edge (umbo to posterior edge) were sequenced using Illumina technology which yielded a total of 61,674,325 reads after adapter trimming and filtering. The raw reads assembled into 179,879 transcripts with an N50 value of 1086bp. A total of 1363 transcripts (321, 223 and 816 in regions 1, 2 and 3, respectively) that differed in abundance in the three mantle regions were identified and putative function was assigned to 54% using BLAST sequence similarity searches (cut-off less than 1e(-10)). Morphological differences detected by histology of the three mantle regions was linked to functional heterogeneity by selecting the top five most abundant Pfam domains in the annotated 1363 differentially abundant transcripts across the three mantle regions. Calcium binding domains dominated region two (middle segment of the mantle edge). Candidate biomineralisation genes were mined and tested by qPCR. This revealed that Flp-like, a penicillin binding protein potentially involved in shell matrix maintenance of the Pacific oyster (Crassostrea gigas), had significantly higher expression in the posterior end of the mantle edge (region one). Our findings are intriguing as they indicate that the mantle edge appears to be a heterogeneous tissue, displaying structural and functional bias., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The calcareous shells secreted by bivalve molluscs display diverse and species specific structural compositions, which indicates possible divergent biomineralization processes. Thus, studying multiple mollusc species will provide a more comprehensive understanding of shell formation. Here, the transcriptomes of the mantle tissues responsible for shell deposition were characterized in three commercially relevant bivalve species. Using high-throughput sequencing and bioinformatics tools, de novo transcriptome assemblies of mantle tissues were generated for the mussel Mytilus edulis, the oyster Crassostrea gigas and the scallop Pecten maximus. These transcriptomes were annotated, and contigs with similarity to proteins known to have shell formation roles in other species were identified. Comparison of the shell formation specific proteins in the three bivalves indicates the possibility of species specific shell proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mya truncata, a soft shell clam, is presented as a new model to study biomineralization through a proteomics approach. In this study, the shell and mantle tissue were analysed in order to retrieve knowledge about the secretion of shell matrix proteins (SMPs). Out of 67 and 127 shell and mantle proteins respectively, 16 were found in both shell and mantle. Bioinformatic analysis of SMP sequences for domain prediction revealed the presence of several new domains such as fucolectin tachylectin-4 pentraxin-1 (FTP), scavenger receptor, alpha-2-macroglobulin (α2 M), lipocalin and myosin tail along with previously reported SMP domains such as chitinase, carbonic anhydrase, tyrosinase, sushi, and chitin binding. Interestingly, these newly predicted domains are attributed with molecular functions other than biomineralization. These findings suggest that shells may not only act as protective armour from predatory action, but could also actively be related to other functions such as immunity. In this context, the roles of SMPs in biomineralization need to be looked in a new perspective., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Allatostatin-type A (AST-A), kisspeptin (KISS) and galanin (GAL) G-protein coupled receptor (GPCR) systems share a common ancestral origin in arthropods and the vertebrates where they regulate metabolism and reproduction. The molluscs are the second most diverse phylum in the animal kingdom, they occupy an important phylogenetic position, and their genome is more similar to deuterostomes than the arthropods and nematodes and thus they are good models for studies of gene family evolution and function. This mini-review intends to extend the current knowledge about AST-A, KISS and GAL GPCR system evolution and their putative function in the mollusc mantle. Comparative evolutionary analysis of the target GPCR systems was established by identifying homologues in genomes and tissue transcriptome datasets available for molluscs and comparing them to those of other metazoan systems. Studies in arthropods have revealed the existence of the AST-A system but the loss of homologues of the KISS and GAL systems. Homologues of the insect AST-AR and vertebrate KISSR genes were found in molluscs but putative GALR genes were absent. Receptor gene number suggested that members of this family have suffered lineage specific evolution during the molluscan radiation. In molluscs, orthologues of the insect AST-A peptides were not identified but buccalin peptides that are structurally related were identified and are putative receptor agonists. The identification of AST-AR and KISSR genes in molluscs strengthens the hypotheses that in metazoans members of the AST-AR subfamily share evolutionary proximity with KISSRs. The variable number of receptors and large repertoire of buccalin peptides may be indicative of the functional diversity of the AST-AR/KISSR systems in molluscs. The identification of AST-A and KISS receptors and ligands in the mantle transcriptome indicates that in molluscs they may have acquired a novel function and may play a role in shell development or sensory detection in the mantle., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Members of the Myidae family are ecologically and economically important, but there is currently very little molecular data on these species. The present study sequenced and assembled the mantle transcriptome of Mya truncata from the North West coast of Scotland and identified candidate biomineralisation genes. RNA-Seq reads were assembled to create 20,106 contigs in a de novo transciptome, 18.81% of which were assigned putative functions using BLAST sequence similarity searching (cuttoff E-value 1E-10). The most highly expressed genes were compared to the Antarctic clam (Laternula elliptica) and showed that many of the dominant biological functions (muscle contraction, energy production, biomineralisation) in the mantle were conserved. There were however, differences in the constitutive expression of heat shock proteins, which were possibly due to the M. truncata sampling location being at a relatively low latitude, and hence relatively warm, in terms of the global distribution of the species. Phylogenetic analyses of the Tyrosinase proteins from M. truncata showed a gene expansion which was absent in L. elliptica. The tissue distribution expression patterns of putative biomineralisation genes were investigated using quantitative PCR, all genes showed a mantle specific expression pattern supporting their hypothesised role in shell secretion. The present study provides some preliminary insights into how clams from different environments - temperate versus polar - build their shells. In addition, the transcriptome data provides a valuable resource for future comparative studies investigating biomineralisation., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Transcriptional profiling not only provides insights into patterns of gene expression, but also generates sequences that can be mined for molecular markers, which in turn can be used for population genetic studies. As part of a large-scale effort to better understand how commercially important European shellfish species may respond to ocean acidification, we therefore mined the transcriptomes of four species (the Pacific oyster Crassostrea gigas, the blue mussel Mytilus edulis, the great scallop Pecten maximus and the blunt gaper Mya truncata) for single nucleotide polymorphisms (SNPs). Illumina data for C. gigas, M. edulis and P. maximus and 454 data for M. truncata were interrogated using GATK and SWAP454 respectively to identify between 8267 and 47,159 high quality SNPs per species (total=121,053 SNPs residing within 34,716 different contigs). We then annotated the transcripts containing SNPs to reveal homology to diverse genes. Finally, as oceanic pH affects the ability of organisms to incorporate calcium carbonate, we honed in on genes implicated in the biomineralization process to identify a total of 1899 SNPs in 157 genes. These provide good candidates for biomarkers with which to study patterns of selection in natural or experimental populations., (Copyright © 2016 Elsevier B.V. All rights reserved.)