Characterisation of the mantle transcriptome and biomineralisation genes in the blunt-gaper clam, Mya truncata

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

Shell matrix proteins of the clam, Mya truncata: Roles beyond shell formation through proteomic study

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

Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.FCT: UID/Multi/04326/2019; European Marine Biological Research Infrastructure Cluster-EMBRIC (EU H2020 research and innovation program) 654008; European Union Seventh Framework Programme [FP7] ITN project 'CACHE: Calcium in a Changing Environment' under REA 60505; NERC Natural Environment Research Council NE/J500173/1info:eu-repo/semantics/publishedVersio

Apport de l’étude du protéome à la compréhension du contrôle de la biominéralisation et de la réponse adaptative de la coquille de mollusques aux modifications environnementales

In this study, the SMPs from four commercially important and divergent bivalve species crassostrea gigas (pacific oyster), Mya truncata (soft shell clam), Mytilus edulis (blue mussel) and Pecten maximus (king scallop) were extracted and analysed using standardized extraction protocol and proteomic pipeline. This enables us to identify critical elements of basic biomineralization tool kit for calcification process irrespective of their shell morphology, mineralogy and microstructure. In addition, it enables the identification of SMPs that are specific to calcite and aragonite mineralogies. The signifiant numbers of SMPs found species-specific were hypothesized as adaptation to their modus vivendi. In fact, the latter proteins possess immunity-related functions and fit into specific pathway, phenoloxidase, suggesting their role in defense against pathogen. The comparative study of shell proteome of mussels living in full marine condition, North Sea and the Iow saline Baltic Sea showed the modulation of the SMPs that constitute the basic biomineralization tool kit. Higher modulation of chitin related proteins and non-modulated protein such as carbonic anhydrase, EGF and fibronectin domain containing proteins points out the impaired scaffold and mineral nucleation process in Baltic mussel. The modulation of immunity related proteins denote the influence of biotic components. These investigations show the functional diversity of SMPs and their roles beyond shell formation in the bivalvesand put forth the idea that shell is dynamic, endowed with both biochemical and mechanical protection.Le processus de biominéralisation confère aux organismes qui le développent une valeur adaptative. La coquille carbonatée des mollusques intègre les fonctions de protection biomécanique à différentes échelles. La coquille résulte de l'association de composés inorganiques et d'une matrice organique protéique, médiatrice du contrôle biologique de la minéralisation. L'analyse du protéome de la coquille chez 4 espèces de bivalves met en évidence deux patrons fonctionnels et leur degré de conservation phylogénétique : l'un lié au contrôle de la minéralisation stricto sensu ; l'autre à la protection immune. L'étude de populations vivant à l'état naturel en mer Baltique, dont les eaux présentent localement de fortes variations ioniques montre que le protéome intègre également l'impact de conditions environnementales limitantes. L'anthropocène impose un rythme adaptatif pressant aux organismes et la modification acido-basique des eaux océaniques est susceptible d'impacter sensiblement les organismes calcifiants. La signature de mécanismes adaptatifs du contrôle biologique de la biominéralisation se traduit dans le protéome de la coquille. Les implications sont particulièrement signifiantes dans un contexte d'intérêt de développement aquacole grandissant

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

11 pagesInternational audienceThe formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods)

Supplementary figures from Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida <i>Elliptio complanata</i> and <i>Villosa lienosa</i>

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