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BACKGROUND: Biomineralization by molluscs involves regulated deposition of calcium carbonate crystals within a protein framework to produce complex biocomposite structures. Effective biomineralization is a key trait for aquaculture, and animal resilience under future climate change. While many enzymes and structural proteins have been identified from the shell and in mantle tissue, understanding biomieralization is impeded by a lack of fundamental knowledge of the genes and pathways involved. In adult bivalves, shells are secreted by the mantle tissue during growth, maintenance and repair, with the repair process, in particular, amenable to experimental dissection at the transcriptomic level in individual animals. RESULTS: Gene expression dynamics were explored in the adult blue mussel, Mytilus edulis, during experimentally induced shell repair, using the two valves of each animal as a matched treatment-control pair. Gene expression was assessed using high-resolution RNA-Seq against a de novo assembled database of functionally annotated transcripts. A large number of differentially expressed transcripts were identified in the repair process. Analysis focused on genes encoding proteins and domains identified in shell biology, using a new database of proteins and domains previously implicated in biomineralization in mussels and other molluscs. The genes implicated in repair included many otherwise novel transcripts that encoded proteins with domains found in other shell matrix proteins, as well as genes previously associated with primary shell formation in larvae. Genes with roles in intracellular signalling and maintenance of membrane resting potential were among the loci implicated in the repair process. While haemocytes have been proposed to be actively involved in repair, no evidence was found for this in the M. edulis data. CONCLUSIONS: The shell repair experimental model and a newly developed shell protein domain database efficiently identified transcripts involved in M. edulis shell production. In particular, the matched pair analysis allowed factoring out of much of the inherent high level of variability between individual mussels. This snapshot of the damage repair process identified a large number of genes putatively involved in biomineralization from initial signalling, through calcium mobilization to shell construction, providing many novel transcripts for future in-depth functional analyses.

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Shell formation and repair occurs under the control of mantle epithelial cells in bivalve molluscs. However, limited information is available on the precise acid-base regulatory machinery present within these cells, which are fundamental to calcification. Here, we isolate mantle epithelial cells from the Pacific oyster, Crassostrea gigas and utilise live cell imaging in combination with the fluorescent dye, BCECF-AM to study intracellular pH (pHi) regulation. To elucidate the involvement of various ion transport mechanisms, modified seawater solutions (low sodium, low bicarbonate) and specific inhibitors for acid-base proteins were used. Diminished pH recovery in the absence of Na+ and under inhibition of sodium/hydrogen exchangers (NHEs) implicate the involvement of a sodium dependent cellular proton extrusion mechanism. In addition, pH recovery was reduced under inhibition of carbonic anhydrases. These data provide the foundation for a better understanding of acid-base regulation underlying the physiology of calcification in bivalves.

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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.

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Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.

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The physiological processes driving the rapid rates of calcification in larval bivalves are poorly understood. Here, we use a calcification substrate-limited approach (low dissolved inorganic carbon, C T) and mRNA sequencing to identify proteins involved in bicarbonate acquisition during shell formation. As a secondary approach, we examined expression of ion transport and shell matrix proteins (SMPs) over the course of larval development and shell formation. We reared four families of Mytilus edulis under ambient (ca. 1865 µmol/kg) and low C T (ca. 941 µmol/kg) conditions and compared expression patterns at six developmental time points. Larvae reared under low C T exhibited a developmental delay, and a small subset of contigs was differentially regulated between ambient and low C T conditions. Of particular note was the identification of one contig encoding an anion transporter (SLC26) which was strongly upregulated (2.3-2.9 fold) under low C T conditions. By analyzing gene expression profiles over the course of larval development, we are able to isolate sequences encoding ion transport and SMPs to enhance our understanding of cellular pathways underlying larval calcification processes. In particular, we observe the differential expression of contigs encoding SLC4 family members (sodium bicarbonate cotransporters, anion exchangers), calcium-transporting ATPases, sodium/calcium exchangers, and SMPs such as nacrein, tyrosinase, and transcripts related to chitin production. With a range of candidate genes, this work identifies ion transport pathways in bivalve larvae and by applying comparative genomics to investigate temporal expression patterns, provides a foundation for further studies to functionally characterize the proteins involved in larval calcification.

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In vivo confocal Raman microscopy (CRM), polarized light microscopy and Fourier transform infrared spectroscopy (FTIR) were used to determine if a significant amount of amorphous calcium carbonate (ACC) exists within larval shells of Baltic mytilid mussels (Mytilus edulis-like) and whether the amount of ACC varies during larval development. No evidence for ACC was found from the onset of shell deposition at 21 h post-fertilization (hpf) until 48 hpf. Larval Mytilus shells were crystalline from 21 hpf onwards and exhibited CRM and FTIR peaks characteristic of aragonite. Prior to shell deposition at 21 hpf, no evidence for carbonates was observed through in vivo CRM. We further analysed the composition of larval shells in three other bivalve species, Mercenaria mercenaria, Crassostrea gigas and Crassostrea virginica and observed no evidence for ACC, which is in contrast to previous work on the same species. Our findings indicate that larval bivalve shells are composed of crystalline aragonite and we demonstrate that conflicting results are related to sub-optimal measurements and misinterpretation of CRM spectra. Our results demonstrate that the common perception that ACC generally occurs as a stable and abundant precursor during larval bivalve calcification needs to be critically reviewed.

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Understanding mollusk calcification sensitivity to ocean acidification (OA) requires a better knowledge of calcification mechanisms. Especially in rapidly calcifying larval stages, mechanisms of shell formation are largely unexplored-yet these are the most vulnerable life stages. Here we find rapid generation of crystalline shell material in mussel larvae. We find no evidence for intracellular CaCO3 formation, indicating that mineral formation could be constrained to the calcifying space beneath the shell. Using microelectrodes we show that larvae can increase pH and [CO32-] beneath the growing shell, leading to a ~1.5-fold elevation in calcium carbonate saturation state (Ωarag). Larvae exposed to OA exhibit a drop in pH, [CO32-] and Ωarag at the site of calcification, which correlates with decreased shell growth, and, eventually, shell dissolution. Our findings help explain why bivalve larvae can form shells under moderate acidification scenarios and provide a direct link between ocean carbonate chemistry and larval calcification rate.

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Biomineralization processes in bivalve molluscs are still poorly understood. Here we provide an analysis of specifically expressed sequences from a mantle transcriptome of the blue mussel, Mytilus edulis. We then developed a novel, integrative shell injury assay to test, whether biomineralization candidate genes highly expressed in marginal and pallial mantle could be induced in central mantle tissue underlying the damaged shell areas. This experimental approach makes it possible to identify gene products that control the chemical micro-environment during calcification as well as organic matrix components. This is unlike existing methodological approaches that work retroactively to characterize calcification relevant molecules and are just able to examine organic matrix components that are present in completed shells. In our assay an orthogonal array of nine 1mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36days to regenerate. Structural observations using stereo-microscopy, SEM and Raman spectroscopy revealed organic sheet synthesis (day 20) as the first step of shell-repair followed by the deposition of calcite crystals (days 20 and 29) and aragonite tablets (day 36). The regeneration period was characterized by time-dependent shifts in gene expression in left central mantle tissue underlying the injured shell, (i) increased expression of two tyrosinase isoforms (TYR3: 29-fold and TYR6: 5-fold) at day 20 with a decline thereafter, (ii) an increase in expression of a gene encoding a nacrein-like protein (max. 100-fold) on day 29. The expression of an acidic Asp-Ser-rich protein was enhanced during the entire regeneration process. This proof-of-principle study demonstrates that genes that are specifically expressed in pallial and marginal mantle tissue can be induced (4 out of 10 genes) in central mantle following experimental injury of the overlying shell. Our findings suggest that regeneration assays can be used systematically to better characterize gene products that are essential for distinct phases of the shell formation process, particularly those that are not incorporated into the organic shell matrix.

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