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 [CO3 2−] 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, [CO3 2−] 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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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...

Abstract. In estuarine coastal systems such as the Baltic Sea, mussels suffer from low salinity which limits their distribution. Anthropogenic climate change is expected to cause further desalination which will lead to local extinctions of mussels in the low saline areas. It is commonly accepted that mussel distribution is limited by osmotic stress. However, along the salinity gradient, environmental conditions for biomineralization are successively becoming more adverse as a result of reduced [Ca2+] and dissolved inorganic carbon (CT) availability. In larvae, calcification is an essential process starting during early development with formation of the prodissoconch I (PD I) shell, which is completed under optimal conditions within 2 days. Experimental manipulations of seawater [Ca2+] start to impair PD I formation in Mytilus larvae at concentrations below 3 mM, which corresponds to conditions present in the Baltic at salinities below 8 g kg−1. In addition, lowering dissolved inorganic carbon to critical concentrations (< 1 mM) similarly affected PD I size, which was well correlated with calculated ΩAragonite and [Ca2+][HCO3-] ∕ [H+] in all treatments. Comparing results for larvae from the western Baltic with a population from the central Baltic revealed a significantly higher tolerance of PD I formation to lowered [Ca2+] and [Ca2+][HCO3-] ∕ [H+] in the low saline adapted population. This may result from genetic adaptation to the more adverse environmental conditions prevailing in the low saline areas of the Baltic. The combined effects of lowered [Ca2+] and adverse carbonate chemistry represent major limiting factors for bivalve calcification and can thereby contribute to distribution limits of mussels in the Baltic Sea.

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, CT) 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 CT (ca. 941 µmol/kg) conditions and compared expression patterns at six developmental time points. Larvae reared under low CT exhibited a developmental delay, and a small subset of contigs was differentially regulated between ambient and low CT 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 CT 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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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 (-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 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 CRM. We further analysed the composition of larval shells in three other bivalve species, , and 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.