Directional fabrication and dissolution of larval and juvenile oyster shells under ocean acidification.

Biomineralization is one of the key biochemical processes in calcifying bivalve species such as oysters that is affected by ocean acidification (OA). Larval life stages of oysters are made of aragonite crystals whereas the adults are made of calcite and/or aragonite. Though both calcite and aragonite are crystal polymorphs of calcium carbonate, they have different mechanical properties and hence it is important to study the micro and nano structure of different life stages of oyster shells under OA to understand the mechanisms by which OA affects biomineralization ontogeny. Here, we have studied the larval and juvenile life stages of an economically and ecologically important estuarine oyster species, Crassostrea hongkongensis, under OA with focus over shell fabrication under OA (pHNBS 7.4). We also look at the effect of parental exposure to OA on larvae and juvenile microstructure. The micro and nanostructure characterization reveals directional fabrication of oyster shells, with more organized structure as biomineralization progresses. Under OA, both the larval and juvenile stages show directional dissolution, i.e. the earlier formed shell layers undergo dissolution at first, owing to longer exposure time. Despite dissolution, the micro and nanostructure of the shell remains unaffected under OA, irrespective of parental exposure history.

Oyster biomineralization under ocean acidification: From genes to shell.

Biomineralization is one of the key processes that is notably affected in marine calcifiers such as oysters under ocean acidification (OA). Understanding molecular changes in the biomineralization process under OA and its heritability, therefore, is key to developing conservation strategies for protecting ecologically and economically important oyster species. To do this, in this study, we have explicitly chosen the tissue involved in biomineralization (mantle) of an estuarine commercial oyster species, Crassostrea hongkongensis. The primary aim of this study is to understand the influence of DNA methylation over gene expression of mantle tissue under decreased ~pH 7.4, a proxy of OA, and to extrapolate if these molecular changes can be observed in the product of biomineralization-the shell. We grew early juvenile C. hongkongensis, under decreased ~pH 7.4 and control ~pH 8.0 over 4.5 months and studied OA-induced DNA methylation and gene expression patterns along with shell properties such as microstructure, crystal orientation and hardness. The population of oysters used in this study was found to be moderately resilient to OA at the end of the experiment. The expression of key biomineralization-related genes such as carbonic anhydrase and alkaline phosphatase remained unaffected; thus, the mechanical properties of the shell (shell growth rate, hardness and crystal orientation) were also maintained without any significant difference between control and OA conditions with signs of severe dissolution. In addition, this study makes three major conclusions: (1) higher expression of Ca2+ binding/signalling-related genes in the mantle plays a key role in maintaining biomineralization under OA; (2) DNA methylation changes occur in response to OA; however, these methylation changes do not directly control gene expression; and (3) OA would be more of a 'dissolution problem' rather than a 'biomineralization problem' for resilient species that maintain calcification rate with normal shell growth and mechanical properties.

Molecular adaptation of molluscan biomineralisation to high-CO2 oceans - The known and the unknown.

High-CO2 induced ocean acidification (OA) reduces the calcium carbonate (CaCO3) saturation level (Ω) and the pH of oceans. Consequently, OA is causing a serious threat to several ecologically and economically important biomineralising molluscs. Biomineralisation is a highly controlled biochemical process by which molluscs deposit their calcareous structures. In this process, shell matrix proteins aid the nucleation, growth and assemblage of the CaCO3 crystals in the shell. These molluscan shell proteins (MSPs) are, ultimately, responsible for determination of the diverse shell microstructures and mechanical strength. Recent studies have attempted to integrate gene and protein expression data of MSPs with shell structure and mechanical properties. These advances made in understanding the molecular mechanism of biomineralisation suggest that molluscs either succumb or adapt to OA stress. In this review, we discuss the fate of biomineralisation process in future high-CO2 oceans and its ultimate impact on the mineralised shell's structure and mechanical properties from the perspectives of limited substrate availability theory, proton flux limitation model and the omega myth theory. Furthermore, studying the interplay of energy availability and differential gene expression is an essential first step towards understanding adaptation of molluscan biomineralisation to OA, because if there is a need to change gene expression under stressors, any living system would require more energy than usual. To conclude, we have listed, four important future research directions for molecular adaptation of molluscan biomineralisation in high-CO2 oceans: 1) Including an energy budgeting factor while understanding differential gene expression of MSPs and ion transporters under OA. 2) Unraveling the genetic or epigenetic changes related to biomineralisation under stressors to help solving a bigger picture about future evolution of molluscs, and 3) Understanding Post Translational Modifications of MSPs with and without stressors. 4) Understanding carbon uptake mechanisms across taxa with and without OA to clarify the OA theories on Ω.