ALBERT

Description: 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 1 mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36 days 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.

Description: 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.

Description: Intracellular pH was measured using the dye BCECF (live cell imaging) in the mantle epithelial cells of the Pacific oyster (C. gigas). Intracellular pH was measured under the presence of modified seawater solutions (low sodium, low bicarbonate) and specific inhibitors for acid-base proteins were used. Value are presented as means.

Description: This data set comprises laboratory measurements of calcium, pH and carbonate concentrations in seawater and beneath the shell (calcification site) to constrain calcium carbonate saturation state dynamics during calcification in larval mussels, under control and acidified conditions. In addition, data on calcium accumulation (flame photometry) during larval calcification was collected. Finally, shell length growth and shell dissolution data under acidified conditions were also collected. This data was collected by Ramesh et al. (accepted) Nature Communications. Data is organized as it appears in figures in the published manuscript. Fig.3 and 4 data are presented in a single table.

Description: Experiments were performed to test the effect of seawater [Ca2+] on the formation rate of the larval prodissoconch I shell in 'Baltic mussels in combination with modified carbonate chemistry. In addition, [Ca2+] of the extrapallial fluid were measured in depdendency of the seawater [Ca2+]. These experimental data were correlated with the [Ca2+] present in the Baltic Sea.

Description: 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.