Supplementary material from "In vivo characterization of bivalve larval shells: a confocal Raman microscopy study"

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.