Inorganic phosphate in growing calcium carbonate abalone shell suggests a shared mineral ancestral precursor

Abstract

The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo.

Fig. 1. Identification of and ACP-like phase in fresh hydrated H. tuberculata larvae.

a Optical microscopy images of H. tuberculata at different stages of development: 48, 72, and 96 hpf. ¹H-³¹P HetCor spectra of b fresh H. tuberculata larvae at 48 hpf and c hydrated synthetic ACP (ACP.H₂O). d Comparison of the ¹H-³¹P HetCor extracted ³¹P rows of H. tuberculata and ACP.H₂O. ¹H-³¹P HetCor spectra recorded for e dry 72 hpf H. tuberculata larvae and f dry ewe bone.

Fig. 2. Localization of the ACP-like phase in abalone larval shell.

a ¹³C DE MAS NMR spectra of unlabelled (black, NS = 620) and ¹³C-labelled (green, NS = 104) of fresh H. tuberculata larvae (72hpf). b Close-up on the carbonate region: ¹³C DE MAS NMR (green) and ¹H-¹³C CP MAS NMR spectrum (blue, t_CP = 750 µs). c {¹H}-¹³C-{³¹P} CP REDOR experiment of ¹³C-labelled fresh 72 hpf larvae. Recoupling time = 36 ms. d {¹H}-¹³C-{³¹P} CP REDOR curve and numerical simulation (see text for details; error bars ±5%).

Fig. 3. Ultrastructure and chemical composition of 48 hpf larval shell of H. tuberculata.

TEM micrographs of the shell sections (double-headed dashed arrow) a–d at low magnification a, c showing two (a, b) or three (c, d) layers. Stars (*) show the less (yellow in a-b) and the more (white in c, d) contrasted layer. At higher magnification b, d, the presence of thin radial oriented particles reminding e in vitro spherulitic apatite observed in cross-section by TEM; f STEM-EDX analysis performed on the larval shell in cross-section showing the STEM micrograph of the analyzed region (left) and images from the related chemical analysis where the presence and the co-localization of phosphate (green) and calcium (red) is revealed.

Fig. 4. Chemical composition of 48 hpf larval by STEM-EELS.

a HAADF image of an area across the 48 hpf larval shell of H. tuberculata. The outer part of the shell is indicated by “out”. b Magnified view of the part of the shell marked with a dashed orange rectangle on (a). c EEL spectra in the energy range corresponding to phosphorus (L₂₃-edge), d carbon (K-edge), calcium (L₂₃-edge) and nitrogen (K-edge) for the positions indicated by the red squares in the corresponding maps (1–5) in (e). The presented data sets correspond to the most common patterns observed in the sample. e Maps obtained from EELS data showing the distributions of phosphorus, calcium, organic species and carbonate in different areas across the larval shell. The region where each feature is commonly found is indicated by the labels I, II, and III in (a). Scale bar = 1 µm for (a), 0.5 µm for (b) and 50 nm for (e).

Fig. 5. Temporal evolution of minerals formed in the presence of inorganic phosphate with Ca/P = 3.

SEM images show the minerals formed after 1 day (a–a1), 3 days (b–b1), 6 days (c–c1), and 9 days (d–d1). The schematic representation of apatite evolution towards calcite with eroded surfaces is showed in (e). FTIR spectra of minerals formed after different reaction time (f).