Calcium Deposits in the Crayfish, Cherax quadricarinatus: Microstructure Versus Elemental Distribution

Abstract

The crayfish Cherax quadricarinatus stores calcium ions, easily mobilizable after molting, for calcifying parts of the new exoskeleton. They are chiefly stored as amorphous calcium carbonate (ACC) during each premolt in a pair of gastroliths synthesized in the stomach wall. How calcium carbonate is stabilized in the amorphous state in such a biocomposite remains speculative. The knowledge of the microstructure at the nanometer level obtained by field emission scanning electron microscopy and atomic force microscopy combined with scanning electron microscopy energy-dispersive X-ray spectroscopy, micro-Raman and X-ray absorption near edge structure spectroscopy gave relevant information on the elaboration of such an ACC-stabilized biomineral. We observed nanogranules distributed along chitin-protein fibers and the aggregation of granules in thin layers. AFM confirmed the nanolevel structure, showing granules probably surrounded by an organic layer and also revealing a second level of aggregation as described for other crystalline biominerals. Raman analyses showed the presence of ACC, amorphous calcium phosphate, and calcite. Elemental analyses confirmed the presence of elements like Fe, Na, Mg, P, and S. P and S are heterogeneously distributed. P is present in both the mineral and organic phases of gastroliths. S seems present as sulfate (probably as sulfated sugars), sulfonate, sulfite, and sulfoxide groups and, in a lesser extent, as sulfur-containing amino acids.

High-resolution structural and elemental analyses of calcium storage structures synthesized by the noble crayfish Astacus astacus

“…The presence and distribution of P were recently determined in Cherax quadricarinatus gastroliths in organic molecules as well as in the mineral phase as ACP (Luquet et al, 2016;Reeder et al, 2013). Phosphorylated proteins have been characterized and hypothesized as playing a role in Cherax gastrolith ACC stabilization Glazer and Sagi, 2012;Glazer et al, 2010;Shechter et al, 2008b).…”

“…In a recent paper (Luquet et al, 2016), in addition to Raman analyses, we have performed elemental analyses on Cherax quadricarinatus gastrolith by Energy Dispersive Xray spectroscopy coupled to Scanning Electron Microscopy (SEM-EDX) and X-ray Absorption Near Edge Structure spectroscopy (XANES) focusing on S. S is present mainly as sulfated sugars and as S-bearing amino acids. Sulfite, sulfoxide and sulfonate were also detected as a minor contribution.…”

“…Immediately following ecdysis, animals were sacrificed by freezing and gastroliths extracted. Cherax quadricarinatus gastroliths were obtained from a rearing located in Chile, as previously described (Luquet et al, 2016).…”

“…Among the non-metal elements, Cl, S and P have been evidenced. Some recent works on invertebrate biominerals have focused on S Dauphin et al, 2003Dauphin et al, , 2005Luquet et al, 2016;Rousseau et al, 2009;Zhang et al, 2011).…”

“…Akiva-Tal et al, 2011;Bentov et al, 2010;Habraken et al, 2015;Luquet et al, 2016;Reeder et al, 2013;Reid et al, 2012;Sato et al, 2011). Nevertheless, it is well known that if the main mineral used by invertebrates for hardening their skeleton is calcium carbonate, numerous other minerals are also found, notably phosphate and silica-based minerals Knoll, 2003;Lowenstam, 1972;Lowenstam and Weiner, 1989;Murdock and Donohue, 2011;Simkiss and Wilbur, 1989).…”