Early pattern of calcification in the dorsal carapace of the blue crab,Callinectes sapidus

Abstract

The pattern of calcium carbonate deposition was observed in the dorsal carapace of premolt (D2-D3) and early postmolt (0-48 h) blue crabs, Callinectes sapidus, using scanning (SEM) and transmission (TEM) electron microscopy. Samples of dorsal carapace for SEM were quick-frozen in liquid nitrogen, subsequently lyophilized, and viewed using secondary and backscattered electrons as well as X-ray maps of calcium. Pieces of lyophilized cuticle were also embedded in epoxy resin and subsequently sectioned and viewed with TEM and SEM. Fresh pieces of dorsal carapace for TEM were also fixed in 2.5% glutaraldehyde in phosphate buffer followed by postfixation in 1% OsO4 in cacodylate buffer. Calcium concentrations were determined using atomic absorption spectrophotometry and quantitative X-ray microanalysis. Calcium accumulation began in the cuticle at 3 h postmolt at the epicuticle/exocuticle boundary and at the distal and proximal margins of the interprismatic septa (IPS). The bidirectional calcification of the IPS continued until the two fronts met at 5-8 h postmolt. The roughly hexagonal walls of the IPS formed a honeycomb-like structure that resulted in a rigid cuticle. The walls of the canal containing sensory neurons also calcified at 3 h, thereby imparting rigidity to the structure and additional strength to the cuticle. Examination of thin sections of lyophilized cuticle and fixed cuticle revealed that the first mineral deposited is more soluble than calcite and is probably amorphous calcium carbonate. The amorphous calcium carbonate is transformed to calcite along a front that follows the original deposition and is probably controlled by a specialized matrix within the IPS. Since amorphous calcium carbonate is isotropic, it would also make the mineral in the exocuticle stronger by an equal distribution of mechanical stress.

Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule

“…This process was first observed in the teeth of chitons where a disordered ferrihydrite precursor transforms into magnetite (2). It has also been observed in different invertebrate phyla (3)(4)(5)(6)(7)(8). Amorphous calcium phosphate was recently identified in the newly deposited fin bones of zebrafish (9).…”

Structure and mechanical properties of selected protective systems in marine organisms

“…Typically, the exocuticle lamellae are denser than those of the endocuticle [133,137]. The crystallinity of the exocuticle not only differs from the endocuticle, but it also exhibits a gradient within it; amorphous calcium carbonate occurs in the proximal end, near the interface with the endocuticle, and transitions to calcite at the distal end [138,139]. In contrast, the endocuticle contains magnesium calcite and amorphous calcium carbonate [139].…”

Magnesium-aspartate-based crystallization switch inspired from shell molt of crustacean

“…Different from the conventional solution growth model (3), latest achievements suggest a multistep strategy of phase transformation and highlight the enrollments of transient amorphous precursors in biomineralization (4). Evidences are the first-formed amorphous calcium carbonate (ACC) in sea urchin spicules (5), crustacean cuticles (6), and corals (7), etc. The participations of amorphous calcium phosphate (ACP) in the early stages during tooth and bone generations and the subsequent transformation into carbonated hydroxyapatite (HAP) have been clarified in vivo (8,9).…”

“…Armadillidium vulgare belongs to terrestrial-dwelling crustacean taxon isopod. Molt of this animal occurs spontaneously as four typical stages: premolt, intra-molt, postmolt, and inter-molt (6,14). The layers in cuticle are deposited and mineralized at distinctly different stages (6,17).…”

“…Molt of this animal occurs spontaneously as four typical stages: premolt, intra-molt, postmolt, and inter-molt (6,14). The layers in cuticle are deposited and mineralized at distinctly different stages (6,17). That is, calcium ions in old cuticle are resorbed and stored in ACC deposits within the ecdysial gap of the first four anterior sternites in premolt.…”