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Biol. Bull. 203: 269-274. (December 2002)
© 2002 Marine Biological Laboratory

Stable Amorphous Calcium Carbonate Is the Main Component of the Calcium Storage Structures of the Crustacean Orchestia cavimana

Sefi Raz1,, Olivier Testeniere2,, Arnaud Hecker2, Steve Weiner1 and Gilles Luquet2,*

1 UMR Université de Bourgogne/CNRS 5548 "Développement-Communication chimique," 6 Bd Gabriel, 21000-Dijon, France
2 Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

* To whom correspondence should be addressed. E-mail: gilles.luquet{at}u-bourgogne.fr

Amorphous calcium carbonate (ACC) is the least stable form of the six known phases of calcium carbonate. It is, however, produced and stabilized by a variety of organisms. In this study we examined calcium storage structures from the terrestrial crustacean Orchestia cavimana, in order to better understand their formation mode and function. By using X-ray diffraction, infrared and Raman spectroscopy, thermal analysis and elemental analysis, we determined that the mineral comprising these storage structures is amorphous calcium carbonate with small amounts of amorphous calcium phosphate (5%). We suggest that the use of amorphous calcium carbonate might be advantageous for these storage structures, which function as reservoirs of ions during the animal molting period. Its high solubility is beneficial for temporary storage of calcium carbonate ions that are subsequently dissolved and used elsewhere. Stabilization of these amorphous minerals is probably due to macromolecular constituents of the organic matrix, and to the magnesium and phosphate present in the mineral phase.

Amorphous minerals constitute a little less than a quarter of all known biominerals (1). When formed with no biological control, these minerals are usually metastable in comparison to their crystalline counterparts. They tend to transform into a more stable crystalline form. In contrast, biologically controlled amorphous minerals are, in most cases, stabilized by organisms for their entire lifetime. There are a few examples in which biogenic amorphous minerals are used as precursor phases for crystalline minerals (1), and two of these involve the formation of a transient amorphous calcium carbonate phase (2,3). Because amorphous minerals are isotropic in polarized light and do not diffract X-rays, their presence in a biological tissue has often been overlooked. It is particularly difficult to identify and characterize them when a crystalline mineral is also present. Biogenic amorphous minerals may thus be much more common than is currently appreciated. Here we identify and characterize the presence of amorphous calcium carbonate and amorphous calcium phosphate (ACP) in one of the more intensively studied biomineralizing systems.

As most amorphous minerals are unstable, they tend to dissolve rather easily. This may be an advantage when frequent replacement of the mineral is required. Most of the crustaceans have mineralized exoskeletons (or cuticles) that are cyclically renewed with exogenous or endogenous calcium carbonate. The use of an amorphous phase as the mineral composing the cuticle, also called carapace, can therefore be advantageous. Indeed, amorphous calcium carbonate has been detected in cuticles, and its presence is associated with large amounts of phosphate (4,5). Nevertheless, the mineral precipitated in the cuticle of most crustaceans is crystalline calcite, with some vaterite also reported (reviewed in 1,6,7).

Frequent renewal of the skeleton requires a reservoir of ions. Crustaceans obtain the calcium they need from their environment, and in addition certain species have developed calcium storage strategies (6,8). The mineral form in which calcium ions are retained in these storage structures is still enigmatic. Electron diffraction of elaborate calcium storage structures, also known as sternal plates, of the terrestrial crustacean Porcellio scaber showed that they do not diffract and are probably composed of amorphous calcium carbonate (9). In some crayfish, gastroliths are reported to be composed of calcium carbonate in an extremely poorly crystalline state, as they do not diffract X-rays or electrons (10).

The terrestrial crustacean Orchestia cavimana stores calcium originating mainly from its old cuticle in diverticula of the midgut (11). The calcium stored in the concretions constitutes about 60% of the cuticle calcium content; the rest originates from the shed cuticle (also known as the exuviae) ingested by the animal just after ecdysis, and from food. Since calcification of this new exoskeleton represents, among other things, a means of defense against environmental pressures, this process must occur as quickly as possible. The calcium is stored for about 15 days in the premolt period (12). After ecdysis, namely the moment when the animal leaves its old cuticle, the calcium is resorbed in less than 48 h to rapidly mineralize the new cuticle in a brief postmolt period (12,13). The exact mechanism of dissolution is not known. We suspect that carbonic anhydrase may be involved. Carbonic anhydrase activity has been detected histochemically at the apical level of the storage organ epithelial cells (14). This enzyme could be responsible for the rapid release of a large amount of calcium ions that may enter the epithelium via Ca2+-2H+ antiports or simply down a concentration gradient (15). Calcium then passes through the epithelial cells by a paracellular pathway as calcified spherules. Other transitory mineralized structures are produced in a dilated intercellular membrane network by precipitation of calcium salts within an organic matrix synthesized by the storage organ cells. They are then resorbed at the basal part of the epithelium, and the Ca2+ released is discharged into the hemolymph through the basal lamina (13,15). After complete dissolution of the concretions, the organic matrix is probably removed via the midgut to which the storage organs are connected, as indicated by studies of orchestin, a well-characterized component of the organic matrix (16,17).

The calcified storage structures, called calcareous concretions, are composed of an organic matrix whose proteinaceous components have been analyzed (16,17). The proteins are synthesized by the storage organ cells within which calcium salts are precipitated. Single spheruliths are firstly elaborated extracellularly in the lumen of the tubular storage organs. The integration of several single spheruliths forms compound spheruliths about 800 µm long and 400 µm in diameter (Fig. 1) in an adult individual. The concretions vary between single spheruliths and compound spheruliths in the 8-mm-long adult storage organs. This facilitates the flexibility required for these jumping animals (18). The concretions were initially considered to be excretion forms. It is now well established that these mineralized structures are transitory calcium deposits (18,19).



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  		Figure 1. Scanning electron micrograph of a compound spherulith of Orchestia cavimana elaborated during the premolt period. Terrestrial amphipod crustaceans (Orchestia cavimana) were reared in the laboratory in subaquatic terraria and staged as described by Graf (12). Calcareous concretions were extracted from the posterior cæca (the paired storage organs) of animals just before or after ecdysis by microdissection under a binocular magnifying glass. The external bright appearance and the translucent appearance of the concretions as observed by light microscopy ensured that they were completely devoid of any surrounding organic matter. Concretions were stored dessicated at 4°C until use.

 
A previous Debye-Scherrer X-ray diffraction experiment performed by Graf (18) led to the conclusion that the concretions in Orchestia are probably poorly crystallized calcite. The nature of the mineralogical form of the calcareous deposits and their characteristics remained, however, unclear. We therefore used X-ray diffraction, thermogravimetric analysis, infrared and Raman spectroscopy, scanning electron microscopy, and energy dispersive spectrometry to characterize the mineral component of these concretions.

Scanning electron microscope observations of the spheruliths that form the concretions (Fig. 1) show that they possess a very smooth surface. Even at high magnification, no sign of crystal structure is evident. This was the first indication that these concretions might be composed of an amorphous phase. We therefore obtained a powder X-ray diffraction analysis of the calcareous concretions (Fig. 2). The pattern reveals a broad peak, consistent with the presence of an amorphous phase, accompanied by sharp peaks, which correspond to calcite. It was, however, noticeable that the intensities of the calcite reflections are very weak, and calcite is therefore not a major component.



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  		Figure 2. X-ray diffraction analysis of the Orchestia calcareous concretions. The samples were finely ground and the powder was subjected to an X-ray beam using the Cu K{alpha} radiation at 40 kV and 30 mA. Measurements were performed with a step size of 0.029° and sampling time of 1.0 s/step using a diffractometer fitted out with an INEL CPS 120 localization curved counter. The base-line rise is due to an amorphous phase, whereas the small sharp peaks correspond to calcite.

 
To further understand the mineralogy of the concretions, we performed IR and Raman spectroscopic analyses. The IR spectra of the concretions both before and after ecdysis are identical and correspond to amorphous calcium carbonate (Fig. 3a1; 20). When we examined the concretions using a Raman imaging microscope, in 9 out of 12 analyses we obtained a spectrum compatible with amorphous calcium carbonate (Fig. 3b1; 21). In these spectra we also note the peak at about 950 cm-1, which is due to the presence of amorphous calcium phosphate (22). In 3 of the 12 analyses we obtained spectra that correspond to amorphous calcium carbonate, with additional peaks that are assigned to crystalline calcite (280 cm-1 and 712 cm-1). It is noteworthy that the calcite peak intensities are weak enough not to mask the amorphous calcium carbonate peaks. This also indicates that the crystalline part of the sample is small. As we do not detect any calcite peaks in the IR spectra, we conclude that the crystalline part makes up less than about 5% of the overall sample. The occurrence of some crystallinity in the Raman measurements is consistent with the data obtained from the X-ray diffraction measurements. As the calcite is present in small amounts and not always present in the samples analyzed, we suspect that it is an artifact caused by the extraction procedure, which involves some dehydration of the sample. This could cause some of the amorphous phase to crystallize. Travis (10) came to the same conclusion.



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  		Figure 3. (a) Infrared spectra of (a1) the concretions after ecdysis, when the storage structures are being resorbed (the IR spectrum of the concretions before ecdysis was identical and therefore is not shown); and (a2) control antler spicules of Pyura pachydermatina composed of stable amorphous calcium carbonate (ACC) (20). The ACC spectrum is characterized by a broad peak of the carbonate bending at 866 cm-1 and a split in the asymmetric stretch of the carbonate ion at 1420 and 1474 cm-1. The additional peak at around 1070 cm-1 is mainly assigned to phosphates. A powdered sample (about 0.1 mg) was mixed with about 10 mg of anhydrous KBr. The mixture was pressed into a 7-mm-diameter pellet. The analysis was performed at 4 cm-1 resolution using a Midac Corporation FTIR spectrometer.  (b) Raman spectra of (b1) one analysis of a concretion before ecdysis, at the end of the storage period; and (b2) control antler spicules of Pyura pachydermatina composed of stable amorphous calcium carbonate (21). The spectra are characterized by a broad peak at 150–250 cm-1 and at about 1080 cm-1. Note that both the sample and the control have an additional peak at about 950 cm-1, which corresponds to ACP. Phosphorus was also detected in the concretions by energy dispersive spectrometry (EDS). For Raman spectroscopy, samples were placed on glass slides and were observed with a Leica microscope at a magnification of 50x, using reflected white light. After focusing on the object, the light source of the microscope was transferred to a diode laser (780 nm). The spectra were scanned for 10 s in the range 100–1200 cm-1, using a Renishaw Raman imaging microscope. For EDS, samples were embedded in a mixture of Buehler ultra-mount powder and liquid. The embedded sample was polished, using a Buehler MINIMET polisher, to obtain a flat sample surface and mounted on an aluminum stub with double-sided carbon tape. The conductivity was further increased using conductive carbon paste between the embedded sample and the aluminum stub, and the sample was carbon coated for scanning electron microscopy (JSM-6400) and EDS (Oxford-ISIS). The analysis was performed using SEMQuant with standards of magnesium oxide for magnesium analysis, wollastonite for calcium analysis, and GaP for phosphorus analysis.

 
We also performed thermogravimetric analysis (TGA) combined with differential thermal analysis (DTA) of the concretions before and after ecdysis (Fig. 4). The curves show water loss of about 15% (w/w), which is similar to other biogenic samples of amorphous calcium carbonate (23), and approximately 30% weight loss at about 780°C, which corresponds to the decomposition of the calcium carbonate to calcium oxide (24).



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  		Figure 4. Thermogravimetric (TGA/DTA) analysis of the concretions. Variations of (a) differential thermogravimetry (DTG, %/min); (b) thermogravimetry (TG, mg); and (c) thermogravimetry (TG, %), as a function of the temperature (°C). After extraction, calcareous concretions were quickly rinsed in absolute ethanol and air-dried. Experiments were performed using a SETARAM TG-DTA92 thermobalance with a rising temperature of 3°C/min under compressed air. Curve b) shows the loss of weight of the sample as temperature increases. Curve c) corresponds to the same loss of weight but expressed as a percentage of the initial weight of the sample. Curve a) corresponds to the percentage of weight loss with time during the increase of temperature. We note three main variations: two small ones (15%) corresponding to the loss of water and organic matter at around 100 and 200°C and a more important one (30%) corresponding to the transformation of calcium carbonate to calcium oxide.

 
A previous X-ray microanalysis detected the presence of phosphorus in the calcareous concretions of Orchestia (15). To determine the elemental composition of the concretions, we performed energy dispersive spectrometry (EDS; for methods see legend Fig. 3). The elemental composition of the concretions was found to be rather uniform. In addition to the expected elements (calcium and oxygen), the concretions contain 3%–6% phosphorus. The phosphorus is most likely in the form of inorganic phosphate, which is probably a component of the amorphous calcium phosphate (as seen in the Raman and IR spectra). Nevertheless we cannot exclude the possibility that some of the phosphorus corresponds to phosphate groups linked to proteinaceous components of the organic matrix. Indeed, we have evidence for one such phosphorylated protein, orchestin (unpubl. results). Small amounts of magnesium (<1%) were also detected in the elemental analyses of the concretions.

We conclude that the concretions from Orchestia cavimana are composed of stable amorphous calcium carbonate together with about 5% amorphous calcium phosphate. The presence of phosphate associated with the amorphous calcium carbonate is widespread (5). Here we show that in Orchestia, at least part of this phosphate is associated with a separate amorphous calcium phosphate phase. The benefit of the amorphous phase is probably its relatively high solubility, so that it can be easily dissolved and transported to the newly formed cuticle.

It is interesting that the amorphous phase of the concretion is stable and does not change during the lifetime of the animal into a more stable crystalline form. Amorphous calcium carbonate, which forms in vitro only in a supersaturated solution, is usually metastable and transforms rapidly into a crystalline stable phase (25). When amorphous phases are stabilized by organisms, components of the organic matrix, such as macromolecules (1, 20,26) or ions such as phosphate or magnesium (21,26–29), are thought to be involved in this stabilization. Both magnesium and phosphate are known to inhibit crystal growth, thus raising the supersaturation levels and allowing the formation of metastable phases such as amorphous calcium carbonate. Several proteins extracted from the cuticle of the blue crab illustrate the importance of macromolecules in similar systems. These proteins were shown to inhibit nucleation of calcium carbonate until about 1 h after ecdysis, when they undergo certain alterations to allow the precipitation of the mineral (30). Interestingly, the macromolecules from the concretions of Orchestia are not transported along with the calcium and carbonate ions to the cuticle, but are preserved in the vicinity of the concretions. Understanding the mineralization function of well-characterized matrix proteins such as gastrolith matrix protein (GAMP) from crayfish gastroliths (31) or orchestin from the calcareous concretions of Orchestia (17) might contribute to a better understanding of how the amorphous calcium carbonate in the concretions forms and is stabilized.

We show here that amorphous calcium carbonate is the main component of the storage structures of a terrestrial crustacean, with amorphous calcium phosphate as a minor component. Identification and characterization of the mineral phase is essential for understanding the processes of biomineralization of the storage structures of Orchestia. These observations support the notion that amorphous calcium carbonate is an important phase in biomineralization, and clarifying how it is formed and stabilized may have broader implications to materials and medical science.


   Acknowledgments
 
Authors are greatly indebted to Marie Mesnier and Denis Chaumont from the "Laboratoire de Réactivité des Solides," Université de Dijon, France, for skillful assistance in X-ray diffraction and TG experiments. We also thank Prof. Lia Addadi for her advice. This work was supported by a United States-Israel Binational Science Foundation grant and by the Minerva Foundation.


   Footnotes
 
Received 20 June 2002; accepted 5 September 2002.

These authors contributed equally to this paper. Back


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