Reciprocal Changes in Calcification of the Gastrolith and Cuticle During the Molt Cycle of the Red Claw Crayfish Cherax quadricarinatus ASSAF SHECHTER 1,3 , AMIR BERMAN 2,3,4 , ALON SINGER 2,3 , AVIAD FREIMAN 1,3 , MOR GRINSTEIN 5 , JONATHAN EREZ 4 , ELIAHU D. AFLALO 1,3 , AND AMIR SAGI 1,3, * 1 Department of Life Sciences, 2 Department of Biotechnology Engineering, 3 National Institute for Biotechnology in the Negev (NIBN), and 4 Ilse Katz Center for Nanoscience and Nanotechnology and Reimund Stadler Minerva Center for Mesoscale Macromolecular Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel; and 5 The Institute of Earth Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Abstract. Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cu- ticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and trans- ported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning elec- tron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chiti- nous lamella. Measurements of pH and calcium levels dur- ing gastrolith deposition showed that calcium concentra- tions in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l -1 ). On the other hand, pH varied greatly, from 8.7  0.15 in the gastrolith cavity through 7.6  0.2 in muscle to 6.9  0.5 in the stomach. Introduction In most crustaceans, the rigid exoskeleton— cuticle— contains much of the stored calcium (Wheatly and Ayers, 1995). Produced by the underlying epidermal cells, the cuticle is composed of four layers (from outer to inner): epicuticle, exocuticle, endocuticle, and membranous layer (Travis and Friberg, 1963b; Raz et al., 2002). The exocu- ticle and endocuticle, which make up most of the cuticle, comprise calcified matrixes of chitin and proteins, with the chitin-protein fibers stacked in layers of continuously changing orientation (Giraudguille, 1984; Roer and Dilla- man, 1984). The thin epicuticle, which is composed of protein, lipids, and calcium but no chitin, has a dense bilaminar organization, with a basal layer that is pervaded by mineral-filled canals normal to the surface. The mem- branous layer also contains chitin and proteins, but it is not calcified (Roer and Dillaman, 1984; Aiken and Waddy, 1992). The main mineral found in the calcified layers of the cuticle is calcium carbonate (Greenaway, 1985; Wheatly and Ayers, 1995; Luquet and Marin, 2004). The growth process in crustaceans demands periodic shedding and replacement of the cuticle during ecdysis. In some crustaceans, molting and ecdysis can take place throughout the life cycle, whereas other species molt only until sexual maturity is reached (Hartnoll, 1982). The prin- cipal source of the calcium required for building the cuticle is seawater, the natural habitat of most crustacean species. Calcium concentration in seawater is about 10 mmol l -1 (Greenaway, 1985). In crustacean species living on land or Received 27 May 2007; accepted 31 October 2007. *To whom correspondence should be addressed. E-mail: sagia@bgu.ac.il Abbreviations: ACC, amorphous calcium carbonate; FTIR, Fourier transform infrared spectroscopy; MMI, molt mineralization index. Reference: Biol. Bull. 214: 122–134. (April 2008) © 2008 Marine Biological Laboratory 122