3 Gastroliths are transient extracellular calcium deposits formed by the crayfish 3 4 Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. 5 Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-3 6 protein microfibrils within which calcium carbonate is deposited. Although gastroliths share 3 7 many characteristics with the exoskeleton, they are simpler in structure and relatively 3 8 homogenous in composition, making them

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... excellent cuticle-like model for the study of 3 9 cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two 4 0 integrated approaches were employed, namely the isolation and mass spectrometric analysis 4 1 of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the 4 2 gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins 4 3 revealed a set of bands at apparent molecular weights of 75-85 kDa, of which peptide 4 4 sequencing following mass spectrometry matched the deduced amino acid sequences of seven 4 5 hemocyanin transcripts. This assignment was then examined by immunoblot analysis using 4 6 anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in 4 7 situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously 4 8