Orchestin, a calcium-binding phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations cyclically elaborated by a terrestrial crustacean.

Orchestia cavimana is a crustacean that cyclically replaces its calcified cuticle during molting cycles in order to grow. Its terrestrial way of life requires storage of calcium during each premolt period, as calcareous concretions, in tubular diverticula of the midgut. During the postmolt period the stored calcium is reabsorbed and is translocated through the storage organ epithelium as calcified small spherules. In a previous study, we sequenced and characterized a remarkable component of the organic matrix of the premolt storage structures, Orchestin, which is a calcium-binding phosphoprotein. In this paper, we analyzed the spatiotemporal expression of the orchestin gene by Northern blotting and in situ hybridization, and its translated product by immunocytochemistry. We found evidence that the gene and the protein are expressed specifically during premolt in the storage organs. More interestingly, we demonstrated that the protein is synthesized also during the postmolt period, as a component of the organic matrix of the calcium resorption spherules. Thus, Orchestin is a matrix component that is synthesized by the same cells to contribute alternately to the elaboration of two different calcifications. These results, in addition to the physical and chemical features of the protein, suggest that Orchestin is probably a key molecule in the calcium carbonate precipitation process leading to the cyclic elaboration of two transitory calcified mineralizations by the crustacean Orchestia.

Characterization and spatiotemporal expression of orchestin, a gene encoding an ecdysone-inducible protein from a crustacean organic matrix.

We report the characterization of a new gene encoding an acidic protein named Orchestin. This protein is a component of the organic matrix of calcium storage structures (calcareous concretions) elaborated during the moulting cycles of the terrestrial crustacean Orchestia cavimana. The deduced molecular mass of Orchestin is estimated to be 12.4 kDa and the pI to be 4.4, whereas the native protein extracted from the calcium deposits migrates as a 23 kDa band on SDS/PAGE. This discrepancy is probably due to the richness of this protein in acidic amino acids (approx. 30%). The protein obtained by expressing the Orchestin cDNA in Escherichia coli presents an electrophoretic mobility of 25 kDa. Antibodies raised against the recombinant protein recognize the 23 kDa native protein exclusively among the organic-matrix components. Spatiotemporal analysis of the expression of the orchestin gene shows that it is expressed only in the storage organ cells when the concretions are elaborated during the premoult period and also, to a smaller extent, during the postmoult period. The translation products are expressed in accordance with the transcript expression during both the premoult and postmoult periods. Study of the hormonal stimulation of orchestin reveals that 20-hydroxyecdysone induces this gene as a secondary-response or late-response gene.

Premolt calcium secretion in midgut posterior caeca of the crustacean Orchestia: Ultrastructure of the epithelium.

Both structural and functional changes are observed within the posterior caeca (PC) of Orchestia during the molt cycle. During the intermolt period, there are two segments which are structurally different: a distal segment lined by type I epithelial cells and a proximal segment lined by type II cells. During molting, the PC cells are active in calcium turnover. Calcium is secreted and stored as calcareous concretions in the caecal lumen during the preexuvial period; then during the postexuvial period it is reabsorbed to mineralize the new cuticle. During the preexuvial period, cellular type III differentiates along the whole length of the PC in poster-anterior sequence and functions in ionic calcium secretion, from the basal part to the cellular apex. During the postexuvial period, this cellular type turns into cellular type IV engaged in calcium reabsorption from successive generations of spherites, from the cellular apex to the basal part. The role played by the caecal epithelium during both formation and reabsorption of the concretions was investigated by experiments in which caeca were transplanted to host pericardial cavities or were blocked by causing an abdominal hernia. The main structural characteristic features of cellular type III are as follows: an extracellular network of channels extends from basal to apical ends; microvilli are long and often apically dilated; multivacuolar complexes are localized in extracellular channels and within dilated tips of microvilli before secretion into caecum lumen; bundles of microtubules are oriented in parallel around the luminal orifices of the extracellular network; ribosomes are abundant in cytoplasm. Cellular type III develops progressively from the distal end of the caecum to the proximal one as the preexuvial period advances and concretions form in the caecum lumen.

Fine structure of the midgut posterior caeca in the crustacean Orchestia in intermolt: Recognition of two distinct segments.

This study carried out on the posterior caeca of Orchestia in intermolt by means of light and electron microscopy shows that the diverticula of the midgut consist of two segments which are different from an anatomical point of view. The distal segment is in close relationship to the dorsal blood vessel, whereas the proximal segment, twice as long as the distal one, only touches the haemocoel. The cells of the distal segment are characterized by a brush border, some apical extrusions, a great number of ribosomes, rough endoplasmic reticulum, often associated with the mitochondria, the matrix of which is clear, high activity of the Golgi complexes, and a great development of extracellular channels. All these features indicate an activity in synthesizing proteins and transport. In the proximal segment, the cells are characterized by a striated border, reduced intercellular space, and especially by a great development of the smooth endoplasmic reticulum sometimes associated with mitochondria having a dense matrix. These diverse features indicate absorption ion and water transport. From an ultrastructural point of view, the posterior caeca of Orchestia cannot be considered homologous to the Malpighian tubules. Whereas during molting the posterior caeca of Orchestia are sites of calcium storage, during intermolt they are probably involved in the processes of water and mineral regulation and excretion.