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Some crustaceans possess exoskeletons that are reinforced with calcium carbonate. In the crayfish Cherax quadricarinatus, the molar tooth, which is part of the mandibular exoskeleton, contains an unusual crystalline enamel-like apatite layer. As this layer resembles vertebrate enamel in composition and function, it offers an interesting example of convergent evolution. Unlike other parts of the crayfish exoskeleton, which is periodically shed and regenerated during the molt cycle, molar mineral deposition takes place during the pre-molt stage. The molar mineral composition transforms continuously from fluorapatite through amorphous calcium phosphate to amorphous calcium carbonate and is mounted on chitin. The process of crayfish molar formation is entirely extracellular and presumably controlled by proteins, lipids, polysaccharides, low-molecular weight molecules and calcium salts. We have identified a novel molar protein termed Cq-M15 from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. Its transcript and differential expression were confirmed by a next-generation sequencing library. The predicted acidic pI of Cq-M15 suggests its possible involvement in mineral arrangement. Cq-M15 is expressed in several exoskeletal tissues at pre-molt and its silencing is lethal. Like other arthropod cuticular proteins, Cq-M15 possesses a chitin-binding Rebers- Riddiford domain, with a recombinant version of the protein found to bind chitin. Cq-M15 was alsofound to interact with calcium ions in a concentration-dependent manner. This latter property might make Cq-M15 useful for bone and dental regenerative efforts. We suggest that, in the molar tooth, this protein might be involved in calcium phosphate and/or carbonate precipitation. [ABSTRACT FROM AUTHOR]

Conversion of one or more amino acids in eukaryotic peptides to the D-enantiomer configuration is catalyzed by specific L/D-peptide isomerases and it is a poorly investigated post-translational modification. No common modified amino acid or specific modified position has been recognized, and mechanisms underlying changes in the peptide function provided by this conversion are not widely studied. The 72 amino acid crustacean hyperglycemic hormone (CHH) in Astacidea crustaceans exhibits a co-existence of two peptide enantiomers with either D- or L-phenylalanine as their third residue. It is a pleiotropic hormone regulating several physiological processes in different target tissues and along different time scales. CHH enantiomers differently affect time courses and intensities of examined processes. The short-term effects of the two isomers on gene expression were examined in the hepatopancreas, gills, hemocytes and muscles of the astacid Pontastacus leptodactylus. Gene expression in muscles and hemocytes was not affected by either of the isomers. Two modes of action for CHH were elucidated in the hepatopancreas and the gills: specific gene induction in both organs by D-CHH, and targeted attenuation caused by both enantiomers in the gills. Consequently, a two-receptor system is proposed for conveying the effect of the two CHH isomers. [ABSTRACT FROM AUTHOR]

The rigid crustacean exoskeleton, the cuticle, Is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle Is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression In the hypodermis underlying the cuticle of the carapace at seven time points along - 1 4 premolt days. Next generation sequencing was used to construct a multitissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysisrevealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure. [ABSTRACT FROM AUTHOR]

Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the Isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85 kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures. [ABSTRACT FROM AUTHOR]

The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ-sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregnlated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle. [ABSTRACT FROM AUTHOR]