A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

Genes encoding putative bicarbonate transporters as a missing molecular link between molt and mineralization in crustaceans.

During their life, crustaceans undergo several molts, which if theoretically compared to the human body would be equivalent to replacing all bones at a single event. Such a dramatic repetitive event is coupled to unique molecular mechanisms of mineralization so far mostly unknown. Unlike human bone mineralized with calcium phosphate, the crustacean exoskeleton is mineralized mainly by calcium carbonate. Crustacean growth thus necessitates well-timed mobilization of bicarbonate to specific extracellular sites of biomineralization at distinct molt cycle stages. Here, by looking at the crayfish Cherax quadricarinatus at different molting stages, we suggest that the mechanisms of bicarbonate ion transport for mineralization in crustaceans involve the SLC4 family of transporters and that these proteins play a key role in the tight coupling between molt cycle events and mineral deposition. This discovery of putative bicarbonate transporters in a pancrustacean with functional genomic evidence from genes encoding the SLC4 family-mostly known for their role in pH control-is discussed in the context of the evolution of calcium carbonate biomineralization.

From sporadic single genes to a broader transcriptomic approach: Insights into the formation of the biomineralized exoskeleton in decapod crustaceans.

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C. quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.

CPAP3 proteins in the mineralized cuticle of a decapod crustacean.

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.

MARS: A protein family involved in the formation of vertical skeletal elements.

Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements.

A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties.

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 also found 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.

Exoskeletons across the Pancrustacea: Comparative Morphology, Physiology, Biochemistry and Genetics.

The exoskeletons of pancrustaceans, as typified by decapod crustaceans and insects, demonstrate a high degree of similarity with respect to histology, ultrastructure, function, and composition. The cuticular envelope in insects and the outer epicuticle in crustaceans both serve as the primary barrier to permeability of the exoskeleton, preventing loss of water and ions to the external medium. Prior to and following ecdysis, there is a sequence of expression and synthesis of different proteins by the cuticular epithelium for incorporation into the pre-exuvial and post-exuvial procuticle of insects and the exocuticle and endocuticle of crustaceans. Both exhibit regional differences in cuticular composition, e.g., the articular (intersegmental) membranes of insects and the arthrodial (joint) membranes of crustaceans. The primary difference between these cuticles is the ability to mineralize. Crustaceans' cuticles express a unique suite of proteins that provide for the nucleation and deposition of calcium carbonate. Orthologs of genes discussed in the present review were mined from a recently completed cuticular transcriptome of the crayfish, Cherax quadricarinatus, providing new insights into the nature of these proteins.

A novel chitin binding crayfish molar tooth protein with elasticity properties.

The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

Binary gene expression patterning of the molt cycle: the case of chitin metabolism.

In crustaceans, like all arthropods, growth is accompanied by a molting cycle. This cycle comprises major physiological events in which mineralized chitinous structures are built and degraded. These events are in turn governed by genes whose patterns of expression are presumably linked to the molting cycle. To study these genes we performed next generation sequencing and constructed a molt-related transcriptomic library from two exoskeletal-forming tissues of the crayfish Cherax quadricarinatus, namely the gastrolith and the mandible cuticle-forming epithelium. To simplify the study of such a complex process as molting, a novel approach, binary patterning of gene expression, was employed. This approach revealed that key genes involved in the synthesis and breakdown of chitin exhibit a molt-related pattern in the gastrolith-forming epithelium. On the other hand, the same genes in the mandible cuticle-forming epithelium showed a molt-independent pattern of expression. Genes related to the metabolism of glucosamine-6-phosphate, a chitin precursor synthesized from simple sugars, showed a molt-related pattern of expression in both tissues. The binary patterning approach unfolds typical patterns of gene expression during the molt cycle of a crustacean. The use of such a simplifying integrative tool for assessing gene patterning seems appropriate for the study of complex biological processes.