The calcitonin-like system is an ancient regulatory system of biomineralization.

Biomineralization is the process by which living organisms acquired the capacity to accumulate minerals in tissues. Shells are the biomineralized exoskeleton of marine molluscs produced by the mantle but factors that regulate mantle shell building are still enigmatic. This study sought to identify candidate regulatory factors of molluscan shell mineralization and targeted family B G-protein coupled receptors (GPCRs) and ligands that include calcium regulatory factors in vertebrates, such as calcitonin (CALC). In molluscs, CALC receptor (CALCR) number was variable and arose through lineage and species-specific duplications. The Mediterranean mussel (Mytilus galloprovincialis) mantle transcriptome expresses six CALCR-like and two CALC-precursors encoding four putative mature peptides. Mussel CALCR-like are activated in vitro by vertebrate CALC but only receptor CALCRIIc is activated by the mussel CALCIIa peptide (EC50 = 2.6 ×10-5 M). Ex-vivo incubations of mantle edge tissue and mantle cells with CALCIIa revealed they accumulated significantly more calcium than untreated tissue and cells. Mussel CALCIIa also significantly decreased mantle acid phosphatase activity, which is associated with shell remodelling. Our data indicate the CALC-like system as candidate regulatory factors of shell mineralization. The identification of the CALC system from molluscs to vertebrates suggests it is an ancient and conserved calcium regulatory system of mineralization.

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis).

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.

Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.

Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches.