Introduction: Bivalve molluscs are abundant in marine and freshwater systems and contribute essential ecosystem services. They are characterized by an exuberant diversity of biomineralized shells and typi- cally have two symmetric valves (a.k.a shells), but oysters (Ostreidae), some clams (Anomiidae and Chamidae) and scallops (Pectinida) have two asymmetrical valves. Predicting and modelling the likely consequences of ocean acidification on bivalve survival, biodiversity and aquaculture makes understand- ing shell biomineralization and its regulation a priority.

Objectives: This study aimed to a) exploit the atypical asymmetric shell growth of some bivalves and through comparative analysis of the genome and transcriptome pinpoint candidate biomineralization- related genes and regulatory long non-coding RNAs (LncRNAs) and b) demonstrate their roles in regulat- ing shell biomineralization/growth.

Methods: Meta-analysis of genomes, de novo generated mantle transcriptomes or transcriptomes and proteomes from public databases for six asymmetric to symmetric bivalve species was used to identify biomineralization-related genes. Bioinformatics filtering uncovered genes and regulatory modules char- acteristic of bivalves with asymmetric shells and identified candidate biomineralization-related genes and lncRNAs with a biased expression in asymmetric valves. A shell regrowth model in oyster and gene silencing experiments, were used to characterize candidate gene function.

Results: Shell matrix genes with asymmetric expression in the mantle of the two valves were identified and unique cis-regulatory lncRNA modules characterized in Ostreidae. LncRNAs that regulate the expres- sion of the tissue inhibitor of metalloproteinases gene family (TIMPDR) and of the shell matrix protein domain family (SMPDR) were identified. In vitro and in vivo silencing experiments revealed the candidate genes and lncRNA were associated with divergent shell growth rates and modified the microstructure of calcium carbonate (CaCO3) crystals.
Conclusion: LncRNAs are putative regulatory factors of the bivalve biomineralization toolbox. In the Ostreidae family of bivalves biomineralization-related genes are cis-regulated by lncRNA and modify the planar growth rate and spatial orientation of crystals in the shell.