Abstract
The physiological processes driving the rapid rates of calcification in larval bivalves are poorly understood. Here, we use a calcification substrate‐limited approach (low dissolved inorganic carbon, C T) and mRNA sequencing to identify proteins involved in bicarbonate acquisition during shell formation. As a secondary approach, we examined expression of ion transport and shell matrix proteins (SMPs) over the course of larval development and shell formation. We reared four families of Mytilus edulis under ambient (ca. 1865 µmol/kg) and low C T (ca. 941 µmol/kg) conditions and compared expression patterns at six developmental time points. Larvae reared under low C T exhibited a developmental delay, and a small subset of contigs was differentially regulated between ambient and low C T conditions. Of particular note was the identification of one contig encoding an anion transporter (SLC26) which was strongly upregulated (2.3–2.9 fold) under low C T conditions. By analyzing gene expression profiles over the course of larval development, we are able to isolate sequences encoding ion transport and SMPs to enhance our understanding of cellular pathways underlying larval calcification processes. In particular, we observe the differential expression of contigs encoding SLC4 family members (sodium bicarbonate cotransporters, anion exchangers), calcium‐transporting ATPases, sodium/calcium exchangers, and SMPs such as nacrein, tyrosinase, and transcripts related to chitin production. With a range of candidate genes, this work identifies ion transport pathways in bivalve larvae and by applying comparative genomics to investigate temporal expression patterns, provides a foundation for further studies to functionally characterize the proteins involved in larval calcification.
Highlights
During bivalve ontogeny, major developmental steps occur rapidly within the first days of life to produce the larval shell, prodisso‐ conch I (PD I)
By rearing mussel larvae under conditions of substrate limitation for calcification and analyzing differential gene expression pat‐ terns, we were able to identify a membrane‐bound transport pro‐ tein potentially involved in HCO3− acquisition, belonging to the solute carrier family 26 (SLC26) family of anion transporters and other candidate genes previously identified in human biomineralization
The present study identifies only a small subset of contigs to be differ‐ entially expressed under substrate limitation. This may be improved by a stronger calcification. Substrate (CT) treatment (>50% decrease relative to control), higher sequencing depth, and level of replication, bivalve larvae may possess a fixed capacity to modify their transcriptomic
Summary
Major developmental steps occur rapidly within the first days of life to produce the larval shell, prodisso‐ conch I (PD I). Within 2 days of development, bivalve larvae precipitate an inorganic aragonitic shell that is almost equivalent to their somatic mass and covers the entire larval body (Waldbusser et al, 2013). Accompanying this transition into PD I larval stage is a 250‐fold in‐ crease in larval calcium content within a few hours (Ramesh et al, 2017). The substrates for calcification (Ca2+ and HCO3−) are not accumulated prior to calcification but are continu‐ ally supplied during larval shell formation, most likely by means of transepithelial transport (Ramesh et al, 2017)
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