Transcriptomic analysis of shell repair and biomineralization in the blue mussel, Mytilus edulis

Abstract

Background: Biomineralization by molluscs involves regulated deposition of calcium carbonate crystals within a protein framework to produce complex biocomposite structures. Effective biomineralization is a key trait for aquaculture, and animal resilience under future climate change. While many enzymes and structural proteins have been identified from the shell and in mantle tissue, understanding biomieralization is impeded by a lack of fundamental knowledge of the genes and pathways involved. In adult bivalves, shells are secreted by the mantle tissue during growth, maintenance and repair, with the repair process, in particular, amenable to experimental dissection at the transcriptomic level in individual animals.

Results: Gene expression dynamics were explored in the adult blue mussel, Mytilus edulis, during experimentally induced shell repair, using the two valves of each animal as a matched treatment-control pair. Gene expression was assessed using high-resolution RNA-Seq against a de novo assembled database of functionally annotated transcripts. A large number of differentially expressed transcripts were identified in the repair process. Analysis focused on genes encoding proteins and domains identified in shell biology, using a new database of proteins and domains previously implicated in biomineralization in mussels and other molluscs. The genes implicated in repair included many otherwise novel transcripts that encoded proteins with domains found in other shell matrix proteins, as well as genes previously associated with primary shell formation in larvae. Genes with roles in intracellular signalling and maintenance of membrane resting potential were among the loci implicated in the repair process. While haemocytes have been proposed to be actively involved in repair, no evidence was found for this in the M. edulis data.

Conclusions: The shell repair experimental model and a newly developed shell protein domain database efficiently identified transcripts involved in M. edulis shell production. In particular, the matched pair analysis allowed factoring out of much of the inherent high level of variability between individual mussels. This snapshot of the damage repair process identified a large number of genes putatively involved in biomineralization from initial signalling, through calcium mobilization to shell construction, providing many novel transcripts for future in-depth functional analyses.

Keywords: Bivalve; Calcium; Haemocytes; Mollusc; Shell matrix proteins.

Fig. 1

The paired valve design for assessing shell repair in Mytilus edulis. A Location of drilled holes on the left valve, and the areas of mantle tissue sampled from both valves. B Typical extent of healing 29 days after drilling. Picture attributions (A) Picture obtained and modified under Creative Commons license (2006) from F. Lamiot, Moule, Miesmuscheln, mussel (anatomia and shell), url: https://commons.wikimedia.org/wiki/File: Moules_Miesmuscheln_mussel3.jpg; (B) from Frank Melzner with permission

Fig. 2

Multidimensional scaling identifies significant contributions of individual variation to gene expression differences in shell repair in Mytilus edulis. MDS plots of expression counts for the filtered set of putative genes in (A) All libraries: Central mantle – left/damaged valve; Central mantle – right undamaged (control) valve: Mantle edge – left/damaged valve; Mantle edge – right undamaged (control) valve, B Central mantle libraries only (C) Mantle edge libraries only

Fig. 3

Differential gene expression in Mytilus edulis mantle tissues during shell repair. Volcano plots detailing differential gene expression between the four mantle tissue libraries. Inset mussel pictures show comparisons detailed in each plot. A Damaged valve: mantle edge versus central mantle, B Control valve: mantle edge versus central mantle, C Damaged central mantle versus control central mantle, D Damaged mantle edge versus damaged central mantle. Dashed lines indicate the FDR value of 0.001. Note: The axis scales are not the same across all plots

Fig. 4

Shell matrix protein homologues identified in Mytilus edulis shell proteomes, transcriptomes, and differential gene expression. For each identified protein or protein domain the columns indicate: Shell proteome: Previously identified shell proteome sequences; Mantle transcriptome: Transcripts previously identified in mantle transcriptome studies; DGE: CM vs. ME: Differential gene expression (DGE) identified in the central mantle (CM) versus the mantle edge (ME); DGE: shell repair in CM: Trajectory of DGE in the central mantle (UP = up-regulation; P = putative shell proteins with no strong sequence similarity to, but with similar functional domains to known SMPs); DGE: Prodissoconch I: Genes differentially expressed in the prodissoconch I in transcriptomic analysis of development. The haemocyte dataset has not been included, as only one domain (C1Q) in common was identified

Fig. 5

Expression of selected sets of differentially expressed genes in central mantle during shell repair in Mytilus edulis. For each differentially expressed gene set (rows) four sets of five columns show the fold expression change in each of the five individuals (001–005). The sets of columns from left to right are: Damaged central mantle, Control central mantle, Damaged mantle edge, and Control mantle edge. The differentially expressed gene sets are grouped and colour coded: Blue: DE genes with sequence similarity to SMPs, ordered by SMP name; Green: DE genes with domains found in SMPs, but no sequence similarity to known SMPs, ordered by domain name; Orange: DE genes containing transmembrane domains; Grey: Non-DE genes with sequence similarity to ATPases of interest