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Background Mollusca, a phylum of highly rich species, possess vivid shell colours, but the underlying molecular mechanism remains to be elucidated. DNA methylation, one of the most common epigenetic modifications in eukaryotes, is believed to play a vital role in various biological processes. However, analysis of the effects of DNA methylation on shell colouration has rarely been performed in molluscs, limiting the current knowledge of the molecular mechanism of shell colour formation. Results In the present study, to reveal the role of epigenetic regulation in shell colouration, WGBS, the “gold standard” of DNA methylation analysis, was first performed on the mantle tissues of Yesso scallops (Patinopecten yessoensis) with different shell colours (brown and white), and DNA methylomes at single-base resolution were generated. About 3% of cytosines were methylated in the genome of the Yesso scallop. A slight increase in mCG percentage and methylation level was found in brown scallops. Sequence preference of nearby methylated cytosines differed between high and low methylation level sites and between the brown- and white-shelled scallops. DNA methylation levels varied among the different genomic regions; all the detected regions in the brown group exhibited higher methylation levels than the white group. A total of 41,175 DMRs (differentially methylated regions) were detected between brown and white scallops. GO functions and pathways associated with the biosynthesis of melanin and porphyrins were significantly enriched for DMRs, among which several key shell colour-related genes were identified. Further, different correlations between mRNA expression levels and DNA methylation status were found in these genes, suggesting that DNA methylation regulates shell colouration in the Yesso scallop. Conclusions This study provides genome-wide DNA methylation landscapes of Yesso scallops with different shell colours, offering new insights into the epigenetic regulatory mechanism underlying shell colour.

Pif is an acid matrix protein that is known to be a key macromolecule in the shell nacre formation of molluscs. However, the investigation into the Pif gene at the whole genome level has seldom been carried out, limiting the systematic understanding of its evolution and function. The Yesso scallop (Patinopecten yessoensis), an ancient bivalve with important economic value and evolutionary status, has suffered severe shell infestation by Polydora that badly damaged shell structures. In this study, a total of eight Pif homologous genes (PyPif1–7 and PyBMSP) were identified in the whole genome of the Yesso scallop with diverse gene structures. Two conserved functional domains, VWA and chitin-binding domains, were detected in all PyPif proteins, although with variation in number of copies. Phylogenetic analysis of the Pifs among different molluscan species suggested the molecular evolution of Pif genes was consistent with taxonomic relationships. High sequence divergence of Pif proteins existed among the different species, but a high level of similarity was found in BMSPs, which own four VWA domains. High expression levels of PyBMSP in the central mantles of the healthy and Polydora shell infested Yesso scallops indicated its important role in shell formation. The other PyPifs (PyPif2, PyPif3, PyPif4, PyPif5, PyPif6) may function when the shell is injured, as significantly increased expression levels of these genes were found in the edge mantles of the infested scallops. In addition, haemocytes may also participate in shell repair by up-regulating the expression of PyPifs. This study provides new insights into the evolution and expression patterns of Pif genes in molluscs, which are valuable for elucidating the mechanism of shell repair and formation.

Porphyrins, one of the most common shell pigments, are by-products of the haem pathway. 5-Aminolaevulinate synthase (ALAS) is the first and rate-limiting enzyme in this pathway and has been well studied in vertebrate species. However, the function of ALAS in shell colouration has been poorly studied in molluscs, which are renowned for their colourful shells. In the present study, an ALAS gene, named PyALAS, was identified through whole-genome scanning in the Yesso scallop (Patinopecten yessoensis), an economically and evolutionarily important bivalve species in which the shell colour represents polymorphism. Two conserved domains were detected in the PyALAS protein sequence, including a Preseq-ALAS domain and a 5-ALAS domain, confirming the identification of PyALAS. Phylogenetic analysis of the ALAS proteins among various invertebrate and vertebrate species revealed a high consistency between the molecular evolution of ALAS and the species taxonomy. PyALAS was ubiquitously expressed in most adult tissues of the Yesso scallop. The left mantle expressed a significantly higher level of PyALAS than the right side in brown scallops, whereas there was no significant difference in white scallops. Significantly different expression levels of PyALAS was also detected between the two different shell colour strains. These data indicate that PyALAS plays an important role in shell colouration in Yesso scallops and the present study provides new insights into the molecular mechanism of shell colouration in molluscs.

The Yesso scallop Patinopecten yessoensis is one of the most important aquaculture species in Asian countries because of its high economic value, but it has suffered severe infection by Polydora in recent years, causing great economic losses. Polydora mainly parasitizes the left valves of the Yesso scallop, especially the region around the adductor muscle, badly damaging the shell structure and making the shell particularly fragile. To investigate the response mechanism of the Yesso scallop to Polydora, a histological study was performed on healthy and diseased Yesso scallop mantle tissues, which are thought to be responsible for shell formation. In the present study, separate histological and histochemical changes in different mantle regions were detected for the first time. In the diseased scallops, abundant mucous cells containing acid mucopolysaccharides were found in the epidermis of the outer fold, along with a great increase in cilia on the inner epidermis, and a significant increase in mucous cells secreting acid mucopolysaccharides was observed in the middle fold and the central mantle, suggesting an accelerated process of secretion and transportation of shell substances. These changes in the different mantle regions were probably responsible for the repair of different shell layers. The present study provides valuable information for the function of mantle tissue in shell formation and is helpful for genetic breeding of disease-resistant scallops in the future.