Identification and characterization of key haem pathway genes associated with the synthesis of porphyrin in Pacific oyster (Crassostrea gigas).

Molluscs exhibit diverse shell colors. The molecular regulation of shell coloration is however not well understood. To investigate the connection of shell coloration with pigment synthesis, we analyzed the distribution of porphyrins, a widespread group of pigments in nature, in four Pacific oyster strains of different shell colors including black, orange, golden, and white. The porphyrin distribution was analyzed in oyster mantles and shells by fluorescence imaging and UV spectrophotometer. The results showed that red fluorescence emitted by porphyrins under the UV light was detected only on the nacre of the orange-shell strain and mantles of orange, black and white-shell strains. Extracts from newly deposit shell, nacre and mantle tissue from orange-shell specimens showed peaks in UV-vis spectra that are characteristic of porphyrins, but these were not observed for the other shell-color strains. In addition, genes of the haem synthetic pathway were isolated and characterized. Phylogenetic analysis of CgALAS, CgALAD, CgPBGD, CgUROS, and CgUROD provide further evidence for a conserved genetic pathway of haem synthesis during evolution. Differential expression of the haem genes expressed in mantle tissues support these findings and are consistent with porphyrins being produced by the orange strain only. Tissue in situ hybridization demonstrated the expression of these candidate genes at the outer fold of C. gigas mantles where shell is deposited. Our studies provide a better understanding of shell pigmentation in C. gigas and candidate genes for future mechanistic analysis of shell color formation in molluscs.