CgPPOX and CgFECH Mediate Protoporphyrin IX Accumulation Predominantly in the Outer Mantle Fold of Pacific Oyster Crassostrea gigas

Mollusk shell is secreted through mantle folds and exhibits color polymorphism predominantly caused by pigments. Mantle tissue consists of three folds, with the outer fold playing crucial roles in shell formation. Among various pigments influencing shell color, porphyrins—especially protoporphyrin IX (PPIX)—significantly contribute to orange-shell phenotype. However, the distinct functions of mantle folds in shell coloration and pigmentation remain poorly understood. In this study, the frozen section of fresh mantles from orange and gold shell-color Pacific oyster Crassostrea gigas was observed to have distinct porphyrin distributions. Significant differences in porphyrin spectrum and precise PPIX concentrations were detected among mantle folds of shell-color strains C. gigas. In addition, key genes from porphyrin pathway: protoporphyrinogen oxidase (PPOX) and ferrochelatase (FECH) were characterized to exhibit distinct expression patterns among mantle folds of different shell-colored oysters. Dual-label fluorescence in situ hybridization analysis of CgPPOX and CgFECH with the PPIX translocator protein (CgTspO) verified the predominant functional location of these genes is the oyster outer mantle fold and periostracal groove. Furthermore, in vivo knockdown of CgPPOX and CgFECH verified their roles in PPIX metabolism, resulting in significant changes in porphyrin metabolic-related gene expression and altered PPIX concentrations in both the outer mantle fold and the newly deposited shell of C. gigas. This study identified the differential mantle fold porphyrin distribution and demonstrated essential roles of CgPPOX and CgFECH in PPIX metabolism, correlating with variations in PPIX content and shell color of C. gigas. The results provide new insights into molecular mechanisms underlying shell-color polymorphism in mollusks.