Dietary 5-aminolevulinic acid induces strain-specific effects on shell color and innate immunity in Pacific oyster (Crassostrea gigas) through divergent porphyrin metabolism

Shell color is a key market trait in marine molluscs, while stress resilience is crucial for intensive aquaculture. This study investigated the 90-day effects of dietary 5-aminolevulinic acid (5-ALA; at 0, 10, 20, and 40 mg/L) on shell pigmentation and innate immunity in two shell-color strains of the Pacific oyster, Crassostrea gigas. The results revealed divergent Protoporphyrin IX (PPIX) utilization strategies. The orange-shell strain prioritized PPIX for shell deposition, thereby enhancing coloration. In contrast, the gold-shell strain redirected PPIX toward heme synthesis, which subsequently activated a systemic defense response. In gold-shell C. gigas, this response included the significant upregulation of immune-related genes such as nuclear factor erythroid 2-related factor 2 (Nrf2), Toll-like Receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and interleukin-17 (IL-17). It was also accompanied by increased activities of the antioxidant Enzymes superoxide dismutase (SOD) and catalase (CAT), elevated glutathione (GSH) levels, and reduced lipid peroxidation (malondialdehyde, MDA). Moreover, 5-ALA induced hypomethylation of CgPPOX and modulated the expression of PPIX transporters (CgTspO, CgABCG2), establishing a novel link between porphyrin metabolism and immunity. An optimal dose of 20 mg/L 5-ALA was identified, which simultaneously improved desirable shell-color traits and physiological resilience. This study provides new insights into the molecular basis of color trait improvement and a theoretical foundation for understanding the mechanisms linking pigment metabolism and immune function in aquatic Animals.