Functional Validation of the Phase I-II-III Detoxification Pathway in Azumapecten farreri using Optimized RNA Interference Technology.

Paralytic shellfish toxins (PSTs) are widely distributed neurotoxins that accumulate and transform in bivalves, but the metabolic mechanisms after exposure of Azumapecten farreri (A. farreri) to PSTs remain unknown. To verify the phase I (cytochrome P450, CYP450)-II (glutathione S-transferase, GST)-III (ABC transport) metabolic genes for PSTs, we used RNA interference (RNAi) to silence target genes (CYP46A1, GSTM1, and ABCF2), measured PSTs content, and performed single nucleotide polymorphism (SNP) screening. Injection of small interfering RNA (siRNA) fragments into the scallop adductor muscle silenced target genes in multiple tissues. The three most effective fragments were selected. Secondary injection of screening fragments prolonged the silencing duration, with a silencing efficiency of 12.0%-100%. Silencing increased PSTs accumulation. Notably, with CYP46A1 silencing, the PSTs accumulation (1016 µg STXeq/kg) reached 3.85 times that of the control group. This silencing also delayed the conversion of highly toxic components (GTX1-4) to components with low toxicity (C1-2), thereby prolonging the PSTs metabolic process. SNP analyses indicated that PSTs exposure increased homozygous mutations in CYP49A1 (511908 site), this site is potentially involved in PSTs metabolism. In summary, this study optimized an RNAi method and confirmed the existence of a three-phase metabolic mechanism in scallops. CYP450 was found to be the main mode of PSTs metabolism. This study provides data regarding PSTs metabolic genes and loci in bivalves, as well as valuable molecular markers for anti-toxin breeding in shellfish.