Integrated Lipidomic, Transcriptomic, and Metabolomic Analyses Reveal the Regulatory Mechanism Underlying Venom Synthesis in Parotoid Glands of Bufo gargarizans.

The parotoid gland secretion of the medicinal toad (Bufo gargarizans) is the primary source of Chansu, a valuable traditional medicine. However, the lack of understanding regarding the biological regulation of venom synthesis limits the optimization of farming practices and quality control. Here, we integrated histology, lipidomics, transcriptomics, and untargeted metabolomics to delineate the regulatory mechanisms governing venom accumulation and biosynthesis, aimed at guiding aquaculture management. Histological results revealed that the parotoid gland is a highly regionalized organ, where structural maturity is positively correlated with toxin storage capacity, providing a morphological basis for determining optimal harvest timing. This structure is underpinned by a distinct lipid profile: high levels of triglycerides supply essential energy for massive venom production, while enriched ceramides and sphingomyelin form a specialized barrier to ensure high-quality venom encapsulation. Transcriptome analysis indicated that the upregulation of steroid and primary bile acid biosynthesis genes (e.g., FDFT1, DHCR7, HSD3B7, CYP27A1, and ACOX2) drives the synthesis of bioactive bufadienolides. Furthermore, we identified key enzymatic regulators─including CYP450s (CYP2C25, CYB561, CYP2K4) and acetylases (AACS, ACAT)─as potential genetic markers for identifying high-yielding venom. Collectively, these findings provide a scientific basis for establishing standardized farming protocols, advancing molecular breeding strategies for this important medicinal species, and achieving sustainable exploitation of toad venom.