Insights on nitrate pollution-induced intestinal dysfunction in turbot (Scophthalmus maximus) revealed by integrated dynamic metabolomics and transcriptomics

Nitrate pollution in aquatic ecosystems has attracted global attention and has toxic effects on marine organisms. However, the precise molecular mechanisms underlying nitrate toxicity in the fish gut remain obscure. To this end, turbot were subjected to nitrate exposure (200 mg/L NO₃-N) for 0, 10, 20, and 30 days to explore nitrate toxicity and metabolic mechanisms in the gut by employing a multi-omics analysis integrating metabolomics with transcriptomics. The metabolomics analysis showed that nitrate exposure resulted in significant changes in the intestinal metabolite network, implying that the intestinal metabolism of turbot was impaired. Metabolites Pathway Analysis (MetPA) results revealed that the metabolic pathways significantly impacted by nitrate exposure included amino-acid metabolism pathways, such as phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, D-glutamine and D-glutamate metabolism, and aminoacyl-tRNA biosynthesis. Additionally, network interaction analysis between key differential metabolites (DMs) and differentially expressed genes (DEGs) identified seven essential amino acids associated with this process. Short Time-series Expression Miner (STEM) analysis determined that six distinct temporal expression patterns exhibited dynamic changes in DMs, mainly enriched in the metabolism of carbohydrates and lipids, indicating an increased energy demand to withstand nitrate stress. Multi-omics analysis revealed that sustained nitrate stress can interfere with protein digestion and absorption, alter collagen anabolism and specific composition of the extracellular matrix (ECM), and ultimately disrupt intestinal homeostasis. Our findings enhance our understanding of nitrate toxicity in fish and offer insights that can improve nitrate management in marine ecosystems.