Integrated Analysis of Transcriptome and Metabolome Reveals Molecular Responses to Ammonia Stress in the Gills of Litopenaeus vannamei Under Low-Salinity Conditions

High ammonia nitrogen stress significantly compromises the survival of Litopenaeus vannamei under low-salinity conditions. However, existing studies predominantly focus on ammonia nitrogen responses under single stressors or normal seawater salinity. The molecular regulatory mechanisms, metabolic remodeling patterns, and key pathway interactions in shrimp subjected to high ammonia nitrogen stress under low-salinity environment remain unclear. In this study, we employed integrated transcriptomic and metabolomic analyses to unveil the underlying molecular responses and metabolic biomarkers in the gills of L. vannamei to ammonia stress under low-salinity conditions. First, L. vannamei underwent low-salinity acclimation from 30‰ to 5‰ salinity and was then reared for one week to acclimate to the experimental environment. Subsequently, shrimp were treated with 42.32 mg/L ammonia nitrogen for a consecutive 96 h period. Integrated transcriptomic and metabolomic analyses elucidated the stress response patterns in the gills of L. vannamei under low-salinity ammonia nitrogen exposure. Specifically, 352, 802, and 140 differentially expressed genes (DEGs) were identified at 12 h, 48 h, and 96 h post-exposure, respectively. GO and KEGG enrichment analyses revealed that the significant DEGs were primarily enriched in six major pathways: autophagy, immune-related pathway, ABC transporter, fatty acid degradation and metabolism, metabolic pathway, and PPAR signaling pathway. Metabolomic profiling identified numerous differentially accumulated metabolites (DAMs) in both positive and negative ion modes, with significantly altered DAMs mainly consisting of organic acids and their derivatives, phospholipids, and other related metabolites. Key DAMs included taurine, guanosine, 1-palmitoyl-sn-glycero-3-phosphocholine, pseudouridine, and betaine. Integrative multi-omics analysis revealed that L. vannamei mediates stress responses by modulating five core pathways under low-salinity/high-ammonia-nitrogen dual stress: fatty acid degradation and metabolism (e.g., acyl-CoA dehydrogenase short chain (Acads), acetyl-CoA acetyltransferase 2 (ACAT2)), autophagy (e.g., autophagy-related protein 101-like (atg101)), immune regulation pathway (e.g., V-type proton ATPase subunit H-like (VhaSFD), actin-5C-like (Act5C)), metabolic pathway (e.g., molybdopterin synthase catalytic subunit-like (Mocs2B), cytochrome P450 2U1-like (Cyp2b1)), and ABC transporter (e.g., ATP-binding cassette sub-family D member 3-like (ABCD3), ATP-binding cassette sub-family B member 10 (ABCB10)). Through characterization of these core pathways, this study reveals the fundamental mechanisms by which L. vannamei responds to high ammonia nitrogen stress following low-salinity acclimation, providing a theoretical foundation for estuarine shrimp farming.