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• Neurotransmitter level was increased in juvenile Eriocheir sinensis after ammonia exposure. • More amino acids were consumed for additional energy supply under ammonia stress. • The glycogen content in the ammonia groups decreased in a dose-dependent manner. • Ammonia stress activated immune defense mechanisms. • High ammonia stress upregulated detoxification genes. Ammonia-N accumulation in the rice-crab co-culture system may have negative effects on the health of juvenile Eriocheir sinensis. In this study, physiological, transcriptomic, and metabolomic analyses were performed to explore the toxic responses in the hepatopancreas of juvenile E. sinensis exposed to 0, 0.75, and 2.99 mmol/L total ammonia-N for 24 h. We observed that the content of most amino acids and glycogen was significantly decreased after ammonia exposure. Acid phosphatase and alkaline phosphatase activities showed marginally increased trends after low ammonia exposure. Transcriptomic analysis indicated that immune defense, detoxification, and metabolic pathways were altered. Metabolomic analysis revealed that ammonia exposure affected energy metabolism and nucleotide metabolism. The combination of transcriptomic and metabolomic analyses revealed that the tricarboxylic acid cycle and amino acid consumption were enhanced for additional energy supply to cope with ammonia stress. Ammonia stress activated the immune defense system in juvenile E. sinensis. Moreover, the upregulation of detoxification genes and the acceleration of glycogen degradation for glucose supply are important adaptive mechanisms in response to high ammonia stress. Notably, ammonia stress may affect the nervous system of juvenile E. sinensis. Thus, our data provide a better understanding of the defensive mechanisms of E. sinensis against ammonia toxicity. [ABSTRACT FROM AUTHOR]