Immune and physiological responses in Penaeus monodon to ammonia-N stress: a multi-omics approach

Abstract

Ammonia-N stress is a significant environmental factor that adversely affects the health and productivity of aquaculture species. This study investigates the effects of ammonia-N stress on the shrimp Penaeus monodon through a combination of biochemical, histological, transcriptomic, and metabolomic analyses. Shrimp were exposed to ammonia-N stress for 12 and 96 hours, and key markers of oxidative stress, nitrogen metabolism, immune response, and overall health were assessed. The results showed that prolonged ammonia-N exposure causes significant hepatopancreatic damage, including atrophy and deformation. Transcriptomic analysis revealed significant changes in gene expression related to apoptosis, immune response, and key metabolic pathways, with particular emphasis on the disruption of innate immune signaling and defense mechanisms. Metabolomic analysis identified disruptions in nucleotide turnover, antioxidant defenses, and fundamental metabolic processes. These findings suggest that ammonia-N stress induces a multifaceted stress response in shrimp, involving oxidative stress, immune activation, and metabolic disturbances. Understanding these immune-related and metabolic mechanisms provides valuable insights into the molecular responses of crustaceans to environmental stress, laying the foundation for assessing the ecological risk of ammonia-N and identifying potential immunological biomarkers for monitoring and mitigating its adverse effects in aquaculture systems.

Keywords: ammonia-N stress; immune response; metabolome; oxidative stress; shrimp; transcriptome.

Figure 1

Biochemical Analysis of Shrimp Exposed to Ammonia-N Stress. (A) Blood ammonia levels. (B) Urea nitrogen levels. (C) GS activity. (D) SOD activity. (E) XOD activity. (F) Adenosine deaminase (ADA) activity. (G) Caspase-3 activity. (H) Caspase-8 activity. (I) AST activity. The lowercase letters represent significant differences between groups, as determined by one-way ANOVA. Different letters indicate statistical significance, while the same letter indicates no significant difference.

Figure 2

Histological Analysis of Hepatopancreas in Shrimp Exposed to Ammonia-N Stress. (A) Control group at 0 hours. (B) 12-hour exposure group. (C) 96-hour exposure group.

Figure 3

Transcriptome Analysis of Shrimp Exposed to Ammonia-N Stress. (A) PCA plot. (B) The number of up-regulated and down-regulated genes among all DEGs in each comparison group. (C) Venn diagram of DEGs at 12 hours. (D) Venn diagram of DEGs at 96 hours. (E) Venn diagram of shared DEGs. (F) KEGG pathway enrichment. (G) GO term enrichment.

Figure 4

GSEA of Transcriptomic Data. (A) Downregulated “Glycerolipid metabolism” pathway at 12 hours. (B) Downregulated “Glutathione metabolism” pathway at 12 hours. (C) Downregulated “Proteasome” pathway at 12 hours. (D) Downregulated “Chemokine signaling” pathway at 12 hours. (E) Upregulated “Phosphatidylinositol signaling system” pathway at 96 hours. (F) Upregulated “Peroxisome” pathway at 96 hours. (G) Downregulated “Protein export” pathway at 96 hours. (H) Downregulated “Complement and coagulation cascades” pathway at 96 hours.

Figure 5

Protein-Protein Interaction (PPI) Networks of DEGs. (A) PPI network for 12-hour exposure. (B) PPI network for 96-hour exposure.

Figure 6

Metabolomic Analysis of Shrimp Exposed to Ammonia-N Stress. (A) OPLS-DA score plot. (B) Permutation test results. (C) Venn diagram of shared differential metabolites. (D) Heatmap of upregulated metabolites at 12 and 96 hours. (E) Heatmap of downregulated metabolites at 12 and 96 hours. (F) KEGG pathway enrichment.

Figure 7

Heatmaps of Time-Dependent Metabolite Changes. (A) Heatmap of upregulated metabolites over time. (B) Heatmap of downregulated metabolites over time.