Transcriptome Analysis Reveals Immune and Antioxidant Defense Mechanisms in the Eriocheir japonica sinensis after Exposure to Ammonia

Abstract

As a key species in freshwater aquaculture, Eriocheir japonica sinensis was subjected to ammonia stress to assess its impact on the hepatopancreas. A total of 4007 differentially expressed genes (DEGs) were identified between control and treatment groups, comprising 1838 upregulated and 2169 downregulated genes. Following exposure to 300 mg/L of ammonia, the oxidative phosphorylation pathway was activated, while the lysosomal pathway was suppressed, thereby influencing immune functions. Thirteen DEGs from these pathways were further validated via qRT-PCR, revealing gene expression changes of one- to two-fold. Both acid phosphatase (ACP) and alkaline phosphatase (AKP) levels in the hepatopancreas and hemolymph initially increased and then decreased, indicating a disruption in immune functionality. Additionally, alanine transaminase (ALT) and triglyceride (TG) levels were measured, alongside catalase (CAT) activity, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content, all of which showed an upward trend, signifying oxidative stress and tissue damage. These results offer critical insights into the antioxidant and immune mechanisms of E. j. sinensis in ammonia-enriched environments.

Keywords: Eriocheir japonica sinensis; ammonia; gene expression; immune response; lysosomal pathway.

Figure 1

HE staining of hepatopancreatic tissue structure of E. j. sinensis under ammonia stress. (A), control group; (B), medium-concentration stress group (300 mg/L); Bm, basement membrane; Bc, secretory cell; Ec, embryonic cell; Va, vacuole; Lu, lumen.

Figure 2

Effects of ammonia stress on non-specific enzyme activity in E. j. sinensis. (A) AKP in hepatopancreas; (B) ACP in hepatopancreas; (C) CAT in hepatopancreas; (D) MDA in hepatopancreas; (E) ALT in hepatopancreas; (F) TG in hepatopancreas. Different letters (a, b, c, d) indicate significant differences (p < 0.05).

Figure 3

Results of cell apoptosis detection. Arrows represent apoptotic cells.

Figure 4

Effect of ammonia stress on non-specific enzyme activity in E. j. sinensis. (A) AKP in hemolymph; (B) ACP in hemolymph; (C) hemocyanin content; (D) CAT in hemolymph; (E) T-AOC in hemolymph; (F) MDA in hemolymph. Different letters (a, b, c, d) indicate significant differences (p < 0.05).

Figure 5

Volcano plot of differentially expressed genes (DEGs) from the transcriptomes of the control and treatment groups. For each unigene, the ratio of expression levels was plotted against the -log error rate. The x-axis represents the fold-change between the 300 mg/L ammonia and PBS control groups, and the y-axis indicates the significance of differential expression. Gray dots indicate genes with no significant difference, while red and blue dots indicate upregulated and downregulated unigenes, respectively (q-value < 0.05 and |log₂ (fold-change)| > 1).

Figure 6

DEG enrichment analysis: (A) GO classification of unigenes from the hepatopancreas transcriptome of E. j. sinensis. Each annotated sequence is assigned to at least one GO term under the categories of biological process, cellular component, or molecular function. (B) KEGG pathway analysis of DEGs in E. j. sinensis hepatopancreas.

Figure 7

KEGG enrichment bubble diagram (A) and GSEA results (B,C) of DEG analysis.

Figure 8

(A) Oxidative phosphorylation pathway; (B) Lysosome pathway.

Figure 9

Validation of RNA-seq data by qRT-PCR. (A) Relative expression profiles of seven selected oxidative phosphorylation pathway genes in the hepatopancreas after ammonia stress compared to the control group. (B) Relative expression profiles of six immune-related genes in the hepatopancreas after ammonia stress. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001.