Comparative Transcriptome Analysis Reveals the Molecular Immunopathogenesis of Chinese Soft-Shelled Turtle (Trionyx sinensis) Infected with Aeromonas hydrophila

Abstract

Although hemorrhagic sepsis caused by Aeromonas hydrophila infection is the dominant disease in the aquaculture of Chinese soft-shelled turtle, information on its molecular pathology is seriously limited. In this study, ninety turtles intraperitoneally injected with A. hydrophila exhibited two different phenotypes based on the pathological symptoms, referred to as active and inactive turtles. Comparative transcriptomes of liver and spleen from these two groups at 6, 24, and 72 h post-injection (hpi) were further analyzed. The results showed that cytokine-cytokine receptor interaction, PRRs mediated signaling pathway, apoptosis, and phagocytosis enriched in active and inactive turtles were significantly different. Pro-inflammatory cytokines, the TLR signaling pathway, NLR signaling pathway, and RLR signaling pathway mediating cytokine expression, and apoptosis-related genes, were significantly up-regulated in inactive turtles at the early stage (6 hpi). The significant up-regulation of phagocytosis-related genes occurred at 24 hpi in inactive turtles and relatively lagged behind those in active turtles. The anti-inflammatory cytokine, IL10, was significantly up-regulated during the tested periods (6, 24, and 72 hpi) in active turtles. These findings offer valuable information for the understanding of molecular immunopathogenesis after A. hydrophila infection, and facilitate further investigations on strategies against hemorrhagic sepsis in Chinese soft-shelled turtle T. sinensis.

Keywords: Aeromonas hydrophila; Chinese soft-shelled turtle; hemorrhagic sepsis; molecular immunopathogenesis.

Figure 1

Pathological symptoms of turtles infected by A. hydrophila. The pathological symptoms on the body surface including (A) white spots near the axillae; (B) abdominal congestion in inactive subgroup turtles. (C) The pathological symptoms of viscera including liver, spleen, and intestines after anatomy of inactive turtles. (D) The active subgroup turtles showed no obvious pathological symptoms and were aggressive and active in feeding and moving. Black arrows indicate the location of pathological symptoms.

Figure 2

Transcriptional relationship between samples and the overall gene expression profiles. (A) Heatmap of correlation value (R square) of 37 libraries from liver or spleen samples. (B) Principal component analysis based on all of the expressed genes, showing 14 distinct groups of samples. (C) The dispersion degree of the gene expression level in a single liver or spleen sample. (D) The significantly up-regulated and down-regulated DEGs identified in livers or spleens from active and inactive turtles at 6, 24, and 72 hpi compared to the control.

Figure 3

KEGG enrichment analysis in AL group turtles at different time periods. The top 20 KEGG pathways are presented here in the form of scatterplots to show the up-regulated and down-regulated DEGs enriched in livers from active subgroup turtles at 6, 12, and 72 hpi. The enrichment factor is the ratio between the DEG number and the number of all genes in a certain gene enrichment term. The sizes of the dots on these plots denote the number of DEGs, while colors correspond to the q value range.

Figure 4

KEGG enrichment analysis in IL group turtles at different time periods. The top 20 KEGG pathways are presented here in the form of scatterplots to show the up-regulated and down-regulated DEGs enriched in livers from inactive subgroup turtles at 6, 12, and 72 hpi. The enrichment factor is the ratio between the DEG number and the number of all genes in a certain gene enrichment term. The sizes of the dots on these plots denote the number of DEGs, while colors correspond to the q value range.

Figure 5

KEGG enrichment analysis in AS group turtles at different time periods. The top 20 KEGG pathways are presented here in the form of scatterplots to show the up-regulated and down-regulated DEGs enriched in spleens from active subgroup turtles at 6, 12, and 72 hpi. The enrichment factor is the ratio between the DEG number and the number of all genes in a certain gene enrichment term. The sizes of the dots on these plots denote the number of DEGs, while colors correspond to the q value range.

Figure 6

KEGG enrichment analysis in IS group turtles at different time periods. The top 20 KEGG pathways are presented here in the form of scatterplots to show the up-regulated and down-regulated DEGs enriched in spleens from inactive subgroup turtles at 6, 12, and 72 hpi. The enrichment factor is the ratio between the DEG number and the number of all genes in a certain gene enrichment term. The sizes of the dots on these plots denote the number of DEGs, while colors correspond to the q value range.

Figure 7

Validation of RNA-Seq results by qPCR. Nine DEGs are randomly selected, and the expressions of genes at different time periods are examined relative to the endogenous control genes (β-actin and GAPDH). The relative expression values are transformed into the log₂ (fold change) form. The results are shown as the mean ± SEM of liver and spleen tissues derived from 3 individual turtles.

Figure 8

The suspected molecular immunopathogenesis of hemorrhagic sepsis caused by A. hydrophila infection in Chinese soft-shelled turtles.