Differentially Expressed Genes and Alternative Splicing Analysis Revealed the Difference in Virulence to American Eels (Anguilla rostrata) Infected by Edwardsiella anguillarum and Aeromonas hydrophila

Edwardsiella anguillarum and Aeromonas hydrophila are two common bacterial pathogens affecting cultivated eels, and the differences in their virulence remain unclear. In this study, after two groups of American eels (Anguilla rostrata) were administered the LD₅₀ dose of E. anguillarum and A. hydrophila, respectively, the histopathology of the liver, trunk kidney, and spleen, as well as transcriptomic RNA sequencing (RNA-seq) analysis of the spleen, was examined at three time points: pre-infection (Con group) and post-infection at 36 h (Ea_36 group, Ah_36 group) and 60 h (Ea_60 group, Ah_60 group). The results showed that the differences in pathological changes were characterized by severe hepatocyte edema at 36 h post-infection (hpi) and hepatocyte atrophy at 60 hpi in the livers of eels infected by A. hydrophila, in contrast to the severe atrophy of glomeruli in the trunk kidneys and numerous bacterial nodules in the spleens of eels infected by E. anguillarum. The RNA-seq results revealed 906 and 77 typical differentially expressed genes (DEGs) in eels infected with E. anguillarum and A. hydrophila, respectively, compared to the control eels. The DEGs between the infected and control groups were predominantly annotated in GO terms related to binding, catalytic activity, membrane part, cell part, and cellular process, as well as in KEGG pathways associated with human diseases and organismal systems. The GO enrichment analysis showed 83 and 146 differential GO terms, along with 32 and 78 differential KEGG pathways in two comparisons of Ea_36 vs Con versus Ah_36 vs Con and Ea_60 vs Con versus Ah_60 vs Con, respectively. Furthermore, the analysis of differential alternative splicing genes (DASs) showed 1244 and 1341 DASs out of 12,907 and 12,833 AS genes, respectively, in the comparisons of Ea_36 vs Ah_36 and Ea_60 vs Ah_60. These DASs were enriched in two common KEGG pathways: “NOD-like receptor signaling pathway” and “necroptosis” which shared 11 hub DASs. Finally, analysis of protein–protein interactions revealed that 91 of 412 cross DASs between Ea_36 vs Ah_36 and Ea_60 vs Ah_60 potentially play an essential role in the difference in virulence of E. anguillarum and A. hydrophila in American eels, with 12 encoded proteins being particularly notable. Together, this study is the first to report a comparative pathogenicity and RNA-seq analysis of E. anguillarum and A. hydrophila in American eels, shedding new light on our understanding of the differences in virulence as revealed by pathological changes, DEGs, and DASs, contributing to more effective control strategies to prevent outbreaks of bacterial infections.