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Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with Streptococcus iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK. One of the DEmiRNAs, pol-3p-10740_175, was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of the latter. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. The results of this study provided the first S. iniae -induced miRNA profile in Japanese flounder and indicated that flounder miRNAs play an important role in antibacterial immunity. • Streptococcus iniae infection altered the expression of 81 miRNAs in flounder. • There were 34 and 58 differentially expressed miRNAs at 6 and 24 hpi, respectively. • The putative target genes of the miRNAs were enriched in various GO/KEGG pathways. • One miRNA, pol-3p-10740_175, negatively regulated dual specificity phosphatase 6. • Overexpression of pol-3p-10740_175 significantly inhibited S. iniae invasion. [ABSTRACT FROM AUTHOR]