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Abstract: Toll-like receptor (TLR) 5 is responsible for the bacterial flagellin recognition in vertebrates. Synergistic role of TLR 5 membrane form (TLR 5M) and TLR 5 soluble form (TLR 5S) have been reported from the study on rainbow trout. This system is regarded as the unique system in teleost fish. However, systemic response of TLR 5 genes in teleost fish has not been fully understood. Hence, we cloned Japanese flounder (Paralichthys olivaseus) TLR 5M and TLR 5S genes and their expressions were analyzed. The coding region of Japanese founder TLR 5M and TLR 5S cDNA were 2670 bp and 1923 bp, encoding 889 and 640 amino acid residues, respectively. The Japanese flounder TLR 5M was composed of an extracellular leucine rich repeats (LRRs), a transmembrane and an intracellular Toll/interleukin-1 receptor (TIR) domains, whereas TLR 5S possessed only the LRR domain. TLR 5M was highly expressed in the gill, head kidney, heart and liver. TLR 5S was highly expressed in the brain, head kidney and heart. Flagellin stimulation (1 and 5 μg/ml) led to strong gene expression of TLR 5S in peripheral blood leukocytes (PBLs) and liver cells. In contrast to TLR 5S, TLR 5M was down-regulated until 3 h after flagellin stimulation in PBLs and liver cells. The flagellin stimulation also resulted in the production of the flounder IL-1β and IL-6 from the liver cells and PBLs. The gene expression of TLR 5M was highly induced in the liver, while TLR 5S gene expression was drastically increased in the intestine following challenge with Edwardsiella tarda. Increased number of TLR 5M- and 5S-expressing cell populations were detected by in situ hybridization in the lamina propria of the intestine and liver after E. tarda infection, respectively. These results imply that the expression of these TLR 5 paralogs in Japanese flounder are differently regulated in the whole body and play important roles in the immune response against bacterial pathogens. [ABSTRACT FROM AUTHOR]

Olive flounder ( Paralichthys olivaceus ) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10 −5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV. [ABSTRACT FROM AUTHOR]