Full length article Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene Omkar Byadgi a , Dinda Puteri a, b , Yan-Horn Lee c , Jai-Wei Lee a , Ta-Chih Cheng a, * a Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan b Department of Fisheries and Marine Science, University of Brawijaya, Malang, Indonesia c Tungkang Biotechnology Research Center, Fisheries Research Institute, Council of Agriculture, Pingtung, Taiwan article info Article history: Received 19 October 2013 Received in revised form 13 December 2013 Accepted 19 December 2013 Available online 27 December 2013 Keywords: Innate immunity Rachycentron canadum Toll-like receptor 9 Photobacterium damselae subsp. piscicida CpG ODNs abstract Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P . damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240e253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P . damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was w3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1b, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P . damselae subsp. piscicida infection and use as an adjuvant in vaccine development. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The Toll-like receptors (TLRs) are used to distinguish self and nonself molecules in innate immunity, which is the first line of defense in vertebrate immune systems. The Toll-like receptor 9 (TLR9) is the receptor for bacterial CpG oligodeoxynucleotides (ODN). The binding between TLR9 and ODN can activate innate immune inflammatory response followed by adaptive immune response to eliminate the invaded bacteria. Although the structures are highly conserved in vertebrates, TLRs recognize the pathogen- associated molecular pattern (PAMPs), which are also highly conserved among different microorganisms. The PAMPs include lipoprotein, dsRNA, flagellin, and unmethylated CpG ODN of microorganism. Therefore, broad microorganisms can be recog- nized by only limited type of TLRs followed by the activation of anti-microbial genes and pro-inflammatory cytokines [1]. Recog- nitions between fish TLRs and their ligands are similar to the rec- ognitions of that in mammals due to the TLRs extra-cellular structures. For example, leucine-rich repeats (LRRs) domains are conserved from fish to mammals during evolution [2e4]. In fish, initiations of signal transduction events inside cytoplasm are similar to those in mammals as TLRs contain highly conserved intracellular domains of Toll/Interleukin 1 receptor (TIR) [5,6]. Signal transfer across the cell membrane to intracellular domain occurs when ligand is recognized by extra cellular domain of TLR with the aid of LRR domain. This event triggers a series of signal transduction pathways in either myeloid differentiation factor 88 (MyD88) dependent or independent pathways to activate the pro- inflammatory cytokines and chemokines genes in the nuclear sub section. Meanwhile, phagocytes migrate to lymphnode to modulate naïve CD4þ T cell differentiate mainly into T helper 1 (Th1) cells and produce Th1 type cytokines, which are important for intracellular bacterial pathogen elimination [7e9]. Therefore, TLRs ligands * Corresponding author. Tel.: þ886 8 7703202x6423; fax: þ886 8 7740446. E-mail address: cheng.tachih@gmail.com (T.-C. Cheng). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.12.017 Fish & Shellfish Immunology 36 (2014) 417e427