Expression of nitric oxide synthase (NOS) genes in channel catfish is highly regulated and time dependent after bacterial challenges Jun Yao, Chao Li, Jiaren Zhang, Shikai Liu, Jianbin Feng, Ruijia Wang, Yun Li, Chen Jiang, Lin Song, Ailu Chen, Zhanjiang Liu ⇑ The Fish Molecular Genetics and Biotechnology Laboratory, Aquatic Genomics Unit, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA article info Article history: Received 4 December 2013 Revised 8 February 2014 Accepted 11 February 2014 Available online 18 February 2014 Keywords: Fish NOS Immune response Disease Nitric oxide synthase Genome abstract Nitric oxide is well known for its roles in immune responses. As such, its synthesizing enzymes have been extensively studied from various species including some teleost fish species. However, the NOS genes have not been characterized in channel catfish (Ictalurus punctatus). In this study, we identified and char- acterized three NOS genes including one NOS1 and two NOS2 genes in channel catfish. Comparing with the NOS genes from other fish species, the catfish NOS genes are highly conserved in their structural fea- tures. Phylogenetic and syntenic analyses allowed determination of NOS1 and NOS2 genes of channel cat- fish and their orthology relationships. Syntenic analysis, as well as the phylogenetic analysis, indicated that the two NOS2 genes of catfish were lineage-specific duplication. The NOS genes were broadly expressed in most tested tissues, with NOS1 being expressed at the highest levels in the brain, NOS2b1 highly expressed in the skin and gill, and NOS2b2 lowly expressed in most of the tested tissues. The most striking findings of this study was that the expression of the NOS genes are highly regulated after bacte- rial infection, with time-dependent expression patterns that parallel the migration of macrophages. After Edwardsiella ictaluri challenge, dramatically different responses among the three NOS genes were observed. NOS1 was only significantly in the skin early after infection, while NOS2b1 was rapidly upreg- ulated in gill, but more up-regulated in trunk kidney with the progression of the disease, suggesting such differences in gene expression may be reflective of the migration of macrophages among various tissues of the infected fish. In contrast to NOS1 and NOS2b1, NOS2b2 was normally expressed at very low levels, but it is induced in the brain and liver while significantly down-regulated in most other tissues. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Nitric oxide (NO), as one of the smallest molecular mediators (30 Da), is known to play essential roles in a variety of immune processes ranging from innate immunity to acquired immunity (Bogdan, 2001; Clark and Rockett, 1996; Moncada et al., 1989; Nathan, 1992). During these processes, nitric oxide could be pro- duced by many immune related cells, including macrophages, monocytes, neutrophils, dendritic cells, natural killer (NK) cells, mast cells, endothelial cells, epithelial cells, vascular smooth mus- cle cells, hepatocytes, and chondrocytes (Aktan, 2004; Bogdan, 1998; MacMicking et al., 1997; Tripathi, 2007). Nitric oxide is a po- tent host defence effector in immune system with antimicrobial activity (Coleman, 2001; Nathan, 1992; Nussler et al., 1993), how- ever, the overproduction of NO can lead to cytotoxic effect including host tissue or cell damages (Aktan, 2004; Alderton et al., 2001; Bogdan, 2001). Nitric oxide is generated by nitric oxide synthase (NOS) using L- arginine as the substrate. In mammals, three NOS genes have been identified and named following the cell types from which they were characterized: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3) (Alder- ton et al., 2001; Bogdan, 1998). Among these three NOS genes, NOS1 and NOS3 are mainly expressed in brain and endothelial cells, respectively, while NOS2 is induced in a variety of immune cells, particularlly in macrophages and epithelial cells. In contrast to the situation in mammals, only NOS1 and NOS2 genes have been identified in teleost fish; NOS3 is absent in all fish whose genome sequences are released (Lepiller et al., 2009; Øyan et al., 2000). NOS1 and NOS2 genes have been characterized in a number of fish species including goldfish (Laing et al., 1996), carp (Saeij et al., 2000), rainbow trout (Laing et al., 1999; Wang et al., 2001), zebrafish (Holmqvist et al., 2000; Lepiller et al., 2009; Poon et al., 2003), red drum (Zhou et al., 2009), killifish (Hyndman et al., http://dx.doi.org/10.1016/j.dci.2014.02.005 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +1 334 844 4054; fax: +1 334 844 9208. E-mail address: liuzhan@auburn.edu (Z. Liu). Developmental and Comparative Immunology 45 (2014) 74–86 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci