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  • Orange spotted grouper Epinephelus coioides is a


    Orange-spotted grouper (Epinephelus coioides) is a coral reef fish with a high commercial value in China and Southeast Asian countries. However, in recent years, various outbreaks of viral diseases have affected the grouper aquaculture industry (Wei et al., 2010). Specifically, Singapore grouper iridovirus (SGIV) infection causes spleen and liver hemorrhage and enlargement, resulting in more than 90% mortality in fish farm and challenge experiments (Qin et al., 2003). As chemokines and their receptors play important roles in inflammation, elucidation of the molecular link between SGIV and chemokine responses should help solve such problems in genetic enhancement programs. Previous studies reported two members of the CXC chemokine family (CXCL8 and CXCL12) and one receptor (CXCR4) in orange-spotted grouper (Hu et al., 2010; Wu et al., 2015; Lin et al., 2012), though systematic identification of orange-spotted grouper chemokines and their receptors has not been conducted, and the responses of these genes in SGIV infection have not been fully elucidated. Therefore, the objective of this work was to identify all possible members of the CXC chemokine subfamily and their receptors in orange-spotted grouper as well as their BX-912 mg in the spleen, liver and kidney after SGIV infection.
    Materials and methods
    Discussion Chemokines are a superfamily of structurally related chemotactic cytokines that serve as key regulators for cell migration and activation, especially under inflammatory conditions (Nomiyama et al., 2008; Zlotnik and Yoshie, 2000; Peatman and Liu, 2007; Chen et al., 2013; Fu et al., 2017a, 2017b). As a major subfamily, CXC chemokine ligands and their receptors play fundamental roles in development, homeostasis, cell migration, neurological development and immune responses (Walz et al., 1987; Yoshimura et al., 1987; Belmadani et al., 2006; Vandercappellen et al., 2008; Bai et al., 2009; Gordon et al., 2009; Alejo and Tafalla, 2011; Chen et al., 2013; Zou et al., 2015; Fu et al., 2017a, 2017b). Although previous studies have investigated CXC chemokine and CXC chemokine receptors in several teleost fish species, such as carp, catfish, rainbow trout, zebrafish, medaka and tetraodon, there is a lack of systematic analysis of CXC BX-912 mg chemokine ligands and their receptors in grouper species (Laing et al., 2002; Savan et al., 2003; Baoprasertkul et al., 2004; Nomiyama et al., 2008, 2011; Chen et al., 2010). In E. coioides, only two CXC chemokine ligands and one receptor were identified in a previous study (Hu et al., 2010; Wu et al., 2015; Lin et al., 2012), most of these sequences were not properly annotated due to the lack of genome information. Given the close interaction between CXC chemokine ligands and receptors, we included genes from all ligands and receptors to further elucidate their immune functions in orange-spotted grouper. In the present study, 9 CXC chemokine ligands and 8 CXC chemokine receptors were identified in E. coioides. As confirmation, we cloned and sequenced the genes and also conducted phylogenetic and syntenic analyses. We further determined their tissue distribution and expression profiles in response to SGIV infection. These results will be valuable for comparative immunological studies and provide insight into their roles in immune responses. Our phylogenetic and syntenic analyses offer explicit annotations for orange-spotted grouper CXC chemokine ligands and receptors, involving straightforward identification and determination of their evolutionary relationships. As shown in phylogenetic trees (Fig. 1, Fig. 2), all orange-spotted grouper CXCL and CXCR genes clustered with their respective counterparts from other teleosts; hence, our annotation of these genes was relatively forthright. In addition, syntenic analyses of CXCL2, CXCL12, CXCR1a, CXCR3 and CXCR4 provided further support for their orthology and evolutionary origins (Fig. 3). Interestingly, we identified a new CXC chemokine receptor in orange-spotted grouper, CXCR1b, and phylogenetic analysis showed that this gene clustered within the CXCR1 clade (Fig. 2). Two CXCR1 genes were found in salmon (Salmo salar) in addition to orange-spotted grouper (Grimholt et al., 2015). The presence of this rare chemokine receptor in orange-spotted grouper may be due to expansion of chemokine receptors. It has been proposed that the chemokine system appeared approximately 650 million years ago upon the emergence of vertebrates because no putative chemokines or chemokine receptors have been identified in any analyzed invertebrate species (DeVries et al., 2006). Moreover, analysis of vertebrate genomes (DeVries et al., 2006; Nomiyama et al., 2010, 2011; Chen et al., 2013) has revealed that chemokine receptors have been evolving rapidly through species-specific gene duplication. Indeed, CXCR1/R2-like genes have been described in several fish species, including common carp (Cyprinus carpio) (Fujiki et al., 1999), rainbow trout (Oncorhynchus mykiss) (Zhang et al., 2002), fugu (Fugu rubripes) (Saha et al., 2007), mandarin fish (Siniperca chuatsi) (Chen et al., 2009), zebrafish (Danio rerio) (Oehlers et al., 2010) and miiuy croaker (Miichthys miiuy) (Xu et al., 2013). The origin of teleost CXCR1/R2 loci is considered to be different from that of mammalian CXCR1/R2, which are highly identical in the same mammalian species, and it had been hypothesized that they were generated by gene duplication early in the amniote lineage (Nomiyama et al., 2013). Thus, the repertoire of chemokine receptors differs among species, even within the same lineage. Additionally, the existence of fish-specific CXCL and CXCR genes, as reported in several fish species such as zebrafish and catfish (Nomiyama et al., 2008; Chen et al., 2013; Fu et al., 2017a, 2017b), was observed in the present study. With minor exceptions, a set of 12 CXC chemokines, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCL10, CXCL15, CXCL16 and CXCL17, are only harbored by mammalian species and are absent in other species, whereas two chemokine ligands (CXCL12 and CXCL14) and receptors (CXCR3 and CXCR4) have been found in all analyzed species, suggesting their evolutionary conservation.