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The ORA (olfactory receptor class A) gene family in teleosts is related to the V1R (vomeronasal 1 receptors) family in mammals and plays a key role in odor detection. Although ORA genes have been identified in several teleosts, their characteristics in mandarin fish (Siniperca chuatsi) have not been explored. In this study, we conducted a comprehensive genomic analysis of the mandarin fish and discovered a complete ORA gene family consisting of five members located on chromosome 2 (ORA1, ORA2, ORA3, ORA4) and chromosome 16 (ORA6). Phylogenetic, synteny, and gene structure analyses revealed typical exon–intron conservation with strong evidence of purifying selection. Tissue expression analysis showed distinct expression profiles for each ORA gene, with some showing sexual dimorphism in specific tissues. The expression of ORA1 and ORA2 in the olfactory epithelium exhibits sexual dimorphism, while ORA3 shows sexual dimorphism in the brain. In situ hybridization confirmed that ORA1, ORA2, ORA3, and ORA6 are expressed in the microvillar sensory neurons of the olfactory epithelium, while ORA4 is expressed in crypt cells. Additionally, molecular docking simulations indicated that the five ORA proteins have a high binding affinity with seven bile acids (LAC, GLAC, CA, TLCA, 3-KLCA, 7-KLCA, and 12-KLCA), with ORAs showing stronger binding affinity with LCA and CA. This study comprehensively characterizes the ORA gene family in mandarin fish, examining its phylogeny, synteny, gene structure, and selection pressure. Furthermore, we found that each ORA displays a distinct expression pattern across multiple tissues, with notable sexual dimorphism, and shows potential binding interactions with specific bile acids and pheromones. Our findings provide valuable insights that enhance the overall understanding of fish ORAs and their potential functions.

Toll-like receptors (TLRs) play a crucial role in the recognition of microbial-associated molecular patterns in the innate immune system. Fish TLRs have undergone significant gene expansion to adapt to complex aquatic environments. Among them, TLR20 from the TLR11 family actively responds to viral and bacterial invasions. Previous studies have reported two TLR20s in grass carp (Ctenopharyngodon idella), and in this study, we revised this conclusion. Based on the latest grass carp genome, we identified a new TLR20 member. These three TLR20s are arranged in tandem on chromosome 9, indicating that they are generated by gene duplication events. They were renamed CiTLR20.1 to CiTLR20.3 based on their chromosomal positions. The CiTLR20s in C. idella exhibit higher similarities with those in Danio rerio, Cyprinus carpio, and Megalobrama amblycephala, and lower similarities with those in other distantly related fish species. Selective pressure analysis revealed low conservation and negative evolution of TLR20s during evolution. The 3D structures of the three TLR20s showed significant differences, reflecting functional variations and different downstream adaptor molecule recruitment. Transcriptome data revealed tissue distribution differences of TLR20s, with TLR20.1 showing relatively low expression levels in all the tissues, while TLR20.2 and TLR20.3 showed higher expression in the head kidney, spleen, and gill. Additionally, TLR20.2 and TLR20.3 actively responded to GCRV-II infection, with higher upregulation of TLR20.2 in response to Aeromonas hydrophila challenge. In conclusion, this study corrected the number of grass carp TLR20 members and analyzed TLR20 from an evolutionary and structural perspective, exploring its role in antiviral and antibacterial defense. This study provides reference for future research on fish TLR20.