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 (), 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 exhibit higher similarities with those in , and , 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 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.
