Duplicated TLRs Possess Sub‐ and Neo‐Functionalization to Broaden Their Ligand Recognition in Crucian Carp ( <i>Carassius auratus</i> )

Toll-like receptors (TLRs) play a crucial role in the immune response to pathogen invasion. The TLR response patterns in teleost are significantly different from those in mammals. In this study, we systematically identified and characterized the TLR family of crucian carp (Carassius auratus). Phylogenetic analysis revealed that the CaTLR family consists of 25 members, divided into six subfamilies, and highlighted their homologous relationships with other species. mRNA expression analysis of TLRs demonstrated that most members exhibited distinct response patterns when challenged with different pathogens or pathogen ligands. Furthermore, we found that the duplicated CaTLR3 and CaTLR5 are capable of cross-sensing the dsRNA analogue poly (I: C) and bacterial flagellin, thereby activating the associated immune response. Additionally, we demonstrated that CaTLR3b, rather than CaTLR3a, functions as a homodimer to detect bacterial flagellin, and we identified the key flagellin binding site at S310 for CaTLR3b. Our findings suggest that the expansion of pathogen recognition patterns through sub- and neo-functionalization of duplicated TLR genes represents an evolutionary strategy for fish to effectively address various pathogens in aquatic environments.