Genome-wide identification, expression analysis of toll-like receptors (TLRs) in black carp (Mylopharyngodon piceus), and the positive role of MpTLR1 against Aeromonas hydrophila infection

As one of the world's “staple” freshwater fish species, black carp ( Mylopharyngodon piceus ) faces critical challenges from bacterial diseases. Toll-like receptors (TLRs), serving as frontline pathogen sensors, play crucial roles in antibacterial defense. This study systematically identified 21 TLR members through genomic analysis, revealing their uneven distribution across 11 chromosomes with evolutionarily conserved synteny in cyprinids. Phylogenetic reconstruction, sequence, and selection pressure assessment demonstrated that TLR subfamilies maintain structural and sequence conservation and undergo strong purifying selection. Tissue-specific transcriptome profiling showed broad TLR expression patterns, with several members exhibiting significant differential expression in Aeromonas hydrophila -resistant populations. Functional validation focused on the most prominently altered MpTLR1 revealed that its knockdown increased infection-induced mortality and exacerbated histopathological damage. Mechanistic investigations demonstrated that MpTLR1 suppression significantly impaired antioxidant enzyme activities (antioxidant capacity and superoxide dismutase), complement C3 levels and lysozyme activity, while downregulating mRNA expression levels of pro-inflammatory cytokines ( TNF-α , IFN-γ2 , and IL-6 ), complement C3, and antimicrobial peptides (hepcidin and liver-expressed antimicrobial peptide 2). Our work provides a systematic characterization of black carp TLRs and establishes MpTLR1's pivotal role in bacterial immunity through coordinating antioxidant defense and inflammatory responses. These findings offer insights for breeding A . hydrophila -resistant black carp populations. • 21 TLR genes were systematically annotated in Mylopharyngodon piceus genome. • TLRs undergo strong purifying selection and are highly conserved in evolution. • MpTLR1 knockdown reduces resistance to A. hydrophila infection in M. piceus individuals.