Molecular characterization and expression analysis of four toll-like receptors genes: TLR3, TLR4, TLR5M and TLR18 in Spinibarbu hollandi under Aeromonas veronii infection.

Abstract

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) central to innate immunity. In teleost fish, TLRs play a pivotal role in defending against diverse microbial invaders, yet their evolutionary adaptation and functional specialization in Spinibarbus hollandi (army fish) remain uncharacterized. This study reports the first molecular cloning and functional analysis of TLR3, TLR4, TLR5M, and TLR18 in S. hollandi. All four TLRs possess conserved intracellular Toll/IL-1 receptor (TIR) domains and extracellular leucine-rich repeat (LRR) domains. Basal expression profiling reveals distinct tissue specificity: TLR3 and TLR5M are highly expressed in the liver, TLR4 dominates in the brain and eye, and TLR18 shows minimal constitutive expression. After Aeromonas veronii challenge, liver TLR5M is most dramatically upregulated and acts as an early-response core gene. Phylogenetic analysis confirms high homology between S. hollandi TLRs and cyprinid orthologs. Molecular evolutionary analysis identifies positive selection sites in the LRR domains of TLR3 and TLR5M, indicative of adaptive optimization for pathogen recognition. These findings clarify TLR-mediated bacterial immunity in S. hollandi and provide candidate targets for aquaculture disease resistance breeding.