Toll-like receptors in fish: evolution, structure, immune signaling, and translational prospects for adjuvant-based vaccine design in aquaculture.

Toll-like receptors (TLRs) are central to innate immunity and the most studied class of pattern recognition receptors (PRRs). Upon recognition of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, fungi, and parasites, strong immune responses that bridge innate and adaptive immunity are initiated. Their ability to sense various ligands has made TLR agonists promising candidates for adjuvant development. Unlike traditional adjuvants, TLR ligands directly activate immune pathways, leading to strong, specific, and lasting protection. In aquaculture, there is a critical need for vaccines to reduce reliance on antimicrobials and to address losses from infectious diseases that threaten global food security. We compared TLRs across different teleost and higher vertebrate species for a comprehensive understanding of these receptors. This review focused on defining nomenclature, exploring its historical and evolutionary significance, and structural modeling using computational methods. Using channel catfish (Ictalurus punctatus) as a model, we depict two- and three-dimensional structures of various TLRs as well as three-dimensional ligand binding simulations of select catfish TLRs and their associated ligands. These in silico models reveal functional domains and interactions that help predict ligand recognition profiles, binding capacity, and downstream signaling potential. By highlighting the structural features that influence TLR-ligand interactions, our work offers a basis for choosing and improving TLR agonists as next-generation adjuvants in fish vaccines. These findings enhance our understanding of fish immunology and beyond, opening opportunities to develop safer and more effective disease-control techniques in aquaculture through a comparative immunological lens. Together, these findings provide a direct framework for selecting and optimizing TLR agonists, particularly flagellin, CpG ODNs, and poly I:C, as next-generation adjuvants in species-specific aquaculture vaccines.