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Toll-like receptors (TLRs) play a vital role in the immune response by recognizing pathogen-associated molecular patterns (PAMPs) and triggering signaling pathways that activate innate immunity. In bony fish, TLR7 is essential for both antiviral and antibacterial defense; however, its interactions with a wide range of ligands and pathogens are still not well understood across various fish species. This study focuses on the identification and characterization of TLR7 in (LrTLR7) and aims to evaluate its response to pathogen challenges and stimulation by PAMPs. To clone the TLR7 gene, RNA was extracted from kidney tissue using a standard protocol, followed by cDNA synthesis with commercial kits. The TLR7 gene was amplified by PCR, and the gel-purified product was cloned into the pGEM-T Easy vector. DNA sequencing and BLAST analysis confirmed the identity of the LrTLR7 gene. The ORF of LrTLR7 cDNA was predicted using ORF-finder, while structural motifs in the encoded protein were identified through SMART. Phylogenetic relationships were analyzed using MEGA7 to construct evolutionary trees. Gene expression profiles of LrTLR7 were evaluated by quantitative real-time PCR (qRT-PCR) across developmental stages, tissues/organs of rohu fingerlings, and during challenges with and infections, as well as LPS and Poly I:C stimulation. Mucosal RBCs and PBLs were isolated using density-gradient centrifugation with HiSep™ LSM 1077 (Himedia, India). Cultured gill (LRG) cells in Leibovitz's L-15 medium were infected with or at a multiplicity of infection (MOI) of 1, following established protocols. LrTLR7 showed the closest phylogenetic affinity to TLR7 in . During embryonic development, LrTLR7 expression surged dramatically (~111-fold, <0.05) in embryos at 120 h post-fertilization (hpf). In juveniles, the gene was ubiquitously expressed across tissues/organs, with peak expression in gills (~2,000-fold). Following infection with or , LrTLR7 gene transcripts in the liver increased sharply at 6 hpi (~93-fold and ~53,000-fold, respectively). In the infected fish, mucosal RBCs showed a ~500,000-fold upregulation ( <0.05), while PBLs exhibited maximal responses at 24 hpi (~5,000-fold for and ~10 million-fold for ). In the LRG cell line, LrTLR7 gene expression rose ~30-fold by 3 hpi. during infection. stimulation with LPS or poly I:C triggered a ~30,000-fold increase in hepatic LrTLR7 expression at 12 h post-stimulation, with kidney tissue showing secondary activation. Mucosal RBCs and PBLs displayed rapid (1-3 h) LrTLR7 upregulation following ligand exposure. Imiquimod and gardiquimod activated LrTLR7-signalling pathways in both and systems, elevating transcription of IRF7 and type I interferon genes. Similar to higher vertebrates, LrTLR7 plays a crucial role in responding to pathogenic invasions and various PAMPs to induce innate immunity. Consequently, TLR7 in fish represents a significant target for immune activation using specific agonists or ligands, which could aid in the prevention of fish diseases.

Toll-like receptors (TLRs) are key pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) and elicit broadly acting innate immune responses. This study accomplished the identification, cloning, and sequencing of Labeo rohita TLR18 (LrTLR18). Structurally, LrTLR18 possesses a signal peptide, six leucine-rich repeats (LRRs), an LRR-C-terminal (LRR-CT) domain, a transmembrane (TM) region, and a Toll/IL-1 receptor (TIR) domain. Phylogenetically, LrTLR18 is evolutionarily closely related to the Schizothorax prenanti and Cyprinus carpio TLR18. The quantitative real-time PCR analysis revealed that LrTLR18 gene was expressed in all tested tissues, and among the tissues, the highest expression was observed in the eye followed by spleen, intestine, and gill in the descending order. During the ontogenic developments, the highest expression of LrTLR18 gene has been detected in the late blastula and gastrula stages. In response to the Aeromonas hydrophila and Edwardsiella tarda infections, LrTLR18 gene was differentially expressed in the blood, kidney, liver, and gills. Labeo rohita gills (LRG) cells in vitro infected with A. hydrophila and E. tarda , and red blood cells (RBCs) and peripheral blood leucocytes (PBLs) isolated from the rohu fingerlings infected with these pathogens revealed significantly ( p  < 0.05) enhanced expression of LrTLR18 gene. Following in vivo and in vitro stimulations with lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C), LrTLR18 gene expression was also significantly ( p  < 0.05) enhanced in various tissues, RBCs, PBLs, and macrophages. Together, these results highlight the important roles of the TLR18 against pathogenic invasions in fish.