Molecular Characterization and Immune Role of TLR7 in Labeo rohita

Saswati Pani Bristy Ganguly Smruti Mahapatra Smruti prajnya Dash RAKESH DAS Ashis Saha Mrinal Samanta ^*

• Central Institute of Freshwater Aquaculture (ICAR), Bhubaneswar, India

TLR7 (Toll-like receptor 7) in Labeo rohita (LrTLR7) displays the characteristic domain organization typical of TLRs and is most closely related phylogenetically to TLR7 in Cyprinus carpio. During early developmental stages, LrTLR7 gene expression is significantly elevated (~ 111-fold, p<0.05) in spawns at 120 hrs post-fertilization. In the juvenile L.rohita, this gene is broadly expressed across various organs and tissues, with the highest expression observed in gill tissues, reaching ~ 2,000-fold. Infection of L. rohita with Aeromonas hydrophila or Edwardsiella tarda leads to a notable increase in LrTLR7 gene expression in the liver, with enhancements of ~ 93-fold and ~53000-fold respectively at 6 hrs post-infection (h.p.i). Furthermore, LrTLR7 gene expression in the mucosal red blood cells (RBCs) also revealed a significant (p<0.05) increase (~500,000-fold) at the same time point following infections with these two pathogens. In the peripheral blood leukocytes (PBLs), the highest induction of LrTLR7 gene expression occurs at 24 h.p.i, showing an increase of ~ 5,000-fold for A. hydrophila and ~10 million-fold for E. tarda. In the L. rohita gill (LRG) cell line, during the pathogenesis of A. hydrophila, LrTLR7 gene expression rises significantly (~30-fold) at 2.30 h.p.i. In-vivo stimulation of juvenile fish with LPS or poly I:C results in a substantial increase (~30,000-fold) in LrTLR7 gene expression in the liver followed by kidney at 12h poststimulation. In the mucosal RBCs and PBLs, significant enhancements of LrTLR7 gene expression occurs within 1-3h after in-vitro stimulation with these ligands. Imiquimod and gardiquimod stimulation activates LrTLR7 signalling pathways both in-vivo and in-vitro, leading to increased expression of IRF7 and Type-I interferon genes. These findings together underscore the crucial immune function of TLR7 in Labeo rohita.