Disruption of TcGNBP3 via RNA interference compromises antibacterial immunity in Tribolium castaneum

Gram-negative bacteria binding proteins (GNBPs) serve as essential pattern recognition receptors in insect innate immunity, detecting pathogen-associated molecular patterns to activate downstream immune responses. This molecular recognition mechanism presents a promising target for pest control strategies. However, the immunological functions of GNBP family members in Tribolium castaneum remain poorly characterized, particularly for those with typical structural features. In this study, we identified and characterized a novel GNBP3 (designated TcGNBP3) from the T. castaneum cDNA library. Structural analysis revealed that TcGNBP3 exhibits the typical domain architecture characteristic of the GNBP family, comprising an N-terminal carbohydrate-binding module 39 (CBM39) domain and a C-terminal glycoside hydrolase family 16 (GH16) domain. Spatiotemporal expression profiling demonstrated peak TcGNBP3 transcript accumulation during the early pupal and late adult developmental stages, with predominant localization in immune-related tissues including the fat body and hemolymph. Bacterial challenges (Escherichia coli or Staphylococcus aureus) induced significant upregulation of TcGNBP3 expression from 6 to 72 h. Molecular docking and ELISA analyses demonstrated TcGNBP3's binding affinity for lipopolysaccharide, peptidoglycan, and β-1,3-glucan, while functional assays confirmed its ability to bind and agglutinate five tested bacterial strains. RNAi-mediated silencing of TcGNBP3 severely compromised the beetles' immune response, suppressing immune-related genes (including transcription factors and antimicrobial peptides), disrupting prophenoloxidase cascade activation, and significantly reducing survival rates upon bacterial infection. These results identify TcGNBP3 as a key immune regulator in T. castaneum, supporting its development as an RNAi-based pest control target.