Disruption of <i>TcGNBP3</i> via RNA interference compromises antibacterial immunity in <i>Tribolium castaneum</i>

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 <i>GNBP</i> family members in <i>Tribolium castaneum</i> remain poorly characterized, particularly for those with typical structural features. In this study, we identified and characterized a novel <i>GNBP3</i> (designated <i>TcGNBP3</i>) from the <i>T. castaneum</i> cDNA library. Structural analysis revealed that TcGNBP3 exhibits the typical domain architecture characteristic of the <i>GNBP</i> 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 <i>TcGNBP3</i> 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 (<i>Escherichia coli</i> or <i>Staphylococcus aureus</i>) induced significant upregulation of <i>TcGNBP3</i> 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 <i>TcGNBP3</i> 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 <i>TcGNBP3</i> as a key immune regulator in <i>T. castaneum</i>, supporting its development as an RNAi-based pest control target.