Functional characterization of TmToll-6 in mediating innate immune responses to systemic microbial infections in Tenebrio molitor.

Toll-like receptors (TLRs) are evolutionarily conserved regulators of innate immunity and extensively characterized in mammals and Drosophila melanogaster; however, their functional roles in other insects remain incompletely defined. In this study, we investigated the immunological function of TmToll-6 in Tenebrio molitor larvae under systemic pathogenic bacterial and fungal challenge. Transcriptional analyses revealed that its expression was strongly induced in the fat body, gut, Malpighian tubules, and hemolymph following exposure to Escherichia coli, Staphylococcus aureus, and Candida albicans. The RNA interference-mediated silencing of TmToll-6 significantly increased larval mortality after E. coli and S. aureus infection, highlighting its essential role in antibacterial defense. Mechanistic evaluation further demonstrated that TmToll-6 knockdown markedly suppressed the antimicrobial peptide (AMP) gene expression, particularly in the fat body and gut, which are the main effector tissues of insect humoral immunity. Although TmToll-6 was strongly induced following C. albicans infection, its silencing produced comparatively limited effects on AMP expression, indicating that antifungal AMP regulation is less dependent on TmToll-6 and may involve partially redundant immune pathways. Therefore, TmToll-6 could function as a pivotal immune regulator in T. molitor through Toll pathway-dependent AMP regulation in a tissue- and pathogen-specific manner. This study highlighted the evolutionary diversification of Toll receptors in coleopteran immunity.