Simple SummaryThe red flour beetle,Tribolium castaneum, is a severe pest that infests stored agricultural products and serves as a widely used model insect for molecular study. While transforming growth factor-β (TGF-β) has been extensively studied in vertebrates, its role in insects remains to be thoroughly investigated. In this study, we report our findings on the impact of TGF-β type II receptor Punt on the antimicrobial peptide (AMP) expression inT. castaneum.PuntRNAi in the larvae led to increased transcript levels of AMP genes via a transcription factor Relish in the IMD pathway, leading to inhibition ofEscherichia coliproliferation. Knockdown ofPuntin larvae also resulted in the splitting of elytra and abnormal compound eye development in adults. WhenPuntwas knocked down during the female pupal stage, it not only increased transcript levels of AMP genes but also had adverse effects on ovarian development, leading to a decrease in fecundity and failure of egg hatching.AbstractThe transforming growth factor-β (TGF-β) superfamily in insects regulated various physiological events, including immune response, growth and development, and metamorphosis. This complex network of signaling pathways involves conserved cell-surface receptors and signaling co-receptors that allow for precisely coordinated cellular events. However, the roles of TGF-β receptors, particularly the type II receptor Punt, in mediating the innate immunity in insects remains unclear. In this study, we used the red flour beetle,Tribolium castaneum, as a model species to investigate the role of TGF-β type II receptor Punt in mediating antimicrobial peptide (AMP) expression. Developmental and tissue-specific transcript profiles revealedPuntwas constitutively expressed throughout development, with the highest transcript level in 1-day female pupae and the lowest transcript level in 18-day larvae. Tissue specific expression profiles showed the highest transcript level ofPuntwas observed in the Malpighian tubule and ovary in 18-day larvae and 1-day female adults, respectively, suggesting Punt might have distinct functions in larvae and adults. Further results indicated thatPuntRNAi in the 18-day larvae led to increased transcript level of AMP genes through transcription factor Relish, leading to inhibition ofEscherichia coliproliferation. Knockdown ofPuntin larvae also led to splitting of adult elytra and abnormal compound eyes. Furthermore, knockdown ofPuntduring the female pupal stage resulted in increased transcript levels of AMP genes, as well as abnormal ovary, reduced fecundity, and failure of eggs to hatch. This study deepens our understanding of the biological significance of Punt in insect TGF-β signaling and lays the groundwork for further research of its role in insect immune response, development, and reproduction.