Analysis of the Toll and spaetzle genes involved in Toll pathway-dependent antimicrobial gene induction in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebriodae)

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus activating the Toll pathway, which in turn induces AMP genes. Previous studies have shown the difference immune ststems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, 9 Toll and 7 spaetzle genes have been identified. To extend our understanding of AMP production by T. castaneum, we conducted functional assays of Toll and spaetzle genes related to Toll pathway-dependent AMP gene expression in T. castaneum under challenge with bacteria or budding yeast using RNA interference and qRT-PCR. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll pathway-dependent immune reactions, whereas spz7 plays a small part.