RNA interference-based characterization of Caspar, DREDD and FADD genes in immune signaling pathways of the red fl our beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Abstract

We previously demonstrated that two immune signaling pathways, Toll and IMD, were concomitantly activated in themodel beetle Tribolium castaneum by challenges to their immune system by several species of microbes, including Gram-positiveand -negative bacteria as well as yeast. This contrasts with the Drosophila immune system in which more specifi c pathway activationdepending on the type of microbe is well established. We suggest that the activation of an indiscriminate immune pathway inT. castaneum is due in part to an unselective recognition of pathogen-associated molecular patterns by the extracellular sensingmodules of the two pathways. In order to obtain a more detailed understanding of the T. castaneum immune pathway, we investigatedwhether potential components of the T. castaneum IMD pathway, Caspar, DREDD and FADD, are involved in immune reactionstriggered by microbial challenges. A sequence analysis of these three genes with the orthologues of other species, includinginsects, mouse and human, indicated that T. castaneum Caspar, DREDD and FADD functioned as immune signal transducers,which are usually induced by microbial challenges. However, these genes were not induced by microbial challenges. To establishwhether these genes are involved in immune reactions, we used RNA interference-mediated knockdown of these genes to assessthe microbial induction levels of the representative read-out antimicrobial peptide genes of the respective classes. The resultsindicated that these genes encode the canonical constituents of the IMD pathway of this beetle. DREDD and FADD infl uencedthe induction of Toll-dependent antimicrobial peptide genes, providing novel crosstalk points between the two immune pathways,which appears to support indiscriminate pathway activation in T. castaneum. Furthermore, the phenotypes of DREDD or FADDknockdown pupae challenged by the two model bacterial pathogens correlated with AMP gene induction in the respective knockdowns,indicating that these intracellular factors contributed to antibacterial host defenses.