Therefore, we hypothesized that T. castaneum should also have Toll and IMD pathways with similar intracellular signal transduction systems as Drosophila. Based on this hypothesis, we

investigated the effects of MyD88 and IMD knockdown on the induction profiles of five representative AMP genes, Everolimus molecular weight three from group I and one each from group II and III, by the three microbes Ec, Ml and Sc. Since T. castaneum has nine Toll or Toll-related genes, we chose MyD88 as a target gene representative for the Toll pathway, closely related genes of which are not found in the genome. Similarly, we adopted IMD, which also does not have any other variants, as a target to repress the IMD pathway. Therefore, using MyD88 and IMD RNAi, we estimated the contributions of the two pathways in induction of the respective AMP genes. MyD88 RNAi weakened the induction of Def3 (group II) and Cec2 (group III) by both Ec and Ml at the two time points employed except for Def3 induction at 24 h post Ml challenge. IMD knockdown attenuated the induction of Att1, Col1, Def2 (group I) and Def3 (group II) by both Ec and Ml except for Def2 induction at 6 h post Ec challenge. Cec2 (group III) seemed to depend partly on IMD at 24 h post Ec or Ml challenge, but the extent was lower than the other four genes. As for the induction by Sc challenge, group I genes were

influenced by both PCI-32765 mw MyD88 and IMD knockdown except for Def2 induction at 6 h while group II and III genes were more affected by MyD88 knockdown. These tendencies of respective AMP genes in

terms of dependence on Toll (MyD88) or IMD pathway were more obvious at 24 h post microbial injection than at 6 h irrespective of the microbial species used. Given these results, we consider that in pupae of T. castaneum the induction of group I AMP genes is regulated mainly by the IMD pathway while that of group III gene is regulated mainly by the Toll pathway, and the group II gene by both pathways. The T. castaneum group I AMP genes exhibited an acute response to Ec, Ml and Sc while the response to Sc were C-X-C chemokine receptor type 7 (CXCR-7) weaker. The group III AMP genes showed a slow and sustained response to Ec, Ml and Sc, and the degrees of response elicited by the three microbes did not vary greatly. Moreover, our results suggested the dependence of group I genes on the IMD pathway and the dependence of group III genes on the Toll pathway. Thus, the group I genes and group III genes of T. castaneum may represent good parallels to frequently-used read-outs of Drosophila IMD pathway (Diptericin) and Toll pathway (Drosomycin) [14]. However, we should note here that these T. castaneum AMP genes were induced by both Ec and Ml to comparable levels. Our present results showed that seven of the nine AMP genes were induced by the five microbes although the induction by Sc was generally weaker. Whereas there were a few exceptions, IMD knockdown inhibited the induction of group I genes by Ec, Ml and Sc, and the induction of group II gene by Ec and Ml.