Induction of group I genes, Att1, Att2, Col1 and Def2 by Ec and Ml were acute and very strong; induction by Ec persisted while that by Ml declined

more rapidly. Def3 (group II) induction by Ec and Ml was also acute as in the cases of group I genes while the degrees of induction was relatively modest in comparison with group I genes. The induction kinetics of group III genes Cec2 and Cec3 were slow and sustained, and the degree of induction by the three microbes was weak or moderate and did not vary as greatly as for group I and II genes. Group IV genes Def1 and Att3 mRNAs were not or very weakly induced by the three microbes. Shrestha and Kim conducted similar AMP gene induction and IMD or Toll knockdown studies using the nine T. castaneum AMP genes as well as four lysozyme genes [40]. Based on the gel analyses of RT-PCR products, these authors mentioned: Cec2, Att3, Def3 and Col1 genes were induced Tyrosine Kinase Inhibitor Library concentration by gram-negative bacteria, but not by gram-positive bacteria; Def1 was induced only by gram-positive bacteria; the other four AMP genes, Cec3, Att1, Att2 and Def2 Doxorubicin nmr were induced by both gram-positive and gram-negative bacteria. As for knockdown experiments targeting IMD or four Toll gene variants, these authors reported: the induction of four

AMP genes that responded only to gram-negative bacteria was inhibited by IMD knockdown; Def1 induction that was induced only by gram-positive bacteria was abolished by knocking down any of four O-methylated flavonoid Toll variants; the induction of remaining four AMP genes that responded to both gram-negative and gram-positive bacteria was insensitive to any of dsRNA treatments. These authors used Ec and Xenorhabdus nematophila as gram-negative bacteria, and Bs and Flavobacterium sp. as gram-positive bacteria, but actually, Flavobacterium is a gram-negative bacterium and Bs possesses DAP-type PG. Thus, elicitors that they used were gram-negative bacteria or a gram-positive bacterium bearing DAP-type PG. There are also several differences between their and our experimental conditions. They used fully grown late instar larvae reared at room temperature while we used pupae that were reared at 30 °C. Incubation time period after microbial challenge

also differed. They used 8 h incubation while we incubated for 6 and 24 h. The most critical differences could be the measurement methods for determining mRNA amounts. We employed qRT-PCR, whereas they used end-point RT-PCR with 35 thermal cycles followed by gel electrophoresis. RT-PCR/gel analysis is simple and easily available but may not supply high-precision quantitative data. Therefore, it may be difficult to compare directly their and our results. As for AMP gene induction by Ec, our results are consistent with their results except that they observed Att3 induction. However, while our results indicate that Ec and Bs showed similar properties as elicitors probably because of their possession of DAP-type PG, they only found the induction of Def1, Att1 and Def2 by Bs.