The fold induction values of Att2 24 h after microbial injection are seemingly high because of the lower basal expression levels found in unchallenged IOX1 concentration animals at 24 h than at 6 h. This is also the case for Col1 ( Fig. 1K and L), Def2 ( Fig. 1O and P) and Def3 ( Fig. 1Q and R). Contrary to the other Atts, the amount of Att3 mRNA did not seem to be changed by Ec, Ml and

Sc ( Fig. 1E and F). Contrasting with the acute responses of Att1 and Att2, Cec2 showed slower and sustained kinetics of induction for the three microbes ( Fig. 1G and H). Ec and Sc seemed to be more effective than Ml in inducing Cec2, but, for all of these microbe treatments, the induced levels of mRNA were relatively low and find more the induction was weak or modest. The kinetics of Cec3 induction was similar to that of Cec2 whereas the basal level of expression was higher, which makes apparent induction degrees more moderate ( Fig. 1I and J). Col1 mRNA was most abundant among the nine AMP mRNAs when pupae were challenged with the microbes ( Fig. 1K and L). It showed an acute response and the profile was similar to those of Att1 and Att2. Induction of Def1 mRNA by the microbes at 6 h was shown to be negligible ( Fig. 1M), and challenge with

Ml or Sc exhibited very weak induction at 24 h ( Fig. 1N). Def2 showed a robust and acute response as in the cases of Att1, Att2 and Col1 ( Fig. 1O and P). The induction profiles of Def3 mRNA are shown in Fig. 1(Q and R). It showed a similar tendency of induction to Att1, Att2, Col1 and Def2 whereas the response to the microbes was appreciably weaker than those found for the four genes. We also observed weak or very weak induction with around 3-fold for Att2, Col1 and Def2 after Parvulin the control PBS injection. These reactions were acute and may reflect the local responses caused by injury. In addition to these non-pathogenic model microbes, we also tested the other two bacteria that showed some pathogenicity to T. castaneum. These are Ecl (gram-negative) and Bs (gram-positive),

the latter of which as well has DAP-type PG. These two bacterial species were used as well in the survival assays ( Fig. 4). The results of AMP gene induction by these two bacteria are included in Fig. 1(A–R). Overall, these two bacteria reasonably elicited similar responses to the cases by Ec since these three bacteria possess DAP-type PG. However, Ecl and Bs challenge resulted in more prolonged responses than Ec challenge. This phenomenon was more obvious in Ecl and seemed to be associated with the degrees of pathogenicity of respective bacteria, which might determine the time point of termination of humoral immune responses. Based on the induction profiles described above, we categorized the nine AMP genes into four groups (Table 3). Group I contains the AMP genes that showed acute and very strong induction by Ec.