Transcriptional Profiling of Midgut Immunity TAK1 (antibody against mouse TAK1; 70% similarity to B. mori TAK1) were detected in the midgut during the wandering stage. Plasma (P) was from larvae injected with E. coli. For each lane, approximately 10 mg cell lysate was loaded. Bars: (B and E) 4 mm; (C and F): 50 mm. doi:10.1371/journal.pone.0043769.g001 a complex with it. However, if JHE is present, JHBP will help JHE to specifically detect the complex for subsequent JH hydrolysis [25]. Two JHBP genes were up-regulated on the wandering stage (Fig. 3A). When larvae on V-3 were injected with 20-E, JHEH1, 3- DE 3a-reductase 1, and 3-DE 3a-reductase 2 were all down- regulated at 24 h as compared to the na¨ıve and buffer injection (Fig. 3C–3E). However, ecdysone oxidase and JHBP1 were down- regulated at 12 h, but up-regulated at 24 h post-injection (Fig. 3F and 3G). JHBP2 was up-regulated at 12 h as compared to the na¨ıve and buffer injection (Fig. 3H). The EcR-USP (EcR: ecdysone receptor; USP: ultraspiracle protein) complex responds to the change of 20-E to initiate metamorphosis in insects [26]. In Tribolium castaneum, EcR and USP mediate midgut remodeling through a 20-E signal [27]. During the wandering stage, the transcription of three EcRs increased to their maximum, and then decreased within 36 h to almost the same level as during the feeding stage (Fig. 4A). The levels of the USP transcript showed a similar pattern (Fig. 4A). The changes in transcript levels of EcR and USP upon 20-E injection also showed a similar change tendency. They increased at different time (Fig. 4B–4E). This indicates that the increasing level of ecdysteroid in the hemolymph also regulates EcR and USP transcription in the midgut. Heat Shock Proteins The transcriptions of several heat shock proteins (HSPs) were up-regulated in the midgut during the wandering stage (Table S1). The qRT-PCR showed that the transcript levels of HSP 22.6, HSP 19.9, HSP 20.4, HSP 25.4, and HSP 70 were up-regulated in the midgut during development (Fig. 5A and 5B). HSP 75 is Figure 2. General statistics on the genes regulated between the feeding stage (V-3:12 h) and wandering stage (W:3 h). (A) The times of different sampling. We selected larvae at 12 h on day 3 of the fifth larval stage (V-3:12 h), or 3 h after the initiation of the wandering stage (W: 3 h), which were dissected for the microarray. The time points for Western blot and qRT-PCR were determined according to the preliminary work with lysozyme. (B) Volcano plots depicting estimated fold change (log2, X-axis) and statistical significance (2log10 P value, Y-axis). Each point represents a gene, and colors correspond to the range of negative log10 P and log2 fold-change values. (C) GO categories of differentially transcribed genes between the feeding and wandering stages. (D) The numbers of up- and down-regulated genes associated with various metabolic events and innate immunity. doi:10.1371/journal.pone.0043769.g002 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43769