Transcriptional Profiling of Midgut Immunity Figure 4. Ecdysone receptors (EcR) and ultraspiracle (USP) proteins are under the control of 20-E. (A) EcR and USP genes were quickly up-regulated when entering the wandering stage. The transcription levels of these genes were low during the feeding stage and the end of the wandering stage. (B–E) 20-E injection induced the transcription of EcR1, EcR3, and USP during the first 12 h compared with naive or buffer injection. EcR2 was up-regulated at 24 h after 20-E injection. *p,0.05; **p,0.001. doi:10.1371/journal.pone.0043769.g004 Immunity Twenty-five genes related to immunity were differentially regulated in the wandering stage (Fig. 2D). They comprise genes encoding five pattern recognition receptors (PRR), eight antimi- crobial peptides (AMP), nine genes belonging to Toll or Imd pathways, and three genes whose protein products probably regulate prophenoloxidase activation. CTL10 and CTL21 that belong to C-type lectins and have humoral and cellular immunity functions [34,38] were significantly up-regulated in the wandering stage. Before pupation, their transcript levels decreased (Fig. 6A). During the wandering stage, the transcription of the lysozyme gene was up-regulated (Fig. 6B). Lysozyme was also detected in the midgut of the wandering stage but not in the feeding stage (Fig. 1G), which is consistent with the previous report in M. sexta midgut [39] and its transcriptional profiling. PGRP-L2 and PGRP-S6 take part in prophenoloxidase activation or antimicro- bial peptide production, according to studies in Drosophila [40]. Beta-1,3-glucan recognition protein 2 (bGRP2) increased in both mRNA and protein levels (Fig. 6A and Fig. 1G). bGRP can specifically bind to bacterial glycan to trigger the PPO activation pathway [41]. Eight AMP genes were significantly up-regulated during the wandering stage (Table S1 and Fig. 2D). Further analysis of the transcription of these AMPs during development confirmed that these AMPs were significantly up-regulated after the insects entered the wandering stage (Fig. 6B–6D). Defensin increased almost 10,000-fold 24 h after the initiation of the wandering stage PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43769