Transcriptional Profiling of Midgut Immunity Figure 3. Genes concerned with regulation of hormones were differentially transcribed in the midgut. (A, B) Transcriptional changes of two JHBP genes, two 3-DE 3a-reductase genes, ecdysone oxidase and JHEH1 were different during development. The two JHBP genes and ecdysone oxidase were up-regulated, but the two 3-DE 3a-reductase genes and JHEH1 were down-regulated in the midgut during the wandering stages. (C–H) Influence of 20-E injection on the transcription of the above genes. JHEH1 and 3-DE 3a-reductase were down-regulated in the midgut when the larvae were injected with 20-E. However, the remaining genes responded to 20-E inconsistently. *p,0.05; **p,0.001. doi:10.1371/journal.pone.0043769.g003 different; it had higher transcript levels during the feeding stage than during the wandering stage (Fig. 5B). When larvae (V-3) were injected with 20-E, the transcription of HSP 19.9, HSP 20.4, HSP 22.6, HSP 25.4, and HSP 70 significantly increased at 24 h post- injection (Fig. 5C–5G). However, 20-E negatively regulates HSP 75 transcription at 12 and 24 h as compared to the na¨ıve and buffer injection (Fig. 5H). HSPs are involved in protecting animals under various stresses and thermal injury and are involved in development [28,29]. HSPs are divided into five groups according to their molecular weights, HSP 100, HSP 90, HSP 70, HSP 60, and small HSP (sHSP) [30]. The molecular weights of sHSPs vary from 12 kDa to 42 kDa [30,31]. sHSPs connect with the cell nuclei, cytoskeleton, and membrane, and can also bind to denatured proteins as chaperonins, preventing irreversible coagulation under stress conditions [32]. In the wandering stage, many metabolism- and transport-related genes were down-regulated (Table S1). This may cause a stress in the midgut for normal physiological functions. The up-regulated transcription of sHSPs (HSP 19.9, HSP 20.4, HSP 22.6 and HSP 25.4) could represent a response by the midgut in an effort to maintain its integrity under this stress. Proteases Serine proteases (SPs) and serine protease homologs (SPHs) have many physiological functions, such as innate immunity, development, digestion, and signal transduction [33,34]. In the silkworm genome, there are 51 SP and 92 SPH genes [35]. The transcription of four SP genes (serine protease 54, trypsinogen-like protein, 35 kD protease and trypsin) and three SPH genes (30 kD protease A precursor, trypsin and trypsinogen-like protein) were down-regulated after entering the wandering stage (Table S1). In addition, 10 genes associated with protein digestion were down- regulated in the wandering stage (Table S1). One gene for protein proteolysis was up-regulated. However, in the prepupal midgut of Heliothis virescens, there are many hydrolytic enzymes [8]. In M. sexta, SPH1 and SPH2 have a very similar amino acid sequence to SP, but have no enzyme activity because of the loss of one or more catalytic residues [36,37]. However, SPH1 and SPH2 have innate immunity functions: M. sexta SPHs are necessary for prophenolox- idase (PPO) activation [34]. The exact physiological functions of SP and SPH in the midgut deserve further study. PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43769