expressed between samples at two different stages using SAM software [51]. Significance was determined with q-value set at 1%, and ratio of at least 1.5 folds for the signal intensity between experimental sample and control. Gene ontology analysis was performed using Molecular Homological Description System 2.0 (MAS 2.0, http://www.capitalbio.com) [52]. The enzyme-cata- lyzed reactions were performed using the online pathway relationship database KEGG (http://www.genome.jp/kegg/) [49]. Immune Challenge V-3 silkworm larvae were injected with 56106 formalin-killed Escherichia coli cells suspended in sterilized 0.85% NaCl for immune challenge for 12 h [53]. Plasma samples were collected from the larvae to detect different immunity proteins by Western blot as positive controls, as previously described [53]. 20-Hydroxyecdysone (20-E) Injection Silkworm larvae on the 3rd day of fifth larval feeding stage (V-3) were injected with 5 mg 20-E (Santa Cruz, CA, USA) per larva [26]. The control larvae were injected with the same volume of solvent buffer. The silkworm larvae injected with 20-E or buffer and na¨ıve larvae were dissected for the midguts as described above for RNA extraction. Quantitative RT-PCR (qRT-PCR) Total RNA was extracted from midguts using Trizol reagent and then treated with RNase-free DNase I. mRNA in 3 mg of total RNA was transcribed into single strand cDNAs using a first strand cDNA synthesis kit (TOYOBO, Osaka, Japan), according to the manufacturer’s protocol. All specific primers were designed using the online Primer3 internet-based interface (http://biotools. umassmed.edu/bioapps/primer3_www.cgi) and are listed in Table S2. qRT-PCR reactions were performed in a 20 ml volume containing 10 ml of 26SYBR Green Master Mix (TOYOBO), 1 ml of cDNA, 1 ml of each primer (10 mM), and 7 ml of H2O. The PCR reaction was performed on a Bio-Rad CF696TM Real-time System using the following program: 95uC for 3 min, followed by 39 cycles of 95uC for 10 s, 55uC for 30 s, and 72uC for 10 s. Ribosomal protein S7 (rps 7) was used as an internal control. All the samples were measured independently three times. The relative transcription abundances (22DDCT) were calculated according to the equation of 22DCT, where DCT was calculated as follows: CT target gene-CT rps 7 [15]. GraphPad Prism software was used to produce figures. Columns represent the mean of individual measurements 6 SEM (n = 3). Significant differences were calculated with an unpaired t-test program by comparing 20- E injection with na¨ıve and buffer injection. SDS-PAGE and Western Blot Analysis Tissues were sonicated in 10 mM Tris-HCl (pH 7.4), and centrifuged at 10,0006g at 4uC for 5 min as previously described [53]. Approximately 10 mg supernatant protein was loaded per lane, and SDS-PAGE and Western blot assay were performed. Antibody against the silkworm lysozyme (a gift from Dr. K. References 1. Charroux B, Royet J (2010) Drosophila immune response: From systemic antimicrobial peptide production in fat body cells to local defense in the intestinal tract. Fly (Austin) 4: 40–47. 2. Nehme NT, Liegeois S, Kele B, Giammarinaro P, Pradel E, et al. (2007) A model of bacterial intestinal infections in Drosophila melanogaster. PLoS Pathog 3: e173. 3. Klein U, Koch A, Moffett DF (1996) Ion transport in Lepidoptera. Biology of the Insect Midgut: 236–259. Transcriptional Profiling of Midgut Immunity Suzuki; 1:5,000) [54], or M. sexta b-GRP2 (a gift from Dr. M. Kanost; 1:2,000) [41], or Mouse TAK1 (Santa Cruz, CA, USA; 1:1,000) was used as the primary antibody, and the AP-conjugated goat anti-rabbit IgG (1:5,000), or AP-conjugated goat anti-mouse IgG (1:5,000) was used as the secondary antibody [53]. In situ Apoptosis Detection: the TUNEL Method Silkworm midguts at different developmental stages were dissected as described above and fixed overnight at 4uC in Bouin’s fluid [53]. Samples were sectioned and deparaffinized as previously described [53]. After deparaffinization and rehydration, the midguts were stained using an In Situ Cell Death Detection kit, TMR Red (Roche, Basel, Switzerland), following the manufac- turer’s instructions and as previously described [49]. DAPI was used to counter-stain nuclei. All images were taken using a fluorescent microscope (Olympus BX51, Japan). BrdU Labeling and Detection Silkworm larvae at the desired age were weighed, anesthetized on ice, and then injected with 0.5 mg/g body weight of BrdU (Invitrogen, San Diego, USA), as previously described [55]. The BrdU-injected larvae were sacrificed to obtain their midguts three hours later. Circulating hemocytes were stained to show the positive signal. Midguts labeled with BrdU were fixed and sectioned and stained by a previously-described immuno-staining method [53]. An anti-BrdU (IgG1) monoclonal antibody produced in mouse (1:100; Invitrogen, San Diego, USA) was used as the primary antibody to detect BrdU-labeled cells for 1 h. Rhoda- mine-conjugated goat anti-mouse IgG1 (1:100; Santa Cruz, CA, USA) was used as the secondary antibody for another 1 h incubation. All images were taken using a fluorescent microscope (Olympus BX51). Histological Staining Insect midguts from different species and at different stages were fixed as described above. Sections (5 mm) were stained with 2% Mayer’s hematoxylin and 1% eosin as described [8]. Supporting Information Table S1 Genes identified by microarray analysis as having $1.5-fold higher expression (fold difference) in larvae at 12 h on 3rd day of 5th larval stage (V-3:12 h) than in the larvae at 3 h after the initiation of wandering (W:3 h) stage. (XLS) Primers for qRT-PCR analysis. Table S2 (XLS) Author Contributions Conceived and designed the experiments: EL QX AL. Performed the experiments: QX AL BY JZ XL. Analyzed the data: QX GX EL PZ CW. Contributed reagents/materials/analysis tools: JG QS. Wrote the paper: EL QX AL BTB. 4. Riddiford LM (1994) Cellular and Molecular Actions of Juvenile Hormone I. General Considerations and Premetamorphic Actions Advances in Insect Physiology 24: 213–274. 5. Riddiford LM, Cherbas P, Truman JW (2000) Ecdysone receptors and their biological actions. Vitam Horm 60: 1–73. 6. Truman JW, Riddiford LM (1999) The origins of insect metamorphosis. Nature 401: 447–452. PLOS ONE | www.plosone.org 13 August 2012 | Volume 7 | Issue 8 | e43769