Transcriptional Profiling of Midgut Immunity Figure 11. Cell proliferation in the midguts of larvae during the feeding and wandering stages. Green labeling indicates a BrdU- incorporating cell. In the normal feeding stage midgut (V-3: 12 h), very few cells incorporated BrdU (A, B). The midgut at 6 h after initiation of wandering (W: 6 h) had more dividing cells (C, D). At the end of wandering stage (W: 24 h), no cells in the midgut incorporated BrdU (E, F), indicating that cell division there stopped. However, hemocytes from the BrdU injected larvae (W: 24 h) were still stained positively (G, H). Images were taken using a green filter using a fluorescent microscope (A, C, E, G) or under DIC (Nomarski) filter (B, D, F, H). Control experiments performed without primary antibody (anti-BrdU) showed no staining (data not shown). Bars: 50 mm. doi:10.1371/journal.pone.0043769.g011 regulated. The down-regulation of these genes may induce the gradual loss of the normal function of the midgut. Some cells in the early wandering stage midgut incorporated BrdU for a brief period, after which no BrdU incorporation by the midgut cells was observed. On the other hand, the amount of apoptosis increased with time during the wandering stage. Therefore, the down- regulated metabolism and transportation, and the imbalance between cell division and cell apoptosis induce the degeneration of the silkworm midgut in the wandering stage. Materials and Methods Insect Feeding and Dissection B. mori larvae (Nistari) were reared on mulberry leaves at 25uC under a 12-h photoperiod. Nistari has a period of 36 h of wandering stage (from the initiation of wandering to the time before pupation). The adult butterfly Pachliopta aristolochiae (Fabricius) was kindly provided by Dr. Haisheng Yin. The times for different sampling of silkworms are shown in Fig. 2A. According to preliminary work, lysozyme, a very important immunity related protein, was found to be at maximum (protein and transcription levels) in the midgut at 24 h after the initiation of the wandering stage (W: 24 h). However, lysozyme already had a very high level of transcription at the beginning of the wandering stage (W: 0 h) but the protein was not visible until 6 h (W: 6 h) later (see Fig. 1G and Fig. 6B for the above information). In order to cover the genes transcription and protein expression changes as much as possible, 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), for dissection for the microarray. The time points for Western blot and qRT-PCR as shown in Fig. 2A were determined according to the preliminary work with lysozyme. To obtain the midgut, the silkworm larvae or moths and butterfly adults were dissected in autoclaved 0.85% NaCl after bleeding. The dissected tissues were washed in fresh 0.85% NaCl three times to remove the hemolymph. The silkworm midguts at the desired ages were dissected in the same way for qRT-PCR, and Western blot assays. Isolated midgut was then pulverized in liquid nitrogen and stored at 280uC in Trizol (Invitrogen, San Diego, USA). Oligonucleotide Microarray RNA isolation, amplification, labeling, hybridization, and microarray imaging and data analysis were performed according to the previously published papers [49,50]. The microarray, designed by the CapitalBio Corporation (Beijing, China), contains 23,022 probes, each 70 nucleotides (70-mer) in length, corre- sponding to the approximately 23,000 known and predicted B. mori genes [50]. Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions. Total RNA (5 mg) was used to prepare the fluorescent dye–labeled cDNA using cRNA Amplification and Labeling Kit (CapitalBio). The labeled cDNAs were dissolved in 80 ml of hybridization solution (36SSC, 0.2% SDS, 56Denhardt’s solution, 25% formamide), and hybridizations were performed in a hybridization chamber (BioMixer TM) overnight at 42uC. Slides were washed two times using washing buffer 1 (0.2% SDS, 26SSC) and 2 (26SSC) respectively at 42uC for 5 min. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software (CapitalBio). Each experimental group was repeated three times. Data were normalized by the LOWESS method. The filtered data were further examined to find genes that are differentially PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e43769