spectrophotometry and subjected to 1.0% formaldehyde dena- tured agarose gel electrophoresis. Then, samples were precipitated in 100% ethanol and sent to CapitalBio Corp for microarray analysis or stored at 280uC for further analysis. Microarray Hybridization and Original Data Normalization. Gene expression analysis was performed using the Affymetrix Silkworm GeneChip kit according to instructions in the Affymetrix GeneChip expression manual. The microarray hybridization and data normalization analysis were performed by CapitalBio Corp [19]. Procedures were performed as described in detail on the website of CapitalBio (http://www.capitalbio.com). Briefly, total RNA was purified using NucleoSpinH RNA clean-up kit (MACHEREY-NAGEL, Germany). Then, formaldehyde dena- turing gel electrophoresis was used to detect the RNA quality. The cDNA targets were prepared from 5 mg of total RNA and were labeled with a fluorescent dye (Cy5 and Cy3-dCTP). Analyses were performed twice per sample, using a dyereversal procedure in which cDNA from the control was labeled with Cy3 and cDNA from Bb induced was labeled with Cy5. In the second analysis, control cDNA was labeled with Cy5 and cDNA from Bb induced was labeled with Cy3. This dye reversal helps to minimize error due to fluor-associated bias. Labeled cDNA were hybridized to the 23k silkworm genome oligonucleotide chip (CapitalBio), which has 22,987 oligonucleotide 70-mer probes. Chips were scanned using a Lux-Scan 10KA dual pathways laser scanner (CapitalBio), and images were analyzed by LuxScan3.0 image analysis software. At least two independent replicates were performed. Data Analysis The microarray data of multiple tissues expression of day 3 of the fifth instar were downloaded from the silkworm genome database (http://www.silkdb.org/microarray/download.html) (10,393 active transcripts) [excel]. The expression data of each gene in each tissue were averaged form four to six repeats. For each gene, if its averaged expression signal was more than 400, it was considered having expression. Genes only showed expression in one tissue or more than 10 times than other tissues were considered as tissues specific genes. The transcript values of infected larvae were subtracted from those of unchallenged control larvae to account for the development-regulated genes in the further steps. The data from the independent experiments were then averaged. Transcripts were selected when they displayed at least a 2-fold change in expression level compared with control larvae. The induced gene ontology analysis was predicted using the online molecule annotation system of CapitalBio Corp (http://www.capitalbio. com/zh-hans/support/MAS). The typical enzyme-catalyzed re- actions were predicted using the online pathway relationship database KEGG (http://www.genome.jp/kegg/). All the data used reporting this report is presented in Table S2. Real Time PCR Confirmation of Microarray Data Seven pairs of primers were designed to confirm microarray data. The primers sequences are listed in additional file 5. The real time RT-PCR confirmation results were performed using the SYBR Premix Ex Taq kit (TaKaRa, China) and each reaction was prepared in 25 ml containing 70 ng cDNA (2 m1), SYBR Premix Ex Taq 12.5 m1, 10 mM each of sense and anti-sense primers 0.5 m1. After 40 cycles of amplification, the results were read by ABI Prism 7000 Sequence Detection System (Applied Biosystems). The real time RT-PCR was performed in duplicate for at least three biological replicates. For internal standardization primers, sw22934 (transcription initiation factor 2 gene) was used [116]. B. mori Host Response to Bb For each pair of primers, cDNA samples of four time points of induced and non-induced were performed. Gene Identification Genes were identified using the proteins of SilkDB (http:// silkworm.swu.edu.cn/silkdb/) to blast homologs in NCBI database (http://www.ncbi.nlm.nih.gov/) and PFAM domain database (http://pfam.sanger.ac.uk/search). E-values less than 1e-5 were used. For most silkworm immune related genes used in this study, we referenced Tanaka et al who identified silkworm innate immunity genes [23]. Scanning Electron Microscopy (SEM) Infected silkworm larvae guts were dissected into 0.9% NaCl medium. The intestinal contents were removed and the guts were washed in 0.9% NaCl medium for twice to wash off all the intestinal contents. The Bb bacteria were cultured in LB solid medium for 36 hours. The cleared guts and Bb bacteria were immediately fixed with 2.5% glutaraldehyde for 2 hours. The fixed samples were rinsed in 0.01 M phosphate buffer (pH 7.4) for 20 minutes for three times. The samples were post fixed in 1% osmium tetroxide for 2 hours and rinsed in ddH2O for 15 minutes for three times. Then, the fixed samples were immersed in a series of ethanol-water washes (30%, 50%, 60%, 70%, 80%, 90% and 100%) for 15 minutes per gradient and immersed in a series of tert-butyl alcohol (50%, 75% and 100%) gradient dehydration for 10 minutes per gradient and immersed in solvents of tert-butyl alcohol: acetonitrile 2:1 and tert-butyl alcohol: acetonitrile 1:1 for 10 minutes respectively. Finally, the samples were kept in 100% acetonitrile solvent. Samples were dried using a critical point drying apparatus and CO2 coated specimens with gold/palladium (60-40) using a sputter coater. Remove the samples from the dryer and attach them to SEM. The samples were observed with Hitachi S-3000N scanning electron microscope (Japan). Hemolymph Absorbance Detection Three infected silkworm hemolymph were got together in 1.5 ml tube at different time points. Three samples were got for each time point. About 10 mg phenylthiourea was added into each sample tube immediately to prevent hemolymph melanization. 490 nm absorbance of hemolymph was detected using DUH 800 UV/Visible spectrophotometer (USA). Supporting Information Table S1 Bb induced enzymes involved in general metabolism of silkworm by KEGG prediction. Found at: doi:10.1371/journal.pone.0008098.s001 (0.06 MB PDF) Table S2 Ratios and anatation of all Bb induced genes. Found at: doi:10.1371/journal.pone.0008098.s002 (0.27 PDF) MB Table S3 Multiple tissues expression data of the induced genes. Found at: doi:10.1371/journal.pone.0008098.s003 (0.17 MB PDF) Table S4 The ratios of genes mentioned in this report. Found at: doi:10.1371/journal.pone.0008098.s004 (0.03 PDF) MB Table S5 Primers used for cloning the 16S rRNA gene clone, real time PCR analysis. Found at: doi:10.1371/journal.pone.0008098.s005 (0.01 MB PDF) PLoS ONE | www.plosone.org 15 December 2009 | Volume 4 | Issue 12 | e8098