Bacillus remains the major pathogenic bacterium isolated in majority of areas for Bombyx mori [17]. Among them, Bacillus bombysepticus (Bb) was first separated from the sick silkworm larvae cadavers and identified by Hartman in 1931 [18]. As a Gram- positive bacterium, Bb can produce spores and parasporal crystals. Bb is a resistant species among silkworm bacterial pathogens in the natural environment. Bb natural infection the silkworm results a typical symptom of a disease: a peutz first appears on its thoracoabdominal region or the first 1–3 abdominal aspect, and then expands to the whole body. So far, however, the pathogenesis of Bb or interaction between Bb and its host silkworm is rarely reported. The silkworm genome contains about 14,623 genes and larvae multiple tissue transcriptional data were obtained using a 22,987 oligonucleotide probe microarray [19–21]. The genome-wide analysis of model insects showed that the numbers of immunity- related genes in A. gamibae and D. melanogaster are greater than those in B. mori and A. mellifera, but their innate immune signal transduction pathways are rather primitive [22,23]. Using the particular advantages of GeneChips, Bb infection can be used to survey the host silkworm genome-wide responses, including its innate immune response to the pathogen at transcriptional level and to provide another detailed comprehension of the interaction between a Bacillus pathogen and its host. The results demonstrated that Bb induced the host strong response. A lot of basal metabolic pathways were significantly modulated. Genes related to poisoning that might be a key to silkworm fuliginosa septicaemia, are also regulated. Furthermore, genes of juvenile hormone synthesis and metabolism related showed upregulation, suggesting that juvenile hormone participate in host modulation during the infection. Moreover, host cellular and systemic immune responses are also induced. Results An Overview of Bb Oral Infection Bb is close to B. cereus and Bt. by its 16S rRNA gene sequence analysis. We cloned a 1.5-kb sequence of the 16S rRNA gene of Bb using universal primers and registered it in GenBank (accession number: GQ281063). Searching GenBank, this is the first registration sequence of bombysepticus species. After comparison of the Bb 16S rRNA gene sequence with 16S rRNA sequences in the NCBI database, the resulting phylogenetic tree clearly indicated that Bb belongs to Bacillus (Fig. 1a). The dendrogram showed that Bb is similar to Bacillus cereus and that Bt. Bacillus anthracis is a distant relative. As a typical species of Bacillus, Bb can produce spores and parasporal crystals (Fig. 1a). The extraordinary versatility of Bacillus species is reflected by their ability to survive in nature. As a consequence, they are virulent toward insects and humans [24]. However, Bb is the first bacterium of this genus found to be highly pathogenic by natural infection for the silkworm in the sericulture production. Bb strain cultured in LB medium is pathogenic to the silkworm. To determine the pathogenicity of the Bb strain cultured in LB medium, survival ratios were obtained by oral infection using silkworm larvae at day 3 of the fifth instar. The results showed that Bb induced .50% mortality within 30 hours after oral infection and the remaining hosts died within about 60 hours under the rearing condition of temperature of 30uC and humidity of 90%. From 20–40 h, there was a significant reduction in survival from about 90% to 10% under this condition. Rearing under the temperature of 25uC and humidity of 70%, the host died much more slowly. However, most of the silkworms died during the two infection conditions, indicating that this Bb line is B. mori Host Response to Bb pathogenic for the silkworm (Fig. 1b). The results demonstrated that the pathogenicity of Bb strain cultured in LB medium warrants further analysis. Comparison of the corpses of larvae dies naturally or due to oral infection showed that that the peutz of natural infection larvae was mainly in the anterior chest, whereas that of oral infection larvae was much larger around the middle chest, caused by substantial microorganism invasion of the digestive tract after oral infection (Fig. 1c). The peutz pattern of Bb oral infection larvae indicated a stronger reaction than natural infection. Bb induces strong silkworm response and changes in expression profiles. Bb oral infection can change the expression of many silkworm genes, as shown by transcriptional analysis. A total of 2,436 genes were modulated, using a 2.0-fold cut-off, between 3 and 24 hours post-infection. At the beginning of the infection (3 hours post-infection, 3 hpi), the number of regulated genes showed a small peak owing to a large-scale microbial attack. At this time, 120 genes were upregulated and 374 were downregulated. At the mid-point of the infection (6 hpi to 12 hpi), Bb was counterattacked by the host defense system and the reproduction level of the surviving mircoorganisms in the silkworm midgut was much slower than in the medium, so the gene expression changes were relatively weak; 124 and 164 genes were modulated at 6 hpi and 12 hpi, respectively. At 6 hpi, the numbers of upregulated and downregulated genes were similar (59 and 65, respectively). At 12 hpi, the number of upregulated genes was approximately half the number of downregulated genes (49 and 115, respectively). At 24 hpi, as bacteria adapt to the internal environment of the host midgut, they enter exponential growth phase and increase bacterial toxic production. Thus, the induced gene number peaked. These genes included many metabolic system- and immune system-related genes. At this time, 1,063 genes were upregulated and 980 were downregulated (Fig. 2a). Overall, the cluster analysis of expression profiles of all induced genes showed time-specific patterns. By average linkage of hierarchical cluster analysis, 12 clusters of gene expression profiles could be defined using Bb oral infection microarray data (Fig. 3). Among the clusters, cluster 1 and cluster 12 were interesting for the significant down- and up-regulation, respectively, of large numbers of genes at 24 hpi. Their mean log2 ratios were close to 21.5 and 1.5. Clusters 8 and 11 were also notable for their dynamic exchange from down- to upregulation from 3 hpi to 24 hpi. Clusters 3 and 6 were significantly upregulated at 3 hpi. Clusters 7 and 4 were significant upregulated at 6 hpi, and cluster 5 was significantly upregulated at 12 hpi. The ratios and annotations of all these genes are shown in Table S2. By GO analysis of these induced genes, their functions could be classified to 14 categories (Fig. 2b) [25–27]. Among these 14 families, catalytic activity, physiological process and cellular process were the largest, with 201, 193 and 182 members, respectively. These data indicate that Bb oral infection of the silkworm could induce a large number of enzymes involved in many basal metabolic pathways. The induced genes were then analyzed further. Bb induces a strong midgut response–by tissue expression analysis. Pathogen infections always have tissue- specific features [28]. Multiple tissue expression data from day 3 of the fifth instar showed that 1,403 of the 2,436 induced genes have multiple tissue expression, indicating that almost all of the silkworm tissues were affected by the infection [19]. At the same time, Bb induced genes that showed some organizational preference, as more genes were highly expressed in the midgut, integument and testis (Fig. 4a). Of the 1,403 genes, 886 (63%) genes were expressed in the midgut (signal value.400), of which PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8098