considered as a non-cell semi-permeable membrane mainly composed of chitins and proteins [53,54]. The PM can promote insect digestion and prevent the invasion of pathogenic microor- ganisms by forming a natural barrier of midgut epithelial cells. After the infection, four PM structural protein genes (domain CBM_14, PF01607), with high tissue expression only in the midgut, were downregulated at 24 hpi (Fig. 6a). Among them, the expression level of A009641 showed a dynamic change from more than twice of up- regulation at 3 hpi to about twice of down-regulation at 24 hpi. The results indicated that, with time, the production of PM proteins decreased, resulting in damage of the PM and allowing passage of the Bb crystal toxin. Thus, the toxins can be observed accumulated on the host intestinal epithelial cells (Fig. 6b). The activated toxin can bind the metallopeptidase receptors of the brush-border membrane vesicles of the host midgut epithelial cells, which allows it to cross the cell membrane [55]. Aminopeptidase N receptors (APNs), members of the zinc metallopeptidase M1 family (PF01433), are a specific type of exonuclease that can digest proteins or peptides from their N- terminal amino acids. Previous studies have shown that the APNs of insects are the receptors for the Cry toxin [56–58]. A total of 16 APNs containing the Peptidase_M1 domain can be identified in the silkworm genome, 3 of which have also been identified as Cry toxin receptors: BmAPN1 (A008059, NCBI No. AF084257 and AF352574), BmAPN2 (A008017, NCBI No. AB011497) and BmAPN4 (A008060, NCBI No. AB013400) [44,46,59]. In this study, nine APN receptors were modulated by the infection (Fig. 6a). Most showed dynamic expression changes, which manifested as a low level of upregulation at 3 hpi and downregulation at 24 hpi. A010679 and A008061 showed more intense upregulation than the other genes; the increase in expression level was approximately double at 3 hpi and 12 hpi. Three of the above-mentioned Cry toxin receptors, however, showed weak upregulation from 3 hpi to 12 hpi than A010679 and A008061. This result reflected the selective binding features of the silkworm APNs. Some genes involved in infiltration balance also showed regulation after the infection. For example, the sodium/calcium exchange protein A000688 was upregulated by a factor of about seven at 3 hpi, indicating that Bb can rapidly create a sodium/ calcium exchange imbalance [60] (Fig. 6a). ABC transporters are involved in the transmembrane export or import of a wide variety of substrates from small ions to macromolecules [61,62]. After Bb oral infection, 12 ABC transporter-encoding genes were regulated. Most were downregulated by the infection, indicating that exchange between the outside and inside of the membrane was weakened. A nucleoplasmin protein, A009067, which is only expressed in the midgut, showed upregulation at 24 hpi. Nucleoplasmin, a nuclear molecular chaperone, is usually considered to be involved in nucleosome assembly, chromatin reconstruction, material transport, and cell apoptosis [63,64]. The upregulation of this gene might indicate increased exchange between the nucleus and cytoplasm after the infection. Finally, the infiltration balance and material exchange of the insect body are disrupted. Pores can be observed in the host intestinal epithelial cells and intestinal epithelial cells microvillus were vacuolated (Fig. 6c). Infiltration balance disorder is the main cause of insect death due to Cry toxin [65]. Thus, these results indicate that Bb can poison the silkworm via a similar mechanism as the Bt Cry toxin. Bb Induced Juvenile Hormone Synthesis and Metabolism-Related Gene Upregulation Juvenile hormone (JH) is the main hormone that regulates maintenance of the physical form of larvae, pigment occurrence in B. mori Host Response to Bb larvae and reproduction activities [66,67]. The silkworm JH biosynthetic pathway can be divided into two main stages: the early steps, up to farnesyl diphosphate (FPP) formation, belong to the mevalonate pathway, and the late steps control the conversion of FPP into JH [68]. JH is then metabolized to JH acid diol and JH diol phosphate [69]. On the other hand, JH can bind to JH- binding proteins and regulate target gene expression. So far, little is known about the pathogen infection and modulation of insect JH. Interestingly, in this analysis, a lot of JH synthesis, metabolism and JH-binding genes were shown to be upregulated after Bb oral infection (Fig. 7). The expression of the HMG-CoA synthase gene (HMGS, A004001), responsible for catalysis of acetoacethl-CoA to HMG- CoA, was increased by nearly three-fold [70]. Phosphomevalonate kinase gene (MevPK, A001556), encoding an enzyme that converts phosphomevalonate to diphosphomevalonate, was upregulated by more than two-fold [68]. Short-chain dehydrogenase gene (SDR, A002886), which converts farnesal to farnesoic acid, was upregulated by more than three-fold [71]. The JH acid methyltransferase (JHAMT) is an enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis pathway in insects [72]. One of the two JHAMT coding gene (A010563) was upregulated from 3 hpi to 24 hpi, whereas the other (A010392) was downregulated from 3 hpi to 24 hpi. At the same time, the Farnesoic acid O-methyltransferase (FAMeT, A002604), which is involved in the conversion of farnesoic to methyl farnesoate, was downregulated, indicating that the JH acid branch pathway might be a method of JH biosynthetic after Bb infection [73]. Similarly, some genes related to JH metabolism were also modulated. Two JH epoxide hydrolase genes (JHEH, A011468 and A013929), involved in the regulation from JH to JH diol, were upregulated at 12 hpi and 6 hpi, respectively [74]. However, JH esterase (JHE, A000772) on HJ to JH acid diol pathway branch, showed downregulation from 3 hpi to 24 hpi. Two genes coding JH diol kinase (JHDK, A008813 and A008815), involved in the conversion of JH diol to JH diol phosphate, both showed more than three-fold upregulation at 24 hpi [69]. JH binding protein (JHBP) can bind to JH and transport JH from the corpus allatum to target tissues [75]. Two genes encoding JHBPs (A011457 and A011458) were upregulated from 3 hpi to 24 hpi. FKBP39, a Drosophila homolog, can bind the immunosuppressive drug FK506 and mediate the binding of the target element of JH and showed a dynamic upregulation from 3 hpi to 24 hpi [76]. The general function of insect JH is to maintain larvae morphology. After a microorganism infection, insects will pupate earlier than normal and lay eggs to preserve their future generations. However, after a pathogenic granulovirus infection, the insect cannot pupate, indicating that the JH concentration is maintained at a high level after the infection [77]. At the same time, we detected a lot of JH-related genes which were also upregulated after NPV oral infection (data not shown). In addition, synthetic JH can improve the production of NPV in insect cells, indicating the role of JH in the reproduction of NPV [78,79]. Hence, we speculate that Bb might active silkworm JH synthesis, metabolism and binding related genes to extend the silkworm larvae stage and provide nutrients for its reproduction, similar to NPV. Bb Induced Silkworm Immune Response Bb can induce silkworm cellular response. Once a pathogenic microorganism crosses the natural barrier of its host, it has to face a strong host immune response. For insects, the non- specific cellular immune response, which is mainly mediated by PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8098