study, showed significant regulation in this analysis [92]. PGRPs are also related to the Toll and Imd signal transduction pathways of the insect innate immune system [93]. Thioester-containing proteins (TEPs) also showed recognition receptor activity in A. gambiae during malaria parasite infection [84]. Unlike A. gambiae, which contains 15 TEPs, the silkworm only contains three TEPs. During Bb infection, only TEP3 (A013565), which has no orthologs in Drosophila and Anopheles, was downregulated at 24 hpi. After microorganism recognition, regulators of the serine protease cascade, including the CLIP serine proteases (CLIPs), serpins (SPNs) and CTLs showed modulation [84]. Expression levels of two of the 15 silkworm CLIPs, CLIP1 (A008668) and CLIP2 (A014603), were regulated [23]. CLIP1, which is only expressed in the integument and head, showed about twice of up- to down- regulation from 3 hpi to 24 hpi. Expression levels of three of the 26 serpins, SPN1(A010213), SPN2 (A004726) and SPN3 (A008831), were regulated [23]. The expression level of SPN1, which is highly expressed in the integument, head and hemocyte, was doubled during the early stages of infection (3 hpi). Finally, melanization effectors, including three prophenoloxidase genes (proPOs, PPOs), were activated. PPO1 (A012764) and the PPO1 subunit (PPO-1S, A012763), which are expressed at very high levels in the hemolymph, were upregulated from 3 hpi to 12 hpi. The PPO-2 subunit (A013115), whish is highly expressed in the hemolymph and head, also showed upregulation from 3 hpi to 12 hpi [94,95]. The real time PCR analysis of PPO1 (A012764) and PPO2 (A013115) supported the above result (Fig. 8c). Activated PPO can catalyze the oxidation of mono- and diphenoles to orthoquinones, which non-enzymatically polymerize to melanin. These results show that many of the melanization pathway genes were upregulated from 3 hpi to 12 hpi, indicating that melanization occurred during the early stages of infection. Also, the dissected body and 490 nm hemolymph absorbance showed that the host hemolymph became markedly darker compared with uninfected insects at the early stage of infection (Fig. 8c). Bb induced host systemic immune response. To combat microorganism infections, the insects rely on multiple innate defence reactions such as local and systemic immune responses [96]. Systemic immune responses involve pathogen recognition, signal transduction, and AMP expression. In this analysis, two short PGRPs were upregulated as expounded in serine protease cascade melanization pathway section. The insect systemic immune response, involves signal transduction of the Toll, Imd, and JAK/STAT pathways [80,97]. However, for the silkworm, little is known about the corresponding pathway by which AMPs are induced. To determine this question, we searched the microarray data of the silkworm Toll, Imd and JAK/STAT signaling pathway genes. However, microarray data did not show any regulation due to its much lower sensitivity (signal values of most of them are less than 400 and therefore can not be identified ‘‘expression’’ to current standards) (Table S6). So, the real-time RT-PCR analysis of the genes indicating the regulation of innate immunity (SPZ1, Toll1, Toll6, Myd88, Tube and Rel of Toll pathway and Hop, Dome and Stat1 of JAK/STAT) was performed (Fig. 9b, c, Table S6). Comparison the microarray data and the real time PCR data, although the degree of modulation was different, the tendency of regulation of most genes were similar. Because previous RT-PCR analysis of the IMD pathway genes showed very weak modulation, we gave up its testing by real time PCR analysis (data not shown). Most of our knowledge of the Toll pathway was obtained from the initial studies in Drosophila, and it has been shown to be activated by fungi and Gram-positive bacterial infection. The Toll receptor is B. mori Host Response to Bb activated upon binding by a cleaved form of spa¨tzle, which is proteolytically processed after activation by secreted recognition molecules (PGRP-SA, GNBP1) [98,99]. Mature spa¨tzle binds as a dimer to Toll, thereby inducing its dimerization at the plasma membrane. This causes the recruitment of three death-domain- containing proteins, MyD88, Tube and Pelle [100–102]. Then, the Rel transcription factors are released from the cytoplasm to the nucleus to active APMs expression [103,104]. In the silkworm, the Toll pathway is conserved in terms of innate immunity [23]. Our real time PCR analysis showed that Spz1, Toll1, Toll6, MyD88, Tube and RelA can be induced after Bb infection at 6 hpi and 12 hpi (Fig. 9b). Spz1, mature peptide injection of which to the silkworm can significantly upregulate transcription of eight antimicrobial peptide genes (attacin 2, cecropin A1, -B1, -D1, gloverin A5, -B, lebocin-3, and moricin-A1), are highly expressed at the beginning of the infection and showed marked upregulation from 3 hpi to 48 hpi [99]. The Toll receptor Toll1, Toll6 also showed upregulation, as did the adapters MyD88 and Tube. MyD88 is only expressed after Bb infection. The Rel transcription factor RelA, which can markedly activate lebocin 4, was upregulated more than 6 times [105]. Thus, we can speculate that the silkworm Toll pathway can be activated by Bb oral infection. The JAK/STAT pathway, originally identified through its role in embryonic segmentation, has three main cellular components in Drosophila: the receptor Domeless, the Janus Kinase (JAK) Hopscotch, and the STAT transcriptional factor [106–108]. This pathway is thought to participate in antiviral and inflammatory responses [109,110]. After Bb oral infection, the membrane receptor Dome was upregulated at 12 hpi (Fig. 9c). Hop and stat1 also showed upregulation 12 hpi. This results illustrated that Bb oral infections can cause a weak JAK/STAT1 pathway response. After that, AMP genes, including those of the attacin, enbocin, gloverin, lebocin and moricin subfamilies, showed upregulation at 24 hpi, indicating that Bb can induce the silkworm systemic immune response. The moricin subfamily, which shows the greatest expression levels in the malpighian tubules, showed the greatest upregulation at 24 hpi; the expression level was increased more than eight-fold. In general, these results show that Bb is recognized by silkworm PGRPs and the signals can be transduced mainly by the Toll pathway, leading to the production of AMPs. Discussion B. bombysepticus is closely related to B. cereus and B. thuringiensis, and is a typical natural pathogen of the silkworm B. mori. The host transcriptional analysis after Bb oral infection presented here will help us to understand the relationship between the Bacillus pathogen and B. mori host. In this report, we found that many pathways involved in the silkworm physiological functions were changed after the infection. First, the basal metabolic pathways were most involved. The results demonstrated that Bb can affect six types of basal metabolic system-related genes, leading to overexpression or reduced expression. These types include genetic information processing and transcription, carbohydrate metabolism, amino acid metab- olism and nitrogen metabolism, nucleotide metabolism, metabo- lism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. During Bb infection, particularly after the pathogen entered the hemolymph, the silkworm has to meet the basic material and energy needs of Bb growth and reproduction, leading to the upregulation of some metabolic pathway genes. During Bb infection, a partial energy originally used for silk protein synthesis will be assigned to microorganism reproduction. A previous study on the functional genomics of Buchnera and the ecology of aphid PLoS ONE | www.plosone.org 12 December 2009 | Volume 4 | Issue 12 | e8098