pathways, pantothenate and CoA biosynthesis pathways and one carbon pool by folate. Eight genes encoding enzymes involved in porphyrin and chlorophyll metabolism were regulated, of which six and two were up- and downregulated, respectively (Fig. 5 and Table S4). They encoded eight types of enzymes in this pathway, including uroporphyrinogen-III carboxy-lyase (EC 4.1.1.37), hydrolases (EC 3.2.1.31), NADP+7, 8-oxidoreductase (EC 1.3.1.33) (Table S1). Five genes related to pantothenate and CoA biosynthesis were modulated, including 39-dephospho-CoA 39-phosphotransferase (EC 2.7.1.24), 2-oxoglutarate aminotransferase (EC 2.6.1.42), (R)- pantothenate 49phosphotransferase (EC 2.7.1.33) and 5, 6- dihydropyrimidine amidohydrolase (EC 3.5.2.2). In addition, three genes related to one carbon pool by folate were also modulated, including tetrahydrofolate ligase (EC 6.3.4.3), dUMP C-methyltransferase (EC 2.1.1.45) and transferases (EC 2.1.2.3). These results show that the three cofactor and vitamin metabolic pathways were sensitive to the infection. Xenobiotics biodegradation and metabolism. Pathways involved in xenobiotic biodegradation including 2,4-dichlorobenzoate degradation, benzoate degradation via hydroxylation and styrene degradation were induced after Bb oral infection; most of these genes were upregulated (Fig. 5, Table S1 and Table S4). The 2,4- dichlorobenzoate degradation pathway-related genes encoding hydrolases (EC 3.1.1.-) and oxygen 4, 5-oxidoreductase (EC 1.13.11.8) were modulated. The expression of oxygen 4, 5- oxidoreductase was increased more than six-fold at 24 hpi, when the bacteria produced large quantities of bacterial toxins within the silkworm host, indicating that oxygen 4,5-oxidoreductase might be involved in the detoxification of bacterial toxins such as parasporal crystal [38]. Four genes involved in benzoate degradation via the hydroxylation pathway were also regulated. In addition to the above- mentioned oxygen 4, 5-oxidoreductase gene, acyl-CoA:acetyl-CoA C- acyltransferase (EC 2.3.1.16), mandelate racemase (EC 5.1.2.2) and lyases (EC 4.1.1-) were also regulated. For example, lyases, which have also been shown to be related to the pyruvate metabolism pathway, were highly expressed in the midgut and malpighian tubules; at 24 hpi, expression levels were more than three times the baseline levels. Thus, we speculated that lyases might be involved in the detoxification of midgut and malpighian tubules. In addition, three genes involved in the styrene degradation pathway were modulated, including acylamide amidohydrolase (EC 3.5.1.4) and 4-maleylacetoacetate cis-trans- isomerase (EC 5.2.1.2). Also, three members of the cytochrome family involved in detoxification, including the cytochrome P450 family 4 (A001003) and cytochrome P450 (A013237, A013241), were upregulated. These results illustrated that Bb infection accelerated the xenobiotics biodegradation and metabolism, probably mainly caused by the released bacterial toxins. Amino acid metabolism and nitrogen metabolism. Amino acids are important molecules in every organism. Pathways of tryptophan metabolism, histidine metabolism, valine, leucine and isoleucine degradation, urea cycle and amino group metabolism, and aminophosphonate metabolism involved in amino acid metabolism and nitrogen metabolism were modulated by the infection (Fig. 5, Table S1 and Table S4). Of the genes involved in amino acid metabolism, more were upregulated than downregulated. For example, 14 genes involved in the tryptophan metabolism pathway, including (S)- 3-hydroxyacyl-CoA: NAD+ oxidoreductase (EC 1.1.1.35), transferases (EC 2.1.1.-), and acylamide amidohydrolase (EC 3.5.1.4), eleven of the 14 genes were upregulated and only 3 were downregulated. These results show that Bb infection promoted the amino acid metabolism. Nitrogen metabolism is a basic pathway that maintains the balance of nitrogen in organisms. After Bb infection, all of the seven nitrogen-pathway regulated B. mori Host Response to Bb genes including NAD (P) +oxidoreductase (EC 1.4.1.3) were downregulated, indicating that nitrogen metabolism was slowed by the infection. Carbohydrate metabolism. Carbohydrate metabolism is a basal metabolic process and provides carbon and energy for organisms [39,40]. Enzyme-encoding genes of basic carbohydrate metabolic pathways were modulated by the infection, including those responsible for pentose and glucuronate interconversions, the citrate cycle (TCA cycle), pyruvate metabolism, the pentose phosphate pathway and butanoate metabolism (Fig. 5, Table S1 and Table S4). Many genes related to these pathways were upregulated. For example, for the pentose and glucuronate interconversions pathways, five genes were induced, including glucuronosyltransferase (EC 2.4.1.17), beta-D-glucuronoside glucuronosohydrolase (EC 3.2.1.31), NAD (P)+ 1-oxidoreductase (EC 1.1.1.21), NADP+ 4-oxidoreductase (EC 1.1.1.10) and L (or D)-ribulose 5- phosphotransferase (EC 2.7.1.16). Four of these five genes were upregulated at 24 hpi. For example, NADP+ 4-oxidoreductase (EC 1.1.1.10) was highly expressed in the fat body, and the expression was increased by a factor of four. The fat body is the site of energy storage, thus we speculated that the energy metabolism of the silkworm fat body was accelerated. Ten genes involved in the TCA cycle were regulated, including CoA ligase (EC 6.2.1.5), NAD+ oxidoreductase (EC 1.1.1.41), oxaloacetate carboxy-lyase (EC 4.1.1.32), and carbon-dioxide ligase (EC 6.4.1.1), however, many of which were downregulated at 24 hpi. Similar to amino acid metabolism, for carbohydrate metabolism, more genes were upregulated than downregulated, indicating that carbohydrate metabolism was accelerated to provide more energy. Similar to Bt, Bb Can Induce Silkworm Larvae Poisoning Related Response As a typical Bacillus, Bb can produce spores and crystal toxins. Bb toxins and spores are thought to induce a similar host response compared to Bt. The poisoning happened in the midgut. First, when Bb enters the midgut of the silkworm, parasporal crystal was released and degraded by proteases of the host midgut. The dissolved monomer of the toxin is activated and poisonous [41–47]. Parasporal crystal, as a type of protein, can be degraded by serine proteases [48]. As expected, the host expression levels of proteases including trypsins, other serine proteases, and zinc carboxypeptidase were regulated (Fig. 6a). Trypsins, as a type of serine protease that can selectively hydrolyze proteins, were considered to be the main hydrolases responsible for Bt toxin hydrolysis in the insect midgut [49,50]. In our study, 20 trypsins were induced after the infection, many of which were upregulated. For example, A008513, with tissue expression only in the midgut, was upregulated by more than 5-fold at 24 hpi. Other serine proteases, the usual role of which is to disrupt macromolecular protein peptide bonds, were also regulated, including seven other serine proteases, including peptidase_S24 (PF00717, A001027 and A012810), peptidase_S28 (PF05577, A012452 and A008167), peptidase_S51 (PF03575, A003141) and peptidase_S9 (PF00326, A006179 and A001272). Among these seven proteases, A001027, A012810, A012452 and A003141 were significantly upregulated at 24 hpi. Three members of the zinc carboxypeptidase family (Peptidase_M14, PF00246), with the general function of hydrolysis of carboxyl terminal amino acids, were only or highly expressed in the midgut and were also upregulated after the infection [51]. The dissolved Bb crystal toxin can damage the silkworm midgut peritrophic membrane (PM) [52]. The PM of the insect midgut is PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8098