upregulated after B. bassiana and NPV oral infection of the silkworm (data not shown). Previous studies have shown that JH expression is beneficial for NPV reproduction. Indeed, after a powerful pathogen infection, JH regulation is an effective method to prevent host insect metamorphosis and provide material and energy for pathogen reproduction. At the same time, we found the ecdysone receptor gene 20E (A006767) was downregulated at 24 hpi. Further studies are required to resolve this issue. Melanin accumulation can form peutz after pathogen infection, and this generally occurs in the insect midgut and integument. Peutz formation after bacterial infection is universal. A Bb natural infection can lead to the formation of a typical peutz of the silkworm thoracic cuticle. In addition to encapsulation, serpin cascade melanization genes and the silkworm tyrosine hydroxy- lase-coding gene (TH, A000563), which can catalyze tyrosine to dopa and then to dopamine melanin, were upregulated from 3 hpi to 6 hpi [113]. Three hours after Bb oral infection, the hemolymph was substantially darker, indicating that the infection signal can be rapidly transduced to the hemolymph from the midgut and that hemolymph melanization occurs during the early stages of infection. Thus, after clotting, encapsulation of bacteria, as well as the melanotic encapsulation caused by the PPO cascade and tyrosine melanization pathway, the cuticle peutz is formed [80]. The hemolymph clots and melanin can accumulate in the chest integument and finally cause cuticle peutz on the silkworm corpse. Furthermore, we also detected 20 cuticle-protein-encoding genes that were upregulated at 24 hpi. These might be related to formation of the cuticle peutz. Bb induced the silkworm systemic immune response. By real time PCR analysis, we found that Bb, as a Gram-positive bacterium, can induce the silkworm Toll pathway, which is similar to results from Drosophila [114]. Most AMPs, including those of the attacin, lebocin, enbocin, gloverin and moricin families, showed upregulation at 24 hpi. At this time, the microorganisms had passed through the midgut into the hemolymph and other organs, becoming increasingly likely to evade the host’s immune responses. After the oral infection, the AMP expression pattern differed to that seen after direct injection; the latter can rapidly induce AMP upregulation in the fat body (data not shown). It remains to be determined in the silkworm whether AMPs are induced by the Toll signal pathway or the interaction with Imd and/or JAK/STAT pathway. Concluding Remarks B. bombysepticus oral infection the host silkworm triggered a strong host response. Basal metabolic pathways were most involved after the infection, including those of genetic information processing and transcription, carbohydrate metabolism, amino acid metabolism and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. Similar to Bt, Bb can induce the modulation of silkworm poisoning- related genes, such as APNs. Interestingly, the host JH synthesis, metabolism and binding-related genes showed to be upregulated after the infection. On the other hand, the silkworm immune responses, including the cellular immune response and melanization and the systemic immune response were also induced. The relationship between Bb and the silkworm can be used as a model to investigate pathogen-host interaction. Materials and Methods Insect Strain The Chinese silkworm strain Dazao was used in this study. The silkworm was reared at a stable temperature of 25uC. The larvae B. mori Host Response to Bb stopped feeding on day 3 of the fifth instar for the infection experiments. Bacterial Strain A strain of bacterium Bb was kindly provided by Professor Yanwen Wang (silkworm Diseases Laboratory of Shandong Agriculture University, China). This strain was separated from the corpses of silkworms that had died due to Bb natural infection in Daiyue district, Taian city, Shandong province, China. 16S rRNA PCR Amplification, Sequencing and Phylogeny The 16S rRNA gene was PCR amplified with universal primers using the Pfu DNA polymerase according to the manufacturer’s instructions (Takara). The PCR product was fully sequenced, and each nucleotide of both strands was read at least twice. Phylogenetic trees were first constructed using the NCBI database and the Blast and tree construction programs. Then, several sequences representing their species were selected for the final tree reconstruction using Mega 4.0 software. Primers used in this report were shown in Table S5. Silkworm Oral Infection by Bb Bacterium For insects, direct bacterium injection to the body cavity is not thought to be a natural infection process. To overcome this limitation, a method of oral infection was developed [115]. In this study, oral infection method was used. Approximately 250 day 3 of the fifth instar larvae were placed in a petri dish without food to ensure hunger before infection. Bacteria were concentrated from an overnight culture in LB medium with 100 mg/ml ampicillin to avoid contamination with other type of bacterium. Then, the bacterial pellet was washed three times with diluted water to extract the bacterial toxin before optical density (OD) assessment. About 125 grams of artificial feed and 50 ml of concentrated bacterial solution (OD600<100) were thoroughly mixed in a beaker and were then cut into fine grains and given to the silkworms. After 3 hours, most of the bacterial meal had been eaten by the larvae. Then, the larvae were divided into four groups and transferred to four large petri dishes and reared with normal artificial feed. One group was raised at 25uC with approximately 70% humidity, and larvae were collected at different time intervals after infection for microarray analysis and real time PCR analysis. The other three groups were raised at 30uC with approximately 90% humidity, and dead larvae were counted at different time intervals for calculation of survival rate. For the survival rate raised under the condition of temperature of 25uC and humidity of 70%, 150 fifth instar larvae were fed with the same concentration of Bb as before, and raised under the condition of temperature of 25uC and humidity of 70%, and dead larvae were counted at different time intervals for calculation of survival rate. For the non-induced control, the same volume of ddH2O was mixed in the feed for the silkworm and the rearing conditions kept the same as Bb induced. Analysis of mRNA Expression Using Oligonucleotide Arrays RNA Extraction. For infection material, three larvae were collected at different time points and snap-frozen in liquid nitrogen immediately as one sample. Three independent samples were got. After homogenation of the larvae in liquid nitrogen, the resulting powders were added to 2.0 ml centrifuge tubes (each containing approximately 0.1 g), TRIzol reagent (Invitrogen) was added and total RNA was extracted according to the manufac- turer’s instructions. The total RNA templates were quantified by PLoS ONE | www.plosone.org 14 December 2009 | Volume 4 | Issue 12 | e8098