A genome-wide survey for host response of silkworm, Bombyx mori during pathogen Bacillus bombyseptieus infection

Abstract

Host-pathogen interactions are complex relationships, and a central challenge is to reveal the interactions between pathogens and their hosts. Bacillus bombysepticus (Bb) which can produces spores and parasporal crystals was firstly separated from the corpses of the infected silkworms (Bombyx mori). Bb naturally infects the silkworm can cause an acute fuliginosa septicaemia and kill the silkworm larvae generally within one day in the hot and humid season. Bb pathogen of the silkworm can be used for investigating the host responses after the infection. Gene expression profiling during four time-points of silkworm whole larvae after Bb infection was performed to gain insight into the mechanism of Bb-associated host whole body effect. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. Similar to Bacillus thuringiensis (Bt), Bb can also induce a silkworm poisoning-related response. In this process, genes encoding midgut peritrophic membrane proteins, aminopeptidase N receptors and sodium/calcium exchange protein showed modulation. For the first time, we found that Bb induced a lot of genes involved in juvenile hormone synthesis and metabolism pathway upregulated. Bb also triggered the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs), including of Attacin, Lebocin, Enbocin, Gloverin and Moricin families, were upregulated at 24 hours post the infection.

Figure 1. Phylogenetic tree of 16S rRNA sequences and survival curve of Bb infection the silkworm.

(a) Phylogenetic tree with the sequences of 16S rRNA gene of Bb and its related species. Bb can produce spores and parasporal crystal. (b) Survival curve of Bb oral infection silkworm on day 3 of the fifth instar. Control 1: ddH₂O feed silkworm rearing under the condition of temperature of 30°C and humidity of 90%. Control 2: ddH₂O feed silkworm rearing under the condition of temperature of 25°C and humidity of 70%. Induced 1: Bb feed silkworm rearing under the condition of temperature of 30°C and humidity of 90%. Induced 2: Bb feed silkworm rearing under the condition of temperature of 25°C and humidity of 70%. (c) Natural infection and oral infection both can cause silkworm cuticle peutz.

Figure 2. General statistics on the genes regulated after Bb oral infection.

(a) The number of up- and down-regulated genes after Bb oral infection at 3 h, 6 h, 12 h and 24 h time points (h: hours post infection). (b) GO categories of total Bb oral induced genes.

Figure 3. Differentially regulated genes during Bb infection.

(a) Clustered expression profiles of regulated 2436 genes taken 2.0-fold cutoff criterion from 3 h to 24 h. For expanded ratios and annotation details, see Table S2. (b) Mean expression values of genes (log₂) located in the defined clusters.

Figure 4. Tissues expression data on day 3 of the fifth instar of the induced genes.

(a) Cluster of multiple tissues expression data of the induced genes. For details of these data, see Table S3. (b) The summary of the multiple tissues expression of the induced genes.

Figure 5. Cluster analysis of basal metabolism pathways.

Detailed expression profiles of functionally pathways related sets of genes. The gene ID of SilkDB are shown on the left (for each gene ID, we used the letter A to substitute letters BGIBMGA to simplify the expression) and the serial number of KEGG database are shown on the right. For a detailed view, see Table S1 and Table S4.

Figure 6. Functional groups of silkworm poisoning related genes.

(a) Cluster analysis of silkworm poisoning related genes referencing to Bacillus thuringiensis toxins. For a detailed view of the cluster ratios, see Table S4. (b) SEM pictures of the silkworm middle gut epithelium after Bb infection. The pictures showed Bb crystals accumulated in the host intestinal epithelial cells and pores were formed in the host intestinal epithelial cells after Bb oral infection. (c) Schematic overview of the process that Bb damage silkworm midgut, into the hemolymph. After oral infection, Bb interred the ingestion of silkworm. The parasporal crystal (PC) produced by Bb could be digested by midgut proteases. The digested PC could pass through the peritrophic membrane (PM) to bind the aminopeptidase N receptors (APNs) of the midgut epithelial cells to damage them. Bb can go to hemolymph from the damaged midgut.

Figure 7. Diagram showing the induced biosynthesis, metabolism and signal transduction related genes of juvenile hormone in the silkworm.

The expression pattern of each gene was indicated near the gene name. Diagram of the pathway is referenced form Schooley and Baker, 1985. For a detailed view of the cluster ratios, see Table S4.

Figure 8. Bb induced silkworm cellular immune response and serine protease cascade melanization pathway.

(a) Cluster of cellular immune response families. (b) Cluster diagram of the induced synthesis of melanin by serine protease cascade melanization pathway related genes. For a detailed view of the cluster ratios, see Table S4. (c) Real time PCR analysis of ppo1 and ppo2 ratios of Bb infected whole larvae comparing to non-induced control and 490 nm absorbance of hemolymph during the infection, and the picture of the hemolymph melanization 3 h after Bb oral infection.

Figure 9. Bb induced silkworm systemic immune response.

(a)Bb can be recognized by short-type of peptidoglycan recognition proteins (PGRPs) PGRP-S2 and PGRP-S5, then, mainly through Toll signal pathway, antimicrobial peptide coding genes were induced (the asterisk indicated a family, for the probe can not distinguish a member out from highly homologous sequences). (b) Toll pathway signal transduction diagrammatic sketch and real time PCR ratios of corresponding genes. (c) JAK/STAT sinal transduction diagrammatic sketch and real time PCR ratios of corresponding genes. For a detailed view of the cluster ratios, see Table S4.