Responses Of Silkworm To Microsporidium Infection And The Activation Of Immune Signaling Pathway

Host–pathogen interactions are complex processes, which have been studied extensively in recent years. Our focus is on Nosema bombycis, which is a pathogen of silkworm pebrine and causes great economic losses to the silk industry. A complete understanding of the host response to infection of N. bombycis and the interaction between them is necessary to prevent this disease. In insects, midgut is a vital organ of digestion and nutrient absorption, and also serves as the first physiological and immune barrier against invading pathogenic microorganisms. Silkworm midgut infected with N. bombycis is a good model to investigate the host responses to microsporidia infection and the interaction between the silkworm and the microsporidium. The infected systems of N. bombycis in BmN cell line was established in this study and we observed the multiplicationin of N.bombycis in BmN cell line, then we investigate the midgut transcriptome profile of the P50 silkworm strain orally infected with N. bombycis at 6, 12, 18, 24, 48, 72, and 96 h post-infection(hpi) based on the Illumina Genome Analyzer platform. At the same time, we measured the transcriptional level of genes involved in signal identification and signal transduction involved in innate immunity post infection with N. bombycis based on the real-time PCR.1. The establishment of infected systems of N. bombycis in BmN cell line. After being treated with 0. 2 mol/ L KOH solution, Spores of N.bombycis were inoculated in BmN cell line. Propagation of N.bombycis was observed. The growth of N.bombycis in host cells was a chronic process. With the increasing of spores, they occupied cytoplasmic space and then cracked cells. Spores released from the infected cells infected other cells until they take up all space of cultures. The establishment of the systems lay the foundation for studies on the genomics and proteomics of N. bombycis.2. Responses of silkworm to infection of microsporidium. Though investigation of the midgut transcriptome profile of midgut of silkworm infected with N. bombycis, we identified 1902 differentially expressed genes, including 1168 up-regulated genes and 734 down-regulated genes. The number of different expressed genes varied at different phase post infection, and was closely related to the life cycle of N.bombycis in the silkworm. These results of DEGs clustering at different time points showed that only 3 genes showed different expression at 5 time points and 7 genes showed different expression at 6 hpi, 24 hpi, 48 hpi and 96 hpi. Most of these genes were involved in physiological metabolism and were down-regulated. The GO analyses showed that the expression of genes encoded proteins with different function varied at different time points. In the early phase, most of these were involved in basal metabolism and protein function, and most of different expression genes were corresponded to cellular component function. The KEGG analyses identified 24 pathway, including Lysine degradation、SNARE interactions in vesicular transport、Folate biosynthesis, etc., as significantly different pathway, which indicated that infection of N. bombycis has great influence on these metabolic pathways. In addition, some genes involved in silkworm innate immune were identified by Digital Gene Expression.3. The induction of immune signaling pathways by infection of N.bombycis. Based on method of real-time PCR, we investigate the expression pattern of genes involved in signal identification and signal transduction in innate immunity post infection with N. bombycis. The results showed that genes involved in signal identification, including BGRP1-4, PGRP, CTL-5, GNBP, were up-regulated post infection, which indicated that N.bombycis could be recognized by silkworm. Many genes involved in Imd, Toll and JAK-STAT signaling pathway were different expressed, which indicated that infection of N.bombycis activated the signaling pathway in silkworm and then induced the expression of immune effect factors.The current study of the responses of silkworm midgut to microsporidium infection and the expression profile of genes incolved in innate immunity not only analysis the resposes of silkworm to the infection of microsporidium in the point of genomics and identified many main metabolic pathways post infection, but also obtain lots of profile of different expressed genes post infection, which provide an overview of an insect’s host response to pathogen infection and pave the way for future studies on the molecularmechanisms of the complex interactions between N. bombycis and the silkworm. In addition, the current study demonstrated that infection of N. bombycis induce the signaling pathway in innate innmunity of silkworm.