Comparative Transcriptome Analysis of Bombyx mori (Lepidoptera) Larval Midgut Response to BmNPV in Susceptible and Near-Isogenic Resistant Strains

Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens causing severe economic losses in sericulture. However, the molecular mechanism of silkworm resistance to BmNPV remains largely unknown. Here, the recurrent parent P50 (susceptible strain) and the near-isogenic line BC9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with BmNPV. A total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (DEGs) were identified after comparing the four transcriptomes. Many DEGs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to BmNPV infection. Moreover, some immunity related genes were also altered following BmNPV infection. Specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing BmNPV infection. These genes were related to transport, virus replication, intracellular innate immune, and apoptosis. Our study provided an overview of the molecular mechanism of silkworm resistance to BmNPV infection and laid a foundation for controlling BmNPV in the future.

Fig 1. Length distribution of unigenes in the assembled transcriptomes.

The x axis represents different groups and treatments and the y axis shows the number of unigenes.

Fig 2. Correlation between gene expression ratios obtained from transcriptome data and RT-qPCR.

(A) Expression ratios (FPKM fold change) obtained from transcriptome data (red) and RT-qPCR (blue). (B) Lineage analysis between the transcriptome and RT-qPCR. The ratios obtained by RT-qPCR (x-axis) were plotted against the ratios obtained by RNA-Seq (y-axis).

Fig 3. Venn diagram showing the DEGs related to BmNPV infection in different resistant strains.

Fig 4. Gene ontology (GO) analysis of DEGs in different comparable groups.

These genes were divided into groups based on cellular component, molecular function, and biological process. The percent of DEGs that could be assigned to the different categories are indicated.

Fig 5. Expression patterns of selected genes related to BmNPV resistance in different resistant strains.

Each row represents a different gene, with blue, yellow and red indicating low, medium and high levels of gene expression, respectively.

Fig 6. Real-time PCR analysis of expression profiles of several genes in B. mori midgut.

After removing genetic background and individual immune stress response genes, 22 differentially expressed genes of interest potentially involved in resistance to NPV infection were obtained. Additionally, 119 genes unique differentially expressed in the isogenic-line BC9 (resistant strain) following BmNPV infection were observed. In order to further conformed the function of these genes, 4 genes from 22 DEGs and 5 genes from 119 DEGs with well reported previously were selected to conduct RT-qPCR.

Fig 7. Hypothesized modal analysis of the roles of the screened DEGs in BmNPV infection pathway.

V-ATPase is activated by LPH to promote viral entry into the cytoplasm, a process which was also effected by PAN1 and otoferlin. BAT1 related channel could serve as an alternative pathway for virus transmembrane transport. The released nucleocapsid is transported into the nucleus with the help of EFP. During replication, EFHP, ASGPA, ALT2, U2AF, ACT5 and ZRDP play an important role in facilitating virus replication. MFS is induced by the virus to increase cell volume leading to cell death. At the same time, the apoptosis process could be triggered by PDK to inhibit BmNPV infection.