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Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm
Published on: April 12, 2017
Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type
Katsuhiko Ito1, Kurako Kidokoro, Sachiko Shimura
1Insect Genome Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. katsuito@affrc.go.jp
Abstract:
Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.
Insights
Bombyx mori densovirus type 1 (BmDNV-1) causes flacherie disease in silkworms, primarily affecting midgut cells. This study details the sequential pathology and viral propagation, revealing significant midgut disruption and virus multiplication correlating with larval death.
Area of Science:
- Virology
- Entomology
- Pathology
Background:
- Bombyx mori densovirus type 1 (BmDNV-1) is a significant pathogen responsible for flacherie disease in silkworms.
- Previous studies have utilized immunohistochemistry, but a comprehensive understanding of the sequential pathological changes and viral propagation in BmDNV-1-infected larvae remains incomplete.
Purpose of the Study:
- To conduct sequential investigations into the pathological features of BmDNV-1-infected silkworm larvae.
- To elucidate the propagation dynamics of BmDNV-1 within the host.
- To correlate viral gene expression with observed histological alterations.
Main Methods:
- Oral infection of newly ecdysed 4th instar Bombyx mori larvae with BmDNV-1.
- Histological examination of midgut tissues at various time points post-infection.
- Quantitative RT-PCR to monitor the expression of two BmDNV-1 genes.
- Bioassays across different larval developmental stages to assess pathogenicity.
Main Results:
- Larval mortality commenced at 9 days post-infection (dpi), with complete mortality by 10 dpi.
- Histological analysis revealed phenotypic alterations in midgut cells starting at 4 dpi, progressing to complete structural disruption by 9 dpi.
- Quantitative RT-PCR indicated BmDNV-1 propagation initiated around 4 dpi, with a gradual increase correlating with histological damage and larval death.
- Pathogenicity was found to be independent of the larval stage or molting process.
Conclusions:
- BmDNV-1 infection leads to progressive pathological changes in the silkworm midgut, culminating in larval death.
- Viral propagation directly correlates with the observed histological damage and disease progression.
- The susceptibility of Bombyx mori to BmDNV-1 is not influenced by developmental stage or molting.
