Molecular detection of microsporidia Vairimorpha ceranae and Nosema bombycis growth in the lepidopteran Sf9 cell line

Abstract

The search for new therapeutics and strategies to suppress microsporidiosis of domesticated insects requires cultivation of honeybee and silkworm parasites in available insect cell cultures as well as reliable methods of their detection. In this study, we infected the commonly used lepidopteran Sf9 cell line with the Vairimorpha ceranae and Nosema bombycis spores to evaluate molecular methods for microsporidia growth assay. The silkworm parasite N. bombycis effectively develops in lepidopteran cells and, according to literature data, its growth can be detected by qPCR analysis of β -tubulin gene copies in infected Sf9 cultures. Here, we used Western blotting with antibodies against N. bombycis β -tubulin to analyze Sf9 cultures infected with the parasite spores and demonstrated the prospects of immunochemical methods to assay its intracellular growth. Analysis of five genes of N. bombycis spore wall and polar tube proteins in infected cultures by reverse transcription (RT) PCR showed that expression of the polar tube protein PTP2 may serve as a specific marker of the parasite growth because only its transcripts were not detected in freshly inoculated Sf9 cells. The honeybee parasite V. ceranae infects Sf9 cells less efficiently. To find a sensitive and specific marker of the growth of this parasite, we analyzed the transcripts of its 13 genes in infected cultures using the same RT-PCR method. The spore wall protein SWP32 gene demonstrated the highest expression at the 4 th -day post infection with V. ceranae spores, alongside with specificity of PCR-amplification, and the absence of transcripts in freshly inoculated cultures. Thus, quantitative PCR analysis of its expression may help to assay the V. ceranae intracellular growth in the Sf9 cell line.