Temporal and spatial viral gene expression in Microplitis demolitor ovaries

Microplitis demolitor (Hymenoptera: Braconidae) is a parasitic wasp of caterpillars in the family Noctuidae, which harbors an endogenized virus, Microplitis demolitor Bracovirus (MdBV). MdBV transfers virulence genes to parasitized host immune cells, allowing for the successful development of wasp progeny. The ovaries of M. demolitor contain a specialized region that produces MdBV, termed the calyx. MdBV replication and packaging occurs within the nuclei of calyx cells in distinct stages of transcription. These stages have previously been described as the “early” stage, which is categorized by the transcription of genes encoding the viral RNA polymerase, and the “late” stage, designated by the transcription of structural genes encoding viral envelope and nucleocapsid proteins. Calyx cells grow in size as more virus is produced and finally lyse, causing virions to accumulate in the calyx lumen. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, we characterized cell type specific gene transcription within M. demolitor ovaries. Single-cell RNA sequencing was used to identify and isolate populations of cells producing viral genes, which were then confirmed to be calyx cells using spatial transcriptomics. Our results suggest that there are three stages of viral replication in calyx cells rather than the two stages previously reported. The single-cell resolution further indicates that early gene expression is turned off in larger calyx cells with late gene expression profiles. Altogether, these findings provide a deeper understanding of the biology and potential origin of calyx cells and report the first transcriptional progression of calyx cells at the single cell level.