Identification of silkworm hemocyte subsets and analysis of their response to BmNPV infection based on single-cell RNA sequencing

A wide range of hemocyte types exist in insects but a full definition of the different subclasses is not yet established. The current knowledge of the classification of silkworm hemocytes mainly comes from morphology rather than specific markers, so our understanding of the detailed classification, hemocyte lineage and functions of silkworm hemocytes is very incomplete. Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of the baculoviruses, which are a major pathogens that specifically infects silkworms and cause serious loss in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of silkworm hemocytes in BmNPV and mock-infected larvae to comprehensively identify silkworm hemocyte subsets and determined specific molecular and cellular characteristics in each hemocyte subset before and after viral infection. A total of 19 cell clusters and their potential marker genes were identified in silkworm hemocytes. Among these hemocyte clusters, clusters 0, 1, 2, 5 and 9 might be granulocytes (GR); clusters 14 and 17 were predicted as plasmatocytes (PL); cluster 18 was tentatively identified as spherulocytes (SP); and clusters 7 and 11 could possibly correspond to oenocytoids (OE). In addition, all of the hemocyte clusters were infected by BmNPV and some infected cells carried high viral-load in silkworm larvae at 3 day post infection (dpi). Interestingly, BmNPV infection can cause severe and diverse changes in gene expression in hemocytes. Cells belonging to the infection group mainly located at the early stage of the pseudotime trajectories. Furthermore, we found that BmNPV infection suppresses the immune response in the major hemocyte types. In summary, our scRNA-seq analysis revealed the diversity of silkworm hemocytes and provided a rich resource of gene expression profiles for a systems-level understanding of their functions in the uninfected condition and as a response to BmNPV.