Abstract
Within the hemolymph, insect hemocytes constitute a heterogeneous population of macrophage-like cells that play important roles in innate immunity, homeostasis and development. Classification of hemocytes in different subtypes by size, morphology and biochemical or immunological markers has been difficult and only in Drosophila extensive genetic analysis allowed the construction of a coherent picture of hemocyte differentiation from pro-hemocytes to granulocytes, crystal cells and plasmatocytes. However, the advent of high-throughput single cell technologies, such as single cell RNA sequencing (scRNA-seq), is bound to have a high impact on the study of hemocytes subtypes and their phenotypes in other insects for which a sophisticated genetic toolbox is not available. Instead of averaging gene expression across all cells as occurs in bulk-RNA-seq, scRNA-seq allows high-throughput and specific visualization of the differentiation status of individual cells. With scRNA-seq, interesting cell types can be identified in heterogeneous populations and direct analysis of rare cell types is possible. Next to its ability to profile the transcriptomes of individual cells in tissue samples, scRNA-seq can be used to propose marker genes that are characteristic of different hemocyte subtypes and predict their functions. In this perspective, the identities of the different marker genes that were identified by scRNA-seq analysis to define 13 distinct cell clusters of hemocytes in larvae of the silkworm, Bombyx mori, are discussed in detail. The analysis confirms the broad division of hemocytes in granulocytes, plasmatocytes, oenocytoids and perhaps spherulocytes but also reveals considerable complexity at the molecular level and highly specialized functions. In addition, predicted hemocyte marker genes in Bombyx generally show only limited convergence with the genes that are considered characteristic for hemocyte subtypes in Drosophila.
Highlights
The blood cells or hemocytes play an important role in the regulation of whole-body homeostasis in insects
The main function of the macrophage-like cells is phagocytosis; while generally belonging to the same category, lepidopteran granulocytes differ from Drosophila plasmatocytes by the presence of specialized inclusions that are released upon activation [10, 11]
ScRNA-seq clusters that represent crystal cells in Drosophila are more isolated in principal component analysis and, in addition, represent a much lower proportion of the total hemocyte population (5% in Drosophila versus 28% in Bombyx; Supplementary Table 1; 4, 5). This indicates that crystal cells in Drosophila correspond to a much more differentiated cell type than oenocytoids in Bombyx, where several clusters have marker genes with relatively log2FC and are closely associated with clusters designated as granulocytes (Table 1; Figure 1)
Summary
The blood cells or hemocytes play an important role in the regulation of whole-body homeostasis in insects. It was hypothesized that the BmNPV-infected cells in clusters 1, 2, 3, 9, 11, 13 and 18 represent pro-hemocytes that were released from the hematopoietic organs in Bombyx larvae to replenish the loss of differentiated hemocytes (granulocytes, plasmatocytes, oenocytoids) in the hemolymph following baculovirus infection [24]. Sequences of marker genes assigned by scRNAseq to specific clusters, on the other hand, contain sufficient information to allow the prediction of their function based on knowledge from better studied model organisms, most notably Drosophila, for which scRNA-seq data of hemocyte types are already available [4, 5, 23]. In the analysis that follows, an attempt is made to predict the function of the hemocyte types in B. mori larvae based on the marker genes that were proposed following scRNA-seq analysis
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