Identification of Silkworm Hemocyte Subsets and Analysis of Their Response to Baculovirus Infection Based on Single-Cell RNA Sequencing

Abstract

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 and a major pathogen that specifically infects silkworms (Bombyx mori) and causes serious losses in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of 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 infectmadion. A total of 20 cell clusters and their potential marker genes were identified in silkworm hemocytes. All of the hemocyte clusters were infected by BmNPV at 3 days after inoculation. Interestingly, BmNPV infection can cause great changes in the distribution of hemocyte types. The cells appearing in the infection group mainly belong to prohemocytes (PR), while plasmatocytes (PL) and granulocytes (GR) are very much reduced. Furthermore, we found that BmNPV infection suppresses the RNA interference (RNAi) and immune response in the major hemocyte types. In summary, our results 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.

Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles

“…The technique of scRNA-seq was also applied to hemocytes of silkworm (Bombyx mori, Lepidoptera) (24). In this study, hemocytes were isolated from 5 th instar larvae at three days after experimental manipulation which consisted either of (1) injection into the body cavity of a high infectious dose of B. mori nucleopolyhedrovirus (BmNPV) (baculovirus infection) or (2) injection of saline solution (wounding).…”

“…After normalization, gene expression data from 22,286 cells were subjected to dimensionality reduction methods such as component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) (25). The R package Seurat (26) was used as a graph-based clustering method to obtain 20 hemocyte cell type clusters and to screen for marker genes in each cluster (24).…”

“…The cluster analysis of scRNA-seq of Bombyx hemocytes revealed that cell clusters associated with baculovirus infection (7 clusters: 1, 2, 3, 9, 11, 13, 18) were clearly separated from the cell clusters of the (wounded) control (9 clusters: 0, 4, 5, 7, 8, 10, 12,16,17), which reflects the severe impact of baculovirus infection on the composition of the hemocyte population (24). In 4 clusters (6,14,15,19), the contribution from both control and BmNPV-infected groups is less polarized (10-30% contributed by cells from BmNPV-infected group) (Table 1).…”

“…The cell clusters that are predominant in the BmNPV-infected group are characterized by much lower number of differentially expressed genes (DEGs) (range: 98 to 181) compared to the control group (range: 170 to 1571) (Table 1) and their morphological features resemble those of pro-hemocytes (24). It was therefore 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).…”

Quantification of new and archived Diaphorina citri transcriptome data using a chromosomal length D. citri genome assembly reveals the vector’s tissue-specific transcriptional response to citrus greening disease

“…Additionally, single-cell RNAseq is the next frontier of understanding insect-pathogen interactions, especially for intracellular symbionts, at the highest resolution. Currently, single-cell RNAseq has been done on very few insects, but the list is expanding [59–62].…”