key: cord-0311558-o2aj2dz9
authors: Feng, Min; Xia, Junming; Fei, Shigang; Wang, Xiong; Zhou, Yaohong; Wang, Pengwei; Swevers, Luc; Sun, Jingchen
title: Identification of silkworm hemocyte subsets and analysis of their response to BmNPV infection based on single-cell RNA sequencing
date: 2020-10-18
journal: bioRxiv
DOI: 10.1101/2020.10.18.344127
sha: 1b8a3f2de42feac90248d208aa150acc24114922
doc_id: 311558
cord_uid: o2aj2dz9

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.

The domesticated silkworm, Bombyx mori, the only truly domesticated insect，has up-regulated DEGs in each cluster, we found that genes involved in immune-related 152 host response processes are enriched in cluster 0, 1, 2, 5, 7, 9, 11, 14, 17, and 18 153 ( Figure 1F , G). We speculate that these hemocyte clusters are the main effectors of 154 silkworm that respond to external stimuli. However, the clusters with the highest 155 numbers of DEGs and enrichment in immune-related host response processes 156 correspond to the clusters that are predominant in control hemocytes (clusters 0, 1, 2, 157 3, 7, 9, 11, 14, 17 and 18) (see further below).

To check that the strategy of combining the hemocytes of the control and 159 BmNPV groups into one set has no effect on the results, the hemocytes of the separate 160 groups of control and BmNPV infection were mapped to the unified set (BmNPV+CK) 161 using cell-specific barcodes. In addition, the hypervariable genes that were identified only detected in the cluster 17 and gloverin 2 was highly expressed in several clusters 186 such as clusters 2, 5, 9, and 14 ( Figure 2D ). AMP genes of the Cecropin B family are 187 highly expressed in hemocyte clusters 5 and 9, especially cluster 9 ( Figure 2D ).

Caspase-8, a molecular switch for apoptosis, necroptosis and pyroptosis (Fritsch et al., 189 2019), was found to be highly expressed in most of the cells of several clusters such 190 as clusters 0, 5 and 9 ( Figure 2E ). Caspase-3, encodes a key enzyme playing an 191 essential role in both exogenous and endogenous apoptotic pathways (D'Arcy, 2019). 192 We found that the silkworm Caspase-3 is only highly expressed in cluster 11, while In conclusion, hemocyte clusters 0, 1, 2, 5, 7, 9, 11, 14, 17 and 18 may be 201 involved in the antiviral responses after BmNPV infection. 203 We further compared the distribution of each silkworm hemocyte cluster in the Figure 3A ). On the other hand, only a few cells were detected in these hemocyte 210 clusters (0, 1, 2, 5, 9, 11, 14, and 17) in the BmNPV-infected sample ( Figure 3B ).

Cluster 5 was absent in the BmNPV-infected sample, while only one cell was detected 212 in clusters 0 and 9 ( Figure 3B ). Correspondingly, few cells of hemocyte clusters 3, 4, were detected in these clusters in the infection group ( Figure 3A , B). Comparing the 215 cellular landscapes, we therefore observe that the cells in the BmNPV-infected group and the control group were for a large part distributed in different clusters ( Figure 3C ).

After analyzing the proportion of control and infected cells in each cluster, it was 218 found that hemocyte clusters which may be involved in the antiviral response (0, 1, 2, 219 5, 9, 11, 14, and 17) showed a striking depletion in the BmNPV-infected sample 220 ( Figure 3D ). On the other hand, clusters 3, 4, 6, 8, 10, 13, 15 and 16 are the main 221 components of the silkworm hemocytes in the BmNPV-infected group ( Figure 3D ).

Within the clusters that dominated the BmNPV sample, however, few highly 223 expressed DEGs were detected ( Figure 1D ). We speculate that this reflects To characterize the host responses against to BmNPV infection, we calculated 271 the differential expression of genes between cell populations of the BmNPV-treated 272 and control samples using Seurat (applied on clusters with more than 25 cells).

Compared to the control sample, it was found that most of the host genes were analysis of DEGs was performed for each cluster, the GO terms "response to 279 stimulus" and "immune system process" and the KEGG pathways "Immune system", Figure 6D ). Thus, we speculate that on the third day after infection with BmNPV, the 297 host's antiviral system in hemocytes is almost in a state of collapse. Figure 8C) . Furthermore, the host immune responses in silkworm hemocytes were 509 inhibited by BmNPV infection at the late stage ( Figure 8D ). In order to replenish the 510 lost hemocytes including GR, PL, OE and SP, the host mobilizes a large number of 511 progenitor cells such as prohemocytes into the circulating hemolymph. However, after 512 the virus infects these progenitor cells, it may prevent them from continuing to 513 differentiate into other types of hemocytes. Therefore, the exhaustion of all blood 514 cells is inevitable ( Figure 8E ). Due to the exhaustion of the main hemocyte subsets 515 and the severe suppression of the immune response, the silkworm's antiviral system 516 collapses causing death ( Figure 8F ).

In summary, our scRNA-seq study on hemocytes in the silkworm represents a 518 rich resource of data that can be mined for future experiments to address the function 519 of these cells during development and in response to pathogenic infection in 520 Lepidoptera. The "marker genes" that were assigned to different clusters need to be Other data analyses including standardization, difference of gene expression, and 592 marker gene screening were also achieved by Seurat.

Notably, scRNAseq data were validated for analysis according to the following 594 QC criteria: 1) 200 < gene counts < 4000 per cell; 2) UMI counts < 30, 000 per cell; 3) 595 percentage of mitochondrial genes < 25%. After normalizing the data using Seurat, 596 gene expression level in scRNA-seq was calculated as log( 1 + ( UMI A ÷ UMI Total ) 597 × 10000 ). corresponding interacting specific antibodies, the PL and GR isolates described above 649 are therefore considered to be impure preparations. Competing financial interests 679 The authors declare that they have no conflicts of financial interest. 

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