Hallmarks of crustacean immune hemocytes at single-cell resolution

Abstract

In invertebrates, hemocytes are the key factors in innate immunity. However, the types of invertebrate immune hemocytes are unclassified due to the limitation of morphological classification. To determine the immune hemocytes of crustaceans, the heterogeneity of hemocytes of shrimp Marsupenaeus japonicus and crayfish Procambarus clarkii, two representative crustacean species, were characterized in this study. The results of single-cell RNA sequencing indicated that shrimp and crayfish contained 11 and 12 types of hemocytes, respectively. Each of different types of hemocytes specifically expressed the potential marker genes. Based on the responses of shrimp and crayfish to the infection of white spot syndrome virus (WSSV) and the challenge of lipopolysaccharide (LPS), four types of immune hemocytes of crustaceans were classified, including semi-granular hemocytes involved in antimicrobial peptide production, granular hemocytes responsible for the production of antimicrobial peptides, hemocytes related to cell proliferation and hemocytes in immunity-activated state. Therefore, our study provided the first classification of crustacean hemocytes as well as of immune hemocytes of crustaceans at the single-cell resolution, which would be helpful to understand the innate immunity of invertebrates.

Keywords: crustaceans; hemocytes; immune response; innate immunity; single-cell RNA sequencing.

Figure 1

Identification of the clusters of shrimp or crayfish hemocytes by single-cell RNA sequencing. (A) Schematic diagram highlighting the experiments for the isolation of shrimp or crayfish hemocytes and the sequencing of single-cell RNAs using the 10×chromium system. (B) The clusters of hemocytes by t-SNE analysis. A total of 14,258 hemocytes were characterized. The different types of hemocytes were indicated with different colors. The axes corresponded to the 2-dimensional embedding produced by the t-SNE algorithm. (C) t-SNE plot of 14,258 hemocytes colored by UMI counts. The hemocytes with greater UMI counts likely had higher RNA content than the hemocytes with fewer UMI counts. The axes corresponded to the 2-dimensional embedding produced by the t-SNE algorithm. Pairs of hemocytes that were close to each other had more similar gene expression profiles than the hemocytes that were distant from each other.

Figure 2

Functional analysis of shrimp hemocyte clusters. (A) Heat map of expression levels of the top 10 highly expressed genes in each hemocyte cluster. Each column represented a hemocyte cluster and each row indicated a gene. (B) Dot plots of genes highly expressed in 11 hemocyte clusters. Color gradient of the dot represented the expression level, while the dot size indicated the percentage of hemocytes expressing the genes. IGFBP, insulin-like growth factor-binding protein.

Figure 3

The shrimp hemocytes responsible for immunity. (A) The clusters of hemocytes from the combined single-cell RNA-seq data of healthy shrimp, WSSV-challenged shrimp and LPS-treated shrimp by t-SNE analysis. A total of 34,244 hemocytes were characterized. The different types of hemocytes were indicated with different colors. The axes corresponded to the 2-dimensional embedding produced by the t-SNE algorithm. (B) t-SNE displaying all identified hemocyte clusters in healthy shrimp, WSSV-challenged shrimp and LPS-treated shrimp. (C) Percentage of hemocyte clusters in healthy, WSSV-challenged and LPS-treated shrimp.

Figure 4

Classification of the clusters of crayfish hemocytes by single-cell RNA sequencing. (A) The clusters of hemocytes of healthy crayfish by t-SNE analysis. A total of 4,381 hemocytes were characterized. The different types of hemocytes were indicated with different colors. The axes corresponded to the 2-dimensional embedding produced by the t-SNE algorithm. (B) Dot plots showing the highly expressing genes in hemocyte clusters. Color gradient of the dot represented the expression level, and the dot size indicated the percentage of hemocytes expressing the genes.

Figure 5

The immunity-associated hemocytes of crayfish. (A) The clusters of crayfish hemocytes using the merged single cell RNA-seq data of healthy crayfish, WSSV-challenged crayfish and LPS-treated crayfish by t-SNE analysis. A total of 18,944 hemocytes were characterized. The different types of hemocytes were indicated with different colors. The percentage of each cluster was shown in the parenthesis. The axes corresponded to the 2-dimensional embedding produced by the t-SNE algorithm. (B) t-SNE displaying all the identified hemocyte clusters in healthy, WSSV-challenged and LPS-treated crayfish. (C) Percentage of each cluster in the hemocytes of healthy, WSSV-challenged or LPS-treated crayfish.

Figure 6

Identification of crustacean immune hemocytes. (A) The morphologically typical hemocyte subgroups of crustaceans. The typical hemocyte subgroups (granular hemocytes, semi-granular hemocytes and hyaline hemocytes) of shrimp and crayfish were determined based on the single-cell RNA sequencing data. The potential marker genes of three subgroups were indicated. (B) The ratio of the immune hemocyte clusters in the total clusters of crustaceans. The immune and non-immune hemocyte clusters were identified based on the single cell RNA-seq data of shrimp and crayfish. (C) The percentage of immune hemocytes in the shrimp and crayfish hemocytes. (D) Immune hemocytes in crustaceans. The immune hemocytes of shrimp and crayfish were assigned to 4 types based on the significant and specific gene expression in each cluster. ALF-like protein, anti-lipopolysaccharide factor-like protein; ALF-A1, anti-lipopolysaccharide factor A1; ALF-C2, anti-lipopolysaccharide factor C2; hcPcSPI2, SGC-specific kazal proteinase inhibitor, Cu/Zn SOD, Cu-Zn superoxide dismutase; PET-15, proliferation zone-enriched transcript 15; a2m, alpha2-macroglobulin; LpR1, lipoprotein receptor 1; ALF, anti-lipopolysaccharide factor; CRP, cortical rod-like protein; TSP, thrombospondin; HemTGase, hemocyte transglutaminase; Vago5, single VWC domain protein 5; FAMeT, farnesoic acid O-methyltransferase; PCNA, proliferating cell nuclear antigen; IGFBP, insulin-like growth factor-binding protein. (E) Dot plots profiling of genes of Vago family in each cluster of shrimp and crayfish. Color gradient of the dot represented the expression level, while the dot size indicated the percentage of hemocytes expressing the Vago genes. (F) Schematic diagram of immune hemocytes in crustacean.