scRNA-seq analysis of C. gigas circulating hemocytes reveals 7 transcriptomic cell clusters

(A) Schematic of the scRNA-seq 10X Genomics Chromium microfluidic technology and bioinformatics processing workflow used. Dissociated hemocytes were collected from a pathogen-free oyster and encapsulated in droplets for processing. After sequencing, the data were processed bioinformatically. (B) Uniform Manifold Approximation and Projection (UMAP) plot for dimensional reduction of the data set and summary of cells and the number of Differentially Expressed Genes (DEGs) in each cluster. The table shows the characteristics (number of cells, percentage of total cells and number of Differentially Expressed Genes in each cluster) of the seven clusters identified. (C) Dot plot representing the ten most enriched DEGs per cluster based on average expression (avg_log2FC). The color gradient of the dot represents the expression level, while the size represents the percentage of cells expressing each gene per cluster.

Top 10 overexpressed genes identified in each transcriptomic cluster.

The first column indicates the gene number according to the annotation. ‘log2FC’ represents the log2 fold change of the gene in the cluster compared to all other cells. ‘Pct1’ is the percentage of cells expressing the gene in the cluster and ‘Pct2’ is the fraction of cells expressing the gene in all other clusters. The description is the annotation of the expressed gene (adjusted p-value < 0.05).

KEGG and Gene Ontology analysis of the gene signature in each cluster.

(A) A synthetic representation of the KEGG pathway analysis is shown. Colored columns represent the 7 transcriptomic clusters. Each row is a KEGG pathway, the colored dot represents the - log(p-value) and the dot size represents the number (count) of enriched genes in each pathway category. The fold enrichment is shown on the x-axis. Panels (B) show the results of Gene Ontology terms (GO-terms) for Biological Processes (BP), Cellular Components (CC) and Molecular Functions (MF) respectively, obtained with the genes of each cluster (Absolute value Log2FC > 0.25 and significant p-value < 0.001) using RBGOA analysis (p-value <= 0.001) for three different ontology universes. Each panel corresponds to one ontology universe, and the analysis highlights enriched and depleted terms. The dot size indicate the proportion of significant GO-terms and the gradient scale the p-adjusted value.

C. gigas naive hemocyte formula and Percoll gradient hemolymph fractionation

(A) Morphology, percentages and characteristics of the 7 cell types identified by MCDH staining. H : Hyalinocyte, ML : Macrophage Like, BBL : Basophilic Blast Like cell, ABL : Acidophilic Blast Like cell, SGC : Small Granule Cell, BGC : Big Granule Cell, VC : Vesicular Cell. Scale bar : 5 µm. For the ML, SGC, BGC, and VC hemocyte types, size refers to the average granule diameter, shape describes the morphology of the granules (e.g., round, ellipsoid), and particularities highlight distinguishing features such as granule color or fluorescence properties observed under specific staining or imaging conditions. (B) Sorting of hemocytes on a discontinuous Percoll gradient. 7 fractions were identified along the gradient at the top of each density cushion (from d=1.0647 at #1 to d= 1.1049 at #7). (C) Representation of the average values (from 5 different fractionation experiments) of the different hemocyte types in the seven percoll gradient fractions compared to the average hemolymph composition of a naive oyster (Total). VC : Vesicular Cells, BGC : Big Granule Cells, SGC : Small Granule Cells, ABL : Acidophilic Blast Like cells, BBL : Basophilic Blast Like cells, ML : Macrophage Like cells and H : Hyalinocytes respectively. (Supp. Fig. S4 for statistics).

Table of the 14 marker genes specific to the different transcriptomic clusters.

Gene number, average Log2FC, pct1/pct2 ratio (percentage of cells expressing this transcript in the cluster divided by the percentage of all other cells expressing this transcript) and cluster number are reported. The description is taken from our annotation and the marker name is derived from the description.

Characterization of molecular markers specific to the different hemocyte morphotypes.

(A) Relative gene expression level of the 14 markers in the various fractions after gradient density sorting. The graphs show the relative expression of genes compared to their expression in total hemocytes in the various fractions (red dotted line). Relative gene expression levels were normalized to the reference gene Cg-rps6 and the 2-ΔCt method was used to calculate relative expression levels, where ΔCt represents the difference between the target gene’s Ct value and the reference gene’s Ct value. Standard deviations were calculated based on four independent experiments. (B) Average percentage of each hemocyte type in the 7 Percoll gradient fractions used to quantify marker gene expression by qPCR. (C) Correlation matrix between the relative gene expression of each marker gene in each fraction and the percentage of each hemocyte type in the same fractions. Values and color scale represent the Pearson correlation coefficient (r) ranging from -1 (inverse correlation) to +1 (full correlation). H : Hyalinocyte, ML : Macrophage Like, BBL : Basophilic Blast Like cell, ABL : Acidophilic Blast Like cell, SGC : Small Granule Cell, BGC : Big Granule Cell, VC : Vesicular Cell. LACC24 : Laccase 24, CLEC : C-type lectin domain-containing protein, EGFL : EGF-like domain-containing protein 8, LEVAR : Putative regulator of levamisole receptor-1, TGC : TGc domain-containing protein, XBOX : X-box binding protein-like protein, MLDP : ML domain containing protein, HMGB1 : High mobility group protein B1, CUBN : Cubilin, GAL : Galectin, CAV : Caveolin, NAT1 : Natterin-1, MOX : DBH-like monooxygenase protein 1, GPROT : G protein receptor F1-2 domain-containing protein.

Phagocytosis, Reactive Oxygen Species production capacity and copper storage of hemocytes.

(A) Images of small granule cells (SGC) and macrophage-like (ML) cells with phagocytosed zymosan particles (panels a & c - red stars) and Vibrio tasmaniensis LMG20012T bacteria (panels b & d - red stars) from whole hemolymph sample. Scale bar : 5 µm. (B) Quantification of the phagocytic activity of zymosan particles by each cell type. The graph shows the result of 3 independent experiments. (C) Luminescence recording to detect the production of Reactive Oxygen Species (ROS). In orange, a biphasic curve was obtained on naive oyster hemolymph after zymosan addition at t = 0 min. In blue, the control condition corresponds to hemocytes without zymosan addition. (D) Graph showing the intensity of ROS production in each Percoll fraction. Normalized burst intensity was calculated from the luminescence peak obtained from each fraction. In blue, no drug was added to the experiment, in orange, ROS production was impaired by the addition of apocynin. (E) NBT (NitroBlueTetrazolium) staining of hemocytes exposed to zymosan particles. Hemocytes morphology after MCDH staining: Macrophage Like (a), Basophilic (b) and Acidophilic (c) Blast cells. NBT staining of the different types of hemocytes (d-f). Red stars show zymosan and bacteria particles. Black arrows indicate Macrophage-Like cells. Scale bar : 10 µm (F) Quantification of NBT-positive cells present in the total hemolymph of oysters exposed to zymosan. (H) UMAP plots showing cells expressing NADPH oxidase found in the scRNA- seq dataset and their expression level. (G) Labeling of intracellular copper stores in C.gigas hemocytes. MCDH (upper panels) and rhodanine (lower panels) staining of oyster hemocytes to reveal copper accumulation. Scale bar : 10µm. For panels (B), (D) and (F) the alphabetic characters displayed above the data points in each plot represent statistically significant differences among the groups, as determined by Tukey’s test following ANOVA. Groups denoted by different letters differ significantly at the p < 0.05 level of statistical significance. H : Hyalinocytes, ABL : Acidophilic Blast-Like cells, BBL : Basophilic Blast-Like cells, ML : Macrophage-Like cells, SGC : Small Granule Cells, VC : Vesicular Cells and BGC : Big Granule Cells.

Hemocyte expression profiles of some antimicrobial peptides.

(A), (B) and (C) Relative gene expression in the 7 Percoll hemocyte fractions of Big-Defensin1 & 2 (Cg- BigDefs), BPI (Cg-BPI) and hemocyte defensin (Cg-Defh), respectively, in comparison to the gene expression level in unfractionated hemolymph. Relative gene expression levels were normalized to the reference gene Cg-rps6. The 2-ΔCt method was used to calculate relative expression levels, where ΔCt represents the difference between the target gene’s Ct value and the reference gene’s Ct value. (D) Correlation matrix between the relative gene expression of BigDefensin1 & 2, BPI and hemocyte defensin gene in each fraction and the percentage of each hemocyte type in each fraction (H : Hyalinocytes, ABL : Acidophilic Blast Like, BBL : Basophilic Blast Like, SGC : Small Granule Cell, ML : Macrophage Like, BGC : Big Granule Cell, VC : Vesicular Cell. Values and color scale represent the Pearson correlation coefficient (r) ranging from -1 (inverse correlation) to +1 (full correlation).

Pseudotime ordering of cells revealed 6 potential differentiation pathways of hemocytes.

(A) UMAP plot of scRNA-seq analysis showing the 7 transcriptomic clusters used for pseudotime analysis. 4 clusters were identified cytologically (SGC for small granule cells - cluster 3, H for hyalinocytes - cluster 2, ML for Macrophage Like - cluster 1 and VC for vesicular cells - cluster 7), cl.4, cl.5, and cl.6 represent clusters 4, 5, and 6, respectively. (B) Graphical representation (UMAP projection) of the Monocle 3 pseudo-time order of the clustered cells. Cluster 4 (cl.4) was used as the origin for the pseudotime analysis. (C) (D) (E) (F) (G) and (H) show the gene expression level of selected marker genes obtained from the monocle3 trajectory analysis at the beginning and end of the modelized differentiation pathways (in red on the UMAP plot) from cluster 4 to hyalinocytes, to cluster 5 cells, to cluster 6 cells, to Vesicular Cells (VC), to Macrophage-Like cells (ML) and to Small Granule Cells (SGC) respectively. The color scale represents the normalized expression level of each gene. (I) Dot plot showing the average expression and the percentage of cells expressing identified transcripts encoding for transcription factors in the scRNA-seq dataset. Expression level expression is expressed in Log2FC and dot diameter represents the percent of cells expressing the gene.

Proposed hemocyte ontology in Crassostrea gigas based on the transcriptomic, cytological and functional results obtained.

Cells are colored according to the same color code as the transcriptomic clusters. Cluster numbers and cell types are indicated. To the left of the cells are the overexpressed transcription factors and to the right are the identified marker genes in each cluster. Functional characteristics of hyalinocytes, macrophage-like cells and small granule cells are marked in red. (AMP : AntiMicrobial Peptide, Burst : ROS production, Phago : phagocytosis)