Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation

  1. Hyeogsun Kwon
  2. Mubasher Mohammed
  3. Oscar Franzén
  4. Johan Ankarklev
  5. Ryan C Smith  Is a corresponding author
  1. Department of Entomology, Iowa State University, United States
  2. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden
  3. Integrated Cardio Metabolic Centre, Department of Medicine, Karolinska Institutet, Novum, Sweden
  4. Microbial Single Cell Genomics facility, SciLifeLab, Biomedical Center (BMC) Uppsala University, Sweden
5 figures, 1 table and 8 additional files

Figures

Figure 1 with 4 supplements
scRNA-seq of An. gambiae immune cells.

(A) Graphical overview of the isolation of mosquito immune cells from naive and blood-fed mosquitoes. Following perfusion, cells were stained to enable processing by fluorescent activated cell …

Figure 1—figure supplement 1
FACS sorting of mosquito immune cell populations.

(A) Representative image of An. gambiae immune cell populations examined by flow cytometry. Distinct sub-populations of cells were separated into four gates: Gates 1–3 as unique populations based on …

Figure 1—figure supplement 2
Quality assessment of scRNA-seq analysis on mosquito immune cells.

The number of transcript reads per cell was used to determine the quality of single-cell data. Using a cutoff of 10,000 reads/cell, we removed 122 cells from our analysis, resulting in a total of …

Figure 1—figure supplement 3
Naive and blood-fed immune cells isolated in FACS analysis.

(A) Mosquito immune cells isolated from naive or blood-fed conditions visualized by t-SNE. (B) Cells from naive and blood-fed conditions were isolated in each of the FACS gatings, except for the …

Figure 1—figure supplement 4
Examination of differentially regulated genes in Clusters 2 and 4 under naive and blood-fed conditions.

Transcripts with significant differences in gene expression between naive and blood-fed conditions are displayed for Cluster 2 (A) and Cluster 4 (B). Black dots represent FKPM values obtained from …

Figure 2 with 8 supplements
Comparative analysis of mosquito immune cells.

(A) Marker gene expression displayed by dot plot across cell clusters. Dot color shows levels of average expression, while dot size represents the percentage of cells expressing the corresponding …

Figure 2—figure supplement 1
Comparisons of immune cell clusters to the An.

gambiae phagocyte proteome. Transcripts expressed in >60% of cells of each immune cell cluster were compared to the 811 proteins identified in the naive phagocyte-enriched proteome previously …

Figure 2—figure supplement 2
Comparisons of Cluster 1 to non-hemocyte cell populations.

Mosquito hemolymph perfusions routinely have contaminants (fat body, etc.) in addition to hemocytes (A). Using enriched gene sets for fat body (B) or muscle cells (C) defined by Raddi et al., 2020, …

Figure 2—figure supplement 3
Expression of hemocyte genes across all cell clusters.

Bubble plots display levels of average gene expression of hemocyte genes with previously described roles in Drosophila or Anopheles, with the dot size representing the percentage of cells expressing …

Figure 2—figure supplement 4
Expression of mosquito immune genes across cell clusters.

Expression patterns of known components of the An. gambiae IMD, Toll, and JAK-STAT pathways, as well as other antimicrobial proteins (AMPs) and complement factors with integral roles in mosquito …

Figure 2—figure supplement 5
Expression of serine protease inhibitors (SRPNs) and CLIP serine proteases across cell clusters.

Expression patterns of SRPN and CLIP family members across immune cell clusters. Each row represents the normalized (averaged) gene expressed of a given transcript across clusters, with differences …

Figure 2—figure supplement 6
Expression of chemosensory genes across cell clusters.

Expression patterns of annotated ionotropic receptors (IRs) (A), gustatory receptors (GRs) (B), odorant receptors (ORs) (C), and odorant-binding proteins (OBPs) (D) across immune cell clusters. Each …

Figure 2—figure supplement 7
Expression of specific tRNAs across cell clusters.

Expression patterns of specific tRNAs across immune cell clusters. Each row represents the normalized (averaged) gene expressed of a given transcript across clusters, with differences between cell …

Figure 2—figure supplement 8
Candidate markers of immune cell clusters and hemocyte sub-types.

Summary of candidate marker genes for each immune cell cluster and putative hemocyte sub-type. Solid lines denote strong differential gene expression indicative of one or more clusters, while dashed …

Figure 3 with 8 supplements
Definition of mosquito immune cell subtypes.

RNA-FISH and gene expression profiles across cell clusters for the ‘universal’ marker, NimB2 (A), the ‘granulocyte’ marker, LRIM15 (B), and ‘oenocytoid’ marker, SCRB9 (C). The percentage of adherent …

Figure 3—figure supplement 1
RNA-FISH of SCRB9+ immune cells.

RNA-FISH images of immune cells labeled with SCRB9 and stained with DAPI. Below is the corresponding phase contrast image of the same fixed cell. Scale bar, 10 µm.

Figure 3—figure supplement 2
Expression profile and RNA-FISH of SCRB3+ immune cells.

(A) Expression of SCRB3 across immune cell clusters. (B) RNA-FISH images of immune cells labeled with SCRB3 and stained with DAPI. Below is the corresponding phase contrast image of the same fixed …

Figure 3—figure supplement 3
Co-hybridization of SCRB9 and LRIM15 labels distinct immune cell populations by RNA-FISH.

RNA-FISH experiments were performed with both SCRB9 and LRIM15 probes, with the resulting percentage of cells expressing either, both, or neither immune cell marker. n, number of individual …

Figure 3—figure supplement 4
Expression of granulocyte and oenocytoid markers to distinguish immune cell sub-types.

Expression of marker genes to distinguish granulocyte (A) or oenocytoid (B) immune cell sub-types. Cluster 5 is included as an outgroup to help distinguish gene expression differences between …

Figure 3—figure supplement 5
Examination of conserved hemocyte markers across Anopheles and Drosophila single-cell studies.

Hemocyte markers defined by functional studies in Drosophila that correspond to ‘universal’, ‘granulocyte/plasmatocyte’, and ‘oenocytoid/ crystal cell’ lineages were compared across recent studies …

Figure 3—figure supplement 6
Examination of mosquito hemocyte markers in Anopheles single-cell studies.

Previously described mosquito-specific hemocyte markers were examined in recent single-cell studies in Anopheles (this study, Raddi et al., 2020) in prohemocyte/granulocyte populations (A) and in …

Figure 3—figure supplement 7
Comparison of immune cell clusters to Raddi et al.

Markers used to define immune cell subtypes (HC1-6) in Raddi et al., 2020 were displayed as bubble plots across immune cell clusters identified in our study (A). Using previously described gene sets …

Figure 3—figure supplement 8
Comparisons of immune cell clusters to previously defined PPO6low and PPO6high hemocyte populations.

To correlate the immune cell clusters identified in our analysis with previously described PPO6low and PPO6high hemocyte populations (Severo et al., 2018), we examined the expression of LysI (A) and …

Lineage analysis of mosquito immune cells.

Using Monocle3, mosquito immune cells were visualized by UMAP to reveal two distinct lineages in pseudotime under naive (A), blood-fed (B), or combined (naïve and blood-fed) samples (C) with the …

Figure 5 with 2 supplements
Lozenge promotes oenocytoid differentiation.

RNA-FISH and gene expression profiles across cell clusters for lozenge (Lz) (A). Scale bar, 10 µm. The percentage of adherent Lz+/NimB2+ or Lz-/NimB2+ cells were examined in naïve adult female …

Figure 5—figure supplement 1
Phagocytic properties of a subset of lozenge (lz)+ cells.

RNA-FISH experiments were paired with phagocytosis assays using the injection of fluorescent beads, which display a subset of lozenge (lz)+ cells with phagocytic properties (bead +) (A). This was …

Figure 5—figure supplement 2
Validation of lozenge knockdown following RNAi.

To disrupt lozenge expression, dsRNA corresponding to GFP (control) or lozenge were injected into adult female mosquitoes and evaluated for knockdown 3 days post-dsRNA injection by qRT-PCR. Data …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (An. gambiae)KeeleHurd et al., 2005;
Ranford-Cartwright et al., 2016
NA
Biological sample (An. gambiae)adult female hemolymphNANAPerfused hemocytes from naïve (sugar-fed) or blood-fed (24 hr post-feeding) mosquitoes
Sequence-based reagentLRIM15 (qRT-PCR primers)Smith et al., 2016AGAP007045F:CGATCCTGATCCTGAACGTGGGCTTC
R:GCAAGCAAGCCACTCACAAATCCTCG
Sequence-based reagentLRIM16A (qRT-PCR primers)Smith et al., 2016AGAP028028F:ATCAGAGTGCAGCACAAGTTGAAGGT
R:TCTCTGTTAGCATAGCGCCTTCGTTC
Sequence-based reagentLz (qRT-PCR primers)This studyAGAP002506F:GCACCGTCAATCAGAACCAA
R:TGCCACTGATCGAATGCTTG
Sequence-based reagentNimB2 (qRT-PCR primers)Kwon and Smith, 2019AGAP029054F:CAATCTGCTCAAATGGCTGCTTCCACG
R:GCTGCAAACATTCGGTCCAGTGCATTC
Sequence-based reagentPPO1 (qRT-PCR primers)Kwon and Smith, 2019AGAP002825F:GACTCTACCCGGATCGGAAG
R:ACTACCGTGATCGACTGGAC
Sequence-based reagentPPO2 (qRT-PCR primers)Kwon and Smith, 2019AGAP006258F:TTGCGATGGTGACCGATTTC
R:CGACGGTCCGGATACTTCTT
Sequence-based reagentPPO3 (qRT-PCR primers)Kwon and Smith, 2019AGAP004975F:CTATTCGCCATGATCTCCAACTACG
R:ATGACAGTGTTGGTGAAACGGATCT
Sequence-based reagentPPO4 (qRT-PCR primers)Kwon and Smith, 2019AGAP004981F:GCTACATACACGATCCGGACAACTC
R:CCACATCGTTAAATGCTAGCTCCTG
Sequence-based reagentPPO5 (qRT-PCR primers)Kwon and Smith, 2019AGAP012616F:GTTCTCCTGTCGCTATCCGA
R:CATTCGTCGCTTGAGCGTAT
Sequence-based reagentPPO6 (qRT-PCR primers)Kwon and Smith, 2019AGAP004977F:GCAGCGGTCACAGATTGATT
R:GCTCCGGTAGTGTTGTTCAC
Sequence-based reagentPPO8 (qRT-PCR primers)Kwon and Smith, 2019AGAP004976F:CCTTTGGTAACGTGGAGCAG
R:CTTCAAACCGCGAGACCATT
Sequence-based reagentPPO9 (qRT-PCR primers)Kwon and Smith, 2019AGAP004978F:TGTATCCATCTCGGACGCAA
R:AAGGTTGCCAACACGTTACC
Sequence-based reagentrpS7 (qRT-PCR primers)Kwon and Smith, 2019AGAP010592F:ACCCCATCGAACACAAAGTTGACACT
R:CTCCGATCTTTCACATTCCAGTAGCAC
Sequence-based reagentSCRB3 (qRT-PCR primers)This studyAGAP005725F:CATCGGGACAGCTACATCCT
R:TTATTGCTGCTACCGTTGCC
Sequence-based reagentSCRB9 (qRT-PCR primers)This studyAGAP004846F:CGATATTCGGCGATGCAACT
R:CACGCATGACACGATTCAGT
Sequence-based reagentGFP (T7 RNAi primers)Kwon and Smith, 2019NAF:TAATACGACTCACTATAGGGAGAATGGTGAGCAAGGGCGAGGAGCTGT
R:CACGCATGACACGATTCAGT
Sequence-based reagentLz (T7 RNAi primers)This studyAGAP002506F:TAATACGACTCACTATAGGGCTGCAACCGTCCCAGAACAACGGC
R:TAATACGACTCACTATAGGGACAAACCGGAGATCGTTGAATTTGG
Sequence-based reagentNimrod B2 (RNA-FISH probe)Advanced Cell DiagnosticsAGAP029054Severo et al., 2018
Sequence-based reagentLRIM15 (RNA-FISH probe)Advanced Cell DiagnosticsAGAP007045regions 2–874 of XM_308718.4
Sequence-based reagentLz (RNA-FISH probe)Advanced Cell DiagnosticsAGAP002506regions 168–1372 of XM_312433.5
Sequence-based reagentSCRB3 (RNA-FISH probe)Advanced Cell DiagnosticsAGAP005725regions 337–1276 of XM_315741.5
Sequence-based reagentSCRB9(RNA-FISH probe)Advanced Cell DiagnosticsAGAP004846regions 402–1306 of XM_001688510.1
Commercial assay or kitStandard macrophage depletion kitEncapsula NanoSciences LLCCLD-8901Control liposomes or clodronate liposomes were used in a 1:5 dilution in 1x PBS
Commercial assay or kitDNA Clean and Concentration kitZymo ResearchD4013
Commercial assay or kitMEGAscript RNAi kitLife TechnologiesAM1626
Commercial assay or kitRevertAid First Strand cDNA Synthesis kitLife TechnologiesK1622
Commercial assay or kitRNAscope Multiplex Fluorescent Detection Reagents
V2
Advanced Cell Diagnostics323110
Software, algorithmSeuratButler et al., 2018
Software, algorithmMonocle3Cao et al., 2019
Software, algorithmalonaFranzén and Björkegren, 2020https://alona.panglaodb.se/
https://github.com/oscar-franzen/alona/
Software, algorithmGraph Pad PrismGraph Pad Software, LLC
OtherFITC-conjugatedWheat Germ Agglutinin (WGA)SigmaL49851:5000
OtherDRAQ5Thermo Fisher Scientific622511:1000
OtherLive/Dead Fixable Dead Cell StainThermo Fisher ScientificL349651:1000
OtherFluoSpheres Fluorescent MicrospheresMolecular ProbesF8821, F8823Red or Green fluorescent fluorospheres for phagocytosis assays
OtherOpal Fluorophore reagentAkoya BiosciencesOpal520 (FP1487001KT), Opal570 (FP1488001KT)1:1000
OtherProLongDiamond Antifade Mountant with DAPILife TechnologiesP36966
OtherPowerUp SYBR Green Master MixApplied BiosystemsA25742
OtherE-RNAihttp://www.dkfz.de/signaling/e-rnai3/idseq.php
OtherDRSC RNA Seq ExplorerTattikota et al., 2020https://www.flyrnai.org/scRNA/blood/
OtherRaddi et al., 2020https://hemocytes.cellgeni.sanger.ac.uk/
OtherThis studyhttps://alona.panglaodb.se/results.html?job=2c2r1NM5Zl2qcW44RSrjkHf3Oyv51y_5f09d74b770c9

Additional files

Supplementary file 1

FKPM values of individual immune cells following scRNA-seq analysis.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp1-v3.xlsx
Supplementary file 2

Differential gene expression in immune cell clusters displaying significant differences between naive and blood-fed cells.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp2-v3.xlsx
Supplementary file 3

Significant markers of immune cell clusters identified by the FindAllMarkers program using the Seurat toolkit.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp3-v3.xlsx
Supplementary file 4

Genes expressed in more than >80% of cells within each respective immune cell cluster.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp4-v3.xlsx
Supplementary file 5

Averaged gene expression of cells within each immune cell cluster.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp5-v3.xlsx
Supplementary file 6

Primers for qRT-PCR and dsRNA-mediated gene silencing.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp6-v3.docx
Supplementary file 7

Primers for RNAi.

https://cdn.elifesciences.org/articles/66192/elife-66192-supp7-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/66192/elife-66192-transrepform-v3.docx

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