(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 …
(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 …
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 …
(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 …
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 …
(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 …
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 …
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, …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
Source data pertaining to data presented in Figure 3D–H.
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.
(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 …
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 …
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 …
Hemocyte markers defined by functional studies in Drosophila that correspond to ‘universal’, ‘granulocyte/plasmatocyte’, and ‘oenocytoid/ crystal cell’ lineages were compared across recent 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 …
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 …
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 …
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 …
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 …
Source data pertaining to data presented in Figure 5B–E.
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 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (An. gambiae) | Keele | Hurd et al., 2005; Ranford-Cartwright et al., 2016 | NA | |
Biological sample (An. gambiae) | adult female hemolymph | NA | NA | Perfused hemocytes from naïve (sugar-fed) or blood-fed (24 hr post-feeding) mosquitoes |
Sequence-based reagent | LRIM15 (qRT-PCR primers) | Smith et al., 2016 | AGAP007045 | F:CGATCCTGATCCTGAACGTGGGCTTC R:GCAAGCAAGCCACTCACAAATCCTCG |
Sequence-based reagent | LRIM16A (qRT-PCR primers) | Smith et al., 2016 | AGAP028028 | F:ATCAGAGTGCAGCACAAGTTGAAGGT R:TCTCTGTTAGCATAGCGCCTTCGTTC |
Sequence-based reagent | Lz (qRT-PCR primers) | This study | AGAP002506 | F:GCACCGTCAATCAGAACCAA R:TGCCACTGATCGAATGCTTG |
Sequence-based reagent | NimB2 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP029054 | F:CAATCTGCTCAAATGGCTGCTTCCACG R:GCTGCAAACATTCGGTCCAGTGCATTC |
Sequence-based reagent | PPO1 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP002825 | F:GACTCTACCCGGATCGGAAG R:ACTACCGTGATCGACTGGAC |
Sequence-based reagent | PPO2 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP006258 | F:TTGCGATGGTGACCGATTTC R:CGACGGTCCGGATACTTCTT |
Sequence-based reagent | PPO3 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP004975 | F:CTATTCGCCATGATCTCCAACTACG R:ATGACAGTGTTGGTGAAACGGATCT |
Sequence-based reagent | PPO4 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP004981 | F:GCTACATACACGATCCGGACAACTC R:CCACATCGTTAAATGCTAGCTCCTG |
Sequence-based reagent | PPO5 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP012616 | F:GTTCTCCTGTCGCTATCCGA R:CATTCGTCGCTTGAGCGTAT |
Sequence-based reagent | PPO6 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP004977 | F:GCAGCGGTCACAGATTGATT R:GCTCCGGTAGTGTTGTTCAC |
Sequence-based reagent | PPO8 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP004976 | F:CCTTTGGTAACGTGGAGCAG R:CTTCAAACCGCGAGACCATT |
Sequence-based reagent | PPO9 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP004978 | F:TGTATCCATCTCGGACGCAA R:AAGGTTGCCAACACGTTACC |
Sequence-based reagent | rpS7 (qRT-PCR primers) | Kwon and Smith, 2019 | AGAP010592 | F:ACCCCATCGAACACAAAGTTGACACT R:CTCCGATCTTTCACATTCCAGTAGCAC |
Sequence-based reagent | SCRB3 (qRT-PCR primers) | This study | AGAP005725 | F:CATCGGGACAGCTACATCCT R:TTATTGCTGCTACCGTTGCC |
Sequence-based reagent | SCRB9 (qRT-PCR primers) | This study | AGAP004846 | F:CGATATTCGGCGATGCAACT R:CACGCATGACACGATTCAGT |
Sequence-based reagent | GFP (T7 RNAi primers) | Kwon and Smith, 2019 | NA | F:TAATACGACTCACTATAGGGAGAATGGTGAGCAAGGGCGAGGAGCTGT R:CACGCATGACACGATTCAGT |
Sequence-based reagent | Lz (T7 RNAi primers) | This study | AGAP002506 | F:TAATACGACTCACTATAGGGCTGCAACCGTCCCAGAACAACGGC R:TAATACGACTCACTATAGGGACAAACCGGAGATCGTTGAATTTGG |
Sequence-based reagent | Nimrod B2 (RNA-FISH probe) | Advanced Cell Diagnostics | AGAP029054 | Severo et al., 2018 |
Sequence-based reagent | LRIM15 (RNA-FISH probe) | Advanced Cell Diagnostics | AGAP007045 | regions 2–874 of XM_308718.4 |
Sequence-based reagent | Lz (RNA-FISH probe) | Advanced Cell Diagnostics | AGAP002506 | regions 168–1372 of XM_312433.5 |
Sequence-based reagent | SCRB3 (RNA-FISH probe) | Advanced Cell Diagnostics | AGAP005725 | regions 337–1276 of XM_315741.5 |
Sequence-based reagent | SCRB9(RNA-FISH probe) | Advanced Cell Diagnostics | AGAP004846 | regions 402–1306 of XM_001688510.1 |
Commercial assay or kit | Standard macrophage depletion kit | Encapsula NanoSciences LLC | CLD-8901 | Control liposomes or clodronate liposomes were used in a 1:5 dilution in 1x PBS |
Commercial assay or kit | DNA Clean and Concentration kit | Zymo Research | D4013 | |
Commercial assay or kit | MEGAscript RNAi kit | Life Technologies | AM1626 | |
Commercial assay or kit | RevertAid First Strand cDNA Synthesis kit | Life Technologies | K1622 | |
Commercial assay or kit | RNAscope Multiplex Fluorescent Detection Reagents V2 | Advanced Cell Diagnostics | 323110 | |
Software, algorithm | Seurat | Butler et al., 2018 | ||
Software, algorithm | Monocle3 | Cao et al., 2019 | ||
Software, algorithm | alona | Franzén and Björkegren, 2020 | https://alona.panglaodb.se/ https://github.com/oscar-franzen/alona/ | |
Software, algorithm | Graph Pad Prism | Graph Pad Software, LLC | ||
Other | FITC-conjugatedWheat Germ Agglutinin (WGA) | Sigma | L4985 | 1:5000 |
Other | DRAQ5 | Thermo Fisher Scientific | 62251 | 1:1000 |
Other | Live/Dead Fixable Dead Cell Stain | Thermo Fisher Scientific | L34965 | 1:1000 |
Other | FluoSpheres Fluorescent Microspheres | Molecular Probes | F8821, F8823 | Red or Green fluorescent fluorospheres for phagocytosis assays |
Other | Opal Fluorophore reagent | Akoya Biosciences | Opal520 (FP1487001KT), Opal570 (FP1488001KT) | 1:1000 |
Other | ProLongDiamond Antifade Mountant with DAPI | Life Technologies | P36966 | |
Other | PowerUp SYBR Green Master Mix | Applied Biosystems | A25742 | |
Other | E-RNAi | http://www.dkfz.de/signaling/e-rnai3/idseq.php | ||
Other | DRSC RNA Seq Explorer | Tattikota et al., 2020 | https://www.flyrnai.org/scRNA/blood/ | |
Other | Raddi et al., 2020 | https://hemocytes.cellgeni.sanger.ac.uk/ | ||
Other | This study | https://alona.panglaodb.se/results.html?job=2c2r1NM5Zl2qcW44RSrjkHf3Oyv51y_5f09d74b770c9 |
FKPM values of individual immune cells following scRNA-seq analysis.
Differential gene expression in immune cell clusters displaying significant differences between naive and blood-fed cells.
Significant markers of immune cell clusters identified by the FindAllMarkers program using the Seurat toolkit.
Genes expressed in more than >80% of cells within each respective immune cell cluster.
Averaged gene expression of cells within each immune cell cluster.
Primers for qRT-PCR and dsRNA-mediated gene silencing.
Primers for RNAi.