Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: CFP fluorescence was examined in whole mount fourth-instar larvae and pupae from PPO6 ( a ) or SPARC ( b ) transgenic lines. Arrowheads highlight presumed hemocytes. Scale bars: 1 mm. Potential differences in CFP expression between larvae and adult mosquitoes were examined by qPCR for both PPO6 ( c ) or SPARC ( d ) transgenic lines. For ( c and d ), expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled ( n = ~10) larvae or adult female mosquitoes, with values relative to rpS7 expression. Significance was determined using a two-tailed unpaired Student’s t -test. Exact P values are displayed in the figure where significant. ns not significant. Additional ex vivo analysis of perfused hemocytes from PPO6 ( e ) or SPARC ( f ) transgenic lines using microscopy was used to examine hemocyte fluorescence. The injection of fluorescent beads (red) prior to perfusion indicates that both PPO6-CFP + and SPARC-CFP + immune cell populations are comprised of phagocytic and non-phagocytic cells. Scale bars: 10 μm. Subpanels demonstrate different representative cell phenotypes. The specificity of CFP expression was examined in hemocytes and carcass samples via qPCR for the PPO6 ( g ) and SPARC ( h ) constructs across individual transgenic lines. The hemocyte-specific expression of NimB2 was used as a positive control for gene expression analysis. Expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled hemolymph perfusions ( n = >30 adult female mosquitoes) or adult female mosquitoes ( n = ~10) following perfusion (carcass), with values relative to rpS7 expression. Significance was determined using multiple two-tailed unpaired t -tests and a Holm–Šídák correction. Adjusted P values are displayed in the figure where significant. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Fluorescence, Transgenic Assay, Expressing, Two Tailed Test, Ex Vivo, Microscopy, Injection, Construct, Positive Control, Gene Expression

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: The percentage of PPO6 + ( a ), SPARC + ( b ), and LRIM15 + ( c ) hemocytes were evaluated under sugar-fed (SF), at 24 h post-feeding (24 h PBF), and at 48 h post-feeding (48 PBF). For each experimental condition in ( a – c ), data display the percentage of CFP+ ( a , b ) or GFP+ cells ( c ) of the total hemocytes collected from individual mosquitoes (dots). n number of individual mosquitoes examined. Data from three independent biological replicates were pooled and displayed as the mean ± SE. Differences in the shading of the bar graphs highlight differences between sugar-fed (light gray) and blood-fed (dark gray) conditions. Statistical analysis was performed using a one-way ANOVA with a Holm–Šídák comparison test. Adjusted P values are displayed in the figure where significant. ns not significant. For each transgenic construct, representative images are displayed at right for each experimental condition. Scale bars represent 10 μm. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Comparison, Transgenic Assay, Construct

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: Flow cytometry analysis was performed on immune cell populations resulting from LRIM15 + , SPARC + , and PPO6 + individual transgenic lines ( a – f ). Representative UMAPs resulting from spectral imaging flow cytometry for LRIM15-GFP ( a ), SPARC-CFP ( c ), and PPO6-CFP transgenics ( e ) display the overall cell populations and the composition of fluorescent cells as an overlay using the established gating for each of the 12 hemocyte subpopulations identified in wild-type mosquitoes. The total number of events for each cell classification are displayed on the right of each figure subpanel. These distributions are summarized in pie charts to display fluorescent cell subtypes for LRIM15-GFP ( b ), SPARC-CFP ( d ), and PPO6-CFP transgenics ( f ). Data were averaged from three independent biological replicates. To determine potential overlap between transgenic markers, crosses were performed to establish either LRIM15 + /SPARC + ( g ) or LRIM15 + /PPO6 + ( h ) genetic backgrounds. For each genetic background, the abundance of GFP + , CFP + , and GFP + /CFP + cells were examined by overlaying fluorescent cell populations on the UMAP and summarized in bar graphs to denote the hemocyte clusters represented for each fluorescent cell subtype. Data display the average of three independent biological replicates. UMAP analysis was performed using the Euclidean distance metric using FlowJo V10.10.0, including the following parameters: DRAQ5 signal, Maximum Intensity Forward Scatter (FSC), Maximum Intensity Side Scatter (SSC), and Maximum Intensity Light Loss. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Flow Cytometry, Transgenic Assay, Imaging

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: The injection of red fluorescent beads prior to perfusion enabled flow cytometry analysis of phagocytosis in mosquito immune cells ( a ) and the identification of multiple phagocytic immune cell phenotypes based on the intensity of bead signal ( b ). This resulted in the identification of non-phagocytic and phagocytic immune cell subtypes ( c ) that were confirmed by imaging ( d ). When phagocytic cells were further distinguished by bead signal intensity, we identified immune cell subtypes with low, medium, or high phagocytic capacity ( e ). Similar experiments with our LRIM15, SPARC, and PPO6 transgenic lines revealed the phagocytic ability of fluorescent immune cells ( f ) and the overall proportion of phagocytic immune cells ( g ), which are displayed as the mean ± SE of three biological replicates. h The phagocytic capacity of each transgenic line is visualized as the percentage of fluorescent cells displaying low, medium, or high bead-positive cells and displayed as the average of three independent biological replicates. Dot plots represent single replicates of three independent biological experiments. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Injection, Flow Cytometry, Imaging, Transgenic Assay