Figure 5. 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 mosquitoes to estimate cell abundance (B). Data were collected from two independent experiments. Asterisks denote significance (****p< 0.0001). To more closely examine the population of Lz+ cells, RNA-FISH experiments were performed double staining for Lz and the oenocytoid marker, SCRB9 (C). The percentage of fixed cells positive for one, both, or neither marker is displayed with representative images. Data are summarized from two independent experiments. To determine the effects of Lz on immune cell populations, the abundance of LRIM15⁺/SCRB9^- (granulocyte) and LRIM15^-/SCRB9⁺ (oenocytoid) cells were evaluated by RNA-FISH after GFP (control)- or Lz-silencing (D). Data represent the mean ± SE of three independent replicates. Significance was determined using Mann-Whitney analysis and is denoted by an asterisk (*p < 0.05); ns, not significant. Since Lz expression has previously been associated with prophenoloxidase (PPO) expression, the expression of all eight genes identified in our scRNA-seq analysis were examined by qRT-PCR in GFP (control) - Lz-silenced mosquitoes (E). Data represent the mean ± SE of three or more independent replicates and were analyzed by a one-way ANOVA and Holm-Sidak’s multiple comparison test using GraphPad Prism 6.0. Asterisks denote significance (***p < 0.001). (F) Summary of Lz-silencing experiments which display a reduction in oenocytoid numbers and a specific sub-set of PPO gene expression which support that Lz is integral to the differentiation of the mosquito oenocytoid lineage.

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 confirmed by a reduction in the percentage of NimB2⁺/Lz⁺ cells following treatment with clodronate liposomes (CLD) when compared to liposome only (LP) controls (B), and further validated by qRT-PCR analysis of lozenge expression from perfused hemocytes (C). Two independent biological experiments were performed for RNA-FISH experiments in (A) and (B). Data were analyzed by Mann–Whitney analysis using GraphPad Prism 6.0. Bars represent mean ± SE. lozenge expression was analyzed using an unpaired t test to determine relative gene expression between LP and CLD treatments. Bars represent mean ± SE of three independent replications. Asterisks denote significance (*p < 0.05, ***p < 0.001). Scale bar, 10 µm.

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 from three experiments were analyzed using an unpaired t test in GraphPad Prism 6.0 to determine significance (**, p<0.01).