Fig. 3: RNAi and in vivo validation of candidate genes involved in clodronate liposome function.

a Genes identified from both clodronate liposome screens were selected based on their enrichment in both screens, expression in mosquito hemocyte populations⁴⁴, and presumed biological function to select for candidate genes for further validation in vivo. Following the injection of gene-specific dsRNAs, the efficiency of RNAi was evaluated in whole mosquitoes via qRT-PCR two days post-injection (b). Expression data from three independent experiments are displayed as the mean ± SEM and compared to GFP controls. Statistical differences were examined using a two-tailed unpaired t-test for each individual gene and compared to controls. Bar graph colors denote differences between the control (dark grey), statistically significant (medium grey), and non-significant (light grey) genes. Exact P values are displayed in the figure where significant. Additional details of the statistical analysis are included in the Source Data file. c To determine the influence of candidate genes on clodronate liposome function, candidate genes were first silenced via the injection of dsRNAs, then control or clodronate liposomes were injected two days post-dsRNA injection. The effects of gene-silencing on clodronate liposome function were assessed by the expression of eater and Nimrod B2 as a proxy to measure immune cell depletion (d). The heatmap summarizes the effects of gene-silencing on the efficacy of clodronate liposome-mediated cell ablation at 8 and 24 h, where non-significant changes in eater and Nimrod B2 expression support that the gene-silenced background impairs clodronate liposome function. Data represent three or more independent experiments. For each RNAi background, expression data were compared between control liposomes and clodronate liposomes. Differences in relative gene expression were examined between GFP controls and each RNAi background using mixed-effects analysis and a Dunnett’s multiple comparison test. Adjusted P values are displayed in the figure where significant. Additional details of the statistical analysis are included in the Source Data file. Images in a and c were created in part using BioRender. Smith, R. (2025) https://BioRender.com/l758x5w and incorporate illustrations created by David Hall using Inkscape. Source data are provided as a Source Data file.