Fig. 4: Clodronate liposome uptake is mediated by phagocytosis.

a Overview of endocytic pathways, clathrin-mediated endocytosis and phagocytosis, with their respective inhibitors. To address the manner by which liposomes undergo uptake, mosquitoes were injected with the respective endocytic inhibitors (a), with the uptake of LP-DiO particles by mosquito hemocytes (immune cells) assessed at 8 h post-injection as the percentage of LP-DiO⁺ hemocytes (b). Data were collected from individual mosquitoes (n; dots) from two independent replicates and examined using Kruskal-Wallis with a Dunn’s multiple comparisons test. Data are displayed as the mean ± SEM with exact P values are denoted in the figure where significant. Additional details of the statistical analysis are included in the Source Data file. c The effects of phagocytosis-inhibition (via cytochalasin D, cytoD) on clodronate liposome efficacy were evaluated using Nimrod B2 expression as a proxy for immune cell ablation. Data from four independent experiments are displayed as the mean ± SEM and were analyzed using multiple unpaired t-tests with a two-stage step-up correction to determine significance. Exact P values are displayed in the figure where significant. Additional details of the statistical analysis are included in the Source Data file. After further confirmation that cytoD treatment reduces the uptake of fluorospheres (d), candidate genes were evaluated for potential phenotypes that similarly influence phagocytosis (e). Data from d and e display the mean ± SEM values collected from individual mosquitoes (n) from two independent replicates. Statistical analysis was respectively performed with individual (two-sided Mann-Whitney) or multiple comparisons (Kruskal-Wallis and Dunn’s multiple comparisons test) to determine significance. Exact P values are displayed in the figure where significant. Additional details of the statistical analysis are included in the Source Data file. Bar graph colors in e denote differences between the control (dark grey), statistically significant (medium grey), and non-significant (light grey) genes. These data contribute to a model (f) suggesting that liposome uptake is mediated by phagocytosis and implicates Traf6 (with AGAP gene ID number in parenthesis) in phagocytic uptake. Summary figures in a and f were created using BioRender: Smith, R. (2025) https://BioRender.com/hfec788; Smith, R. (2025) https://BioRender.com/ljy5a6i. Source data are provided as a Source Data file.