Fig. 3

The midgut of a newly emerged female mosquito undergoes a rapid species-specific maturation process characterized by epithelial proliferation and/or endoreplication. The guts of 1, 2, and 3-day old sucrose-fed Aedes aegypti and Anopheles gambiae were dissected immediately following 24 h on a diet supplemented with EdU (administered, respectively, from pupal water to 24 h, 24–48 h, and 48–72 h), and are depicted after Click-iT EdU-labeling (green), anti-PH3 (red) and DAPI (blue) staining. Day 0 guts were dissected within six hours of emergence and stained with DAPI only (A) (scale bar = 50 µm). B Quantification of PH3-positive cells revealed higher levels of mitosis at 24 h post-emergence in Ae. aegypti but not An. gambiae. C In both species, EdU incorporation primarily occurred over the first 24 h post emergence. Plots depict the percentage of EdU-positive cells accumulated in the posterior midgut region of interest over 24 h of exposure in 1-, 2-, and 3-day-old mosquitoes, alongside percentages from mosquitoes which received EdU continuously (initially in pupal water, subsequently in sucrose) for 72 h post-emergence (cumulative). Results are three biological replicates. Values on top indicate mean values, and error bars are SEM. Brown-Forsythe and Welch ANOVA (analysis of variance) test for multiple comparisons was utilized. Dunnett’s T3 multiple comparisons test for multiple comparisons; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001. D Flow cytometry data from mosquitoes continuously exposed to EdU from the pupal phase showed that the cell populations of dissected posterior midguts at day 3 were significantly altered compared to day 0 in both An. gambiae and Ae. aegypti. Representative histograms are shown. Cell count in the Y-axis is normalized to the total number of cells. Ploidy is indicated at the top of each peak, and gating strategy with diploid controls can be found in Fig. S2. E Cell percentage relative to each peak of DNA content was plotted as stacked bar plots, equalizing values to a 100%. F In each cell population, percentages of EdU-positive cells were plotted (median with 95% CI) in stacked bars. Percentages of EdU-positive cells, from each cell population based on nuclei size, show that the cell populations ≥ 32C had the largest percentages of EdU incorporation. For flow cytometry, samples consisted of pools of 40 posterior midguts at day 0 and 25 posterior midguts at day three, n = 9 samples per condition, from at least three biological replicates. Brown-Forsythe and Welch ANOVA (analysis of variance) test for multiple comparisons was utilized to compare EdU positive percentages between ploidy groups. Groups were assigned ID as follows: 2C = a, 4C = b, 8C = c, 16C = d, 32C = e. Letters at the top of each bar indicate which groups are significantly different using Dunnett’s T3 multiple comparisons test for multiple comparisons