Fig. 4
Oral infection with Pseudomonas entomophila induces mitosis and ploidy changes in Anopheles gambiae and Aedes aegypti. Mature, 5-day-old females were infected with a sucrose-baited solution containing P. entomophila (OD600 100) and EdU, thereafter maintained on sucrose/EdU and dissected at 24- or 72-h post-infection (PI). Guts were treated with a Click-iT cocktail to label EdU (green) and stained with an anti-PH3 antibody (red) and DAPI (blue). Representative images of An. gambiae and Ae. aegypti at each timepoint are shown (A) (scale bar = 50 µm). For PH3 quantifications (B) and 24-h EdU pulse experiments (C) midguts were dissected from mosquitoes that were maintained on EdU from 0–24 h (day 1), 24–48 h (day 2) or 48–72 h (day 3) PI. EdU-positive cells were quantified in the posterior midgut region of interest (ROI). Cumulative EdU incorporation in the ROI in unchallenged (UC) mosquitoes and mosquitoes orally infected with P. entomophila/sucrose/EdU and maintained on sucrose/EdU over the full 72 h PI was also quantified. Results are from at least three biological replicates. Values on top indicate mean values. Statistics: Mann–Whitney test; *, **, and *** respectively indicate P values of < 0.05, < 0.001, and < 0.001. Ae. aegypti accumulated the greatest number of EdU-positive cells in the first day, with the rate of incorporation falling on the third day, while the rate of incorporation remained steady in An. gambiae across all three days. This was confirmed also by an EdU/BrdU switch assay, where after the first 24 h PI, mosquitoes were switched from EdU (green) to BrdU (red) (D). Most of the cells in the Ae. aegypti posterior midgut incorporated EdU, confirming that there was a period of intensive DNA synthesis during the first 24 h PI. In An. gambiae, the incorporation of EdU and BrdU was comparable, confirming a more modest but sustained response. Flow cytometry data showed that the cell populations from non-infected mosquitoes (sugar-fed) and infected mosquitoes were significantly different from each other. Representative histograms are shown in (E). Cell count in the Y-axis is normalized to the total number of cells. Ploidy is indicated at the top of each peak. Relative percentages of the total number of events were graphed as stacked bar plots to present the portion of each population relative to the total (F). Samples consisted of pools of 8 posterior midguts, n = 9 samples per condition, from at least three biological replicates