Fig. 6

Human blood triggers rapid DNA synthesis in the midgut epithelium of Anopheles gambiae and Aedes aegypti leading to increases in ploidy. Adult mosquitoes were fed on human blood supplemented with EdU and continuously maintained on a diet of sucrose/EdU for up to 72 h prior to dissection. 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 the posterior midgut region of interest (ROI) at 2, 12, 24 and 72 h post-blood meal (PBM) are shown (A), scale bar = 50 µm. B-C Quantification of EdU-positive cells using flow cytometry at 2,12, 24 and 72 h post-blood meal also shows early incorporation of EdU after the blood meal in An. gambiae and Ae. aegypti. D-E Quantification of PH3-positive cells in the posterior midgut after 2, 4, 6, 12, 24, 36, 48 and 72 h did not reveal any timepoint at which proliferation was high enough to account for the DNA synthesis observed in An. gambiae. Results are from at least three biological replicates. Values on top indicate mean values, and error bars are SEM. F Flow cytometry analysis shows that upon blood feeding, larger ploidy cells (> 32C) were generated in An. gambiae, effecting a persistent change to epithelial structure. G Ae. aegypti mosquitoes also showed a significant increase of 16C and > 32C cells, but this effect appeared to be transient as the cell population at 72 h post-blood meal reverted to the ploidy profile observed in the sugar-fed epithelium, with the larger cells generated during the peak of the digestive process appearing to be lost