Fig. 2: Effect of hemocyte depletion in blood-feeding mosquitoes, midgut epithelium integrity and intestinal cell proliferation.

a Survival curve of NT, LPSM-injected, and CLD-injected mosquitoes fed on blood. Each experiment consisted of at least three biological replicates pooled together to generate the graph. Data are represented as percentage ± SE. n = 35 (NT), n = 25 (LPSMs), n = 31 (CLDs). Statistical analysis was performed using the log-rank (Mantel–Cox) test. ****p < 0.0001; ns not significant. b Mosquito phenotype 48 h after blood feeding. Note that NT and LPSM-injected mosquitoes have a normal abdomen, with a regular shape of the midgut containing a blood bolus. Some CLD-injected mosquitoes have blood spread along the abdomen, indicating a rupture of the midgut epithelium. c, d Smurf assay to measure the midgut epithelium integrity. c Smurf phenotype rate and mortality of NT, LPSMs-injected, and CLDs-injected mosquitoes. Data were generated from three biological replicates; they are represented as percentages in a bar graph. n = 84 (NT), n = 89 (LPSMs), n = 77 (CLDs). Statistical analyses of smurf phenotype and mortality rates were performed with Fisher´s exact test, ****p < 0.0001; ns not significant. d Mosquito phenotype of Smurf (left) and Non-Smurf (Right). e Representative image of immunofluorescent staining detecting phosphorylation of serine 10 of histone H3 (PH3) in sugar- and blood-fed NT, LPSM-injected, and CLD-injected mosquitoes. Red stain indicates anti-PH3 antibody; blue, nuclei stained with DAPI; PH3+ cells are indicative of cell division, bar scale = 50 μm. f Index of PH3+ cells per midgut in NT, LPSM-injected, and CLD-injected mosquitoes at 24 h post-blood feeding. Data were generated from three biological replicates; each dot indicates the number of PH3+ cells per midgut, presented as mean ±SD in a bar graph. n = 11 (NT), n = 10 (LPSMs), n = 11 (CLDs). Statistical analysis of PH3+ cell index was performed by ANOVA test followed of Tukey´s multiple comparation test. *p < 0.05; **,p < 0.01; ns not significant (g) Heatmap of Log2 (-fold change) of cell proliferation-related genes in the whole mosquito and midguts. Basal expression is indicated by the color white, upregulation by red, and downregulation by blue; cells contain Log2 values with SD. Significant changes were determined by ANOVA test, followed of Tukey´s multiple comparation test. *p < 0.05; ns not significant, (n = 3). Data and detailed statistical analysis of the -fold changes are available in the Source Data file and Supplementary Figs. 7k–m, 8k–m. b All experiments were performed three times. Source data are provided as a Source Data file. Schematic illustrations of the experimental strategy in the Fig. (2a, c, g) were created by Victor Cardoso-Jaime using Microsoft PowerPoint Version 16.92.