FIG 3.

Mosquito feeding promotes the degradation of the midgut basal lamina. Using a fluorescein-labeled collagen hybridizing peptide (CHP) to detect degraded collagen, midgut basal lamina integrity was examined temporally at 3, 6, 18, 24, and 48 h following blood or protein feeding (A). Heat-treated midguts (70°C for 10 min in 1× phosphate-buffered saline [PBS]) were used as a positive (+) control sample. The CHP fluorescence signal was quantified with ImageJ for each sample, and used to determine the relative fluorescence at each time point following blood feeding (B) or protein feeding (C). CHP binding analysis was performed in three independent experiments under blood-fed conditions and in two independent experiments with protein feeding. For each time point, three or more midgut samples were examined by fluorescence microscopy with images analyzed using ImageJ. Relative fluorescence was calculated using the 0-h time point as the baseline measurement, then examined across multiple time points using a one-way analysis of variance (ANOVA) with a Holm-Sidak multiple-comparison test using GraphPad Prism 7 software. Asterisks denote significance (***, P < 0.001; ****, P < 0.0001).