*3.2. E*ff*ect on Oocyst Development of Heparin Administered to Mosquitoes by Sugar Meal*

Heparin-Cy5 fed in the sugar meal to female *A. stephensi* mosquitoes was detected in the midgut of the insects for up to 72 h after administration (Figure 2a–d and Figure S2). Heparin effect on ookinete to oocyst transition was then assessed in live mosquitoes, by offering them heparin by sugar feed during 48 h before infecting them by direct bite to a *P. berghei* ANKA-GFP-parasitized mouse (Figure 2e). Unfed mosquitoes were removed, and eight days later, mosquitoes were dissected, and GFP-expressing oocysts were counted. The prevalence of infection (PI, percentage of mosquitoes with ≥1 oocyst) and the infection intensity (II, number of oocysts per midgut) were not significantly affected when compared to untreated controls up to heparin concentrations in the sugar feed of 50 mg/mL (Figure 2f). It is likely that most of the sugar feed might be pushed out by the blood meal, in which case heparin would not interact with ookinetes. This result led us to explore new strategies to ensure the presence of heparin at the moment of ookinete development in the mosquito midgut by including heparin in a *Plasmodium*-infected blood meal.

‐ **Figure 2.** Effect on ookinete development of heparin fed to mosquitoes by sugar meal. (**a**,**c**) Fluorescence detection in (**a**) intact abdomen and (**c**) dissected midgut of heparin-Cy5 fed to *A. stephensi* female mosquitoes in a sugar meal. (**b**,**d**) Bright field images of the microscope fields in panels **a** and **c**, respectively. (**e**) Depiction of the method for sugar feed used in mosquito assays. (**f**) Effect on parasite development of heparin delivered by sugar swaps. ns: not significant.
