*3.3. E*ff*ect on Oocyst Development of Heparin Administered to Mosquitoes by Blood Membrane Feeding*

‐ ‐ ‐ Heparin-Cy5 fed to female *A. stephensi* mosquitoes by whole blood MFAs was detected in the midgut of the insect for at least 24 h after administration (Figure 3a–d and Figure S3). Often, Cy5 fluorescence was only faintly observed in the dissected midgut (Figure 3a,b), but it intensified after having pushed out the blood bolus (Figure 3c,d). This might result from light being absorbed or screened by the compacted blood bolus. Heparin activity on ookinete to oocyst transition was then assessed with this method of administration. Heparin was added to the blood of mice infected with *P. berghei* ANKA-GFP, which was then offered to female *A. stephensi* by MFA (Figure 3e). Unfed mosquitoes were

removed, and eight days later, mosquitoes were dissected, and oocysts were counted. A significant decrease of PI and II was observed in mosquitoes fed with heparin-containing infected blood samples (Figure 3f,h–k). PI was 38% and 23% for respective MFA heparin concentrations of 5 µg/mL and 500 µg/mL, compared to 52% for the heparin-free control, whereas the mean II for the same samples was, respectively, 24.22 ± 65.10, 0.95 ± 4.12, and 36.38 ± 89.57 oocysts per midgut.

‐ ‐ μ ‐ **Figure 3.** Effect on ookinete development of heparin fed to mosquitoes by membrane feeding assay (MFA). (**a**–**d**) Fluorescence detection of heparin-Cy5 fed to *A. stephensi* female mosquitoes by blood feed. (**a**,**b**) Whole dissected midgut and (**c**,**d**) magnification of the same midgut with the blood bolus pushed away. (**b**,**d**) Bright field images of the microscope fields in panels **a** and **c**, respectively. (**e**) Depiction of the MFA method used in mosquito assays. (**f**) Effect on parasite development of non-modified heparin delivered by MFA. (**g**) Effect on parasite development of hypersulfated heparin delivered by MFA. ns: not significant. (**h**–**k**) Fluorescence images of representative mosquito midguts from the MFA 500 µg/mL non-modified heparin group (**h**,**i**) and from the MFA control group (**j**,**k**); the fluorescence signal is shown alone (**i**,**k**) and merged with bright field images of the midgut contours **h**,**j**).

 μ When a modified heparin with higher proportion of sulfated residues in the polysaccharide chain (hypersulfated heparin) was offered to mosquitoes by MFA, a significant decrease in PI and II

μ μ

was observed with as little as 5 µg/mL of heparin (Figure 3g). PI was 19% and 28% for respective hypersulfated heparin concentrations of 5 µg/mL and 500 µg/mL compared to 56% for the control, whereas the mean II for the same samples was, respectively, 1.53 ± 5.56, 1.74 ± 4.61, and 16.29 ± 31.94 oocysts per midgut. Although the extensive dialysis performed at the end of the heparin sulfation process should have removed any residual byproduct, future research has to rule out potential interferences of trace chemicals on the mechanism of oocyst formation. No impact on mosquito viability was observed for any of the heparins studied here (data not shown).

The observation that PI in blood feeding assays was significantly lower than in sugar meal experiments might be explained by the presence of sodium citrate, which is a calcium chelator used to prevent blood coagulation. Since the induction of exflagellation in *Plasmodium* requires calcium [17], sodium citrate could have a synergistic effect with heparin potentiating its inhibitory effect on oocyst formation. Heparin is also a potent calcium chelator which binds ca. one Ca2<sup>+</sup> ion per average disaccharide [18]. At the high 50 mg/mL heparin concentration of sugar meal assays, the calcium binding capacity of heparin was comparable to that of the sodium citrate amount used in MFAs. Although in these experiments no effect of heparin was seen on ookinete development, the suspected immiscibility of sugar feed and blood meal calls for caution before drawing any conclusions regarding the suspected inhibitory effect of calcium chelators on *Plasmodium* development in the mosquito.
