*3.4. E*ff*ect of Heparin on Fertilization*

When blood from *P. berghei*-infected mice was put into culture to obtain ookinetes and heparin was added to the culture either at the moment of blood extraction (t0) or 1 h later (t1), no significant effect was observed in the number of ookinetes produced when compared with untreated control cultures (Figure 4). However, the fold-increase in ookinete numbers when heparin was added at t<sup>0</sup> (ookinetes relative to normalized control: 3.32 ± 2.26 and 2.26 ± 1.18 for 5 µg/mL and 500 µg/mL heparin, respectively), though non-significant due to the high dispersion of the results, could indicate that heparin enhances fertilization. This effect might operate through heparin interactions with coagulation factors, which would facilitate gamete motility. Heparin use in MFAs was previously recommended over other anticoagulants such as EDTA, as better infection rates were obtained [19]. Consistently, no effect on ookinete numbers was observed when heparin was added at t1, when fertilization has already occurred [20]. These results indicated that the inhibitory activity of heparin on *Plasmodium* mosquito stages is not exerted during fertilization or zygote maturation. Although sodium citrate was not used in ex vivo assays, the presence of 500 µg/mL heparin bound a significant amount of calcium, and yet, exflagellation was not affected. However, the potential role of calcium sequestration on this part of the parasite's development deserves further exploration. ‐ ‐ μ μ ‐ μ

**Figure 4.** Effect of heparin in the ex vivo development of ookinetes. (**a**) Depiction of the method used for the ex vivo growth of ookinetes. The parasite development scheme has been adapted from Kuehn and Pradel [20]. (**b**) Effect of heparin on ex vivo ookinete maturation analyzed by flow cytometry (see Figure S4 and Table S1). ns: not significant.
