Li X.Jutzeler K.Abatiyow B.Rezakhani N.Haile M.Leeb A.Patel H.Kappe S.Nosten F.Cheeseman I.H.Ferdig M.Anderson T.J.C.Vaughan A.Kumar S.Mahidol University2026-05-082026-05-082026-04-29Proceedings of the Royal Society B Biological Sciences Vol.293 No.2069 (2026)https://repository.li.mahidol.ac.th/handle/123456789/116582Malaria parasites are obligately sexual hermaphrodite protozoans, with gamete fusion occurring in the mosquito midgut, followed by meiosis and recombination. Parasite populations display a range of mating structures, from predominantly selfing to highly outcrossed; yet, the fitness consequences of selfing versus outcrossing remain poorly understood. This project investigated gamete fusion dynamics within mosquitoes and compared the fitness of selfed and outcrossed zygotes. We generated fluorescently labelled clones of NF54 (mCherry; African) and NHP4026 (GFP; Thai), crossed these parasites and genotyped 8540 oocysts from 435 mosquitoes collected 7-14 days post-infection. In two independent crosses, we observed decreasing proportions of outcrossed oocysts and increasing selfing over time. This pattern is consistent with the faster maturation of outcrossed oocysts compared with selfed oocysts. Our results suggest a substantial fitness advantage of outcrossing, potentially due to the removal of deleterious mutations accumulated during asexual replication in the vertebrate host. We also found that selfed NF54 oocysts were larger than either outcrossed or selfed NHP4026 oocysts, which may influence sporozoite production and transmission potential. Fluorescently labelled parasites provide clear resolution of mating patterns, temporal dynamics and transmission potential in mosquitoes. Importantly, faster maturation of outcrossed parasites may maximize levels of recombination in transmitted malaria parasite populations.Environmental ScienceBiochemistry, Genetics and Molecular BiologyAgricultural and Biological SciencesMedicineImmunology and MicrobiologyArticleSCOPUS10.1098/rspb.2026.01162-s2.0-1050374261511471295442049225