Ookluc, a new tool to study Plasmodium transmission

Background: Malaria eradication requires new strategies of control. Targeting the different parasite stages is imperative, and impeding human to mosquito parasite transmission will be key for eradication. Gametocytes, the sexual stage infectious to mosquitoes, differ in morphology and biology between the most important Plasmodium species. Crucially, five distinct morphological and biochemical gametocyte stages are identified in P. falciparum, with mature banana-­‐shaped stage V gametocytes only appearing in the circulation 10 days after infection. Mature P. vivax gametocytes, on the other hand, appear in the circulation as early as 3 days after infection, and it isn’t clear whether there is a fast progression through distinct biochemical stages, or if these stages do not exist in P. vivax. Thus, studies using P. falciparum gametocytes are not be easily translated to P. vivax. The morphology and biological behavior of P. vivax gametocytes are more similar to those of P. knowlesi or the rodent species P. berghei and P. yoelii. Hence we propose that these species are best vivax malaria models for transmission studies. Methods: Here we present the generation of a P. bergheiparasite, named Ookluc, engineered to express luciferase activity only in zygote and ookinete stages. AnLuc gene was inserted into the P. bergheigenome, under the control of a zygote and ookinete-­‐specific promoter. Ookluc kinetics of fertilization was tested in vitro in conversion assays with ookinete medium. Results: Ookluc blood stages express minimum background luciferase, and in conversion assays, where gametocytes form gametes and fertilize, luciferase activity augments 50x after 6 hours, when the zygote is fully formed, and 1000x after 24 hours, when ookinetes are formed. The conversion assay can be performed in as little as 80μl in 96-­‐well plates, and is compatible with plate luminometers. Thus the assay is HTS scalable. Moreover, the Ookluc bears no drug-­‐resistance cassettes, and can be further genetically modified for reverse genetics studies. Conclusion: We present a valuable tool for identifying genes involved in transmission, or finding new transmission blocking drugs, which will likely be more easily translated to P. vivax thanthose found in studies using P. falciparum.