Development of a robust and reproducible method for the in vitro cultivation of Plasmodium falciparum gametocytes for mosquito infectivity studies

In vitro gametocytogenesis is a prerequisite for studying the sexual stages of the human malaria parasite Plasmodium falciparum. Procedures for the high-yield production of P. falciparum gametocytes stages I to V, and for artificially triggering male gametogenesis which is the next step of the life cycle inside the mosquito vector, are described. Gametocytes of P. falciparum are established from continuously maintained cultures of asexual blood stage parasites. In order for gametocytes to comprise a substantial proportion of parasitized erythrocytes, it is necessary for asexual stage parasites to reach a high density and become stressed in vitro before conversion to gametocyte production. This occurs typically 6-8 days after the dilution of a culture. Gametocytes then require at least a further 8 days to reach maturity. Cultures for gametocyte production are therefore maintained for a minimum of 14 days without further dilution with fresh uninfected erythrocytes in order to obtain a large proportion of mature and infectious gametocytes. We routinely perform gametocyte feeds to mosquitoes using cultures propagated continuously for the preceding 14-17 days. Here, a robust and reliable method for production of high levels of infectious gametocytes is described. This is evidenced by the demonstration of the in vitro transformation of male gametocytes into gametes by the process of exflagellation.