Purpose and aims: The comprehensive goal for this project is to utilize single-cell genome-wide transcriptional profiling to broaden our understanding of the mechanisms involved in sexual stage development of the malaria parasite, an event that is responsible for disease transmission. Plasmodium falciparum is responsible for the vast majority of malaria-induced deaths with an annual toll upward of 500.000 and where the vast majority of diseased are children ages one through five. Despite intense efforts to eliminate malaria, only moderate to low success has been achieved, thus highlighting the necessity to expand and refine current eradication strategies, in particular those targeting the inhibition of transmission of the disease. In order to eradicate malaria from regions of high endemicity, transmission blocking i.e. inhibition of the spread of the disease from one person to another, is a key factor to success. Due to a current lack of scientific knowledge of the biology behind the sexual stage of the P. falciparum lifecycle inside its human host and during development inside the mosquito vector, there is an overall limitation in the basis for selection of targets for the development of novel drug- or vaccine-interventions targeting the malaria transmission stages. Studies of global gene expression during P. falciparum development inside the mosquito have to date not been possible to perform due to; the lack of methods to synchronize mosquito stage parasites, and the lack of molecular tools available to target isolated events during mosquito stage development. Thus, the capacity of combining P. falciparum transmission assays using Anopheles gambiae mosquitos at the BSL3 insectary (SUMF) at the MBW, Stockholm University, with single-cell RNA-seq we will for the first time be able to perform targeted gene expression analyses of development during this bottleneck in the P. falciparum life cycle. We are collaborating with the MSCG, SciLifeLab Uppsala to sort these different cell types using FACS based isolation of single cells in 384-well plates pre-dispensed with lysis buffer. We further perform micromanipulation coupled to image capture to sort cells sem automatically, to enable morphological analysis with each single cell transcriptome. We are currently isolating and transcriptionally profiling single parasites during sexual stage development: gametocyte development in vitro, which naturally occurs inside the human host, and early zygote development, which occurs in the mosquito midgut. We anticipate that this will provide the malaria field with novel candidates for de novo drug or vaccine interventions but also immensely increase our general knowledge on malaria transmission stage biology.