PROJECT SUMMARY We have pioneered a human-liver chimeric mouse model that supports Plasmodium falciparum liver stage development and transition to blood stage infection and used this model in the first P01 to successfully conduct more genetic crosses than have ever been achieved in the past and recovered hundreds of recombinant progeny. This has been pivotal to map genetic traits regulating important parasite phenotypes such as drug resistance. The biology of drug resistance is overwhelmingly studied in asexual blood stages because it is at this stage that drug resistance is of clinical relevance. However, for drug resistant parasites to spread through a human population, parasites must develop in a mosquito vector for onward transmission to human hosts and spread in the human population. Upon human infection, parasites must then be able to complete liver stage development and transition back to the blood stage infection to cause clinical disease and for onward transmission. This is extremely relevant to the evolution and spread of drug resistance since recombination could speed the acquisition and spread of resistance-associated genes in the field by introgression of genes that (i) enhance transmission fitness and (ii) pre-erythrocytic infection fitness, into drug-resistant parasite populations. This P01 Research Project will use genetic crosses to understand genetic factors involved in parasite fitness across the mosquito stages and pre-erythrocytic stages of the life cycle. We will determine if and how, after mating of two distinct parasite strains, particularly drug resistant and drug sensitive strains, recombinant progeny utilize fitness advantages to outcompete parent strains. Using distinctly fluorescent parental lines, we have already shown that both recombinant and parent oocysts are readily formed in the mosquito midgut. We will thus determine if superior recombinant parasite fitness during sporogony and/or sporozoite salivary gland colonization increases the success of human host infection and identify the genetic loci that control these fitness traits. We have also shown that recombinant parasite progeny can have enhanced fitness in human liver cell infection when compared to parent strains. We will thus identify the genetic factors that determine sporozoite infection for human hepatocytes using parents and recombinant parasites which show differences in levels of infectivity for human hepatocytes and differences in successful establishment of the intra-hepatocytic replication niche. Finally, we will determine genetic factors that enhance liver stage growth and duration of maturation into exo-erythrocytic merozoites and thereby give a head start in initiating blood stage infection. This work will unravel factors controlling parasite fitness during the mosquito stages and pre-erythrocytic stages of development and will show how these may enhance the spread of drug resistant parasites.