ABSTRACT Drug resistance mutations disrupt key physiological processes resulting in severe fitness costs; compensatory mutations are expected to evolve to restore fitness. Such compensatory mutations are extensively studied in bacterial pathogens, but are much less well understood in P. falciparum. This application aims to redress this by using three complimentary approaches to investigate compensation. First, we will exploit reverse genetic approaches to investigate the role of an amino acid transporter (pfaat1) in quinolone drug resistance evolution. Like the well-studied chloroquine resistance transporter, pfaat1 is situated on the food vacuole membrane, and several lines of compelling population genomic and experimental data suggest that interplay between these two transporters may play a role in drug resistance evolution. We will test competing hypothesis that mutations in this locus compensate for reduced fitness of CQ-resistant parasites, or play a role in response to other quinolone drugs. Genetic mapping methods (e.g. linkage mapping, genome wide association) typically aim to link parasite genotype with drug resistance; these approaches are poorly suited to identifying compensatory loci as these may have no influence on IC50. The second aim will use a modification of an alternative well-proven approach to identify resistance genes (in vitro drug selection) to specifically target compensatory loci. However, rather than selecting with drugs, we will introduce known resistance mutations onto a drug sensitive background, and then culture these parasites in the absence of drug pressure, to select for mutations that restore parasite fitness. We hypothesize that this will provide a powerful approach to understand the mutations and biochemical pathways that can compensate for pfcrt mutations underlying piperaquine resistance and kelch13 mutations underlying artemisinin resistance. Monitoring longitudinal evolution within parasite populations in the field provides a complimentary approach to identify compensatory loci: this can identify variants showing parallel rapid increase in allele frequency with known resistance loci. The final aim in this proposal will examine genes showing similar patterns of allele frequency change to kelch13 on the Thai-Myanmar border. We will test the hypothesis that these are enriched for genes involved for those playing a compensatory role in drug resistance evolution.