Abstract
Seasonal movement of less-immune people from low- to high- transmission regions increase malaria risk and may introduce parasite strains to both areas. This study examined Plasmodium falciparum genetic diversity and connectivity between low-transmission highlands and endemic lowlands in Ethiopia to assess the contribution of seasonal agricultural migration in sustaining transmission. P. falciparum qPCR-positive dried blood spots collected from highland health facilities and lowland agricultural worksites were sequenced using multiplexed amplicon sequencing. Complexity of infection (COI) and infection pairwise relatedness were estimated and used for clustering analysis. Lowland populations (seasonal workers and residents) had higher COI and polyclonal infection rates (mean COI 2.62, 62%, n = 581) than highland residents (mean COI 2.00, 40%, n = 599). Similar expected heterozygosity (He ≈ 0.4) was observed, and P. falciparum infections from worksites showed high genetic connectivity between highland and lowland populations, with extensive parasite sharing, including 27 related clusters in highland cases and 10 in seasonal workers. Integrating parasite genomic data with epidemiological information revealed strong connectivity and low genetic differentiation between these regions linked by seasonal migration. These findings highlight how agricultural mobility likely drives parasite diversity and gene flow, implicating its role in sustaining malaria transmission.