Heterogeneous distribution of avian malaria parasites across mosquito species: disproportionate involvement of Culex restuans in the Midwestern United States.

backgroundAvian malaria parasites and related haemosporidians are diverse and widespread vector-borne parasites that circulate within complex host-vector networks. Despite their ecological importance, their diversity and circulation within mosquito communities remain poorly understood in temperate North America. The Midwestern United States, located along major North American migratory flyways, supports diverse avian habitats and mosquito assemblages, providing an ideal setting to investigate these interactions. Here, we characterized the occurrence, phylogenetic diversity, and mosquito-lineage associations of avian haemosporidians in mosquitoes.

methodsA total of 233 pools comprising 6170 unfed female mosquitoes from seven species were collected from 38 protected natural areas in northern, central, and southern Illinois. Samples were screened for avian haemosporidian DNA by nested PCR targeting the mitochondrial cytochrome b gene. Positive samples were analyzed by multiplex PCR and sequencing to identify parasite lineages and assess potential mixed infections. Phylogenetic relationships were reconstructed, and mosquito-lineage associations were visualized using a bipartite network. Infection rates were estimated using maximum likelihood estimation (MLE) and minimum infection rate (MIR), and factors associated with infection prevalence in pooled samples were evaluated using pooled-binomial regression.

resultsOf 233 pools, 44 (18.9%) were positive for avian haemosporidians. Eleven lineages were identified, including nine Plasmodium and two Haemoproteus lineages; one Plasmodium lineage was novel. pTUMIG03 (Plasmodium unalis) was the most frequently detected lineage, whereas pSYAT05 (P. vaughani) showed the broadest mosquito distribution. Culex restuans harbored the highest lineage diversity and infection rate among the mosquito species examined. Mosquito species was a significant predictor of infection prevalence estimated from pooled samples, and Cx. restuans was most strongly associated with avian Plasmodium detection. Additionally, a deer-associated non-avian Plasmodium lineage was incidentally detected in two pools.

conclusionsMosquito populations in Illinois harbor a diverse assemblage of avian haemosporidians, with heterogeneous lineage distributions across mosquito taxa. Culex restuans emerged as the species most strongly associated with avian Plasmodium prevalence, suggesting an important role in local enzootic circulation. These findings expand current knowledge of avian haemosporidian diversity in North American mosquito communities and provide a basis for future studies integrating mosquito surveillance, avian host sampling, and vector competence experiments.