Ecology of Disease Vector Program 1. Expanding our investigations of windborne mosquitoes (Huestis et al. 2019: Nature Yaro et al. 2022: Frontiers in Epidemiology), describing high-altitude migration across 7 mosquito genera comprising of 50 species. At altitude, females outnumbered males 6:1, and >90% of the females have taken at least one bloodmeal prior to their high-altitude flight. Based on their aerial densities, millions of mosquitoes engage in high-altitude flight across Mali alone. Fourteen of these mosquito species had been reported as vectors to 25 pathogens in West Africa, e.g., malaria, dengue, and Rift Valley fever. Our recent results reveal high infection rates of mosquitoes at altitude with arboviruses, plasmodia, and filariae, including dengue virus and the zoonotic West Nile and M'Poko viruses. For example, infectiousness rate with plasmodium was 2.8%. Fourteen species of avian plasmodia and 2 filariids were identified by sequencing. A manuscript describing these groundbreaking results is in preparation. 2) Aerial sampling across 5 ecozones from the Sahel to the equatorial forest yielded 56 mosquito species among 760 identified specimens (ongoing). Diversity and abundance were higher above perennial ecozones, i.e., equatorial forest than above the Sahel. Low ecozone specificity indicated that most species move between neighboring ecozones, but movement between the equatorial forest to the Sahel >750 km also occurs. 4) A new a collaboration is ongoing with the West-African CRIED led by Drs. Scott Weaver (UTMB) and Mawlouth Diallo (IPD) to evaluate if arboviruses use windborne mosquitoes at altitude to spread from enzootic sylvatic foci to distant sites where they cause outbreaks. We have trained a Senegalese team and assisted in the setup of an aerial sampling station in Kedougou, Senegal. Aerial and ground sampling has started synchronically with our team in Mali and Senegal. Hundreds of mosquitoes were collected at altitude from a station in Kedougou, Senegal and Bantako, Mali while thousands were collected at ground level. Currently the specimens are being analyzed. 5. A new An. stephensi surveillance program is underway in Mali targeting likely ports of entry (airport, truck main routes). Additionally, we are investing in advanced identification of mosquitoes in Mali using AI visual system (IDx) and molecular tools. 6) Using our novel mosquito marking method (Faiman et al. 2021), we have measured extensive movement of mosquitoes across the entire village that undermined assessment of transmission blocking vaccine. In a subsequent study, we measured movement between villages up to 12 km away. Results based on DNA tags have been generated and are ready for final analysis. 7. A new project on Climate Change and malaria transmission has being initiated (application submitted to NIH ITCCH) hopefully be started if funded. Understanding Hemoglobinopathies in Malian Children and Adults The CRLD is a clinic, day hospital, and research center in Bamako, Mali that provides specialized care for more than 12,000 children and adults with sickle cell disease. The LMVR has established a collaboration with the CRLD to conduct clinical observational studies into the pathophysiology of sickle cell disease and the underlying mechanisms by which genetic and acquired hemoglobinopathies such as HbS, HbC, alpha thalassemia, and iron deficiency protect against severe malaria. To support this goal, we are building capacity to conduct clinical and laboratory research at the CRLD. Our natural history protocol has enrolled >4,000 individuals with sickle cell disease. This includes 50% SS, 40% SC, and 10% SB genotypes. The exceptional diversity of hemoglobin genotypes provides unique opportunities to characterize its function in the vascular system. Comparison of anti-RH5 antibodies from human clinical trials in the UK with antibodies elicited by repeated malaria infection We have compared the concentration and characteristics of antibodies to P. falciparum RH5, a leading blood-stage malaria vaccine candidate, resulting from clinical trials in nave volunteers with those elicited by natural infection in Malian adults and children. To address this, we first conducted a serological study of PfRH5 antibodies in over 400 sera from Malian individuals of varying ages at the peak of the transmission season and stratified them into groups by age (Willcox et al., Cell Rep. Med.). While nearly all participants showed antibodies to the P. falciparum FVO parasite extract, antibody prevalence to PfRH5 was limited and the concentrations were very low; antibodies to the other antigens in the invasion complex (CyRPA and RIPR) were so low as to preclude further affinity purification. For additional characterization and comparison of the Malian antibodies, we compared the concentration of infection-induced anti-RH5 with titers induced by vaccination. The vaccination titers in nave volunteers in the UK were approximately 200-fold higher than those from Malians. This shows that PfRH5 is immunogenic when presented in a proper context. In addition, the infection-elicited RH5 antibodies differed in binding site specificity and avidity from the vaccine-elicited ones. We then tested the interaction of Malian IgGs with both human monoclonal and polyclonal antibodies; in general the interactions were additive or synergistic in a parasite growth inhibition assay and interference was not seen. Overall, the pre-existing antibodies to PfRH5 in those living in endemic areas are likely to interact positively with vaccine-induced antibodies, supporting the transition of PfRH5 to efficacy studies in the field (Willcox et al.). Effect of the microbiota on Anopheles gambiae malaria transmission and insecticide resistance. The hypothesis that some bacteria in the mosquito microbiota are major determinants of Plasmodium sporozoite infection in field-collected mosquitoes is under investigation. The microbiota composition of Anopheles gambiae s.l. mosquito females infected with P. falciparum sporozoites was compared with that of uninfected females. Preliminary metagenomics results indicate seasonal differences in the microbiota, but no difference based on infection status. We currently investigate the impact of the microbiota on insecticide resistance. We have been collecting sand flies from distinct biotopes within Fansira Dierobougou, collecting over 570 Phlebotomus sand flies. The gut of each female Ph. duboscqi was dissected, and DNA extracted. Molecular determination of infection status, blood meal source and gut microbial communities PCR and 16S sequencing is underway. We obtained IRB approval for the study of human skin microbiota from CL cases and have obtained swabs from multiple lesions and control uninfected skin of 7 CL cases. Extraction of DNA and 16S sequencing is underway.