Ecology of vector mosquitoes 1. Expanding our investigations of windborne mosquitoes (Huestis et al. 2019: Nature Yaro et al. 2022: Frontiers Epidemiology), describing high-altitude migration across 7 mosquito genera comprising of 50 species. At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one bloodmeal prior to their departure. We estimate that millions of mosquitoes engage in high-altitude flight across Mali alone. Fourteen of these windborne mosquito species had been reported as vectors to 25 pathogens in West Africa, e.g., malaria, dengue, and Rift Valley fever (Yaro et al. 2022). Our recent results revealed high infection rates of mosquitoes at altitude with arboviruses, plasmodia, and filariae. For example, infectiousness rate with plasmodium was 2.8%. High altitude mosquitoes were found infected with human-zoonotic viruses: West Nile and MPoko. 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 36 mosquito species (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) Following these results, we established a collaboration 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. Recently, we trained a Senegalese team and assisted in the setup of an aerial sampling station in Kedougou, where aerial sampling have started synchronically with our team in Mali. 5. Tracking mosquitoes marked by deuterium (2H) from the end of the wet season until the beginning of the subsequent wet season supported that aestivation is a major persistence mechanism of An. coluzzii, contributing at least 20% of the adults at the onset of rain (Faiman et al. 2022: Nature Ecol. Evol.). 6. We have initiated surveillance of An. stephensi in Mali. 7) 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. 8. An epidemiological project, "Evaluating the determinants of the spread of COVID-19 between and within rural communities in Mali, West Africa based on blood-fed mosquitoes" is described separately (AI001328-01). Establishing Clinical and Laboratory Research Infrastructure at the Centre de Recherche et de Lutte Contre la Drepanocytose (CRLD) for studies of Hemoglobinopathies in Malian Children and Adults 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 2,905 individuals with sickle cell disease. This includes 1436 SS, 1132 SC, and 337 SB genotypes. We have also obtained approval from the local IRB to study malaria chemoprevention in children with sickle cell disease. 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.). Anopheles gambiae microbiota and malaria transmission 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 Plasmodium falciparum sporozoites was compared with that of uninfected females. Mosquitoes were collected in houses by insecticide spraying. The mosquito thorax was tested for presence of P. falciparum sporozoites by CSP ELISA and DNA was extracted for microbiome analysis with 16s ribosomal DNA sequencing. We are currently analyzing the metagenomics data. Evaluation of the occurrence of P. vivax near Bamako is ongoing. So far, one infected individual was identified. We started collecting sand flies from distinct biotopes within Fansira Dierobougou and each female is being processed to assess infection status and to determine the microbial communities influencing vector competence and transmission of CL. We also submitted for approval the IRB to initiate the study of human skin microbiota from CL cases. We finalized a study on visceral leishmaniasis in Tchad. Dr. Israel Demba Kodindo analyzed specimens by PCR confirming endemicity of the disease in his country for the first time.