Vaccines that block transmission of malaria to mosquitoes have the potential to be critical tools in the drive to eliminate or eradicate malaria. Assays to estimate the potential benefit of these vaccines need to be standardized and validated in malaria exposed populations. The focus this year was on two assays conducted in the field: direct skin feeding assays (application of unfed mosquitoes to a human volunteer) and experimental hut assays (collection of mosquitoes from the home of a volunteer). These assays were conducted under NIAID protocol 15-I-0044 a Phase 1 study on the safety and immunogenicity of Pfs230 and Pfs25, a transmission blocking vaccine against Plasmodium falciparum. In total 2,006 direct skin feeding assays were performed with an infection rate of 2.9%. This number of feeding assays is unprecedented and required a significant increase in mosquito production, maintenance and dissection. Specimens were preserved from each feeding assay to utilize in the development of molecular assays to accurately determine parasite development as a means to assess transmission blocking activity. Current molecular markers being tested against field samples are the parasite and gametocyte markers 18s and Pfs25 with future work to be conducted using the ookinete specific genes SOAP and WARP. Currently direct skin feeding assays are conducted on all trial volunteers and one of the goals of this project is to confirm parasite gene signatures that predict infectivity to mosquitoes allowing feeds to be conducted only on those individuals who are truly infectious at a given timepoint. As part of this work, peripheral blood samples were collected from all individuals at every direct skin feeding to test against a panel of gametocyte marker genes up in infectious, down in infectious, and constant housekeeping. Work is ongoing to test these marker genes and evaluate whether they could be a robust predictor of infectivity. To date, over 400 experimental hut assays have been conducted on volunteers enrolled in a number of different clinical studies in Mali: NIAID protocols 11-I-N143, 14-I-N159 (closed studies), 13-I-N109, and 15-I-0044. Samples from some of these studies have been analyzed for feeding fidelity i.e. genetic comparison of the identity of the mosquito bloodmeal source with the genetic profile of the individual sleeping in the hut. Feeding fidelity is crucial to determine the validity of using experimental huts as a TBV efficacy surrogate. In Dongubougou, mosquito feeding fidelity was found to be 80% but only 62% in Bancoumana. In the 2015 season, the experimental hut collection procedures were extended to include a live catch of mosquitoes in the hut followed by a spray catch of dead mosquitoes. Live bloodfed mosquitoes were maintained in the laboratory for seven days for oocyst dissection; dead mosquitoes were preserved for genetic fidelity typing. Relatively low numbers of mosquitoes were collected in the live catch compared to the spray catch (n=38 vs n=140) and only 1 infected mosquito was observed at the time of dissection. This low abundance of mosquitoes coupled with lower than expected feeding fidelity suggests that experimental huts are not an optimal assay to use as a TBV efficacy endpoint. Fidelity of the 2015 transmission season mosquitoes are ongoing but no further experimental hut assays are planned at this time.