Using a non-destructive sugar-feeding assay for sporozoite detection and estimating the extrinsic incubation period of Plasmodium falciparum in mosquito vectors

Despite its epidemiological importance, the time Plasmodium parasites take to achieve development in the vector mosquito (the extrinsic incubation period, EIP) remains poorly characterized. A novel non-destructive assay designed to estimate EIP in single mosquitoes, and more broadly to study Plasmodium – Anopheles vectors interactions, is presented. The assay uses small pieces of cotton wool soaked in sugar solution to collect malaria sporozoites from individual mosquitoes during sugar feeding to monitor infection status over time. This technique has been tested across four natural malaria mosquito species of Africa and Asia, six parasite isolates of Plasmodium falciparum , and across a range of temperatures relevant to malaria transmission in field conditions. We find that monitoring individual infectious mosquitoes is feasible, although due to the frequency of mosquito sugar feeding and inter-individual variation in infection intensity, there is inherent risk that this technique will result in some false negatives. The sensitivity rate ranged from 0.27 to 0.81 depending on mosquito species and on infection intensity in mosquitoes used to collect saliva. Using this non-destructive technique, the estimated median extrinsic incubation period of P. falciparum at 27°C was 11 to 14 days depending on mosquito species and parasite isolate. Long-term individual tracking also revealed that sporozoite transfer onto cotton wool can occur at least until day 40 post-infection. In addition to contributing to a better understanding of EIP and mosquito to human transmission with implications for improving epidemiological models, this technique also allows to link different transmission traits at the mosquito individual level. As one example, we found a significant relationship between EIP and mosquito lifespan, with short individual EIP associated with short mosquito lifespan. Correlations between mosquito/parasite traits often reveal trade-offs and constraints and have important implications for understanding the evolution of parasite transmission strategies. ### Competing Interest Statement The authors have declared no competing interest.