RT Journal Article SR Electronic T1 Testing a non-destructive assay to track Plasmodium sporozoites in mosquitoes over time JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.08.22.554268 DO 10.1101/2023.08.22.554268 A1 Oke, Catherine E. A1 Reece, Sarah E. A1 Schneider, Petra YR 2023 UL http://biorxiv.org/content/early/2023/08/22/2023.08.22.554268.abstract AB Background The extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP.Methods We tested a non-destructive assay to quantify sporozoites of two rodent malaria species, Plasmodium chabaudi and Plasmodium berghei, expelled throughout 24hr windows, from sugar-feeding substrates using quantitative PCR.Results The assay can quantify sporozoites from sugar-feeding substrates, but the prevalence of parasite positive substrates is low. Multiple methods to increase the detection of expelled parasites (running additional technical replicates; using groups rather than individual mosquitoes) did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent.Conclusions We reveal successful detection of expelled sporozoites from sugar-feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion, or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explain Plasmodium’s highly variable EIP and improve understanding of malaria transmission dynamics.Competing Interest StatementThe authors have declared no competing interest.