Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission
Fig 4
Identification of malaria transmission-blocking compounds.
The open-source MMV Pathogen Box was screened in a barcoded assay for P. falciparum transmission using a luminescent reporter parasite. Stage V gametocytes were preincubated with test compound at 10 or 20 μM as indicated in S1 Data and fed to A. stephensi mosquitoes through an Asaia barcoded blood meal. Eight days after feeding, infection status was determined by a luminescence assay. Barcodes were retrieved from infected (luciferase positive) and uninfected (luciferase negative) mosquitoes and quantified. (A) Oocyst intensities in mosquitoes from 9 experimental runs that were used to screen a collection of 400 compounds. The figure shows luminescence activities in individual mosquitoes. The red dotted line indicates the threshold (average background + 5σ) that was used to discriminate infected from uninfected mosquitoes. (B) Assay controls. The figure shows the percentage of the total barcode signal that associated with the uninfected phenotype for barcodes in control wells containing vehicle (0.1% DMSO) or 10 μM atovaquone. (C) Proportion of barcode in the uninfected mosquitoes for all compounds tested. Colours indicate the origin of the compound sets that compose the Pathogen Box. The red dotted line indicates the threshold for selection of active compounds (≥80% of the total barcode signal derived from uninfected mosquitoes). (D) Compounds selected for confirmation experiments. The lower left corner of each panel indicates the proportion of barcode signal that was retrieved from uninfected mosquitoes. The lower right corner indicates the percentage reduction in oocyst intensity that was observed in an SMFA. All compounds except for 2 compounds indicated with a red dot showed transmission-blocking activity in the SMFA. Compounds identified by a blue dot were selected for full dose–response analysis. (E) Dose–response analysis in SMFA for selected compounds. All test concentrations were analysed in duplicate in replicate feeders. For each feeder, infection status of individual mosquitoes was analysed through luminescence analysis. The figure shows oocyst intensities expressed as relative light units for individual mosquitoes. The solid lines indicate the fitted dose–response curves. Dashed lines indicate the luminescence background level in uninfected mosquitoes. Underlying data can be found in S1 Data.