Fig. 2.

Bioluminescence imaging of oocysts in mosquito midguts. a Representative fluorescence images of dissected midguts from PbmCherry, PbFLuc, and PbNLuc-infected mosquitoes. b Average oocyst numbers in PbmCherry, PbFLuc and PbNLuc-infected midguts at days 7–9 post-feed. c Luminescence ratio obtained from 50 mosquitoes infected with PbFLuc or PbNLuc lines (PbFLuc set as 1). d Comparative analysis of prevalence of PbNLuc infection in mosquitoes. Luminescence values of PbNLuc-infected mosquitoes at days 7–9 post-feed, prior to oocyst rupture and sporozoite egress, and at day 15 post-feed, following initial sporozoite egress events. e At day 9 post-feed, 200 mosquitoes were sorted by fluorescence into ‘infected’ and ‘uninfected’. Based on these data, the prevalence of infection was calculated. The same mosquitoes were dissected and the midguts lysed for bioluminescence assays and infection prevalence calculation. f Scatter plot showing correlation and 95 % confidence interval range (CI) of PbNLuc bioluminescence values to fluorescence-based semi-automated counts. 100 individual PbNLuc-infected mosquitoes (represented by each dot) were dissected, and their midguts extracted. Oocyst numbers for each individual midgut were quantified using a semi-automated fluorescence-based macro (y-axis). Midguts were then lysed and imaged by luminescence (x-axis). g Scatter plot showing correlation and 95 % CI range of PbFLuc bioluminescence values to fluorescence-based semi-automated counts. Experimental setup as described for (f). [Graphs are the result of triplicate experiments. Luminescence values are expressed as photons per second (p/s); *p < 0.05; ***p < 0.001; error bars in graphs represent standard deviations (SD)]