Fig. 3
Bioluminescence imaging of sporozoites in mosquito salivary glands. a Representative images obtained by fluorescence microscopy, of dissected salivary glands from PbFLuc, and PbNLuc-infected mosquitoes. b Average sporozoite numbers per mosquito at days 16–28 post-feed, in groups of PbFLuc and PbNLuc-infected mosquitoes. c Luminescence ratio of PbFLuc and PbNLuc sporozoites. Four sets of 50,000 sporozoites from 10 separate mosquitoes each, for either PbFLuc or PbNLuc, were isolated from the salivary glands, and their luminescence acquired. The value for PbFLuc luminescence was set to a value of 1, and the value of PbNLuc luminescence expressed in relation to the PbFLuc value. d Correlation of luminescence values of PbNLuc and PbFLuc, and detection limit obtained by imaging 1–106 sporozoites (Square at axis shows no signal from PbFLuc at low sporozoite densities). 20 independent mosquitoes were dissected, and their sporozoites diluted to the specific values shown in the figure. R2 corresponds to values over the detection limit of each respective parasite line. e Images from 3 independent dilutions of sporozoites (obtained from pools of 20 independent mosquitoes each). Measurements of different sporozoite concentrations were made in separate plates to avoid under-detection, or signal saturation in the presence of extremely high luminescence values. f Scatter plot showing correlation and 95 % CI range of PbNLuc luminescence values of 100 individual mosquitoes to manual counts in a Neubauer chamber. 100 individual PbNLuc-infected mosquitoes (represented by each dot) were dissected, and their salivary glands extracted. Sporozoite numbers for each individual mosquito dissected were quantified manually using a Neubauer chamber (y-axis). Sporozoites were then lysed and imaged by luminescence (x-axis). g Representative IVIS image of individual dissected and disrupted salivary glands (salivary glands from one uninfected mosquito is used as control in every row) Blue and red outlines show, respectively, minimum and maximum detected sporozoite loads. (luminescence values are expressed as photons per second (p/s) (linear or logarithmic) [***p < 0.001; error bars in all graphs represent standard deviations (SD)]