The detectability of sporozoites by molecular methods and oocysts by immunolabeling.

A. qPCR performance for P. falciparum sporozoites. Serial dilutions of sporozoites (x-axis) were prepared in PBS and assessed in octuplicate on a single plate to determine qPCR COX-1 limit of detection and quantification. Dots represent sample cycle threshold (left y-axis), and bars coefficient of variation (right y-axis). For each serial dilution Ct sample positivity is shown as the percentage (%) of total tested. B. The relationship between oocyst density versus infection prevalence from 457 membrane feeding experiments using cultured gametocytes. Colours represent regular feeds (red) and those selected for performing experiments on the extrinsic incubation period (EIP; green) or sporozoite expelling experiments (blue). C. Immunofluorescence staining with 3SP2-Alexa 488 anti-CSP. Empty sheet and intact oocyst. D. Violin plots of oocysts staining - day 8 post infection by mercurochrome (purple) and - day 18 by 3SP2-Alexa 488 anti-CSP immunostaining (yellow) for cultured parasites. Box plots show interquartile range, whiskers show the 95% intervals.

Extrinsic Incubation Period in high versus low infected mosquitoes.

A. Total sporozoites (SPZ) per mosquito in body plus salivary glands (x-axis) were binned by infection load <1k; 1k-10k; 10k-50k; >50k and plotted against the proportion of mosquitoes (%) that were sporozoite positive (y-axis) as estimated from an additive logistic regression model with factors day and SPZ categories. 120, 120, 40 mosquitoes were dissected on day 9 (blue), 10 (dark green), 11 (light green) respectively (Supplementary information S2). Error bars show the 95% confidence intervals. B. Violin plots of sporozoite density (SPZ) in single oocysts dissected on day 9 (purple) and day 10 (green). The box indicates the interquartile range (IQR) (Q1 and Q3 quartiles) and the median. Lines extending Q1 and Q3 quartiles indicate the range of the data within 1.5 times IQR.

Sporozoite expelling in relation to infection burden in Anopheles stephensi mosquitoes infected with cultured gametocytes.

A. Binning of mosquitoes by total sporozoite load and expelling prevalence (N=186) B. The number of ruptured oocysts stained by 3SP2-Alexa 488 anti-CSP and fluorescent microscopy (X-axis) in relation to total salivary gland sporozoite density (Y-axis), assessed by COX-1 qPCR; ρ=0.80 (CI: 0.74 - 0.85, p<0.0001). The red dot indicates a mosquito which had 9 ruptured oocysts but only 126 residual salivary glands sporozoites while expelled 1567 sporozoites. Considering the high number of ruptured oocysts in the midgut it is possible that some lobes of salivary glands were missed during dissection and sporozoite load was underestimated by qPCR (95% CI: 0.17, 0.50). C. Total sporozoite density (residual salivary gland sporozoites + sporozoites expelled, X-axis) in relation to the number of expelled sporozoites (Y-axis) by COX-1 qPCR ρ=0.35 (CI: 0.17 - 0.50, p=0.0002). The dotted line on the x-axis shows the threshold of qPCR detection of 20 sporozoites.

Sporozoite expelling in relation to infection load in Anopheles coluzzii mosquitoes infected by naturally acquired gametocyte infections in Burkina Faso.

(A) Direct feeding (blue) vs magnetic-activated cell sorting (MACS; green). Bars show the infection prevalence for each of the 7 gametocyte carriers. Scatter plots with median lines show the midgut oocyst density as result of direct feeding (blue) and MACS (green). (B) Binning of total sporozoite load and expelling prevalence (N=25). (C) Scatter plot of absolute numbers of ruptured oocyst (sheet) density assessed by fluorescent microscopy vs total salivary gland sporozoite density assessed by COX-I qPCR; ρ=0.90 (95% CI: 0.80 - 0.95). The line represents the fitted linear regression line and the grey shaded area is the 95%CI. (D) Scatter plot of absolute numbers of total sporozoite density (residual salivary gland sporozoites + sporozoites expelled) and sporozoites expelled into the artificial skin assessed by COX-I qPCR; ρ=0.70 (CI: 0.52 - 0.82). The line represents the fitted linear regression line and the grey shaded area is the 95% CI.

Clonal complexity of P. falciparum infections in salivary glands and artificial skins following probing by mosquitoes infected by gametocyte carriers who were naturally infected in Burkina Faso.

Clonal data for three donors: 01646 IR (top row), 01661 NJ (middle row) and 011690 CC (bottom row). Left panel shows scatter plots for the association between sporozoite salivary gland load and expelled sporozoites in skin, with a Spearman’s rank correlation across samples from each donor. Right panel shows clonal data for each donor sample in a heatmap plot. The colored numbers on y-axes correspond with the color of the sample in the scatter plot. Purple indicates the presence of a clone in the salivary gland only, turquoise indicates the presence of a clone in the skin only and green indicates the presence of a clone in both salivary gland and skin.