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 assesses 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). 120, 120, 40 mosquitoes were dissected on day 9 (blue), 10 (dark green), 11 (light green) respectively. 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. 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. 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. 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 (orange). 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 (purple). The line represents the fitted linear regression line and the grey shaded area is the 95% CI.