A transmission bottleneck for malaria? Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes

It is currently unknown whether all Plasmodium falciparum infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. Sporozoites were quantified by highly sensitive qPCR; intact and ruptured oocysts by fluorescence microscopy following antibody staining of circumsporozoite protein. In laboratory conditions, higher total sporozoite burden in mosquitoes was associated with a shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of P. falciparum infected An. stephensi mosquitoes expelled sporozoites into artificial skin. The medians of expelled and residual salivary gland sporozoites were 136 (IQR: 34-501) and 23,947 (IQR: 9127-78,380), respectively. There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load ({rho}=0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and the number of sporozoites expelled ({rho}=0.35; p=0.0002). In Burkina Faso, An. coluzzii mosquitoes were infected by natural gametocyte carriers. Among mosquitoes that were salivary gland sporozoite positive, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR: 171-2969) and harbored a median of 45,100 residual salivary gland sporozoites (IQR: 20,310-164,900). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load ({rho}=0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled ({rho}=0.7; p<0.0001). Mosquito salivary glands in Burkina Faso harbored 1-3 distinct parasite clones; several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.