DOI: 10.5061/dryad.dbrv15f89
title: Data from: Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes

description: 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&lt;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 (ρ=0.8;
 p&lt;0.0001) and a weaker positive correlation between salivary gland
 sporozoite load and the number of sporozoites expelled (ρ=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 (ρ=0.9; p&lt;0.0001) and a positive correlation
 between salivary gland sporozoite load and the number of sporozoites
 expelled (ρ=0.7; p&lt;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.
description: This work involves data collected with in vitro cultured
 gametocytes and gametocytes circulating in naturally infected individuals.
 Parasite development in mosquitoes was examined in relation to parasite
 burden in mosquitoes. Data collected centered around the expelling of
 sporozoites by mosquitoes infected with high and low infection burden.
 Parasites were quantified primarily by qPCR, supplemented by
 microscopy.
description: # "Quantification of sporozoite expelling by Anopheles mosquitoes
 infected with laboratory and naturally circulating P. falciparum
 gametocytes "
 [https://doi.org/10.5061/dryad.dbrv15f89](https://doi.org/10.5061/dryad.dbrv15f89) Datasets are linked to individual display figures in the article. ## Description of files #### File Name: Data\_for\_Figure\_1B\_oocyst\_density\_and\_prevalence.xlsx **Description of file:** Data that is generated in mosquito feeding assays with cultured gametocytes in Nijmegen The Netherlands. The set contains three conditions: standard, routine feedings for generating infectious mosquitoes; EIP, feedings performed to conduct EIP experiments in the paper aiming for with low and high infected mosquitoes; Skin, feedings performed to conduct Skin experiments in the paper aiming for low and high infected mosquitoes. **Variables:** * Condition: Standard, Skin, EIP * Oocyst density: Mean of oocysts counted in at least 10 mosquitoes that derived from the same cage * Prevalence infection %: Mean percentage of infected mosquitoes that contained at least one oocyst --- #### File Name: Data\_for\_Figure\_2\_infection\_intensity\_and\_time\_to\_sg\_positivity.xlsx **Description of file:** Generated data for low and high infected mosquitoes, with cultured gametocytes, processed on different days post infection to determine EIP for mosquitoes reared under laboratory/controlled conditions (Nijmegen, The Netherlands). **Variables:** * Day: Day post infection, either day 9, 10 or 11 * Experiment: Data collected in independent experiments number 6, 7 or 8 * SG load: Load/number of SG (Salivary gland) SPZ by targetting COX-1 in qPCR * SG (pos=1/neg=0): Positive or negative salivary gland * Mosquito Carcass load: Signal/load - number of parasites - measured in the carcass * Total parasite load: Signal/load - number of parasites - measured in dissected salivary glands --- #### File Name: Data\_for\_Figure\_2B\_Sporozoite\_densities\_increase\_with\_oocyst\_age.xlsx **Description of file:** Data collected on day 9 or day 10 post infection, with mosquitoes infected with cultured gametocytes in Nijmegen The Netherlands. Infected mosquitoes with only one oocyst were selected for this experiment. The oocyst was removed from midgut and processed in qPCR targetting COX1.\ **Variables:** * Day: Day post infection, either day 9 or 10 * Single oocyst: midgut containing 1 oocyst * Sporozoites density: Density of sporozoites within one oocyst, quantified by qPCR targetting COX1 --- #### File Name: Data\_for\_Figure\_3\_Artificial\_skin\_feeding\_cultured\_gametocytes.xlsx **Description of file:** Data generated for low and high infected mosquitoes with cultured gametocytes (strain NF54 and NF135), in Nijmegen The Netherlands. **Variables:** * Sample Number: Labeling of collected samples * Skin Exp: Experiment, group of infected mosquitoes within the same experiment all processed on the same day * Strain: NF54 or NF135 * Condition (feed): Describes if the mosquito ingested a blood meal and if it was fully fed (FF), partially fed (PF), unfed (UN) or only probed (unfed but probing) * Oocyst sheet: Number of oocysts sheets observed after immunolabeling * Intact Oocyst: Number of intact oocysts observed after immunolabeling * SG load (COX1): Dissected salivary glands processed by qPCR targetting COX1 for quantification of SPZ density * Skin (COX1): Processed skin and quantified SPZs by qPCR targetting COX1 * Notes: Description of labeling --- #### File Name: Data\_for\_Figure\_4\_Artificial\_skin\_feeding\_natural\_infected\_Burkina\_Faso.xlsx **Description of file:** Data generated with infected mosquitoes, using natural infected gametocytes carriers in Burkian Faso. The skin feeding data is presented on tab one. To generate high infected mosquitoes, wth low density cariers, gametocytes were enriched with MACS, donor/MACS information can be found on tab 2. tab: Skin Feedings tab: all MACS experiments **Variables:** tab: Skin Feedings * Sample No. : Labeling of collected samples * Skin Exp: Experiment, group of infected mosquitoes within the same experiment all processed on the same day * Strain: Donor used to infected mosquitoes * Condition (feed): Describes if the mosquito ingested a blood meal and was fully fed (FF), partially fed (PF), unfed (UN) or probed (unfed but probing) * Oocysts sheets: Number of oocysts sheets observed after immunolabeling * Oocyst intact: Number of intact oocysts observed after immunolabeling * Oocyst(sheets+intact) combined : Total number of oocysts present on the midgut * sg_load: Dissected salivary glands processed by qPCR targetting COX1 for quantification of SPZ density * sporo expelled into skin: Processed skin for quantification of expelled SPZs, by qPCR targetting COX1 * total sporo load skin + SG: Total number of spz of salivary gland and skin sample combined * notes: Description of labeling --- #### File Name: Data\_for\_Figure\_5\_Clonal\_complexity\_of\_P.\_falciparum\_infections\_in\_Burkina\_Faso.xlsx **Description of file:** Data generared for identifying clonality of infection from natural gametocyte cariers in Burkina Faso, that were used to infected mosquitoes for conducting skin feeding experiments.\ **Variables:** * ID: Sample numbering according to centre in Burkina Faso * Donor: Donor used to infected mosquitoes * where: Sample from mosquito salivary gland (SG) or Skin * density: SG spz density of parasites quantified by qPCR targetting COX1 * CloneA: Clone with unique DNA sequence, labeled A * CloneB: Clone with unique DNA sequence, labeled B * CloneC: Clone with unique DNA sequence, labeled C * CloneD: Clone with unique DNA sequence, labeled D * CloneE: Clone with unique DNA sequence, labeled E * CloneF: Clone with unique DNA sequence, labeled F * CloneG: Clone with unique DNA sequence, labeled G * CloneH: Clone with unique DNA sequence, labeled H * CloneI: Clone with unique DNA sequence, labeled I * CloneJ: Clone with unique DNA sequence, labeled J * Notes:Description of labeling