Biopharma Insights Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
Total time
~10–14 days (gametocyte culture preparation through oocyst enumeration)
Model organism
Anopheles mosquitoes; Plasmodium falciparum
Steps
1
Identify critical reagents for malaria infection assay
Review and source all required reagents, media, and culture supplements for gametocyte preparation and mosquito feeding protocols.
▶ 00:36
2
Prepare Plasmodium falciparum gametocyte culture
Culture and mature P. falciparum gametocytes to infectious stage using standard in vitro protocols and appropriate media conditioning.
▶ 01:10
3
Feed mosquitoes parasite-containing bloodmeal
Use membrane feeding apparatus to allow Anopheles mosquitoes to ingest gametocyte-infected human blood under controlled temperature conditions.
▶ 05:10
4
Dissect and isolate mosquito midguts
Remove midguts from infected mosquitoes at appropriate timepoints post-feeding using sterile dissection techniques.
▶ 10:14
5
Estimate oocyst loads in midgut tissue
Prepare and examine midgut samples microscopically to quantify oocyst numbers and determine infection phenotype.
▶ 12:41