Quantifying Plasmodium Sporozoite Production

To quantify how food treatment affected sporozoite production for each infected mosquito, we performed genomic DNA extraction and qPCR analysis for Plasmodium genomes in midguts saved from oocyst dissection. Plasmodium genomic DNA was extracted from midguts using the E.Z.N.A. MicroElute Genomic DNA kit (Omega Bio-Tek, as per the manufacturer’s protocol). DNA was eluted in 20 μL of molecular grade water, and the number of parasite genomes present in midguts was quantified using a previously developed qPCR assay [47 (link)]. Briefly, reactions were run on an ABI Prism 7500 Sequence Detection System (TaqMan). Initial denaturation was 20 seconds at 95°C followed by 40 cycles of a three-second 95°C denaturation period and a 30-second 60°C period of annealing and extension. Primers and probes were designed to amplify DNA from several Plasmodium species. We constructed standard curves for P. yoelii genome detection by extracting DNA from a known number of infected mouse red blood cells using the BloodPrep kit (Applied Biosystems) on the ABI Prism 6100 Nucleic Acid Prep Station (as per the manufacturer’s protocol). Parasite production per oocyst was evaluated by dividing the total number of parasite genomes by the number of oocysts quantified for each midgut. We used both sporozoite production per midgut and per oocyst as measures of the efficiency of parasite replication.