Single-cell transcriptomics to define Plasmodium falciparum stage-transition in the mosquito midgut

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 hours post blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors, and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early-to-late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. Single-cell data can be visualized interactively via https://mubasher-mohammed.shinyapps.io/shinyapp/ In-house bash, R code scripts and data that were implemented in this study are available on GitHub https://github.com/ANKARKLEVLAB/Single-cell-P.falciparum-midgut . Overall design: Plasmodium falciparum NF54 gametocyte producing cell line were committed to sexual stages (gametocytes) and pooled to 1% gametocytaemia and 50% hematocrit. Gametocyte were membrane fed to Anopheles gambiae adult mosquitoes and dissected at time points; 2, 4, 8, 12 and 20 hours post infection, parasites were then isolated using Micromanupulation based on Anti-Pfs25 antibody conjugated to Alexa-flour-488 anti-mouse IgG (H+L) and Hoechst 33342 stain. cDNA libraries were prepared using SMART-seq2 protocol and ScRNA-seq high-through put data were then analyzed using various computational tools.