Using scRNA-seq to identify transcriptional variation in the malaria parasite ookinete stage

The crossing of the mosquito midgut epithelium by the malaria parasite motile ookinete form represents the most extreme population bottleneck in the parasite life cycle and is a prime target for transmission blocking strategies. However, we have little understanding of the clonal variation that exists in a population of ookinetes in the vector, partially because the parasites are difficult to access and are found in low numbers. Within a vector, variation may result as a response to specific environmental cues or may exist independent of those cues as a potential bet-hedging strategy. Here we use single-cell RNA-seq to profile transcriptional variation in Plasmodium berghei ookinetes across different vector species, and between and within individual midguts. We then compare our results to low-input transcriptomes from individual Anopheles coluzzii midguts infected with the human malaria parasite Plasmodium falciparum. Although the vast majority of transcriptional changes in ookinetes are driven by development, we have identified candidate genes that may be responding to environmental cues or are clonally variant within a population. Our results illustrate the value of single-cell and low-input technologies in understanding clonal variation of parasite populations

Impact of mating on Anopheles coluzzii in response to infection

Mating causes dramatic changes of female physiology, behaviour, and immunity in many insects, often inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20E. This hormone, also induced by bloodfeeding, plays a major role in activating vitellogenesis for egg production. In this thesis, I present data showing that the mating status of an Anopheles coluzzii female influences her midgut bacterial load and bacterial composition. Furthermore, I show that her susceptibility to Plasmodium falciparum infection is also enhanced upon mating especially when infection intensity is high. I find that mating status has a major impact on the midgut transcriptome, but only under sugar-fed conditions; once females have bloodfed, the transcriptional changes that are still observable as induced by mating are masked. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, I mimicked mating by injecting 20E into virgin females, finding that these females have significantly increased infection intensity compared to controls. I carried out further RNAseq to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. I find that 11% of the genes upregulated by 20E are in common with genes upregulated by mating. Together, these findings suggest that male Anopheles mosquitoes might contribute to malaria transmission by influencing female midgut bacterial loads and by potentially increasing P. falciparum susceptibility in females.Open Acces