Fig. 3: The sporozoite journey in the mosquito.

a, b UMAP representation of single-cell sporozoite transcriptomes at different stages of the sporozoite developmental journey colored by cluster assignment (a) and sorted stage (b). The arrow in B represents the direction of development. c Sporozoites aligned along pseudotime. Overall, the pseudotime trajectory correlates with the direction of development indicated by the time of sample collection in the y axis. d Heatmap showing the mean expression of differentially expressed (DE) genes over pseudotime, arranged into six pseudotime clusters (pt1–pt6). Each row in the heatmap represents the average expression of all genes in one cluster (the number of genes in each cluster, N, is indicated). Genes in each cluster show similar expression dynamics along the developmental trajectory. e Gene modules representative of enriched GO terms (see Supplementary Data 4 for Bonferroni-adjusted p values) in pseudotime clusters are shown. Transient expression of CRMP4, p41, and MSP1 (GO term anchored component of plasma membrane in d) correlates with the release of sporozoites into the hemocoel. f UMAP representations of the complete life cycle (as shown in 1b) with the expression (log counts) of CRMP4, p41, and MSP1 highlighted. g 3D rendering of a 12-day oocyst immunostained with anti-CSP (green), anti-MSP1 (red), and Hoechst (blue). The image captures the egress of sporozoites (CSP positive) from the oocyst. Released sporozoites express MSP1 on their surface. Note that the lack of signal in sporozoites that remain inside the oocyst is likely a result of poor antibody penetration across the oocyst wall. Scale bar, 10 µm. The image is representative of 6 egress events (out of 9), each releasing hundreds of sporozoites, captured from 19 infected midguts in two independent infections. h Expression of highly variable genes (HVG) across sporozoite developmental stages. HVGs were independently determined for each stage using M3Drop with a false discovery rate (FDR) < 0.05. Only HVGs that are also associated with the pseudotime trajectory are shown. HVGs are colored by their stage association, as per (b). i Expression of DE ApiAP2 TFs over pseudotime. AP2-Tel/SP3 and AP2-O5 expression levels steadily decline from an earlier peak in ooSpz until egress (marked by the C2–C1 transition and expression of CRMP4/MSP1/P41). Three ApiAP2 TFs are transiently expressed before egress, whereas another two are upregulated after egress.