Fig 1.
scRNAseq analysis of trypanosome developmental stages in tsetse.
(A) A schematic of the trypanosome life cycle and collections of the single parasite transcriptomes from midgut (MG; blue), proventriculus (PV; turquoise) and salivary glands (SG; pink) from different time points and strains. The number of parasites that passed QC at each collection is shown in parentheses. Trypanosomes show two conformations: trypomastigote with kinetoplast (small black dot) posterior to nucleus (e.g. bloodstream form, procyclic, metacyclic) and with kinetoplast anterior to nucleus (e.g. epimastigote). (B) A UMAP of the 388 cells that passed QC across collections, coloured by tissue of origin. (C) The UMAP coloured by cluster assignment. (D) A heatmap of the top significant marker genes from each of the five clusters that had marker genes (AUROC >0.75 & adjusted p-value < 0.01).
Fig 2.
mVSG expression in fly-derived trypanosomes.
(A) The genomic context of 11 mVSGs identified in strains 1738 and J10. The rectangles with a solid black outline represent the mVSG and are coloured to match Fig 2D. The sequences of each mVSG can be found in S1 File. (B) The transcript abundance of mVSG across 388 fly-derived trypanosome cells on the UMAP coloured by the logged sum of the mVSG counts in each cell and sized by the number of different mVSG detected in that cell. (C) The breakdown of mVSG expression per cluster. C5 had the highest proportion of cells expressing mVSG and the greatest proportion of those cells expressing multiple mVSG. (D) A barchart of all cells expressing mVSG (>1 read) organised by cluster and strain. Strain-specific expression of mVSG was seen at high levels in C5, which is primarily composed of strain J10.
Fig 3.
Pseudotime trajectory analysis of developing salivary gland parasites.
Strain 1738 parasites collected from the salivary gland at day 21, 24 and 40 pi were used to map fine-scale changes in gene expression over development. (A-D) A UMAP of the 161 strain 1738 salivary glands parasites coloured by global cluster assignment from Fig 1 (A), day PI (B), attachment treatment (C) and pseudotime assignment (D). (E) A heatmap of 20 clusters of genes differentially expressed over the pseudotime trajectory from (D).
Fig 4.
Classification of hybrid progeny.
Souporcell was used to assign genotypes based on SNPs found between the two strains. The two genotype assignments (0,1) were each primarily composed of one of the strains based on fluorescent identification with FACS (strain 1738 GFP+/RFP- = cluster 0; strain J10 GFP-/RFP+ = 1), and the potential hybrid progeny were classified as inter-genotypic doublets (clusters 0/1 and 1/0). The likelihood ratio of cluster 0 assignment is shown for each of the three sorted populations (A). The UMAP of day 24 mixed- and single-infection experiments coloured by strain assignment and shaped by Fig 1 cell cluster assignment (B) and infection treatment (C).
Fig 5.
Expression of surface antigens and genes involved in sexual reproduction throughout development in the tsetse fly.
We observed co-expression of procyclic surface antigen genes and HAP2 in early parasite development in the salivary glands (A) and this general pattern of expression was also seen in proventricular forms (mesocyclics) (C2) as well as putative epimastigotes (C4) that also had high expression of BARPs (B). Immunofluorescence assays confirmed that these surface proteins corresponded to their transcriptional profiles and were present on the epimastigote and sexual stages (C).
Fig 6.
Expression of Tb927.10.12080 coincides with sexual forms.
(A) Co-expression of Tb927.10.12080 with genes encoding HAP2 and GEX1, proteins associated with gamete and nuclear fusion in eukaryotes, and the surface antigen genes GPEET and EP1; co-expression is seen in a subset of cells from C3 (Fig 1D). (B) T. brucei strain 1738 GFP::Tb927.10.12080–3’UTR transcribed from the procyclin promotor in the salivary gland (SG) and proventricular forms (PV). (C) Diagram showing major cell types observed during meiosis in T. brucei (adapted from [10]); nuclei are shown in black (4C or 2C DNA contents) or grey (1C, haploid) and kinetoplasts are shown as smaller black dots. Values beneath are the numbers and percentages of cells recorded for each cell type; both 1K1N and 2K1N gametes are included in the gamete total. Full data are presented in S7 Table. (D) Trypanosomes from tsetse fly salivary gland spill-out 16–21 days pi with T. brucei strain 1738 expressing GFP::Tb927.10.12080–3’UTR transcribed from the procyclin promotor. Left to right: phase contrast, DAPI, GFP::Tb927.10.12080–3’UTR. The scale bar represents 10 μm.