Infection rates (%) of slender T. brucei in teneral tsetse flies, with and without NAG supplementation.

Flies of both genders were fed 4 slender cells per bloodmeal, with or without 60 mM N-acetyl-glucosamine (NAG). Slender cells were harvested and checked for PAD1 signal to confirm slender identity before infection (< 1% PAD1 positive). Infections were performed in triplicates with approximately 20 flies/replicate. The midgut (MG), proventriculus (PV) and salivary glands (SG) of all flies were dissected after 35 days to check for infection. Bar graphs show mean infection rates across replicates, with individual dots representing infection percentages per replicate. Fisheŕs exact test was used on the mean infection rates to determine significance. ns = not significant (p > 0.05). Infection rates are shown for both sexes (A), male (B), and female flies (C).

Infection rates (%) of slender and stumpy T. brucei in non-teneral tsetse flies.

Non-teneral flies were infected between 144 and 168 hpe with either slender (orange) or stumpy (blue) parasites, after receiving two non-infectious bloodmeals beforehand. The tdTomato NLS-GFP:PAD1 3’UTR cell line enabled FACS to separate stumpy (PAD1 positive, GFP in nucleus) and slender (PAD1 negative, no nuclear fluorescence) prior to infection. Infections were performed in quadruplets with roughly 20 flies/replicate. Midgut (MG), proventriculus (PV), and salivary glands (SG) of all flies were dissected after 30-35 days to check for parasite presence. Bar graphs show mean infection rates across replicates, with individual dots representing infection percentages per replicate. Fisheŕs exact test was used on the mean infection rates to determine significance; ns = not significant (p > 0.05). Infection rates are presented for both sexes (A), males (B), and females (C).

RNA sequencing of slender and stumpy trypanosomes differentiating into procyclic forms, performed in biological triplicates.

A: Principal Component Analysis (PCA) showing the transcriptional progression to procyclic forms for slender (blue/green) and stumpy (orange/red) cells. The trajectories remain distinct until converging at 72hrs. B: Number of differentially expressed genes that are upregulated in slender compared to stumpy forms at corresponding time points during differentiation. Genes with an absolute log2FC > 2 and an adjusted p-value < 0.01 were classified as differentially expressed. Corresponding volcano plots and detailed gene counts can be found in Supplementary Figure 5. C: Differentially expressed genes that are upregulated between offset time points during slender and stumpy differentiation. The offset comparison aligns slender 15 hrs with stumpy 0 hrs, where only 22 genes are differentially expressed. Genes with an absolute log2FC > 2 and an adjusted p-value < 0.01 were classified as differentially expressed. Corresponding volcano plots and detailed gene counts can be found in Supplementary Figure 6.

Slender and stumpy bloodstream forms must activate distinct pathways to transform into the procyclic form in the tsetse fly.

Differentiation to the PAD1-positive (green), cell cycle-arrested, short stumpy form can be triggered by either SIF or ES-attenuation(Zimmermann et al., 2017). Stumpy forms have a 2-3 day window to be ingested by a tsetse fly before they perish. When a tsetse fly takes a blood meal, it can ingest both slender and stumpy forms. Once in the fly’s midgut, both forms begin their transformation into the procyclic fly form. During the initial 15 hrs, slender forms shift towards stumpy gene expression before diverging again. Stumpy forms need to reactivate the cell cycle, fully switch to proline metabolism, and elongate both their cytoskeleton and flagella (Supplementary Figure 7A). Slender forms must activate the essential PAD1 pathway, complete the switch to proline metabolism, and change to a procyclin coat (Supplementary Figure 7B). By 72 hrs into differentiation, both slender and stumpy forms have transitioned into the procyclic form. This Figure was adapted from Schuster et al. 2021.