{"paper_id":"330c4e7c-25f0-4875-873b-03bb5e1f52ef","body_text":"Research advance: Unexpected plasticity in the life cycle of Trypanosoma brucei \nCarina Praisler 1ⴕ, Jaime N. Lisack 1ⴕ, Anna Sophie Kreis 1, Laura Hauf 1, Johanna Krenzer 1, \nFabian Imdahl2, Markus Engstler1* \n \n1Department of Cell and Developmental Biology, Biocenter, Julius-Maximilians-Universitaet, \nWuerzburg, Germany; 2Helmholtz Institute for RNA -based Infection Research, Helmholtz -\nCenter for Infection Research, Wuerzburg, Germany \n \nⴕ These authors contributed equally to the work  \n* for correspondence: markus.engstler@biozentrum.uni-wuerzburg.de  \n \n \nAbstract  \nWe have previously shown that  the slender form of Trypanosoma (T.) brucei is able to infect \nteneral tsetse flies, develop to the first fly form, which is the procyclic form, and complete the \nlife cycle in the insect vector (Schuster et al., 2021). Further, analysis of the transmission index \n(TI; defined as the number of salivary gland infections relative to the number of midgut \ninfections) revealed a higher TI for slender as compared to  stumpy forms under laboratory \nconditions, which include d the addition of N-acetyl-glucosamine (NAG) to the infective \nbloodmeal.  \nThese findings challenge the prevailing view of the life cycle, according to which only stumpy \nforms are considered infective to tsetse flies.  \nHere, we show that slender trypanosomes can infect both male and female tsetse flies , \nirrespective of their teneral status, in the absence of supplements in the bloodmeal.  \nAdditionally, an RNA-sequencing time course was performed on both slender and stumpy cells \nduring their transition into procyclic forms. This analysis revealed that slender and stumpy form \ntrypanosomes remain transcriptionally distinct  throughout differentiation  into the procyclic \nform. Furthermore, while the protein associated with differentiation 1 (PAD1) remains essential \nfor the transition, slender cells do not require expression of other hallmark stumpy form traits, \nsuch as cell cycle arrest or the shortening of their flagella or microtubule corset. Instead, slender \ntrypanosomes are able to transition directly into procyclic forms.     \nTaken together, these findings demonstrate that while slender cells of T. brucei follow distinct \nroutes to become the procyclic form, they are capable of infecting both teneral and non-teneral \ntsetse flies, thereby contributing to the transmission and spread of these African parasites.    \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nIntroduction:  \nIn the bloodstream of a mammalian host, two main forms of Trypanosoma (T.) brucei can be \nobserved, the long slender and the short stumpy bloodstream form (bsf). \nThe slender cell is proliferative  whereas the stumpy cell has undergone  cell-cycle arrest in \nG1/G0 phase. During division, slender cells release peptidases, most significantly \noligopeptidase B and metallocarboxypeptidase  1(Tettey et al., 2022) , generating a pool of \noligopeptides in the blood and tissues around them. These essential peptides, together with \npossible other unknown components, are part of the molecular cocktail collectively called the \nStumpy Induction Factor (SIF) (Vassella et al., 1997; Reuner et al., 1997; Bossard et al., 2013; \nMoss et al., 2015; Rojas et al., 2019) . After exceeding a certain threshold, SIF causes the \nproliferative slender cells to change into the cell-cycle arrested stumpy forms (Vassella et al., \n1997; Reuner et al., 1997). This transition is accompanied by shortening of the flagellum, cell-\ncycle arrest and other molecular changes , such as  the remodelling of the mitochondrion to a \ncristate structure and expression of Krebs cycle enzymes. The trypanosomes also begin to \nexpress the Protein Associated with Differentiation 1  (PAD1), a member of the carboxylate -\ntransporter protein family, on their surface . PAD1 functions as a transducer of the signal that \ntriggers differentiation into the procyclic form, the first fly form that develops in the tsetse \nmidgut (Reuner et al., 1997; Dean et al., 2009).  \nCell-cycle arrest is lethal for stumpy forms, as they die, if they are not taken up by the tsetse fly \nwithin a few days. Thus, their purpose in the life cycle of T. brucei has long been debated, with \nthe two most accepted theories being: \n1. Stumpy forms are needed for the regulation of the parasitaemia in the host. \n2. Due to pre-adaptation for life in the insect vector, they are the only form able to infect \nthe tsetse fly (Robertson, 1912; Wijers & Willett, 1960; Fenn & Matthews, 2007; \nMacGregor et al., 2012; Matthews et al., 2015; Silvester et al., 2017). \nIn 2021, our laboratory reported unexpected plasticity in the life cycle of T. brucei. We found \nthat even a single slender cell can infect the tsetse fly without exhibiting morphological and \nbiochemical manifestations that define a stumpy cell (Schuster et al., 2021).  \nInstead, slender cells turn on the essential PAD1 pathway (Dean et al., 2009) and transit directly \ninto procyclic forms - all whilst continuously dividing. We suggested that the ability of slender \nbloodstream forms to infect the tsetse fly vector would , at least in part, solve the transmission \nparadox, which refers to the low blood parasitaemia observed in chronically infected hosts and \nthe small bloodmeal volume of tsetse flies, both of which make it unlikely that a stumpy form \nwould be ingested (Capewell et al., 2019).  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nHowever, our original study was criticised (Matthews & Larcombe, 2022; Ngoune et al., 2025) \nfor mainly four reasons:  \n(1) As routinely done in tsetse laboratories, we supplemented all infectious bloodmeals with \nthe immune -suppressive chemical N-acetyl-glucosamine (NAG), which is known to \nenhance tsetse midgut infections for stumpy forms but has no effect on subsequent \nsalivary gland infections (Peacock et al., 2012). It was argued that this treatment allowed \nslender forms to infect the fly.  \n(2) We used teneral flies (which have not yet taken a bloodmeal and younger than 3 days) \nfor all infection experiments. This led to the question if the slender trypanosomes might \njust be able to infect young flies.  \n(3) The use of male flies is common practice for studying trypanosome infections. \nNevertheless, could it be possible that slender trypanosomes can infect male but not \nfemale tsetse flies?  \n(4) We had shown that slender trypanosomes express the differentiation marker pad1 while \nbecoming procyclic. This was taken as a proof that slender cells must turn into stumpy \ncells before becoming procyclic.  \nTo address the critique further than already done (Lisack et al., 2022), we have systematically \nconducted additional experiments. We found that slender form trypanosomes can indeed infect \nboth teneral and non -teneral flies (i. e. flies that have taken  at least one non -infectious \nbloodmeal and are more than 72 hours post eclosion (hpe)), even in the absence of immune \nsuppressing chemicals.  \nUsing an RNA-sequencing time course, we confirmed that slender forms are able to transition \ndirectly to procyclic forms without becoming stumpy forms.  \nTaken together these new findings further highlight the plasticity in the life cycle of T. brucei \nas well as the ability of slender trypanosomes to contribute to the spread of the se parasites to \nnew hosts.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nResults  \nIt has been shown that N-acetyl-glucosamine (NAG) aids stumpy trypanosome infection of the \nfly midgut, while not influencing the number of subsequent salivary gland infections (Peacock \net al., 2006, 2012).  \nTo exclude the possibility that use of the lectin-inhibitor NAG rendered the tsetse fly midgut \nartificially permissive to slender trypanosome infections, tsetse flies were infected with slender \ncells in either untreated blood or NAG-supplemented blood.  \nAs described in Schuster et al., a stumpy marker cell line (NLS-GFP:PAD1 3´UTR) was used \nto confirm that less than 1% PAD1-positive cells were present in any slender culture.  \nInfection rates in the midgut and proventriculus showed negligible differences between non -\nsupplemented and NAG -supplemented infections  (Figure 1A and Supplementary Figure 1 ). \nMidgut infections reached 11.6% with and 9. 2% without NAG ( Figure 1A) while \nproventriculus infections occurred in 9.4% and 8.3%, respectively (Figure 1A). Salivary gland \ninfections were observed in 3.6% of flies infected with and 0.9% without NAG, however, this \ndifference was not statistically significant (p > 0.05) (Figure 1A).  \nThe use of male flies is common  practice for studying tsetse infections, as they show higher \nsalivary gland infection rates than female flies (Jackson, 1949; Maudlin, 1990; Maudlin et al., \n1991). However, because female flies have a longer lifespan , they cannot be disregarded as \npotential important contributors to parasite transmission  in the wild (Maudlin et al., 1990) . \nTherefore, the influence of fly sex on infection rates was assessed by using the same dataset as \nin Figure 1A (Figure 1B and C). Using NAG-supplemented blood, 8.0% of male flies exhibited \nmidgut, 5.8% proventriculus  and 3.6% salivary gland infection s. In the absence  of NAG, \nmidgut, proventriculus, and salivary gland infections were observed in 5.5%, 4.6%, and 0.9% \nof male flies, respectively (Figure 1B). Fisher´s exact test revealed no significant differences in \ninfection rates between NAG -supplemented and non -supplemented groups for male flies \n(Figure 1B).  \nAmong female flies, 3.6% harboured midgut and 3.7% proventriculus infections, regardless of \nNAG supplementation (Figure 1C). No salivary gland infections were identified in female flies \n(Figure 1C). The total percent age of fly infections and their  corresponding replicates  are \nprovided in Supplementary Figure 1.  \nIn conclusion, NAG has a negligible effect on slender infections in tsetse flies and even low \nnumbers of slender cells can infect flies without the aid of immune suppressing compounds.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n  \n \n                           \n \n \n \n \n \n \n \nFigure 1: Infection rates (%) of slender T. brucei in tsetse flies, with and without NAG supplementation. Flies of both genders \nwere fed 4 slender cells per bloodmeal, with or 60 mM N-acetyl-glucosamine (NAG). Slender cells were harvested and checked \nfor PAD1 signal to confirm slender identity before infection (< 1% PAD1 positive). Infections were performed in triplicates  \nwith approximately 20 flies/replicate. The midgut (MG), proventriculus (PV) and salivary glands (SG) of all flies were dissected \nafter 35 days to check for infection. Bar graphs show mean infection rates across replicates, with individual dots representing \ninfection percentages per replicate. Fisher´s exact test was used on the mean infection rates to determine significance. ns = not \nsignificant (p > 0.05). Infection rates are shown for both sexes (A), male (B), and female flies (C).  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nAlthough infection rate s for teneral flies are relatively low, commonly ranging between 10-\n30%, they are more susceptible to trypanosome infection than non-teneral flies (Peacock et al., \n2012). Consequently, the use of teneral flies is standard practice in T. brucei infection \nexperiments, and w e have exclusively used them in  our previous infections. It is  possible, \nhowever, that in nature, older flies contribute more to the spread of trypanosomes than younger \nflies. As such, it was of interest to test if slender trypanosomes could also infect non-teneral \nflies (Van Hoof L. et al., 1937; Wijers, 1958; Otieno et al., 1983; Walshe et al., 2011; Matthews \n& Larcombe, 2022). Therefore, to ensure our flies would survive for at least 30  days to allow \ninfection assessment, we defined our non -teneral flies as those infected between 144 and  \n168 hours post eclosion (hpe). These flies were fed two non-infectious bloodmeals prior to the \nthird infectious bloodmeal, all spaced at least two days apart.  \nFor these experiments, we again used the cell line containing the NLS -GFP:PAD1 3´UTR \nstumpy reporter and a cytoplasmic red fluorescent protein (td Tomato) (Reuter et al., 2023). To \nensure pure populations (Schuster et al., 2021), fluorescence activated cell sorting (FACS) was \nperformed to separate PAD1-positive (stumpy) from PAD1-negative (slender) cells. To confirm \nsorting success, populations were subsequently examined by fluorescence microscopy for \nPAD1 signal. Stumpy cells were also sorted to remove any slender cells and to keep conditions \nconstant.   \nAdditionally, the growth of sorted slender cells was monitored in vitro to ensure that sorting did \nnot affect parasite fitness by causing any stress (Quintana et al., 2021) (Supplementary Figures \n3 and 4). Only then did we proceed with infections, using either sorted slender or sorted stumpy \ncells to infect male and female, teneral or non-teneral flies. With the confidence of having pure \nslender or stumpy populations, we increased parasite concentrations  to 1x106 cells/ml for \ninfection. Flies were dissected 30 days post infection and their organs examined for parasite \npresence.  \nInfection data show that slender trypanosomes were able to establish infections in non -teneral \nflies at rates comparable to stumpy forms (Figure 2A and Supplementary Figure 2) . Midgut \ninfections were detected in 6.4% of flies infected with slender and 6.6% of flies infected with \nstumpy cells. Similarly, 5.1% and 4.4% of flies exhibited proventriculus infections, and 3.8% \nand 1.5% salivary gland infections,  respectively. Statistical analysis using Fisher´s exact test \nrevealed no significant difference for any fly organ in slender or stumpy infections.  \nConsistent with published data on stumpy infections (Peacock et al., 2012), male flies showed \nthe highest infection rates (Figure 2B). Importantly, we found that non-teneral female flies can \nalso be infected with slender cells. Midgut infections were observed in 9.5% of non-teneral \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nfemale flies infected with slender cells and 5.6% with stumpy cells ( Figure 2C). Although \noverall numbers were low, it is noteworthy that only slender cells caused any salivary gland \ninfections in non-teneral female flies (Figure 2C). A table of all percentages of fly infections, \nreplicates, and sex can be found in Supplementary Figure 2.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \nFigure 2: Infection rates (%) of slender and stumpy T. brucei in non-teneral tsetse flies. Non-teneral flies were infected between \n144 and 168  hpe with either slender (orange) or stumpy (blue) parasites, after receiving two non -infectious bloodmeals \nbeforehand. The tdTomato NLS-GFP:PAD1 3´UTR cell line enabled FACS to separate stumpy (PAD1 positive, GFP in nucleus) \nand slender (PAD1 negative, no nuclear fluorescence) prior to infection. Infections were performed in quadruplets with roughly \n20 flies/replicate. Midgut (MG), proventriculus (PV), and salivary glands (SG) of all flies were dissected after 30-35 days to \ncheck for parasite presence. Bar graphs show mean infection rates across replicates, with individual dots representing infection \npercentages per replicate. Fisher´s exact test was used on the mean infection rates to determine significance; ns = not significant \n(p > 0.05). Infection rates are presented for both sexes (A), males (B), and females (C).  \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nWe have previously shown that slender and stumpy forms both differentiate to procyclic forms \nwith comparable kinetics (Schuster et al. 2021). To assess whether slender cells \ntranscriptionally transition through a stumpy state or differentiate directly into the procyclic \nform, we performed RNA sequencing of slender and stumpy parasites throughout \ndifferentiation. For this, we used t he same cell line as in Schuster et al. (2021), containing an \nEP1:YFP fusion protein and NLS-GFP:PAD1 3´UTR stumpy reporter. In vitro differentiation \nof both slender and stumpy cultures was induced by glucose depletion, addition of cis-aconitate, \nand temperature drop to 27°C (Mowatt & Clayton, 1987; Richardson et al., 1988; Roditi et al., \n1989; Ziegelbauer et al., 1990; Matthews & Gull, 1994; Dean et al., 2009).  \nSlender or stumpy cells were collected in triplicates at five time -points throughout \ndifferentiation, 0, 8, 15, 24, and 72 hours (hrs), and subjected to paired-end sequencing (Figure \n3). Hierarchical clustering identified two biological replicates as outliers, which were excluded \nfrom further analysis.   \nPrincipal component analysis (PCA) was performed, with the 1 st principal component \naccounting for 32.2% and the 2nd principal component for 11.1% of total variance (Figure 3A).  \nWhen comparing corresponding timepoints between slender and stumpy forms , for example, \nslender 8 hrs versus stumpy 8 hrs, both cell types initially exhibit a high number of differentially \nexpressed genes. This number gradually decreases, reaching zero at 72  hrs (Figure 3B; \nSupplementary Figure 5). Interestingly, when comparing all time points, an additional \nconvergence with only 22 differentially expressed genes is observed between slender 15 hrs \nand stumpy 0 hrs (Figure 3C; Supplementary Figure 6). \nAt first glance, this might suggest that slender cells transition into the stumpy form at this point \nbefore proceeding to differentiate into procyclic forms in the same way as stumpy cells . \nHowever, comparison of subsequent timepoints reveals a different pattern: at slender 24 hrs and \nstumpy 8 hrs, the number of differentially expressed genes increases markedly  to 316. This \nelevated number remains relatively consistent until both cell types converge at 72  hrs, where \nno differentially expressed genes are detected (Figure 3B; Supplementary Figures 5 and 6). \nIn line with these findings, Gene Ontology (GO) enrichment analysis reveals that gene \nexpression profiles of slender and stumpy cells throughout differentiation are associated with \ndistinct molecular functions and biological processes. In slender cells, the most significant GO \nterms are related to extracellular structure and matrix organization, glycolytic processes and \nproteolysis (Supplementary Figure 7). In contrast, stumpy cells show enrichment for genes that \nare involved in RNA processing, ribosome biogenesis as well as cellular component biogenesis, \nconsistent with them re -entering the cell cycle to become procyclic forms  (Supplementary \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nFigure 7). Genes associated with the procyclic form, like the procyclin surface proteins EP1 \nand EP2, as well as pyruvate phosphate dikinase (PPDK), start with low expression levels for \nboth, slender and stumpy forms. However, expression increases to procyclic levels by only 8 hrs \nin stumpy cells but more gradually in slender cells, reaching comparable levels only by 72 hrs \n(Supplementary Figure 8). \nCollectively, these data indicate that differentiation in slender and stumpy trypanosomes \nproceeds via distinct gene expression programs. Rather than following a shared trajectory, each \nform activates a unique set of genes to transition into the first fly form, the procyclic form \n(Figure 4). \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nFigure 3: RNA sequencing of slender and stumpy trypanosomes differentiating into procyclic forms. A: Principal Component \nAnalysis (PCA) showing the transcriptional progression to procyclic forms  for slender (blue/green) and stumpy ( orange/red) \ncells. The trajectories remain distinct until converging at 72hrs. B: Number of differentially expressed genes between slender \nand stumpy forms at corresponding time points during differentiation . Genes with an absolute log2FC  > 2 and an adjusted \np-value < 0.01 were classified as differentially expressed. Corresponding volcano plots and detailed gene counts can be found \nin Supplementary Figure 5. C : Differentially expressed genes between offset time points  during slender and stumpy \ndifferentiation. The offset comparison aligns slender 15 hrs with stumpy 0 hrs, where only 22 genes are differentially expressed. \nGenes with an absolute log2FC > 2 and an adjusted p-value < 0.01 were classified as differentially expressed.  Corresponding \nvolcano plots and detailed gene counts can be found in Supplementary Figure 6. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \n \n \n \n \n \n \n \n \nFigure 4: Slender and stumpy bloodstream forms must activate distinct pathways to transform into the procyclic form in the \ntsetse fly. Differentiation to the PAD1-positive (green), cell cycle-arrested, short stumpy form can be triggered by either SIF or \nES-attenuation(Zimmermann et al., 2017) . Stumpy forms have a 2 -3 day window to be ingested by a tsetse fly before they \nperish. When a tsetse fly takes a blood meal, it can ingest both slender and stumpy forms. Once in the fly's midgut, both forms \nbegin their transformation into the procyclic fl y form. During the initial 15 hrs, slender forms shift towards  stumpy gene \nexpression before diverging again. Stumpy forms need to reactivate the cell cycle, fully switch to proline metabolism, and \nelongate both their cytoskeleton and flagella  (Supplementary Figure 7A). Slender forms must activate the essential PAD1 \npathway, complete the switch to proline metabolism, and change to a procyclin coat (Supplementary Figure 7B). By 72 hrs into \ndifferentiation, both slender and stumpy forms have transitioned into the procyclic form. This Figure was adapted from Schuster \net al. 2021. \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nDiscussion  \nHistorically, the lack of molecular markers to distinguish slender and stumpy forms resulted in \nconflicting reports on their transmissibility to the tsetse fly. Early experiments indicated that \nhigher trypanosome levels in mammalian blood correlated with more infected flies. Since \nstumpy forms arise through a density -dependent mechanism, increased b loodstream \nconcentrations inevitably meant more stumpy forms in mammalian hosts, an uncontrollable \nfactor in these early studies (Robertson, 1912; Van Hoof, 1947; Baker & Robertson, 1957; \nWijers, 1958).  \nMoreover, the discovery of the secreted quorum sensing factor SIF in the 1990s reinforced the \nnotion that this pathway serves to ensure the presence of stumpy forms (Reuner et al., 1997; \nVassella et al., 1997). It was also shown that stumpy forms begin developing traits needed for \nsurvival in the fly midgut, such as an elaborated mitochondrion and expressing enzymes \nassociated with the Krebs cycle  (Vickerman, 1965; Brown et al., 1973; Hamm et al., 1990; \nReuner et al., 1997). Previous research also suggested that the stumpy form was more adapted \nto life in the tsetse fly, pointing to their resistance to proteolytic stress and acidic conditions in \nvitro (Nolan et al., 2000) . Later, however, it was shown that the tsetse midgut is actually  an \nalkaline environment, making the stumpy resistance to acidic conditions mute in the case of \nmidgut survival (Liniger et al., 2003).   \nTaken t ogether, it is understandable why early studies concluded that only stumpy form \ntrypanosomes could infect tsetse flies. This assumption, however, gave rise to the transmission \nparadox: under conditions of low parasitaemia during chronic infections, how can sufficient \nnumbers of stumpy forms be maintained to ensure parasite transmission and sustain the disease \ncycle?  \nThe ability to study the differences between slender and stumpy cells changed upon the \ndiscovery of the first stumpy specific molecular marker, the protein associated with \ndifferentiation 1 (PAD1), in 2009  (Dean et al., 2009) . Using the PAD1 marker, it was shown \nthat skin -resident trypanosomes reveal a locally increased proportion of stumpy forms , \nproviding a possible solution to this transmission paradox. In mice, the proportion of stumpy \ncells in the skin ranged from 8 - 80%, suggesting that these parasites might significantly \ncontribute to infection dynamics (Capewell et al., 2016).  \nOur recent study using artificial human skin models  revealed that trypanosomes freshly \ndeposited into the skin by  tsetse flies develop into a  quiescent form that cannot reinfect the \nvector again (Reuter et al., 2023). Although this finding does not address parasites that migrate \nback into the skin from circulation, it highlights the complex dynamics occurring within tissue. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nUltimately, the extent to which skin -resident populations contribute to tsetse transmission \nremains unclear, as direct quantification currently is not feasible.   \nIn 2021, we offered an alternative explanation for the transmission paradox, namely that slender \nform trypanosomes can also infect and complete the life cycle in tsetse flies. All bloodstream \nform trypanosomes taken up in a bloodmeal ha ve the potential to infect a mammalian host  by \nestablishing salivary gland infection in tsetse flies  (Schuster et al., 2021) . While all our data \nsupported this conclusion, some questions were raised, which we have now answered.  \nTo mimic more natural conditions, we conducted infection experiments using slender forms \nwithout immune-suppressors, both male and female flies, and non-teneral flies.  \nThe definition of a non-teneral and teneral fly is of great importance here.  \nThe age of a teneral fly ranges anywhere from 8 to 72 hours post eclosion (hpe) , with the \nimportant criterion that they have not taken up any blood (Otieno et al., 1983; Walshe et al., \n2011). Thus, when infecting teneral flies, their first bloodmeal is the infectious one. In contrast, \nwe infected our non -teneral flies between  144 - 168 hpe, having received two regular \nbloodmeals, each two days apart, before the third infectious feeding.   \nWhile it could be argued that it would be better to use even older flies, the lifespan of the tsetse \nmust be taken into consideration  to ensure survival through the 30  days required for T. brucei \nto complete development in the vector . It has been suggested that  tsetse flies in the wild may \nlive longer than their laboratory raised counterparts, though estimated and observed life spans \nvary widely. The mean life span ranges from 35 to 178 days for female, and only 21 to 28 days \nfor male flies (Jackson, 1949; Maudlin et al., 1999; Vale & Torr, 2005; Haines et al., 2020) . \nRegardless of the individual lifespan of a fly, a trypanosome infection is permanent. \nWe have demonstrated, in a statistically well -controlled manner, that a single slender \ntrypanosome is capable of infecting the tsetse fly. Here, we unambiguously show that, in the \nabsence of immunosuppressive treatment, slender forms can establish infections in tsetse flies, \nirrespective of the fly’s age or sex.  \nThe study by Ngoune et al. (2025) does not disprove this finding (Ngoune et al., 2025). In their \ninfection experiments, only 63% of the “stumpy” cells expressed PAD1, meaning that 37% \nmust, by definition, have been slender forms. This heterogeneity complicates the interpretation \nof their results. It is also worth noting that Ngoune et al. used the AnTat 1.1E strain, which they \nmaintained in culture without methylcellulose. We have observed that, during adaptation to \nmethylcellulose-free medium, our bona fide pleomorphic AnTat 1.1 strain gradually loses its \ndevelopmental competence. This could account for the heterogenous “stumpy” population \nreported by Ngoune et al. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nMoreover, more than 40% of the adult male flies in their study did not survive the 28-day period \nrequired before dissection, further complicating the interpretation of their infection data. \nIn response to our original work Matthews & Larco mbe postulated that “molecular \ncharacteristics” (PAD1 in particular)  define a stumpy cell and that the eponymous \nmorphological changes (e.g. stumpy formation) are no longer valid criteria (Matthews & \nLarcombe, 2022). Although irreversible cell cycle arrest is still an accepted hallmark of stumpy \ntrypanosomes, we cannot exclude that in the tsetse fly, the dividing slender population arrests \nthe cycle and then transitions to the procyclic form. This scenario is of some theoretical interest, \nespecially in view of the  ongoing discussion about shallow vs. deep cell cycle arrest, e.g. in \nstem cell quiescenc e (Urbaìn & Cheung, 2021).  Therefore, we decided to conduct an RNA \nsequencing time course that would compare the transcriptional landscape of slender and stumpy \nbloodstream populations during differentiation to the procyclic form  (Figure 3) . Using \nfluorescent activated cell sorting , slender cultures were sorted to exclude any PAD1-positive \ncells, while stumpy cultures were sorted to include 100% PAD1 expressing cells. \nAt the outset, more than 300 genes were differentially expressed between the two bloodstream \nforms. However, after 72 hours (hrs) under differentiation conditions, no significant differences \nin gene expression remained; both slender and stumpy cells had transitioned into procyclic \nforms. Importantly, stumpy forms appear transcriptionally primed for rapid progression, while \nslender cells activate a distinct and temporally delayed gene expression program  \n(Supplementary Figure 8). \nThe transient similarity observed at the specific timepoint, slender 15 hrs and stumpy 0  hrs, \nmay reflect a brief convergence in gene expression, but is not indicative of a shared \ndevelopmental pathway. Instead, the sustained differences in transcriptional profiles and \nenriched GO terms  (Supplementary Figures 5, 6 and 7)  argue for fundamentally different \nregulatory mechanisms underlying the commitment to procyclic differentiation. \nThese data argue against a linear progression from slender to stumpy to procyclic. Instead, they \nsuggest that slender forms transiently activate a subset of genes also expressed in stumpy cells \nbut then follow a distinct transcriptional trajectory before ultimately converging with the \nstumpy pathway at 72 hrs, when both forms adopt a procyclic identity (Figure 3). Thus, slender \nand stumpy forms remain transcriptionally distinct for at least the first 24 hrs of differentiation \n(Figure 3B). At this point, their transcriptomes do not correspond clearly to any of the three \ncanonical forms - slender, stumpy, or procyclic. These findings demonstrate that slender forms \ncan differentiate directly into procyclic forms without passing through a bona fide stumpy stage, \ni.e. without cell cycle arrest.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nIf slender forms can adopt a distinct transcriptional trajectory towards the procyclic state in \nvitro, there is no reason to assume that T. brucei could not use both routes - via stumpy or \ndirectly from slender - in the tsetse fly as well.  \nAdditionally, there is one more observation that needs to be taken into account. We have shown \nthat even small numbers of monomorphic trypanosomes strain 427 - incapable of differentiating \ninto stumpy forms - can establish stable midgut infections in tsetse flies yet fail to progress to \nsalivary gland colonization (Schuster et al., 2021). These monomorphic cells do not respond to \nStumpy Induction Factor (Reuner et al., 1997; Vassella et al., 1997) and fail to upregulate PAD1 \n(Dean et al., 2009) , remaining locked in the proliferative slender bloodstream form. Thus, \nslender trypanosomes are indeed capable of infecting the tsetse midgut, even if they are \nmonomorphic. The PAD pathway is not strictly required for colonization of the midgut.  \nHowever, activation of the PAD pathway is essential for the generation of procyclic \ntrypanosomes that are competent to complete development and colonize the salivary glands.  \nIn retrospect, our results are perhaps less unexpected than initially assumed.  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nMaterial and Methods  \nCell line \nThe pleomorphic Trypanosoma brucei brucei strain EATRO 1125 (serodome AnTat 1.1) (Le \nRay et al., 1977) with an NLS-GFP PAD1 3´UTR molecular marker and an additional tdTomato \nfluorescence sequence (Reuter et al., 2023) was used for infection experiments and cultured as \npreviously described (Schuster et al., 2021) . For RNA sequencing experiments the same cell \nline was used with an additional EP1:YFP fusion protein (Schuster et al., 2021).   \n \nFly infection  \nTsetse flies of the species Glossina morsitans morsitans  were kept as previously described \n(Schuster et al., 2021). \nTeneral flies received their first , and infectious,  bloodmeal 24-72 hours post-eclosion (hpe). \nThey were infected with four trypanosomes per bloodmeal, either untreated or supplemented \nwith 60 mM N-acetyl-glucosamine (NAG).  \nNon-teneral flies (144-168 hpe) were given  two non-infectious bloodmeals , each two days \napart, prior to the infectious bloodmeal containing 1x106 cells/ml of either slender or stumpy \nparasites. Prior to infection, fluorescence activated cell sorting was performed to ensure 100% \nslender or stumpy population.   \n \nImmunofluorescence  \nCells were prepared, fixed and stained as previously described (Schuster et al., 2021). \n \nFluorescence activated cell sorting  \nCell sorting was performed using the FACS Aria III (BD biosciences, Franklin Lakes, USA). \nCells were harvested, resuspended in pre-warmed TDB at 1x107 cells/ml, transferred to FACS \ntubes through a 35 µm cell strainer cap. Cells were first gated based on the tdTomato signal to \nexclude debris and residual medium. Subsequently, GFP fluorescence from the NLS-GFP PAD1 \n3´UTR reporter was analyzed. Depending on the desired population, gates were set to isolate \neither PAD1-positive (stumpy) or PAD1 -negative (slender) cells with high purity. For each \nexperiment, 1x10 6 cells were sorted using a 100  µm nozzle at the lowest sorting speed to \nminimize mechanical stress. Sorted cells were coll ected into 15  ml tubes containing pre -\nwarmed FCS, resulting in a final FCS concentration of 15%  (v/v). After sorting, cell motility, \nconcentration, and PAD1 signal were checked by microscopy.   \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nBulk RNA sequencing   \nBoth slender and stumpy trypanosomes were differentiated into procyclic forms as previously \ndescribed. The 0 hour (h r) timepoint was  collected immediately before differentiation was \ninduced, and additional samples were taken at 8, 15, 24, 72  hrs following addition of cis-\naconitate.   \nCells were harvested from biological triplicates, resuspended in  1 ml pre -warmed PBS \ncontaining 10 % FCS, and transported to the Helmholtz Institute for RNA Infection biology \n(HIRI) Würzburg in a 37°C incubation chamber.  \nUpon arrival, cells were washed twice with pre -warmed PBS to remove FCS in 1  ml of pre-\nwarmed PBS 15 min prior to sorting, Calcein-AM violet was added at a final concentration of \n1 µM to label viable cells.  \nLive cells were sorted using a FACS Aria III cytometer (BD Biosciences) . 1000 cells per \nreplicate were deposited into single wells of a 48 well-plate containing 2.6 µl of 1x lysis buffer \n(Takara) and 0.01 µl of RNase inhibitor (40 U/µl; Takara). Sorting was performed in triplicates \nfor each timepoint, and plates were immediately placed on ice and stored at -80°C.   \nLibrary preparation  and sequencing were performed as previously described (Müller et al., \n2018). Very briefly, lysates were supplemented with 0.2  µl ERCC Spike -in Control Mix 1 \n(Thermo Fisher Scientific) at a 1:20,000,000 dilution. Libraries were prepared using the \nSMART-Seq v.4 Ultra Low Input RNA Kit (Takara), utilizing a quarter of the recommende d \nreagent volumes. PCR amplification was performed for 27 cycles, and cDNA was purified with \nAgencourt AMPure XP beads (Beckman Coulter) and 15  µl elution buffer (Takara). Library \nquantification was carried out using a Qubit 3 Fluorometer and dsDNA Hs Assa y kit (Life \nTechnologies), while quality assessment was performed by using a 2100 Bioanalyzer with High \nSensitivity DNA kit (Agilent). 0.5 ng of cDNA was used as input for the Nextera XT (Illumina) \ntagmentation-based library preparation protocol. The reaction was performed at one quarter of \nthe recommended volumes , with a 10 -minute tagm entation step at 55  °C, and a 1 -minute \nextension step during PCR. Libraries were pooled and sequenced in paired -end mode with 2x \n75 cycles using Illumina's NextSeq 500. \n \nAnalysis of bulk RNA-sequencing data  \nBulk RNA -sequencing analysis was performed according to scripts and online resources \npublished by Berry et al., 2021 using RStudio (version 4.02). Read demultiplexing and quality \ncontrol were performed  with FASTQC (version 0.12.1,  (Andrews, 2012) ). Adaptors were \nremoved and reads were trimmed using Fastp (version 0.23.4, (Chen, 2023)).  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nThe T. b. brucei TREU 972 reference genome was obtained from the Tritryp database \n(TritrypDB). rRNA was removed (Aslett et al., 2010) , and, to remove redundancy, only one \nrepresentative from each gene group was retained while others were masked. Gene groups were \ndefined based on sequence similarity using CD-Hit software (Li & Godzik, 2006). \nKallisto was used for read alignment, and genes with ≥ 3 counts per million (cpm) in at least \ntwo samples were retained for downstream analysis. Data normalization was performed using \nthe EdgeR package in RStudio (Robinson et al., 2009; Team R.C., 2021). \nSL152 (slender 15 hrs, 2nd replicate) and SL241 (24 hrs, 1st replicate) were considered outliers \nbased on hierarchical clustering distance using the Minkowski metric (Lee & Willcox, 2014). \nDifferentially expressed genes (DEGs) were identified using DESeq2, EdgeR, and Limma \npackages in RStudio (Love et al., 2014; Ritchie et al., 2015; Robinson et al., 2009; Team R.C., \n2020), applying significance criteria of log2 fold change > 1 and p value < 0.01. \n \nStatistics  \nTwo-tailed Fisher´s exact tests were performed for all fly infection data using GraphPad prism \nversion 9.0.0 for Windows (Graphpad software, San Diego, California USA, \nhttp://www.graphpad.com).  \n \nAcknowledgements  \nThe authors would like to thank the fly team from the Zoology I Department of the University \nof Würzburg for their expert care and maintenance of the tsetse flies.   \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nWijers, D. J. B. (1958). Factors that may influence the infection rate of Glossina \npalpalis with Trypanosoma gambiense. Annals of Tropical Medicine and \nParasitology, 52(4), 385–390. https://doi.org/10.1080/00034983.1958.11685878 \nWijers, D. J. B., & Willett, K. C. (1960). Factors that May Influence the Infection Rate \nof Glossina Palpalis with Trypanosoma Gambiense. Annals of Tropical Medicine \n& Parasitology, 54(3), 341–350. \nhttps://doi.org/10.1080/00034983.1960.11685996 \nZiegelbauer, K., Quinten, M., Schwarz, H., Pearson, T. W., & Overath, P. (1990). \nSynchronous differentiation of Trypanosoma brucei from bloodstream to \nprocyclic forms in vitro. European Journal of Biochemistry, 192(2), 373–378. \nhttps://doi.org/10.1111/j.1432-1033.1990.tb19237.x \nZimmermann, H., Subota, I., Batram, C., Kramer, S., Janzen, C. J., Jones, N. G., & \nEngstler, M. (2017). A quorum sensing-independent path to stumpy development \nin Trypanosoma brucei. PLoS Pathogens, 13(4). \nhttps://doi.org/10.1371/journal.ppat.1006324 \n  \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \nNo. of flies \ninfected \nFly sex No. of \nslender \ncells/ml  \nConcentration \nof NAG \nDays until \ndissection \nNo. of flies \ndissected \nMG PV SG \n37 male 200  0 mM 35 16 0 0 0 \n19 female 200  0 mM 35 17 1 1 0 \n24 male 200  0 mM 35 24 5 4 1 \n24 female 200  0 mM 35 22 2 2 0 \n24 male 200  0 mM 35 21 1 1 0 \n9 female 200  0 mM 35 9 1 1 0 \n \nNo. of flies \ninfected Fly sex \nNo. of \nslender \ncells/ml \nConcentration \nof NAG \nDays until \ndissection \nNo. of flies \ndissected MG PV SG \n37 male 200  60 mM 35 35 3 3 2 \n19 female 200  60 mM 35 18 1 1 0 \n26 male 200  60 mM 35 24 1 1 1 \n27 female 200  60 mM 35 27 1 1 0 \n26 male 200  60 mM 35 26 7 4 2 \n9 female 200  60 mM 35 8 3 3 0 \n \nSupplementary Figure 1: Absolute numbers of tsetse fly infections using slender bloodstream forms \nof T. brucei, with or without the addition of N-Acetyl-Glucosamine (NAG). Both, male and female \nflies were infected with blood containing 200 slender cells  per ml of blood , either untreated or \nsupplemented with  the immun e-suppressing chemical , N-Acetyl-Glucosamine (NAG , 60mM). \nTsetse flies have an estimated drinking volume of 20 µl (Gibson & Bailey, 2003), which results in \nan uptake of 4 parasites per bloodmeal. All flies were dissected 35 days post i nfection, and their \nmidgut (MG), proventriculus (PV), and salivary glands (SG) examined for parasite presence. Prior \nto infection, slender cells of the tdTomato NLS-GFP:PAD1 3´UTR line were verified to lack pad1 \nexpression, confirming pure slender identity (< 0.05% PAD1 positive).  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \nNo. of \nflies \ninfected \nFly sex No. of \nslender \ncells/ml  \nConcentration \nof NAG \nDays until \ndissection \ninfectious \nfeed \nNo. of \nflies \ndissected \nMG PV SG \n20 male 1x106 0 mM 30 3rd  17 0 0 0 \n20 male  1x106 0 mM 30 3rd 19 2 2 2 \n21 male 1x106 0 mM 35 3rd 21 5 4 2 \n20 male 1x106 0 mM 35 3rd 19 0 0 0 \n20 female 1x106 0 mM 30 3rd 20 1 0 0 \n21 female 1x106 0 mM 30 3rd 21 2 2 2 \n20 female 1x106 0 mM 35 3rd 20 0 0 0 \n20 female 1x106 0 mM 35 3rd 20 0 0 0 \n \nNo. of \nflies \ninfected \nFly sex No. of \nstumpy \ncells/ml  \nConcentration \nof NAG \nDays until \ndissection \ninfectious \nfeed \nNo. of \nflies \ndissected \nMG PV SG \n20 male 1x106  0 mM 38 3rd  20 2 2 1 \n21 male  1x106  0 mM 36 3rd 16 1 1 1 \n23 male 1x106  0 mM 35 3rd 19 2 2 0 \n14 male 1x106  0 mM 35 3rd 11 0 0 0 \n20 female 1x106  0 mM 38 3rd 19 1 0 0 \n20 female 1x106  0 mM 36 3rd 20 1 0 0 \n23 female 1x106  0 mM 35 3rd 19 0 0 0 \n15 female 1x106  0 mM 35 3rd 13 2 1 0 \n \nSupplementary Figure 2: Absolute numbers of infections in non-teneral tsetse flies with either \nslender or stumpy T. brucei cells. Male and female flies were infected with untreated blood \ncontaining 1x106 cells/ml. The tdTomato NLS-GFP:PAD1 3´UTR cell line enabled FACS-based \nseparation of stumpy (PAD1 positive, GFP in nucleus ) and slender (PAD1 negative, no nuclear \nfluorescence) forms prior to infection.  All non-teneral flies were 144-168 hours post eclosion (hpe) \nand had already received two non-infectious bloodmeals prior to the infectious feed.  Flies were \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\ndissected 35 days post infection, and their midgut (MG), proventriculus (PV), and salivary glands \n(SG) examined for parasite presence.   \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n   \n \n \n \n \n \n \n \n \nSupplementary Figure 3: Slender T. brucei cells of the tdTomato NLS-GFP:PAD1 3´UTR line do \nnot express PAD1 or exhibit stress responses following fluorescence activated cell sorting (FACS). \nFACS was used to ensure pure slender populations (pad1 negative) prior to infection . A: \nImmunofluorescence (IF) images of slender cells immediately after FACS, confirming sorting \nC \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nsuccess. Parasites were fixed in 4% Paraformaldehyde (PFA) , stained with DAPI (blue) , and \nlabelled with an anti-PAD1 antibody (orange); scalebar: 20 µm. B: High-resolution IF image of a \nsingle slender trypanosome post sorting, showing clear absence of  nuclear pad1 signal; \nscalebar: 10 µm. C:  Growth curves comparing  slender cells post sorting (green) and untreated \nslender cells (purple), indicating no growth impairment due to sorting.  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\nSupplementary Figure 4: Fluorescence activated cell sorting  (FACS) was used to isolate stumpy \ncells of the tdTomato NLS-GFP:PAD1 3´UTR line, ensuring a pure stumpy population  prior to \ninfection. A: Stumpy cells from a SIF -induced stumpy culture (grown to 5x10 5 cells/ml and kept \nfor 48 hours), after FACS to confirm sorting success. Cells were fixed in 4% PFA immediately after \nsorting, stained with DAPI (blue) , and labelled with an anti -PAD1 antibody (orange) ; \nscalebar: 20 µm. B: High-resolution IF image of a single stumpy trypanosome displaying \ncharacteristic stumpy morphology and strong pad1 signal (orange); scalebar: 10 µm.   \n \n  \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n   \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nSupplementary Figure 5: V olcano plots showing differential gene expression between stumpy (red) \nand slender (blue) T. brucei forms during in vitro differentiation to the procyclic form at 0, 8, 15, \n24 and 72  hrs after induction. Differentiation was initiated by adding cis-aconitate, lowering the \ntemperature to 27°C, and depleting glucose in the medium . Each black dot represents one gene. \nGenes within the coloured boxes show 2x up-regulation in slender (blue dotted line) or 2x up-\nregulation in stumpy (red dotted line) cells, with a p- value ≤ 0.01 (grey dotted line). Red and blue \nboxes highlight significantly upregulated genes in stumpy or slender, respectively, with the exact \n slender stumpy \n0hrs 533 314 \n8hrs 310 362 \n15hrs 325 292 \n24hrs 272 175 \n72hrs 0 0 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\ngene counts listed in the accompanying table. logFC= log2 Fold Change ; hrs = hours after the \naddition of cis-aconitate.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \n \n \n \n \n \n \nSupplementary Figure 6: V olcano plots showing differential gene expression of stumpy (red) and \nslender (blue) T. brucei forms following in vitro  differentiation to the procyclic form . \nDifferentiation was initiated by  adding cis-aconitate, lowering the temperature to 27°C, and \ndepleting glucose. An offset comparison – based on proximity in the PCA plot (Figure 3A) - aligns \nslender cells at 15 hours (hrs) with stumpy cells at 0 hrs. Each black dot represents one gene. Genes \nwithin the coloured boxes show 2x up-regulation for slender (blue dotted line) or 2x up-regulation \nfor stumpy (red dotted line) cells , with a p-value ≤ 0.01 (grey dotted line). Red and blue  boxes \nhighlight significantly upregulated genes in stumpy or slender, respectively, with the exact numbers \nof differentially expressed genes listed in the accompanying table. Notably, slender cells at 15 hrs \nexhibit a similar gene expression profile to that of  stumpy trypanosomes after 0  hrs, before \n slender stumpy \nstumpy 0hrs vs slender 8hrs 135 50 \nstumpy 0hrs vs slender 15hrs 15 7 \nstumpy 8hrs vs slender 24hrs 178 138 \nstumpy 15hrs vs slender 24hrs 178 138 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\ndiverging again at later time points . logFC= log2 Fold Change ; hrs = hours after the addition of \ncis-aconitate.  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \n \n \nSupplementary Figure 7: During differentiation into the procyclic form, slender and stumpy \nparasites exhibit gene expression profiles associated with distinct biological processes and \nmolecular functions. Gene Ontology (GO) enrichment analysis between corresponding time points \nidentified genes with a log2 fold change of at least 1 (indicating 2x expression) and a p -value of \n≤ 0.01 for either slender (orange) or stumpy (purple) forms at 0, 8, 15, 24, and 72 hours  \n(Supplementary Figure 5) . GO annotations were  sourced from the TriTryp.org database  \nA \nB \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n(TriTryp.org) and refined using Revigo. The most significantly enriched GO terms for each time \npoint are shown. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint \n\n \nSupplementary Figure 8: Dot plots showing \nexpression levels of genes associated with the \nprocyclic form  - EP1 (A), EP2 (B) and \npyruvate phosphate dikinase (PPDK)  (C) – in \nslender (orange) and stumpy ( blue) forms \nduring in vitro differentiation. Differentiation \nwas induced by the  addition of cis-aconitate, \nreduction of temperature to 27°C, and glucose \ndepletion. Stumpy forms reach procyclic -like \nexpression levels by approximately 8  hours, \nwhereas slender forms display a more gradual \nincrease. RNA-sequencing was performed in \ntriplicates; each coloured dot represents one \nreplicate (1000 cells) and the  black line \nindicates the mean expression value.  \n \nA \nC \nB \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 25, 2025. ; https://doi.org/10.1101/2025.08.20.671235doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}