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Protistology 17(4): 233-243 (2023) | doi:10.21685/1680-0826-2023-17-4-4 PPOtÎStOlOây
Original article
Microscopic study of developmental stages of a Trypanosoma theileri-like trypanosome in axenic culture
Marina N. Malysheva, Alexei Yu. Kostygov* and Alexander O. Frolov*
Zoological Institute of the Russian Academy of Sciences, 199034 St. Petersburg, Russia
| Submitted September 27, 2022 | Accepted November 14, 2023 |
Summary
The development of a Trypanosoma theileri-like trypanosome was studied using light and electron microscopy in a laboratory culture maintained at room temperature. In addition, a three-dimensional reconstruction was made based on serial images obtained by electron microscopy. Three cell morphotypes were identified: elongated epimastigotes, pear-shaped epimastigotes and spheromastigotes. A distinctive feature of this trypanosome is a huge number of glycosomes and acidocalcisomes in the cytoplasm of elongated epimastigotes. Comparison of the forms documented in culture with those from the vector as well as analysis of data from the literature have led us to the conclusion that development of this trypanosome in the culture (at least partially) reproduces that in the vertebrate host.
Key words: axenic culture, ultrastructure, glycosomes, acidocalcisomes, 3D reconstruction
Introduction
Trypanosoma (Megatrypanum) theileri is one of the first trypanosome species described in mammals (Hoare, 1972). This cosmopolitan parasite infects cattle and a wide range of other bovids, such as water buffalo, zebu, European bison, and various antelopes (Schlafer, 1979). Genetically, these trypanosomes are very diverse and, along with parasites of sheep, goats, chevrotain and deer, they represent a so far unresolved species complex (Kostygov et al., 2022). For simplicity, all flagellates detected in cattle are referred to as T. theileri, while those from other ruminant hosts can either be formally assigned to one of the previously described species or (more frequently) be termed T. theileri-like trypanosomes.
https://doi.org/10.21685/1680-0826-2023-17-4-4
© 2023 The Author(s)
Protistology © 2023 Protozoological Society Affiliated with RAS
In general, horseflies and deerflies (Tabanidae) are considered the main vectors for this species complex, although some lineages are likely specialized for transmission by keds (Hippoboscidae) or mosquitoes (Culicidae) (Garcia et al., 2020; Brotankova et al., 2022; Kostygov et al., 2022). There are also a few records of T. theileri-like trypanosomes from other blood-sucking dipterans such as sandflies, blackflies and tsetse flies (Votypka et al., 2015; Ngomtcho et al., 2017; Calzolari et al., 2018; Brotankova et al., 2022).
Most infections by T. theileri-like trypanosomes are asymptomatic, and, therefore, this species is usually regarded as non-pathogenic (Hoare, 1972). In adult animals that are not weakened by concomitant infections, parasitemia is very low; therefore,
Corresponding author: Alexander O. Frolov and Alexei Yu. Kostygov. Zoological Institute RAS, 199034 St. Petersburg, Russia; [email protected], [email protected]
it is almost impossible to detect the flagellates on smears (Dirie et al., 1990; Fernandez et al., 2010). One of the most reliable and sensitive methods for detecting T. theileri-like trypanosomes in mammalian blood is their cultivation in a nutritional medium (Herbert, 1965; Wells, 1971; Schlafer, 1979). Not surprisingly, many works have been devoted to the cultivation of the parasites isolated from the blood of the vertebrate host as well as the use of such cultures in various functional studies (Wink, 1979; Townsend et al., 1982; Oyamada et al., 1995; Lee et al., 2013). Recently, T. theilerihas been used as a model object for the development of trypanocidal drugs (Fentahun, 2020; Fentahun and Paeshuyse, 2021). At the same time, information on the ultrastructure ofthese parasites is very scarce and often devoted only to the issue of their interaction with cells in a tissue culture, on which they are grown (Townsend and Duffus, 1982; Griebel et al., 1989; Oyamada et al., 1995; Rodrigues et al., 2003; Lee et al., 2013). In this work, we studied the development of a trypanosome from the Trypanosoma theileri species complex in an axenic culture isolated from a tabanid vector using light and electron microscopy.
Material and methods
Culture origin and maintenance
The culture KrSl7 has been isolated from the hindgut of the deerfly Chrysops divaricatus (Taba-nidae: Chrysopsinae) collected in the vicinity of Lakhdenpokhya town (Karelia, 61°31' N; 30°12' E) in 2018 (Ganyukova et al., 2018). This isolate corresponds to the provisional species Trypanosoma sp. Ttha (from the TthI clade ofthe T. theileri species complex), which most likely parasitizes cervids (Kostygov et al., 2022).
The flagellates were cultivated in a biphasic nutrient medium consisting of blood agar overlaid with the RPMI-1640 medium (Sigma-Aldrich, United Kingdom) supplemented with 10% fetal serum (Biolot, St. Petersburg, Russia) and a ready-to-use mixture of streptomycin and penicillin (Sigma-Aldrich, St. Louis, USA). Purification ofthe culture from accompanying mycelial and yeast-like fungi was carried out in a device described previously (Podlipaev and Frolov, 1987). The culture was always maintained at room temperature.
Light microscopy
Smears prepared from the culture were air-dried, fixed with 96% ethanol for 20-30 min, and afterwards stained with Giemsa stain for 30 min at pH 6.8. They were examined at * 1,000 magnification with a DM2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany). Photos were taken using a UCM0S14000KPA 14-Mpx camera (ToupTek, Hangzhou, China) mounted on the microscope. The obtained images were used to perform standard measurements (cell length and width, nucleus length, length of the free flagellum, as well as distances between the anterior end and nucleus and/or kinetoplast) using ImageJ v. 1.53e software (Abramoff et al., 2004).
Electron microscopy
For TEM, a pre-precipitated 6-day culture was fixed with 1.5% glutaraldehyde in 0.1 M cacodylate buffer (1 h, 0 °C) and post-fixed with 2% 0s04 in 0.1 M cacodylate buffer (1 h, 0°C). Cells were dehydrated in a series of alcohols ofincreasing concentration and propylene oxide and embedded in a mixture of Epon and Araldite. Thin sections (60 nm) were obtained on a Leica UC-6 ultramicrotome, stained with a saturated aqueous solution ofuranyl acetate (1 h) and lead citrate following a previously described protocol (Reynolds, 1963) and examined under a Morgagni 268-D microscope (FEI Company, Hillsboro, Oregon, U.S.A.).
Reconstructions and subsequent 3D data analyses were performed using IMOD software package (Kremer et al., 1996) following the procedure described earlier (Frolov et al., 2022). The serial sections used for these reconstructions are shown in Supplementary Figs S1 and S2.
Results
Light microscopy
In the smears prepared from the culture KrSl7, the trypanosome cells were represented by three major morphotypes: elongated epimastigotes, pear-shaped epimastigotes and spheromastigotes (Fig. 1, A—B, I—J and M, respectively). The morphogenetic relationships between these cell variants could be
Fig. 1. Morphology of Trypanosoma sp. Ttha in axenic culture (Giemsa staining, light microscopy). A, B — Elongated epimastigote; C—E — fission of elongated epimastigotes; F—H — transitional elongate-to-pyriform epimastigote forms; I, J — pyriform epimastigote; K, L — fission of pyriform epimastigotes; M — spheromastigote; N — fission of spheromastigotes. Scale bars: A—N — 10 ^m.
easily revealed due to the presence of transitional forms (Fig. 1, F—I). All cell types had well-developed free flagella, while a conspicuous undulating membrane was present only in epimastigotes. Active division occurred in all three morphotypes and included both binary (Fig. 1, C, D, K) and multiple fission (Fig. 1, E, L, N). Measurements for the three morphotypes are presented in Table 1.
The elongated epimastigotes had pointed anterior and posterior ends. The nucleus was located in the middle or displaced to the anterior half of the cell (Fig. 1, A, B). The kinetoplast was situated near the nucleus, either anteriorly or laterally to it. The length of these cells varied from 11.4 to 28.3 (18.3+4.0 on average) ^m.
The pear-shaped epimastigotes formed from the elongated ones by gradual reduction of the postnuclear part of the cell with parallel swelling of the area around the nucleus (Fig. 1, F—J). Their posterior end was rounded, while the anterior one was stretched. The latter formed a conspicuous undulating membrane. The nucleus was located in the central part of the cell, and the kinetoplast accompanied it as in the previous morphotype (Figs. 1, I, J). The length ofthe pear-shaped epimastigotes was 5.7-12.8 (9.5+1.9 in average) ^m.
The spheromastigotes were rounded cells with a long free flagellum (Fig. 1, M). They originated from pear-shaped epimastigotes by retraction ofthe rostrum (stretched anterior end). Their proportion
Table 1. Morphometry of three cell types in the culture KrSl7. All measurements are in |jm (M+SD (min-max)).
Elongated epimastigotes Pyriform epimastigotes Spheromastigotes
Cell length 18.3±4.0 (11.4-28.3) 9.5±1.9 (5.7-12.8) 5.9±2.0 (3.7-8.2)
Cell width 3.1±0.1 (1.9-4.2) 5.3±1.2 (3.5-8.0)
Nucleus length 3.0±0.4 (2.2-3.8) 2.3±0.4 (1.5-3.2) 2.3±0.4 (1.7-3.1)
K-A1 5.8±1.2 (4.1-9.9) 5.7±1.3 (3.2-8.9)
N-A2 5.4±1.4 (2.3-8.5) 5.0±1.7 (1.9-8.0)
Flagellum length 11.8±6.3 (3.9-26.0) 11.7±6.8 (3.8-28.6) 14.2±6.5 (3.8-25.3)
Sample size 35 31 16
1 distance between the kinetoplast and the anterior end of the cell
2 distance between the nucleus and the anterior end of the cell.
in the culture was low (1-2%), but this did not prevent observation of dividing forms. These cells measured 3.7-8.2 (5.9+2.0 in average) ^m in diameter.
Electron microscopy
All three morphotypes observed in the culture featured the same set of organelles, but differed in the abundance of acidocalcisomes and glycosomes: elongated epimastigotes and transitional elongate-to-pyriform ones had considerably larger numbers of these organelles than the other two morphotypes (Fig. 2). The nucleus was usually round and harbored a large, somewhat eccentrically located nucleolus (Fig. 2, B, C). The rod-like kinetoplast sizing 0.75—1 ^m in length and 0.26—0.34 ^m in width was situated nearby and its DNA was very tightly packed (Fig. 2, A, c). None of the cell types harbored a discernible glycocalyx.
The flagellum had a well-developed paraflagellar rod that appeared within the flagellar pocket at the level of the terminal structure of the transition zone (?). After leaving the short laterally opening pocket, the flagellum attached to the plasmalemma. These details could also be observed in spheromastigotes (Fig. 2, C). In the immediate vicinity ofthe flagellar pocket, there was a contractile vacuole with a system of afferent canals, the so-called spongiome (Fig. 2, C).
The single mitochondrion was extensively ramified: it formed wide diverticula lying deep in the cytoplasm (Fig. 2, A), and two relatively narrow arms stretching under the plasmalemma and producing a network of lateral offshoots (Fig. 3, A, B). The Golgi complex was located close to the nucleus and was represented by a stack of10 or more flattened cisternae and numerous associated
vesicles (Fig. 3, A). The endoplasmic reticulum was well developed (Fig. 2, B, C). A few lipid inclusions, representing rounded vacuoles (0.2—1.3 ^m in diameter) with homogeneous contents of light gray color, were detected in different parts of the cell (Figs 2, B; 3, B). Autophagosomes were often found in cells (Fig. 3, B).
Numerous acidocalcisomes ranging in size from 0.2 to 0.6 ^m were unevenly distributed throughout the cytoplasm with noticeable clusters near the contractile vacuole, kinetoplast and in the posterior portion of the cell (Figs 2; 3, D; 4, B, C). Depending on the amount and distribution of the contents, acidocalcisomes could sometimes appear as completely transparent or solid black vacuoles. However, in most cases, the electron-dense contents formed either a small spot-like accumulation at the periphery or a relatively thin layer of varying density and thickness on the inner surface of the acidocalcisome membrane. All the characterized diversity of these organelles could be present in a single cell. In the posterior portion of the cells, acidocalcisomes were accompanied by quite large (0.5—1 ^m in diameter) rounded or oval membrane-bound organelles with granular contents of unequal density, indicated by us as dense structures (Figs 2, A and 3, D). In serial sections, we could observe an exchange of contents between these structures and acidocalcisomes (Fig. S3).
The cells in the culture had a large number of glycosomes, which in individual sections looked single or arranged in large groups up to a few dozens (Figs 2, A—C and 3, C). The glycosome profiles were rounded, oval, club- or dumbbell-shaped and significantly varied in sizes: 0.2—1.7 ^m in length and 0.1—0.2 ^m in width. These organelles were often observed in proximity to acidocalcisomes (Figs 2, A; 4, B; Fig. S3).
Fig. 2. Ultrastructure of the three Trypanosoma sp. Ttha morphotypes. A — Elongated epimastigote; B — pyriform epimastigote; C — spheromastigote. Abbreviations', ac — acidocalcisome; cv — contractile vacuole; ds — dense structures; er — endoplasmic reticulum; fl — flagellum; fp — flagellar pocket; gl — glycosome; kp — kinetoplast; li — lipid droplet; mi — mitochondrion; nu — nucleus; pr — paraflagellar rod; sp — spongiome. Scale bars: A—C — 2 ^m.
The 3D reconstruction of a fragment of an elongated epimastigote allowed for a more detailed characterization of the structure of the glycosomal complexes (Fig. 4, A). The center of such a complex was located near the mitochondrion and, apparently, was represented by cisterns of the endoplasmic reticulum, which produced glycosomes. The gly-cosomes, newly formed in the central part of the complex, were elongated, while the products of their division were smaller and shifted to the periphery. According to our estimates based on the reconstructed central fragment of an elongated epimastigote, the number of glycosomes in this cell type exceeded 500. In the reconstructed elongated epimastigote, numerous (at least 100) acido-calcisomes were situated near the glycosomes (Fig. 4, B; Video S1). In the pyriform epimastigote, glyco-
somes and acidocalcisomes were less abundant with the respective numbers being about 50 and 20 (Fig. 4, C; Video S2). The latter organelles were concentrated mostly near the nucleus and the flagellar pocket. The lower number of these two types of organelles was compensated by a larger volume of the intensively branching mitochondrion (Fig. 4, B; Video S2).
Discussion
When cultivating the T. theileri-like trypanosome Ttha, we expected to observe the developmental stages from the vector. Indeed, the culture was isolated from the deerfly and was maintained at room temperature, which is quite different from
Fig. 3. Cytoplasmic organelles of Trypanosoma sp. Ttha in culture. A — Golgi complex; B — autophagosome; C — glycosomes; D — dense structures and acidocalcisomes. Abbreviations: ac — acidocalcisome; ag — Golgi complex; aph — autophagosome; ds — dense structures; fl — flagellum; gl — glycosome; li — lipid droplet; mi — mitochondrion; nu — nucleus; pr — paraflagellar rod. Scale bars: A — 0.5 ^m; B—D — 1 ^m.
that of the vertebrate host body. However, our observations revealed that the cells in the culture are distinct from those previously described in the tabanid intestine (Böse and Heister, 1993; Kostygov et al., 2022). The development of this trypanosome in the vector is accompanied by a successive change of the following developmental stages: elongated epimastigotes, pear-shaped epimastigotes, and metacyclic trypomastigotes. The unattached elongated epimastigotes appear in the tabanid midgut for a short time (1—4 days after infection) and in relatively small numbers (Böse and Heister, 1993; Ganyukova et al., 2018; Kostygov et al., 2022). In the ileum (hindgut), elongated epimastigotes give rise to attached pear-shaped epimastigotes, which rapidly divide and produce metacyclic trypomastigotes. The latter morphotype was not observed in the culture studied here. As for the spheromastigotes, although their presence in the vector hindgut was claimed in previous works (Böse and Heister, 1993; Ganyukova et al., 2018), a more
thorough analysis showed that the cells assigned to this morphotype had signs of degradation and represented dead metacyclic cells (Kostygov et al., 2022). Conversely, the genuine spheromastigotes, which we observed in the culture, did not show any signs of apoptosis and actively divided.
The differences between the development in the tabanid gut and in the culture concerned not only the presence of particular morphotypes, but also their abundance and morphology. Thus, in the culture elongated epimastigotes represented one of the two dominating cell types, while they were rather ephemeral in the vector intestine. All morphotypes in the culture were significantly larger and had longer flagella than the corresponding cells in the vector (Kostygov et al., 2022). At the ultrastructural level, there were further differences. In contrast to the cells from culture, the pyriform epimastigotes and metacyclics from the gut did not have glycosomes, while acidocalcisomes were rare (Kostygov et al., 2022). The kinetoplast in the trypanosomes from
Fig. 4. Three-dimensional reconstructions of Trypanosoma sp. Ttha cells. A, B — Elongated epimastigote (14 sections); C — pyriform epimastigote (15 sections). Abbreviations: ac — acidocalcisome; fl — flagellum; gl — glycosome; kp — kinetoplast; mi — mitochondrion; nu — nucleus.
the vector showed an organization distinct from the compact rod-like shape observed in cultural cells. There were a rectangular profile with striped appearance because of uneven condensation of DNA in epimastigotes, and a barrel-shaped profile with patchy DNA distribution in metacyclic try-panosomes. Interestingly, the rare trypomastigotes found outside the intestine (supposedly escaping from the gut due to its occasional rupture) contained moderate numbers of both acidocalcisomes and glycosomes. However, they still differed from the cells in culture by the organization ofthe kinetoplast and a thick layer of glycocalyx (Kostygov et al., 2022).
In a previous study of a T. theileri-like trypano-some isolated from the sandfly Phlebotomus perfili-ewi, the culture contained epimastigotes (according to published photos, they could be classified as elongated and pyriform) and, reportedly, amastigotes (Calzolari et al., 2018). The authors claimed that the cultural forms were larger compared to the cells in the sandfly gut. According to the photos published in this work, the cultural forms had rod-like kinetoplasts, lipid droplets, rare acidocalcisomes, but neither glycosomes nor a discernible glycocalyx (Calzolari et al., 2018). Importantly, this trypanosome and the one investigated here belong to two different species and even to the different clades of the T.
theileri complex (TthlI versus TthI). In addition, the culture isolated from the sandfly was maintained in a monophasic Schneider Drosophila medium (with fetal bovine serum). These factors could be responsible for the observed morphological differences.
The large number of acidocalcisomes (but not glycosomes) has been previously reported in cultures of T. theileri isolated from cattle and water buffaloes (Rodrigues et al., 2003; Lee et al., 2010). As judged by the hosts, they represented different species from the one studied here, but belonged to the same TthI clade. Thus, the similarity in the abundance of acidocalcisomes is probably explained by the relatedness of these species. Interestingly, in one of these works, T. cruziand T. rangeli (belonging to the subgenera Schizotrypanum and Aneza, respectively) were grown under the same conditions and their epimastigotes showed significantly smaller numbers of acidocalcisomes than the same type of cells in T. theileri. In trypomastigotes ofthe two former species, these organelles were even more scarce (Rodrigues et al., 2003).
Acidocalcisomes were first described in trypano-somatids and, as their name suggests, have an acidic environment and store calcium ions (Docampo et al., 1995). These organelles are distinct from lysosomes, reservosomes, and other vacuoles of similar sizes
(Scott et al., 1997; Scott et al., 1998; Miranda et al., 2000). In addition to calcium, acidocalcisomes are able to deposit Mg2+, Na+, K+, Zn2+ and phosphorus compounds (Docampo, 2016). They can contact with various cellular structures, such as the nucleus, mitochondrion, contractile vacuole, subpellicular microtubules and lipid inclusions (Miranda et al., 2000). Here we additionally revealed their association with glycosomes, as well as the close connection to the organelles provisionally termed dense structures, which probably participated in the genesis of acidocalcisomes.
Acidocalcisomes play an important role in os-moregulation, autophagy, resistance to environmental changes during the life cycle and other processes (Docampo, 2016). The diversity we observed in the structure of acidocalcisomes was previously described in T. theileri(Rodrigues et al., 2003; Lee et al., 2010), as well as in T. cruzi (Miranda et al., 2000) and apparently reflects the dynamics of using ions for cellular processes in the observed cells. We believe that the large number of acidocalcisomes in elongated epimastigotes and transitional elon-gated-to-pyriform ones is associated with active metabolic processes in these cells accompanying morphological transformations. In particular, the autophagy, for which acidocalcisomes are needed, ensures reduction in the number of some organelles as well as the total volume of the cell.
Glycosomes also play an important role in the metabolism of trypanosomatids. These multicom-ponent organelles originated from peroxisomes and are specific to kinetoplastids and diplonemids, therefore these two groups of flagellates were collectively named Glycomonada (Haanstra et al., 2016; Bauer and Morris, 2017). Glycosomes contain numerous metabolic enzymes, of which the most well known are those for glycolysis, hence the name of these organelles (Opperdoes, 2010; Quiñones et al., 2020). It has been previously demonstrated that changes in their abundance are associated with parasite differentiation (Michels et al., 2006; Szoor, 2010). Thus, in T. brucei, the bloodstream forms possess more glycosomes compared to the parasites in the insect vector. Changes in glycosome number are mediated by pexophagy (peroxisome/glycosome autophagy) and biogenesis (Haanstra et al., 2016; Quiñones et al., 2020). Glycosome biogenesis occurs in two ways: by division of pre-existing glycosomes and by de novo formation of these organelles from the endoplasmic reticulum (Bauer and Morris, 2017). Our 3D reconstruction of the glycosomal
complex shows that it has a single center, most likely representing the cisterns of the endoplasmic reticulum, from which glycosomes are produced by budding and can reach extremely large numbers (over 500). In previous works, the number of glycosomes in non-dividing trypanosome cells in a culture were estimated as 30—40 for T. brucei evansi, 50 for T. cruzi and 65—75 for T. brucei brucei (Soares and De Souza, 1988; Tetley and Vickerman, 1991; Hughes et al., 2017). To the best of our knowledge, the largest number (up to 340 glycosomes) has been found in trypomastigotes of T. brucei taken directly from the bloodstream (Opperdoes, 2010). The proximity of glycosomes to mitochondria, which we documented here, is also likely related to their biogenesis. Indeed, it has been shown that in mammalian cells pre-peroxisomes are formed by fusion of two types of vesicles, originating from the endoplasmic reticulum and mitochondria, and then maturate by importing various proteins from the cytoplasm (Kim, 2017; Sugiura et al., 2017).
The activity ofmetabolic processes occurring in cells of the studied culture is also evidenced by a well-developed transport system, consisting of the branched endoplasmic reticulum and the Golgi apparatus, as well as the presence of autopha-gosomes. Autophagy is known to play an important role in trypanosomatid cell differentiation (Herman et al., 2006; Alvarez et al., 2008; Brennand et al., 2011).
The sum of available facts unambiguously indicates that the forms we described in the culture of the T. theileri-like trypanosome Ttha represent developmental stages from the vertebrate host. Indeed, they are distinct from those observed in the vector and are characterized by abundant glycosomes and acidocalcisomes like bloodstream forms of other trypanosomes. Apparently, due to typically low parasitemia, such cells are usually not observed. Nevertheless, in an old publication cited in the Hoare's monograph on mammalian trypanosomes, elongated epimastigotes and unequal division producing a pear-shaped epimastigote and a spheromastigote have been imaged for T. theileri from bovine blood (Hoare, 1972). Thus, cultivation in biphasic media represents a valuable method to study the elusive forms of these trypanosomes developing in ruminant hosts. Moreover, since the developmental stages from the vectors can be indiscernible even for distantly related species of the T. theileri complex (Kostygov et al., 2022), their comparison in culture seems to be more promising in revealing interspecific differences.
Acknowledgements
This work was funded by the Russian Science Foundation grant 21-14-00191, the cultivation of the trypanosome an its analysis using light microscopy was supported by the State Assignments for the Zoological Institute RAS 122031100260-0 and 122031100281-5. Research was completed using equipment of the 'Taxon' Core Facilities Centre at the Zoological Institute of the Russian Academy of Sciences (St. Petersburg, Russia).
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Supplementary material
Fig. S1. Serial sections of a fragment of a Trypanosoma sp. Ttha elongated epimastigote used for 3D reconstruction. Scale bar: 2 ^m.
Fig. S2. Serial sections of a fragment of a Trypanosoma sp. Ttha pyriform epimastigote used for 3D reconstruction. Scale bar: 2 ^m.
Fig. S3. Serial sections of an elongated epimastigote of Trypanosoma sp. Ttha fragment showing contact of dense structures and acidocalcisomes. Numbers indicate the order of the sections. Scale bar: 2 ^m.
Video S1. Animated 3D reconstruction of an elongated Trypanosoma theileri-like epimastigote fragment.
Video S2. Animated 3D reconstruction of a pyriform epimastigote Trypanosoma theileri-like fragment.
