Search

Artemisinins - the frontline antimalarial drug class - are compromised by emerging resistance, putting at risk the lives of hundreds of thousands of people each year. Resistance is associated with mutations in a malaria parasite protein, called Kelch 13 (K13). Recent work suggests that K13 is located at the cytostome (cell mouth) that the parasite uses to take up hemoglobin. Here we explore the proposal that K13 mutations confer artemisinin resistance by dampening hemoglobin endocytosis. This model suggests that the resultant decrease in hemoglobin-derived heme reduces artemisinin activation, which is sufficient to enable parasite survival in the early ring stage of infection. A fuller understanding of the resistance mechanism will underpin efforts to develop alternative antimalarial strategies.

The ultrathin structure of carnivorous flagellate Metromonas grandis Larsen et Patterson has been investigated. The predator captures the whole cell of the prey (bodonids or chrysomonads). Cytostome as a cell pocket has not been found. The cell, besides the flagella and small parts of the folds, is surrounded by a fibrous layer 0.05–0.12 µm in thickness which consists of thick and thin bent shafts. The plasmalemma is beneath this layer. Two unequal flagella come out of a small pocket. The long flagellum bears multiple, very thin mastigonemes (hairs) 0.8–1.0 µm in length; the short flagellum is reduced and is covered by rare mastigonemes. The transversal plate is raised above the cell surface, surrounded by osmiophilic muff. Helix is absent. The root system is simplified and consists of two microtubular bands which originated near the kinetosome of the long flagellum. The kinetosomes lie parallel to each other and are interconnected with at least one fibrous bridge. Cylindrical extrusomes (trichocysts) 1.0–1.4 µm in length and 0.12–0.08 µm in diameter have been found in the cytoplasm. Trichocysts are divided by a diaphragm and contain a wheel-shaped structure with 13 spokes visible in cross sections. Contractile vacuole is absent. The resemblance of Metromonas grandis to M. simplex (Griessmann, 1913) Larsen et Patterson, 1990, Metopion fluens Larsen et Patterson, 1990 cryomonadids, and other predatory flagellates is discussed.

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.

Endocytosis in Trypanosoma cruzi is mainly performed through a specialised membrane domain called cytostome-cytopharynx complex. Its ultrastructure and dynamics in endocytosis are well characterized in epimastigotes, being absent in trypomastigotes, that lack endocytic activity. Intracellular amastigotes also possess a cytostome-cytopharynx but participation in endocytosis of these forms is not clear. Extracellular amastigotes can be obtained from the supernatant of infected cells or in vitro amastigogenesis. These amastigotes share biochemical and morphological features with intracellular amastigotes but retain trypomastigote's ability to establish infection. We analysed and compared the ultrastructure of the cytostome-cytopharynx complex of intracellular amastigotes and extracellular amastigotes using high-resolution tridimensional electron microscopy techniques. We compared the endocytic ability of intracellular amastigotes, obtained through host cell lysis, with that of extracellular amastigotes. Intracellular amastigotes showed a cytostome-cytopharynx complex similar to epimastigotes'. However, after isolation, the complex undergoes ultrastructural modifications that progressively took to an impairment of endocytosis. Extracellular amastigotes do not possess a cytostome-cytopharynx complex nor the ability to endocytose. Those observations highlight morpho functional differences between intra and extracellular amastigotes regarding an important structure related to cell metabolism. TAKE AWAYS: T. cruzi intracellular amastigotes endocytose through the cytostome-cytopharynx complex. The cytostome-cytopharynx complex of intracellular amastigotes is ultrastructurally similar to the epimastigote. Intracellular amastigotes, once outside the host cell, disassembles the cytostome-cytopharynx membrane domain. Extracellular amastigotes do not possess a cytostome-cytopharynx either the ability to endocytose.

The present review addresses basic aspects of the biology of the pathogenic protozoa Trypanosoma cruzi and some comparative information of Trypanosoma brucei. Like eukaryotic cells, their cellular organization is similar to that of mammalian hosts. However, these parasites present structural particularities. That is why the following topics are emphasized in this paper: developmental stages of the life cycle in the vertebrate and invertebrate hosts; the cytoskeleton of the protozoa, especially the sub-pellicular microtubules; the flagellum and its attachment to the protozoan body through specialized junctions; the kinetoplast-mitochondrion complex, including its structural organization and DNA replication; glycosome and its role in the metabolism of the cell; acidocalcisome, describing its morphology, biochemistry, and functional role; cytostome and the endocytic pathway; the organization of the endoplasmic reticulum and Golgi complex; the nucleus, describing its structural organization during interphase and division; and the process of interaction of the parasite with host cells. The unique characteristics of these structures also make them interesting chemotherapeutic targets. Therefore, further understanding of cell biology aspects contributes to the development of drugs for chemotherapy.

The parasite Trypanosoma cruzi is the etiological agent of Chagas disease and chronically infects upwards of 7 million people in the Americas. Current diagnostics and treatments remain grossly inadequate due, in part, to our general lack of understanding of this parasite’s basic biology. One aspect that has resisted detailed scrutiny is the mechanism employed by this parasite to extract nutrient resources from the radically different environments that it encounters as it transitions between its invertebrate and mammalian hosts. These parasites engulf food via a tubular invagination of its membrane, a strategy used by many protozoan species, but how this structure is formed or functions mechanistically remains a complete mystery. The significance of our research is in the identification of the mechanistic underpinnings of this feeding organelle that may bring to light new potential therapeutic targets to impede parasite feeding and thus halt the spread of this deadly human pathogen., Of the pathogenic trypanosomatids, Trypanosoma cruzi alone retains an ancient feeding apparatus known as the cytostome-cytopharynx complex (SPC) that it uses as its primary mode of endocytosis in a manner akin to its free-living kinetoplastid relatives who capture and eat bacterial prey via this endocytic organelle. In a recent report, we began the process of dissecting how this organelle functions by identifying the first SPC-specific proteins in T. cruzi. Here, we continued these studies and report on the identification of the first enzymatic component of the SPC, a previously identified orphan myosin motor (MyoF) specifically targeted to the SPC. We overexpressed MyoF as a dominant-negative mutant, resulting in parasites that, although viable, were completely deficient in measurable endocytosis in vitro. To our surprise, however, a full deletion of MyoF demonstrated only a decrease in the overall rate of endocytosis, potentially indicative of redundant myosin motors at work. Thereupon, we identified three additional orphan myosin motors, two of which (MyoB and MyoE) were targeted to the preoral ridge region adjacent to the cytostome entrance and another (MyoC) which was targeted to the cytopharynx tubular structure similar to that of MyoF. Additionally, we show that the C-terminal tails of each myosin are sufficient for targeting a fluorescent reporter to SPC subregions. This work highlights a potential mechanism used by the SPC to drive the inward flow of material for digestion and unveils a new level of overlapping complexity in this system with four distinct myosin isoforms targeted to this feeding structure. IMPORTANCE The parasite Trypanosoma cruzi is the etiological agent of Chagas disease and chronically infects upwards of 7 million people in the Americas. Current diagnostics and treatments remain grossly inadequate due, in part, to our general lack of understanding of this parasite’s basic biology. One aspect that has resisted detailed scrutiny is the mechanism employed by this parasite to extract nutrient resources from the radically different environments that it encounters as it transitions between its invertebrate and mammalian hosts. These parasites engulf food via a tubular invagination of its membrane, a strategy used by many protozoan species, but how this structure is formed or functions mechanistically remains a complete mystery. The significance of our research is in the identification of the mechanistic underpinnings of this feeding organelle that may bring to light new potential therapeutic targets to impede parasite feeding and thus halt the spread of this deadly human pathogen.

We used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to observe the oral organelle, cytopharynx, and subpellicular structure of a Dileptus sp. The main results were as follows: (a) the cytostome was located on the ventral surface of the base of the beak, surrounded by a periportal matrix that integrated 135 microtube bundles. When these microtube bundles contract, radially arranged into a disk, the cytostome was closed. When these microtube bundles were stretch, they fell into the cytostome and opens. The diameter of the cytostome was about 16 μm regardless of its closure or opening, indicating that the contraction or elongation of these microtube bundles did not change the size of the cytostome, which was only related to whether it blocked the cytostome, thus determining the opening and closing of the cytostome. There were many microtube bundles on two sides of the feeding trough, which could widen or narrow the feeding trough and facilitate beak feeding. (b) The cytopharynx was basket-like without a bottom with a diameter of about 6 μm and was woven from two kind fibers about 0.08 and 0.19 μm. (c) There were two types of extrusomes under the pellicle. Using transmission electron microscopy，the Type I extrusomes showed narrow and long egg shape, its cross section was circular which is composed by various electronic density of concentric. Using the scanning electron microscope, they were two slightly thin clavate, the length was about 5 μm, the diameter of the middle section was about 0.75 μm, and the diameter of the two ends was about 0.32 μm, they were distributed abundantly between the microtubule fasciculi which were located on both sides of the gap on the feeding groove. Using transmission electron microscopy, the Type II extrusomes showed egg shape. Using the scanning electron microscopy, they were about 1.6 × 0.8 μm in size, they were distributed abundantly under the body pellicle while rarely the proboscis. In addition, many different of developmental stages two types of extrusomes could be also seen in the cytoplasm. (d) There were very well-developed fibrous systems under the pellicle that were woven from fibers about 0.14 μm in diameter that attached to the pellicle and bound some organelles in the cytoplasm (e.g., mitochondria, extrusomes) and other structures to the cytoplasm and maintained cell morphology. The results of this study not only supplement and enrich the morphological contents of the Dileptus sp., but also provide the basis for the study of the taxonomy of the Dileptus sp. It also provides a new method for researchers to explore the morphology and structure of ciliate cells under the cortex by SEM.

The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 7 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease continues to receive insufficient attention and resources in order to be effectively combatted. Unlike the other parasitic trypanosomatids that infect humans (Trypanosoma brucei and Leishmania spp.), T. cruzi retains an ancestral mode of phagotrophic feeding via an endocytic organelle known as the cytostome-cytopharynx complex (SPC). How this tubular invagination of the plasma membrane functions to bring in nutrients is poorly understood at a mechanistic level, partially due to a lack of knowledge of the protein machinery specifically targeted to this structure. Using a combination of CRISPR/Cas9 mediated endogenous tagging, fluorescently labeled overexpression constructs and endocytic assays, we have identified the first known SPC targeted protein (CP1). The CP1 labeled structure co-localizes with endocytosed protein and undergoes disassembly in infectious forms and reconstitution in replicative forms. Additionally, through the use of immunoprecipitation and mass spectrometry techniques, we have identified two additional CP1-associated proteins (CP2 and CP3) that also target to this endocytic organelle. Our localization studies using fluorescently tagged proteins and surface lectin staining have also allowed us, for the first time, to specifically define the location of the intriguing pre-oral ridge (POR) surface prominence at the SPC entrance through the use of super-resolution light microscopy. This work is a first glimpse into the proteome of the SPC and provides the tools for further characterization of this enigmatic endocytic organelle. A better understanding of how this deadly pathogen acquires nutrients from its host will potentially direct us toward new therapeutic targets to combat infection.

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cyto s tome-cyto p harynx c omplex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.

The cell mouth is a property of the vast majority of free-living and endosymbiotic/epibiotic ciliates of the class Oligohymenophorea. Cytostome, however, naturally absents in the whole endosymbiotic subclass Astomatia and was naturally or experimentally lost in a few members of the subclass Hymenostomatia. This poses a question of how homoplastic might be the lack of oral structures in the oligohymenophorean evolution. To address this question, we used two mitochondrial genes, five nuclear markers, and detailed morphological data from an enigmatic mouthless ciliate, Clausilocola apostropha, which we re-discovered after more than half of a century. According to the present phylogenetic analyses, astomy evolved at least three times independently and in different time frames of the oligohymenophorean phylogeny, ranging from the Paleozoic to the Cenozoic period. Mouthless endosymbionts inhabiting mollusks (represented by Clausilocola), planarians (Haptophrya), and annelids ('core' astomes) never clustered together. Haptophrya grouped with the scuticociliate genus Conchophthirus, 'core' astomes were placed in a sister position to the scuticociliate orders Philasterida and Pleuronematida, and Clausilocola was robustly nested within the hymenostome family Tetrahymenidae. The tetrahymenid origin of Clausilocola is further corroborated by the existence of mouthless Tetrahymena mutants and the huge phenotypic plasticity in the cytostome size in tetrahymenids.

Three new cyrtophorian ciliates isolated from coastal areas of China were described based on morphological and genetic data. The Chlamydodon mnemosyne-like species Chlamydodon similis sp. n. differs from its congeners mainly by its number of somatic kineties. Chlamydodon oligochaetus sp. n. is distinguished from its congeners mainly by having fewer somatic kineties, and/or an elongated body shape. Chlamydodon crassidens sp. n. is characterized mainly by an inverted triangular body shape, a posteriorly interrupted cross-striated band (5–6 μm wide), and a large cytostome. Moreover, we provided small-subunit (SSU) rDNA sequences of C. similis sp. n. and C. oligochaetus sp. n. Maximum Likelihood (ML) and Bayesian inference (BI) consistently placed C. similis sp. n. as a sister to C. paramnemosyne, but showed different branching position of C. oligochaetus sp. n., which may be due to a low taxon sampling in the Chlamydodontidae and/or an insufficient resolution of the marker gene at species-level. This article is protected by copyright. All rights reserved.

SUMMARYNumerous studies have been conducted on the cellular morphology of Cryptocaryon irritans. However, details regarding the tomont stage of its life cycle remain lacking. In this study, we investigated the morphology of the tomont stage throughout encystment and cell division using light and electron microscopy. Results showed that there was no secretion of encystation-specific secretory vesicles or extrusomes during formation of the cyst wall. Instead, the synthesis and construction of the C. irritans cyst wall materials may involve molecular events at the pellicle. The somatic cilia and the cytostome were present during encystment and covered by the newly formed cyst wall. New somatic cilia were continuously created between old cilia and showed various lengths during cell division, a process that was similar to morphogenesis in many free-living ciliates. During cell division inside the tomont, dividing daughter cells formed temporary cell chains with no oral primordia before separating from each other into dissociative tomite precursors. The process of cell division may not be accompanied by stomatogenesis, and new oral primordia in offspring cells likely formed before the dividing cell chains split into dissociative spherical tomites. Mitochondrial autophagy was observed in encysting C. irritans cells. Numerous endosymbionts and Golgi structures were observed in the tomont cytoplasm. Cellular metabolic activity in the C. irritans tomont was quite high, with large amounts of materials or cellular organelles potentially being synthesized and prepared for the following infective theront stage.

We report the discovery of three new species of freshwater metopid ciliates, Pileometopus lynni gen. et sp. nov., Castula flexibilis gen. et sp. nov., and Longitaenia australis gen. et sp. nov. Based on morphologic features and the 18S rRNA gene phylogeny, we transfer two known species of Metopus to the new genus Castula, as C. fusca (Kahl, 1927) comb. nov. and C. setosa (Kahl, 1927) comb. nov. and another known species is herein transferred to the new genus Longitaenia, as L. gibba (Kahl, 1927) comb. nov. Pileometopus is characterized by a turbinate body shape, a dorsal field of densely spaced dikinetids, a bipartite paroral membrane, and long caudal cilia. A distinctive morphologic feature of Castula species is long setae arising over the posterior third of the body (as opposed to a terminal tuft). Longitaenia spp. are characterized by an equatorial cytostome and long perizonal ciliary stripe relative to the cell length. Based on phylogenetic analyses of 18S rRNA gene sequences, we identify and briefly discuss strongly supported clades and intraspecific genetic polymorphism within the order Metopida.

The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle‐filled tubule running along the anterior–posterior axis of the cell, which is termed the multivesicular tubule (MVT) lysosome. Alongside, the MVT lysosome is one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle‐related changes in MVT lysosome organization; however, these only provided snapshots and did not connect the changes in the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s), we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, this suggests that the Leishmania MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx‐like feature., The Leishmania lysosome has an atypical structure, consisting of a vesicle‐filled tubule called the multivesicular tubule (MVT) lysosome. We examined the dynamics of this structure through the cell cycle using mNeonGreen tagged protein markers; the MVT lysosome elongated during the cell cycle and then disassembled before cytokinesis. The disassembly of the MVT lysosome coincided with a reduction in endocytosis rate.

Three new entodiniomorphid species, Buetschlia minuta n. sp., Buetschlia cirrata n. sp., and Charonina elephanti n. sp., were described from the hindgut of Asian elephants (Elephas maximus) from Gaziantep, Turkey. B. minuta n. sp. has an ovoid body shape with a truncated anterior end and a rounded posterior end, an adoral ciliary zone surrounding the cytostome, somatic ciliary rows in the anterior two thirds of the body, an ovoid macronucleus without a constant position, and a concretion vacuole in the anterior one third of the body. B. cirrata n. sp. has an ovoid body shape with the anterior end truncated and the posterior end rounded, an adoral ciliary zone surrounding the cytostome, unevenly distributed somatic cilia, an ovoid macronucleus without a constant position, and a concretion vacuole in the anterior one third of the body. C. elephanti n. sp. has an ovoid body shape with both ends rounded, an ovoid macronucleus without a constant position, two buccal ciliary zones, an adoral ciliary zone, a vestibular ciliary zone, three somatic ciliary zones, a dorsal ciliary zone, two posterior ciliary zones, dorsal and ventral, and a vestibulum with a Y-shaped infraciliature.

Protozoa parasitism has evolved many times independently in the history of eukaryotes. Some organelles were reduced, and specific invasion apparatuses were developed by adapting to the host. These apparatuses help to penetrate host cells and escape the immune system. The thesis summarizes the findings of the invasion apparatus and invasion of four selected unicellular protozoan parasites of genus Plasmodium, Microsporidia, Ichthyobodo and Rozella. Each chapter includes general knowledge of the parasite, its structure and life cycle. Keywords: Apicomplexa, Plasmodium, Microsporidia, Ichthyobodo, Rozella, merozoite, glideosom, moving junction, polar tube, germination, cytostome, zoospore, germ tube

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.

In recent years, the release of nanomaterials pollutants to water bodies, to a great extent, attributed to anthropogenic activities. Their impacts on aquatic organisms as well as nanomaterial monitoring and bioremediation using organism have drawn much attentions. However, studies on relationship of nano-contaminants and aquatic organisms are very scarce. Our results showed that titanium dioxide nanoparticles (TiO2-NPs) and Multi-walled carbon nanotubes (MWCNTs) caused an obvious cell decreases on the whole, but a significant increase at 48 h TiO2-NPs exposure, indicating a resistant mechanism in ciliates for nano-toxic. Besides, MWCNTs was more toxic to Pseudocohnilembus persalinus than that of TiO2-NPs in terms of EC50 value. It is firstly found that P. persalinus ingested and released TiO2-NPs through cytostome and cytoproct, which might be the reason that TiO2-NPs less toxic than MWCNTs. The significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) enzyme activities and expression levels were evaluated by reactive oxygen species ROS generation, which demonstrated that P. persalinus antioxidant defense enzyme played roles on nano-toxic resistant in ciliates. Moreover, the integrated biomarker response (IBR) was also determined, which demonstrated that MWCNTs had comparatively higher values than those of TiO2-NPs after higher concentration exposure to ciliates. In addition, it was confirmed by the present work that sod, gst and cat played different roles on immunity, and the sensitivity of cat gene expression to these two nanomaterials exposure was dissimilar. Damages of shrunk as well as losses of cilia on the cell surface caused by TiO2-NPs and MWCNTs exposure in P. persalinus using SEM revealed possible physical hazards of aggregated nanomaterials. Our findings will be helpful to understand the effect mechanisms of NPs on ciliates, and also demonstrated the possibility of P. persalinus as bio-indicator of nanomaterials in aquatic and potentials on bioremediation.

Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum -infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N -ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation.

Paratrypanosoma confusum is a monoxenous kinetoplastid flagellate that constitutes the most basal branch of the highly diverse parasitic trypanosomatids, which include human pathogens Trypanosoma and Leishmania. This makes Paratrypanosoma uniquely informative for the evolution of obligatory parasitism from free-living lifestyle and the evolution of human parasitism in some trypanosomatid lineages. It has typical promastigote morphology but also forms surface-attached haptomonads and amastigotes. Haptomonads form by attachment to a surface via a large bulge at the base of the flagellum, which is then remodeled into a thin attachment pad associated with flagellum shortening. Promastigotes and haptomonads multiply by binary division, and the progeny of a haptomonad can either remain attached or grow a flagellum and resume swimming. Whole genome sequencing and transcriptome profiling, in combination with analysis of the cell ultrastructure, reveal how the cell surface and metabolism are adapted to parasitism and how characteristic cytoskeletal features are conserved. Our data demonstrate that surface attachment by the flagellum and the flagellar pocket, a Leishmania-like flagellum attachment zone, and a Trypanosoma cruzi-like cytostome are ancestral features, while evolution of extant trypanosomatids, including the human parasites, is associated with genome streamlining and diversification of membrane proteins.

This thesis is composed of two topics both concerning diverse and obligatory trypanosomatid parasites. First part deals with identification of new Trypanosoma species identified in blood meal of tsetse flies caught in Dzanga-Sangha Protected Areas, Central African Republic, and identification of feeding preferences of tsetse flies. The second part concerns extraordinary monoxenous trypanosomatid Paratrypanosoma confusum which constitutes the most basal branch between free-living Bodo saltans and parasitic trypanosomatids. This thesis helped to elucidate morphology and biology of this deep branching trypanosomatid. Using genome and transcriptome sequencing and comparative bioinformatics approaches enabled search for ancestral genes shared with free-living bodonids and confirmed genome streamlining in trypanosomatids.

We investigated the live morphology, infraciliature, and small subunit rRNA gene sequences of an unusual population of Stentor polymorphus without symbiotic algae that was isolated from the southeastern region of Brazil. The morphological and molecular data confirmed the identity of this strain as S. polymorphus. The Brazilian S. polymorphus organism is 850-2,000 μm in length in vivo and has colorless cortical granules, a moniliform macronucleus with 6-12 nodules, somatic ciliature composed of 50-60 kineties, a single contractile vacuole located to the left of the cytostome, and a conspicuous oral pouch, and it does not build a lorica. Based on the phylogenetic analyses, the Brazilian S. polymorphus was located within a cluster consisting of four other S. polymorphus sequences, with high support values using both the Bayesian inference and maximum likelihood algorithms. Our study presents the first report of a S. polymorphus population without its symbionts under natural conditions. On the basis of our findings, we propose that the presence or absence of symbiotic algae should not be used as a taxonomic character for the identification of Stentor species.

SUMMARYCells of Cryptocaryon irritans at different life history stages were studied using both light and electron microscopy. The characteristics of several organelles were revealed for the first time at the ultrastructural level. It was confirmed that the cytostome of trophonts, protomonts and theronts was surrounded by cilium–palp triplets rather than ciliary triplets. The nematodesmata underlying the circumoral dikinetids were single bundles, whereas these were always paired in Prorodontids. Toxicysts were present in late-stage tomonts and theronts, but were absent in trophonts and protomonts. We posited that toxicysts might play a role in infection and invasion of host-fish tissue by theronts. The adoral brosse was unlike that of any other family of the class Prostomatea based on its location and morphology. Membranous folds were present in trophonts, protomonts and theronts. These folds were longer and more highly developed in C. irritans than in exclusively free-living prostome ciliates suggesting that they might be linked to parasitism in C. irritans. Trophonts, protomonts and theronts had multiple contractile vacuoles. The basic ultrastructure of the contractile vacuole of C. irritans was similar to that of other kinetofragminophoran ciliates. They might play different roles in different stages of the life cycle since their ultrastructure varied among trophonts, protomonts and theronts.

We describe three new phagotrophic euglenoid species by light microscopy and 18S rDNA and Hsp90 sequencing: Scytomonas saepesedens; Decastava edaphica; Entosiphon oblongum. We studied Scytomonas and Decastava ultrastructure. Scytomonas saepesedens feeds when sessile with actively beating cilium, and has five pellicular strips with flush joints and Calycimonas-like microtubule-supported cytopharynx. Decastava, sister to Keelungia forming new clade Decastavida on 18S rDNA trees, has 10 broad strips with cusp-like joints, not bifurcate ridges like Ploeotia and Serpenomonas (phylogenetically and cytologically distinct genera), and Serpenomonas-like feeding apparatus (8–9 unreinforced microtubule pairs loop from dorsal jaw support to cytostome). Hsp90 and 18S rDNA trees group Scytomonas with Petalomonas and show Entosiphon as the earliest euglenoid branch. Basal euglenoids have rigid longitudinal strips; derived clade Spirocuta has spiral often slideable strips. Decastava Hsp90 genes have introns. Decastava/Entosiphon Hsp90 frameshifts imply insertional RNA editing. Petalomonas is too heterogeneous in pellicle structure for one genus; we retain Scytomonas (sometimes lumped with it) and segregate four former Petalomonas as new genus Biundula with pellicle cross section showing 2–8 smooth undulations and typified by Biundula (=Petalomonas) sphagnophila comb. n. Our taxon-rich site-heterogeneous rDNA trees confirm that Heteronema is excessively heterogeneous; therefore we establish new genus Teloprocta for Heteronema scaphurum.

The cytostome-cytopharynx complex is the main site for endocytosis in epimastigotes of Trypanosoma cruzi. It consists of an opening at the plasma membrane surface – the cytostome - followed by a membrane invagination - the cytopharynx. In G1-S cells, this structure is associated with two specific sets of microtubules - a quartet and a triplet. Here, we used electron microscopy and electron tomography to build 3D models of the complex at different stages of the cell cycle. The cytostome-cytopharynx is absent in late G2 and M phase cells, while early G2 cells have either a short cytopharynx or no visible complex, with numerous vesicles aligned to the cytostome-cytopharynx microtubules. The microtubule quartet remains visible throughout cell division (albeit in a shorter form), and is duplicated during G2/M. In contrast, the microtubule triplet is absent during late G2/M. Cells in cytokinesis have an invagination of the flagellar pocket membrane likely to represent early stages in cytostome-cytopharynx assembly. Cells in late cytokinesis have two fully developed cytostome-cytopharynx complexes. Our data suggest that the microtubule quartet serves as a guide for new cytostome-cytopharynx assembly.

Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to nonreducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi., SCOPUS: ar.j, info:eu-repo/semantics/inPress

Mesodinium rubrum Lohmann is a photosynthetic marine ciliate that has functional chloroplasts of cryptophyte origin. Little is known about the oral ultrastructure of M. rubrum compared with several reports on the sequestration of nuclei and plastids from prey organisms, such as Geminigera cryophila and Teleaulax species. Here, we describe the fine structure of the oral apparatus of a M. rubrum strain from Gomso Bay, Korea. The cytopharynx was cone-shaped and supported by 20-22 ribbons of triplet microtubules. At the anterior end of the cytopharynx, an annulus anchored small cylinders composed of 11 microtubules. The small cylinders were spaced at regular intervals, each reinforced by one set of the triplet microtubules. At the opening of the cytostome, larger 14-membered microtubular cylinders were set adjacent to the small, 11-membered microtubular cylinders, each pair surrounded by separate membranes, however, only the large cylinders extended into the oral tentacles. There were 20-22 oral tentacles each having one to five extrusomes at its tip. At the anterior end of the oral apparatus, microtubular bands supporting the cytostome curved posteriad, extending beneath the cell cortex to the kinetosomes of the somatic cirri. The microtubular bands were connected by striated fibers and originated from kinetosomes anchored by fibers. Each cirrus consisted of eight cilia associated with 16 kinetosomes. The ultrastructure of M. rubrum from Korea provides information useful for taxonomic characterization of the genus Mesodinium and relevant to developing a better understanding of the acquisition of foreign organelles through phagocytosis by M. rubrum.

The ultrastructure of the marine predatory flagellate Metromonas simplex Larsen et Patterson was studied. The cell is surrounded by a low-contrast fibrous layer composed of thin hairs covered by a thin bilayer membrane and an outer layer of thin short fibers. The plasmalemma lies under these layers. The predator captures whole cells of the prey, usually bodonids or chrysomonads. The cytostome as a cell pocket is undetectable. The long flagellum bears very thin mastigonemes (hairs) with lengths of 0.8–1.0 μm; the short flagellum is naked and reduced in length. The transitional zone lacks spirals or other additional elements. The transversal plate is elevated on the cell surface. The flagellar root system is very simple and has one microtubular band which originates near the kinetosomes. The latter are parallel to each other and interconnected by fibrous bridges. The vesicular nucleus, Golgi apparatus, and endoplasmic reticulum are of typical structures. The oval mitochondria of 0.6–2.5 μm contain lamellar cristae. The cylindrical extrusomes (trichocysts) found in the cytoplasm have lengths of 1.0–1.4 μm and diameters of 0.12–0.08 μm. The trichocysts have a wheel-shaped structure with 13 spokes visible in cross-sections. The contractile vacuole is absent. The similarity that M. simplex shares with Metopion fluens Larsen et Patterson, cryothecomonads, and other predatory flagellates is discussed.

An oligohalobic peritrichous ciliate, Epistylis chlorelligerum Shen, 1980, was collected from a ditch in Hangzhou, China. The morphology, oral infraciliature, and morphogenesis of the species were studied using living and protargol-impregnated specimens. Zooids of E. chlorelligerum are 160-230 × 50-60 μm in vivo, and characterized by green-colored endoplasm containing symbiotic algae. The oral infraciliature presents a well-developed filamentous reticulum linked to the circular fiber of the cytostome; the outer two rows of P3 extend adstomally over P1 and usually enfold it. During binary fission, one daughter cell inherits most part of the old buccal apparatus and the reorganized haplokinety and germinal kinety (Hk' and G'), and new buccal apparatus of the other daughter cell is mostly developed from the original germinal kinety (G) and haplokinety (Hk): new peniculi 2, 3 (2P2, 2P3), new haplokinety (2Hk), and new germinal kinety (2G) are formed from G, while the new peniculus 1 (2P1) and its peristomial extention (2Pk) originate from Hk. The epistomial membrane can be observed until the two sets of buccal apparatus begin to separate from each other.

A novel free free-living phagotrophic flagellate, Rictus lutensis gen. et sp. nov., with two heterodynamic flagella, a permanent cytostome and a cytopharynx was isolated from muddy, low oxygen coastal sediments in Cape Cod, MA, USA. We cultivated and characterized this flagellate with transmission electron microscopy, scanning electron microscopy and molecular phylogenetic analyses inferred from small subunit (SSU) rDNA sequences. These data demonstrated that this organism has the key ultrastructural characters of the Bicosoecida, including similar transitional zones and a similar overall flagellar apparatus consisting of an x fiber and an L-shape microtubular root 2 involved in food capture. Although the molecular phylogenetic analyses were concordant with the ultrastructural data in placing R. lutensis with the bicosoecid clade, the internal position of this relatively divergent sequence within the clade was not resolved. Therefore, we interpret R. lutensis gen. et sp. nov. as a novel bicosoecid incertae sedis.

Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypo- mastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior re- gion, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the pos- terior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also accumulate lysosomal hydrolases, modulating digestive activity. Although T. cruzi infective forms are unable to uptake molecules, its lysosome related organelles represent a potential targets for anti-parasitic chemotherapy.