The complete coding region of the maxicircle as a superior phylogenetic marker for exploring evolutionary relationships between members of the Leishmaniinae.

The mitochondrial DNA (mtDNA) is a potentially valuable phylogenetic marker given its presence across all eukaryotic taxa and its relative conservation in structure and sequence. In trypanosomatids, a homologue of the mtDNA referred to as the maxicircle DNA, is located within a specialised structure in the single mitochondrion of the trypanosomatids called the kinetoplast; a high molecular weight network of DNA composed of thousands of catenated minicircles and a smaller number of larger maxicircles. Unique to the kinetoplastid protists, the maxicircle component of this complex network could represent a desirable target for taxonomic inquiry that may also facilitate exploration of the evolutionary history of this important group of parasites. The aim of this study was to investigate the phylogenetic value of the trypanosomatid maxicircle for these applications. Maxicircle sequences were obtained either by assembling raw sequence data publicly accessible in online databases (i.e., NCBI), or by amplification of novel maxicircle sequences from trypanosomatid DNA using long-range (LR) PCR with subsequent Illumina sequencing. This procedure facilitated the generation of nearly complete maxicircle sequences (i.e., excluding the divergent region) for numerous dixenous and monoxenous trypanosomatid species. Annotation of each maxicircle sequence confirmed that their structure was conserved across all taxa examined. Phylogenetic analyses confirmed that Z. australiensis showed a greater genetic relatedness with the dixenous trypanosomatids of the genera Leishmania and Endotrypanum, as opposed to members of the monoxenous genera Crithidia and Leptomonas. Additionally, molecular clock analysis supported that the dixenous Leishmaniinae appeared approximately 75 million years ago during the breakup of Gondwana. In line with previous studies, our results support the Supercontinents hypothesis regarding the origin of dixenous Leishmaniinae. Ultimately, we demonstrate that the maxicircle represents an excellent phylogenetic marker for studying the evolutionary history of trypanosomatids, resulting in trees with very high bootstrap support values.

Morphological discordance of the new trypanosomatid species phylogenetically associated with the genus crithidia.

Three new species of monoxenous parasites from the Neotropical Heteroptera are described on the basis of the ultrastructure of cells in culture, as well as gene sequences of Spliced Leader (SL) RNA, glyceraldehyde phosphate dehydrogenase (GAPDH) and small subunit (SSU) rRNA. The results have highlighted a striking discrepancy between the morphological (dis)similarities and the phylogenetic affinities among the insect trypanosomatids. Although each of the new species is characterized by a distinct set of morphological characters, based on the predominant promastigotes observed in culture, each of them has been provisionally assigned to the genus Leptomonas pending the future revision of this genus. Yet, instead of the phylogenetic affinity with the other members of this polyphyletic genus, the new species are most closely related to Crithidia species. Thus, the extremely long promastigotes of Leptomonas acus sp. n. and the unique morphological features found in Leptomonas bifurcata sp. n. sharply contrast with their respective relatives C. fasciculata and C. deanei both of which are typical choanomastigotes. The results clearly show that the current classification at the genus level is misleading and needs to be revised. The phylogenetic clades potentially representing the candidate new genera of monoxenous trypanosomatids have started to emerge from the presented analyses.

Structural and biochemical characterisation of the paraflagellar rod of Crithidia fasciculata.

The trypanosomatid Crithidia fasciculata possesses an intraflagellar structure known as the paraflagellar or paraxial rod which runs from a point 1 to 2 micrometer distal to the basal body to the flagellar tip. In longitudinal section the paraflagellar rod was composed of three "sets" of parallel filaments arranged in a lattice. In cross section it consisted of two electron dense "plaques", one near the flagellar membrane, the other near the axoneme, separated by 6 to 7 fibrous elements. The position of the paraflagellar rod in relation to the axonemal central pair remained static along the length of the flagellum and was the same in all flagella examined. The paraflagellar rod was anchored to the axoneme by a regular array of 5 to 7 nm diameter links. These rod/axoneme links were sensitive to trypsin digestion enabling the rod to be separated from the axoneme. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the paraflagellar rod consisted mainly of two proteins, PFR1 (76 000 Daltons) and PFR2 (68 000 Daltons). The isoelectric points of these two proteins were remarkably similar. A PFR-enriched fraction was obtained by prolonged dialysis of demembranated flagella against a low concentration buffer. The paraflagellar rod and the central pair of singlet microtubules went into solution, leaving only the outer doublets intact. The relevance of these results to the study of the role of the paraflagellar rod in flagellar motility were discussed.

Ultrastructural and biochemical analysis of the relationship of Crithidia deanei with its endosymbiont.

Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.

Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas.

Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion. This organelle is host to a variety of intriguing and unique features, including RNA editing. We examined the characteristics of protein import into mitochondria of Trypanosoma brucei. Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids. The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent. Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes. The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria. Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids). Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms. These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes.

Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata.

Particulate fractions obtained from Trypanosoma cruzi and Crithidia fasciculata by different procedures were subjected to isopycnic centrifugation in sucrose gradients, in order to determine the subcellular localization of phosphoenolpyruvate carboxykinase (PEPCK) in both organisms, and of malic enzyme (ME) I in T. cruzi. The more clear-cut results were obtained with T. cruzi by breaking the cells by grinding in a mortar with silicon carbide and using a gradient from 0.4 to 2.0 M sucrose, whereas with C. fasciculata, the best procedure was disruption of the cells by digitonin treatment and potter homogenization and use of a gradient from 1.1 to 2.0 M sucrose. PEPCK banded together with the glycosomal marker hexokinase in both organisms; there was a clear separation from the mitochondrial markers, oligomycin-sensitive Mg2+-APTase and citrate synthase. PEPCK showed a latency of 24% in the enriched 'glycosoma' fraction of T. cruzi. ME I from T. cruzi, on the other hand, banded together with the mitochondrial markers. These results indicate that PEPCK and ME are present in different subcellular compartments, a fact significant for the prevention of a futile cycle between C4-dicarboxylic acids and C3-monocarboxylic acids, which might take place if both enzymes functioned in the same compartment.

The effect of short-wavelength ultraviolet light on antigens, lectin receptors and the ultrastructure of Crithidia fasciculata.

Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this organelle were present in almost all flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite.

UV-induced growth inhibition in the trypanosomatid Crithidia fasciculata.

The effects of short ultraviolet (253 nm) radiation on the growth of the trypanosomatid flagellate Crithidia fasciculata were studied in liquid medium and nutrient agar plates. Cell duplication was completely inhibited after exposure of the flagellates to doses equal to or higher than 50 J/m2. The UV-induced lag period was dose-dependent. Survival was reduced to 1% after exposure of the parasites to 200 J/m2. Ultrastructural changes after the lag period were studied by transmission electron microscopy. Changes of the kinetoplast network structure and sometimes of the mitochondrial matrix were observed. The existence of DNA repair mechanisms in this protozoan is discussed.

The paraxial structure of the flagellum of trypanosomatidae.

The flagella of several Trypanosomatidae (Trypanosoma cruzi, Herpetomonas samuelpessoai, Leptomonas samueli, Herpetomonas megaseliae, and Crithidia harmosa) were studied. Besides the axoneme, they have a filamentous, latticelike structure, the paraxial or paraflagellar rod. This structure was not observed in the flagellum of the intracellular spheromastigote form of Trypanosoma cruzi, but it appeared when the transformation of epimastigote and trypomastigote stages occured. Cross sections of the flagella show that the paraxial structure maintains a fixed position relative to the axonemal microtubules, being localized in a region comprised between doublets 4 and 7. Flagella of T. cruzi and H. samuelpessoai were isolated using the detergent lubrox PX. Most of them did not have a flagellar membrane. However, the paraxial structure remained associated with the axoneme. By negative staining, short projections were seen connecting the paraxial structure to the axonemal microtubules. The paraxial structure did not stain with phosphotungstic acid as occurs with the peripheral doublet microtubules, but it is formed by microfilaments longitudinally oriented in relation to the axoneme crossed in two directions by oblique filaments which make an angle of 45 degrees with the longitudinal ones.

[Study of DNA-containing structures in the nucleus and cytoplasm of the Crithidia flagellates using the Miller method]. / Izuchenie DNK-soderzhashchikh struktur iadra i tsitoplazmy zhgutikonostsev Crithidia metodom Millera.

Weakly condensed interphase chromosomes made of chromatin, and the tightly packed kinetoplast DNA (kpDNA) of a single mitochondrion of Crithidia (Kinetoplastidea, Trypanosomatida) flagellates were studied on the Miller-type spread preparations by electron microscopy. Chromatin of organisms lysed at low ionic strength conditions for 5-10 min unfolds up to 10-nm nucleosomal filaments and 20-nm chromatin fibers. The initial indications of kpDNA decompaction become visible after a 10-13 min dispersion of lysed flagellates. However, at least a 15 min long procedure is required for well-defined identification of intrakinetoplast structures. In this case, the kinetoplast looks like a heterogeneous network disposed close to the kinetosome of a single flagellum. Cells with diameters of 162.5 nm and contour wall length of 510 nm dominated within the network. With the prolongation of the dispersion time up to 20 min both these parameters increased up to 218 and 686 nm, respectively. Further prolongation of the treatment up to 60 min results in wall disruption in many cells. Within these cells, some isolated circular kpDNA molecules appear with the contour length of 588-792 nm. The circles of this size correspond to individual minicircles of the Crithidia kpDNA. Partly unfolded maxicircles of the kpDNA can be found only at early stages of dispersion (10 min). Special features of compaction of DNA-containing structures in both the nucleus and cytoplasm of Crithidia are discussed.

Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium.

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis.

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.

Crithidia deanei: influence of parasite gp63 homologue on the interaction of endosymbiont-harboring and aposymbiotic strains with Aedes aegypti midgut.

The present study demonstrates that the endosymbiont of Crithidia deanei influences the expression of surface gp63 molecules. Ultrastructural immunocytochemical analysis shows the presence of the gp63-like protein in the protozoan flagellum and flagellar pocket, either attached to shed membranes or in a free form. This molecule is glycosylphosphatidylinositol (GPI) anchored to the plasma membrane as demonstrated by phospholipase C (PLC) treatment and cross-reacting determinant detection by immunoblotting. The gp63 molecule mediates the adhesive process of the protozoan to Aedes aegypti explanted guts, since the binding was reduced by pre-incubating the C. deanei parasites (wild and aposymbiotic strains) with anti-gp63 antibodies, PLC or PLC followed by anti-gp63 antibodies incubation. In addition, the number of wild C. deanei bound to A. aegypti explanted guts was twice as that of aposymbiotic parasites. Flow cytometry assays revealed that the reactivity of the wild strain with anti-gp63 antibodies was approximately twice as that of the aposymbiotic strain. We may conclude that higher expression of surface gp63 by the wild strain of C. deanei may positively influence this interaction, posing a prominent advantage for the endosymbiont-containing trypanosomatids.

Evidence for a sliding-resistance at the tip of the trypanosome flagellum.

Motility in trypanosomes is achieved through the undulating behaviour of a single "9 + 2" flagellum; normally the flagellar waves begin at the flagellar tip and propagate towards the base. For flagella in general, however, propagation is from base-to-tip and it is believed that bend formation, and sustained regular oscillation, depend upon a localised resistance to inter-doublet sliding - which is normally conferred by structures at the flagellar base, typically the basal body. We therefore predicted that in trypanosomes there must be a resistive structure at the flagellar tip. Electron micrographs of Crithidia deanei, Herpetomonas megaseliae, Trypanosoma brucei and Leishmania major have confirmed that such attachments are present. Thus, it can be assumed that in trypanosomes microtubule sliding at the flagellar tip is resisted sufficiently to permit bend formation.

The relative efficiency of various fluids in the rapid freezing of protozoa.

The cooling efficiencies of various fluids at low temperature were compared by measuring the temperature decay in 3 microliter water samples plunged into them. A simple model of cooling was used in order to discuss the results. Liquid ethane was found to produce a cooling rate of 660 KS-1, about twice that of liquid propane, while ethanol was almost as effective as ethane between 273 to 223 K. The heat-transfer coefficient of liquid ethane was estimated to be between 1500 and 5000 W m-2 K-1, depending on the physical state assumed for the water sample. Samples of flagellated organisms, after being frozen rapidly in the above way, were freeze-substituted by the method of Barlow & Sleigh (1979). Although this fixation did not give good definition of the microtubules of the flagellar axoneme, it exhibited reasonable tissue preservation in thin sections of the cell body. The fixation method resulted in preserved flagellar wave shapes, which were observed under the light microscope and in critical-point dried cells examined by scanning and conventional electron microscopy. It was concluded (a) that methods for preserving the wave shape of the flagellum and for preserving its internal structure may not be compatible, and (b) that although the present cooling method (with ethane) approaches the speed required to arrest a flagellar wave, further improvements in the speed of the method are desirable.

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence.

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.

Multiple signals in STEM.

A brief description is given of the eight image signals that can be simultaneously recorded in digital form using an appropriate data recording system and high resolution STEM; this involves a set of detectors covering bright and dark field. Details are provided of the collection and manipulation of image data for STEM, including on-line estimation of off-focus distance, on-line assessment of astigmatism, on-line signal to noise ratio in any or all detectors; examples of all three procedures are given. The use of the data recording system is discussed together with some aspects of image display, including the use of colour. A method is introduced of providing, using the appropriate hardware, a prescribed colour map (image intensity converted to colour) using replaceable PROMS. Two off-line procedures are considered. One is the Karhunen-Loeve transform and its application to bringing together the information contained within the multiple signals, and the other is the discrete cosine transform by which the storage problem for digital image storage may be reduced, according to the image, by between 15 and 30 times.

Flagellar wave reversal in the kinetoplastid flagellate Crithidia oncopelti.

Living Crithidia oncopelti cells swim through their environment by means of tip-to-base waves on their single flagellum. The cells are able to re-orient themselves by using a short burst of asymmetrical base-to-tip waves. All points on a flagellum are capable of initiating waves. Placing a population of cells in a medium of high viscosity initially produces a large number of organisms beating in the reverse mode. An individual cell has a random "switching" behavior. Viscosity affects the frequency of forward and reverse waves in different ways. The concentration of free Ca++ ions determines the direction of wave propagation in reactivated axonemes. Calmodulin may play a role in mediating the Ca++ dependence of wave direction.

Coated pits and coated vesicles in unicellulars (Tetrahymena, Crithidia) and in Hydra.

Coated pits and coated vesicles were observed in the ciliated unicellular Tetrahymena, in the flagellated unicellular Crithidia, and in Hydra. In the unicellulars the coated structures localized for the most part near to the origin of cilia or flagella, and many were present around the Golgi complex, whereas in Hydra they occurred in nonspecific random locations. Since the membrane receptors of the unicellulars serve originally as food receptors, the coated pits and vesicles are presumably involved in food selection, and represent as such the primitive form of the receptor-mediated ligand internalization mechanism operative in higher organisms.