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Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic protists of the group Metamonada, mitochondria are present in reduced forms (also termed hydrogenosomes or mitosomes) and a complete loss of mitochondrion in Monocercomonoides exilis (Metamonada:Preaxostyla) has also been reported. Within metamonads, retortamonads from the gastrointestinal tract of vertebrates form a sister group to parasitic diplomonads (e.g. Giardia and Spironucleus) and have also been hypothesized to completely lack mitochondria. We obtained transcriptomic data from Retortamonas dobelli and R. caviae and searched for enzymes of the core metabolism as well as mitochondrion- and parasitism-related proteins. Our results indicate that retortamonads have a streamlined metabolism lacking pathways for metabolites they are probably capable of obtaining from prey bacteria or their environment, reminiscent of the biochemical arrangement in other metamonads. Retortamonads were surprisingly found do encode homologs of components of Giardia's remarkable ventral disk, as well as homologs of regulatory NEK kinases and secreted lytic enzymes known for involvement in host colonization by Giardia. These can be considered pre-adaptations of these intestinal microorganisms to parasitism. Furthermore, we found traces of the mitochondrial metabolism represented by iron‑sulfur cluster assembly subunits, subunits of mitochondrial translocation and chaperone machinery and, importantly, [FeFe]‑hydrogenases and hydrogenase maturases (HydE, HydF and HydG). Altogether, our results strongly suggest that a remnant mitochondrion is still present., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400-580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 μm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm -1 . Secondary Harmonic Generation highlights the highly ordered structure of the flagella., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pathogenicity, evolutionary history, and unusual cell organization of diplomonads are well known, particularly for Giardia and Spironucleus; however, behavior of these aerotolerant anaerobes is largely unknown. Addressing this deficit, we studied behavior of the piscine diplomonad Spironucleus vortens (ATCC 50386) in in vitro culture. Spironucleus vortens trophozoites from Angelfish, Pterophyllum scalare, were maintained axenically in modified liver digest, yeast extract, and iron (LYI) medium, at 22 °C in the dark, and subcultured weekly. Cultures were monitored every 1-2 d, by removing an aliquot, and loading cells into a hemocytometer chamber, or onto a regular microscope slide. We observed three distinct swimming behaviors: (i) spontaneous formation of swarms, reaching 200 μm in diameter, persisting for up to several min in situ, (ii) directional movement of the swarm, via collective motility, and (iii) independent swimming of trophozoites to form a band (aggregation), presumably at the location of optimal environmental conditions. These behaviors have not previously been reported in Spironucleus. The observation that flagellate motility can change, from individual self-propulsion to complex collective swarming motility, prompts us to advocate S. vortens as a new model for study of group behavioral dynamics, complementing emerging studies of collective swimming in flagellated bacteria., (© 2018 International Society of Protistologists.)

Background: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living., Results: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes., Conclusions: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that Trepomonas has adapted secondarily to a free-living lifestyle. We believe that similar studies of other groups where free-living taxa are nested within parasites could reveal more examples of secondarily free-living eukaryotes.

Investigation of a series of nutrient-supplemented thixotropic gels at successive dilutions that impede the trajectories of a highly vigorous motile flagellated protist, Spironucleus vortens, provides insights into both its swimming characteristics and a means for its immobilization. The progress of movement of this organism through the solidified growth medium was monitored by the in situ reductive production of a formazan chromophore from a dissolved tetrazolium salt. The physical properties of the gels were measured using an Anton Paar rheometer. The test parameters and measurements included: angular frequency, complex viscosity, complex shear modulus, shear rate and rotational recovery. These rheological characteristics affected the forward velocity of the organism through the gels, during and after multiple resetting, information potentially useful for determination of the dynamic characteristics of flagellar movement and propulsion rates of the organism. Application to separation of single cells, individuals of distinct sizes or the differing species from mixed cultures of motile and non-motile organisms or less actively swimming species was evident. These applications can be used when isolating the parasite from the intestinal contents of its host or from faecal pellets., (© 2015 The Authors.)

This study assessed the effects of Hexamita salmonis (Moore) on metabolism of rainbow trout Oncorhynchus mykiss (Walbaum) and its effect on the host's susceptibility to infectious pancreatic necrosis virus (IPNV) after antiparasitic treatment. Rainbow trout naturally infected with H. salmonis were treated with 10 mg metronidazole kg fish(-1) per day, and their physiological recovery was assessed through measuring resting metabolism on the 7th, 14th, 21st and 28th day after treatment. In addition, we exposed the naïve fish to H. salmonis and measured the resting metabolism (oxygen consumption as mg O2 kg(-1) per hour) on the 10th, 20th and 30th day after the exposure to assess the variation in metabolic rates after infection. Significantly lower rates of metabolic activity (P < 0.05) were anticipated 20 days after infection with H. salmonis compared with the fish infected with H. salmonis for 10 days or with the parasite-free fish. Similarly, the treated fish needed about 20 days to fully recover from hexamitiasis. The susceptibility of rainbow trout to IPNV remained unchanged in the presence of H. salmonis. Weight loss was significantly higher (P < 0.05) in infected than that in the parasite-free fish. Fish should be examined regularly for H. salmonis and treated immediately whether found to prevent economic losses and excessive size variation., (© 2013 John Wiley & Sons Ltd.)

The diplomonad genera are here represented by three highly diverse species, both free-living (Hexamita inflata), and parasitic (Spironucleus vortens and Giardia intestinalis). All three are moderately aerotolerant flagellates, inhabiting environments where O2 tensions are low and fluctuating. Many diplomonads are opportunistic pathogens of avian, terrestrial and aquatic animals. Hexamitids inhabit deep waters and sediments of lakes and marine basins, S. vortens commonly infects the intestinal tract of ornamental fish, particularly of cichlids and cyprinids, and G. intestinalis, the upper intestinal tracts of humans as well as domestic and farm animals. Despite these very different habitats, their known physiological and biochemical characteristics are similar, but they do differ in significant respects as their lifestyles and life cycles demand. They have efficient O2 scavenging systems, and are highly effective at countering rapid O2 fluctuations, or clustering away from its source (except for G. intestinalis when attached to the jejunal villi). Their core metabolic pathways (glycolysis using pyrophosphate), incomplete tricarboxylic acid cycle (lacking α-ketoglutarate dehydrogenase), and amino acid metabolism (with an alternative energy-generating arginine dihydrolase pathway as a possibility in some cases), largely conform to those of other protists inhabiting low-O2 environments. Mitochondrial evolutionary reduction to give hydrogenosomes as seen in Spironucleus spp. has proceeded further to its minimal state in the mitosomes of G. intestinalis. Understanding of essential redox reactions and the maintentence of redox state, especially in the infective encysted stage of G. intestinalis provide increasing possibilities for parasite control. To this aim a plethora of new synthetic chemicals and natural products (especially those from garlic, Allium sativum) show promise as replacements for the highly effective (but potentially toxic to higher organisms) 5-nitroimidazoles (e.g., metronidazole) in the treatment and/or prevention of dimplomonad infection in humans and animals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to withstand high fluctuations in O₂ tensions during its life cycle. In the current study, we further investigated the O₂ scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O₂ electrode measurements revealed that the S. vortens ATCC 50386 strain was more O₂ tolerant than a freshly isolated S. vortens intestinal strain (Sv1). In contrast to the related human diplomonad, Giardia intestinalis, RP-HPLC revealed the major non-protein thiols of S. vortens to be glutathione (GSH, 776 nmol/10⁷ cells) with cysteine and H2S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dismutase and NADH oxidase (883 and 37.5nmol/min/mg protein, respectively), but like G. intestinalis, lacks catalase and peroxidase activities. Autofluorescence of NAD(P)H and FAD alongside the fluorescence of the GSH-adduct in monochlorobimane-treated live organisms allowed the monitoring of redox balances before and after treatment with inhibitors, metronidazole and auranofin. H₂O₂ was emitted into the exterior of S. vortens at a rate of 2.85 pmol/min/10⁶ cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H₂O₂ release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O₂ consumption by S. vortens (ajoene oil), or significantly depleted the intracellular GSH pool of the organism (allyl alcohol and DADS). Hence, antioxidant defence mechanisms of S. vortens may provide novel targets for parasite chemotherapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The mitochondria have arisen as a consequence of endosymbiosis of an ancestral α-proteobacterium with a methane-producing archae. The main function of the canonical aerobic mitochondria include ATP generation via oxidative phosphorylation, heme and phospholipid synthesis, calcium homeostasis, programmed cell death, and the formation of iron-sulfur clusters. Under oxygen-restricted conditions, the mitochondrion has often undergone remarkable reductive alterations of its content and function, leading to the generation of mitochondrion-related organelles (MROs), such as mitosomes, hydrogenosomes, and mithochondrion-like organelles, which are found in a wide range of anaerobic/microaerophilic eukaryotes that include several medically important parasitic protists such as Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Cryptosporidium parvum, Blastocystis hominis, and Encephalitozoon cuniculi, as well as free-living protists such as Sawyeria marylandensis, Neocallimastix patriciarum, and Mastigamoeba balamuthi. The transformation from canonical aerobic mitochondria to MROs apparently have occurred in independent lineages, and resulted in the diversity of their components and functions. Due to medical and veterinary importance of the MRO-possessing human- and animal-pathogenic protozoa, their genomic, transcriptomic, proteomic, and biochemical evidence has been accumulated. Detailed analyses of the constituents and functions of the MROs in such anaerobic pathogenic protozoa, which reside oxygen-deprived or oxygen-poor environments such as the mammalian intestine and the genital organs, should illuminate the current evolutionary status of the MROs in these organisms, and give insight to environmental constraints that drive the evolution of eukaryotes and their organelles. In this review, we summarize and discuss the diverse metabolic functions and protein transport systems of the MROs from anaerobic parasitic protozoa., (Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Lateral gene transfer (LGT) is an important evolutionary mechanism among prokaryotes. The situation in eukaryotes is less clear; the human genome sequence failed to give strong support for any recent transfers from prokaryotes to vertebrates, yet a number of LGTs from prokaryotes to protists (unicellular eukaryotes) have been documented. Here, we perform a systematic analysis to investigate the impact of LGT on the evolution of diplomonads, a group of anaerobic protists., Results: Phylogenetic analyses of 15 genes present in the genome of the Atlantic Salmon parasite Spironucleus barkhanus and/or the intestinal parasite Giardia lamblia show that most of these genes originated via LGT. Half of the genes are putatively involved in processes related to an anaerobic lifestyle, and this finding suggests that a common ancestor, which most probably was aerobic, of Spironucleus and Giardia adapted to an anaerobic environment in part by acquiring genes via LGT from prokaryotes. The sources of the transferred diplomonad genes are found among all three domains of life, including other eukaryotes. Many of the phylogenetic reconstructions show eukaryotes emerging in several distinct regions of the tree, strongly suggesting that LGT not only involved diplomonads, but also involved other eukaryotic groups., Conclusions: Our study shows that LGT is a significant evolutionary mechanism among diplomonads in particular and protists in general. These findings provide insights into the evolution of biochemical pathways in early eukaryote evolution and have important implications for studies of eukaryotic genome evolution and organismal relationships. Furthermore, "fusion" hypotheses for the origin of eukaryotes need to be rigorously reexamined in the light of these results.

The hexamitid flagellate Spironucleus vortens, previously reported from Pterophyllum scalare from Florida, was found in the intestine of Leuciscus idus in Norway. The flagellate was cultivated and studied by scanning and transmission electron microscopy. Identification was based on a suite of ultrastructural features unique for S. vortens: compound lateral ridges, a swirled posterior end, and a distinctive microtubular cytoskeleton. Microfibrillar structures with a periodicity of 0.13 microm in the right peripheral part of the compound lateral ridges were shown to be responsible for the distinctive rope-like appearance of the peripheral ridge seen in scanning electron micrographs, and not previously reported for S. vortens. The present results show a wide geographic distribution and a wide temperature tolerance for S. vortens. The flagellate was successfully cultivated at 5 degrees C and 15 degrees C, having previously been cultivated between 2-34 degrees C. Spironucleus vortens is believed to be endemic in Norwegian waters, but an introduction hypothesis is also discussed. The similarity is striking between S. vortens and S. elegans, previously described from amphibians and fish in Europe, and the possibility of conspecificity is believed to be high.

Phylogenetic analysis of five partial planktic foraminiferal small subunit (SSU) ribosomal (r) DNA sequences with representatives of a diverse range of eukaryote, archaebacterial, and eubacterial taxa has revealed that the evolutionary origin of the foraminiferal lineage precedes the rapid eukaryote diversification represented by the "crown" of the eukaryotic tree and probably represents one of the earliest splits among extant free-living aerobic eukaryotes. The foraminiferal rDNA sequences could be clearly separated from known symbionts, commensals, and food organisms. All five species formed a single monophyletic group distinguished from the "crown" group by unique foraminiferal specific insertions as well as considerable nucleotide distance in aligned regions.

We have sequenced the small ribosomal subunit RNA gene of the diplozoan Trepomonas agilis. This provides the first molecular information on a free-living archezoan. We have performed a phylogenetic analysis by maximum likelihood, parsimony, and distance methods for all available nearly complete archezoan small subunit ribosomal RNA genes and for representatives of all major groups of more advanced eukaryotes (metakaryotes). These show Diplozoa as the earliest-diverging eukaryotic lineage, closely followed by microsporidia. Trepomonas proves to be much more closely related to Hexamita, and, to a lesser degree, to Spironucleus, than to Giardia. The close relationship between the free-living Trepomonas on our trees and the parasites Hexamita inflata and Spironucleus refutes the idea that the early divergence of the amitochondrial Archezoa is an artefact caused by parasitism. The deep molecular divergence between the three phagotrophic genera with two cytostomes (Hexamita, Trepomonas, Spironucleus) and the saprotrophic Giardia that lacks cytostomes is in keeping with the classical evidence for a fundamental difference in the symmetry of the cytoskeleton between the two groups. We accordingly separate the two groups as two orders: Distomatida for those with two cytostomes/cytopharynxes and Giardiida ord. nov. for Giardia and Octomitus that lack these, and divide each order into two families. We suggest that this fundamental divergence in manner of feeding and in the symmetry of the cytoskeleton evolved in a free-living diplozoan very early indeed in the evolution of the eukaryotic cell, possibly very soon after the origin of the diplokaryotic state (having two nuclei linked together firmly by the cytoskeleton) and before the evolution of parasitism by distomatids and giardiids, which may have colonized animal guts independently. We discuss the possible relationship between the two archezoan phyla (Metamonada and Microsporidia) and the nature of the first eukaryotic cell in the light of our results and other recent molecular data.

In vivo experiments with a clone of the intestinal flagellate Spironucleus sp. originating from the laboratory European hamster (Cricetus cricetus), and the comparison with a spontaneous infection from laboratory bred European hamsters suggest high specificity of this clone for the homologous host. Only the Syrian golden hamster (Mesocricetus auratus) could be infected experimentally, though the mean intensity of infection was lower. Other heterologous recipients, mice and rats of one outbred and one inbred strain each, could not be infected. Even immunodeficient mice (athymic and C.B-17-scid) remained uninfected after inoculation of 5 x 10(5) cysts per mouse. This is the second Spironucleus clone, after the rat isolate (Schagemann et al., 1990), with a high level of host specificity suggesting heterogeneity within the genus Spironucleus.