Redescription and molecular phylogeny of the freshwater metopid, Castula strelkowi (Jankowski, 1964) from the Czech Republic and synonymization of Pileometopus with Castula.

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula.

Redescription and molecular characterization of Loxocephalus luridus Eberhard, 1862 based on Czech populations: Implications for order Loxocephalida Jankowski, 1980.

The phylogenetic and taxonomic affinities of lineages currently assigned to the non-monophyletic ciliate order Loxocephalida Jankowski (1980) within subclass Scuticociliatia Small (1967) remain unresolved. In the current study, we redescribe the morphology of the type species, Loxocephalus luridus Eberhard (1862) based on two Czech populations and include the first scanning and transmission electron microscopy images of the species. We provide the first 18S rRNA gene sequences for L. luridus and consider its phylogenetic position. Our results support the separation of Dexiotricha from Loxocephalus; however, the former genus is recovered as non-monophyletic. The monophyly of genus Dexiotricha and that of Loxocephalus + Dexiotricha is rejected. Loxocephalus luridus, together with Dexiotricha species, nests within a fully supported clade with Conchophthirus species, long presumed to belong to the Pleuronematida. Haptophrya is recovered as sister to this clade. The monophyly of the Astomatia Schewiakoff (1896) including Haptophrya is rejected. No clear morphologic synapomorphy is identified for the fully supported clade consisting of Haptophrya, Dexiotricha, Loxocephalus, and Conchophthirus.

The association of bacterial agents and flagellated protozoans in the etiopathogenesis of subspectacular abscesses in snakes.

OBJECTIVES: To describe the etiology, clinical pathology, and treatment of subspectacular abscesses (SAs) in 16 snakes. ANIMALS STUDIED: Sixteen snakes from private captive collections that were presented at a veterinary teaching hospital and diagnosed with SAs. PROCEDURES: Exudate was collected from SAs via percutaneous aspiration or by performing a partial spectaculectomy and submitted to direct light microscopic examination and/or microbiological examination. RESULTS: Bacterial isolates were cultured from exudate samples in 12 out of 16 snakes and comprised Pseudomonas aeruginosa isolates that showed identical antimicrobial susceptibility patterns in seven cases. Direct light microscopic examination demonstrated the presence of flagellated protozoa in the exudate in 10 out of 16 snakes and in all of these snakes, concurrent oropharyngeal flagellated protozoan infection could be demonstrated. Combined flagellated protozoan and bacterial SA infection was demonstrated in six snakes. Comparison of small subunit ribosomal RNA gene sequences of flagellated protozoans that were detected in the exudate of SAs from four snakes revealed they represented the parabasalians Monocercomonas colubrorum and Hypotrichomonas acosta in one and three cases, respectively. In all snakes, successful treatment of the SAs was achieved following surgical debridement of the subspectacular space combined with antimicrobial treatment. CONCLUSION: Bacteria as well as flagellates are considered to reach the subspectacular space through ascending infection from the oral cavity via the lacrimal duct. Although opportunistic bacterial infection presumably constitutes the predominant cause of SAs in snakes, the role of flagellate infection in the pathogenesis of this disorder should be further elucidated.

Lighting lantern above Psalteriomonadidae: Unveiling novel diversity within the genus Psalteriomonas (Discoba: Heterolobosea).

Psalteriomonadidae are a small family of anaerobic free-living protists belonging to Heterolobosea, Discoba. We cultured 74 new strains of mostly amoeboid Psalteriomonadidae obtained from mainly freshwater habitats and sequenced their 18S rRNA gene. Based on the phylogenetic analysis and genetic distances, we report multiple novel species, four of which we formally describe based on the light-microscopic morphology (Psalteriomonas minuta, P. australis, P. fimbriata, and P. parva). We also examined the ultrastructure of two Psalteriomonas species using transmission electron microscopy. We transfer Sawyeria marylandensis into the genus Psalteriomonas and synonymize Sawyeria with Psalteriomonas. In addition, we studied the flagellate stage of P. marylandensis comb. nov. for the first time, using light and scanning electron microscopy.

Evolution: No end in sight for novel incredible (heterotrophic) protists.

The microbial eukaryotes known as protists are of immense importance for our understanding of eukaryotic biology. Although it is often difficult to convince funding bodies to sponsor research projects aimed at finding new protist lineages, such discoveries usually provide new and fundamental insights into cell and evolutionary biology, and ecology.

Anaeramoeba pumila sp. nov. and Anaeramoeba sp. OCE22C represent two novel types of symbiosis of Anaeramoebae and prokaryotes.

Anaeramoebae is a recently described phylum of anaerobic, marine amoebae, and amoeboflagellates belonging to the Metamonada supergroup. So far, six species have been described based on light microscopic morphology and sequences of the SSU rRNA gene. Here we present three new strains of Anaeramoeba with a description of their morphology, ultrastructure, and phylogenetic position based on the analysis of SSU rRNA gene sequences. Two of the strains represent a new species, Anaeramoeba pumila sp. nov., that has the smallest cells of all known Anaeramoeba species, and one that represents a species from the newly recognized Anaeramoeba flamelloides complex. Anaeramoebae are known to have a syntrophic relationship with prokaryotes. Our strains display two novel, remarkable types of symbioses, previously unknown from Anaeramoebae.

Single cell transcriptomics reveals UAR codon reassignment in Palmarella salina (Metopida, Armophorea) and confirms Armophorida belongs to APM clade.

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

The protist cultural renaissance.

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans.

Archamoebae comprises free-living or endobiotic amoebiform protists that inhabit anaerobic or microaerophilic environments and possess mitochondrion-related organelles (MROs) adapted to function anaerobically. We compared in silico reconstructed MRO proteomes of eight species (six genera) and found that the common ancestor of Archamoebae possessed very few typical components of the protein translocation machinery, electron transport chain and tricarboxylic acid cycle. On the other hand, it contained a sulphate activation pathway and bacterial iron-sulphur (Fe-S) assembly system of MIS-type. The metabolic capacity of the MROs, however, varies markedly within this clade. The glycine cleavage system is widely conserved among Archamoebae, except in Entamoeba, probably owing to its role in catabolic function or one-carbon metabolism. MRO-based pyruvate metabolism was dispensed within subgroups Entamoebidae and Rhizomastixidae, whereas sulphate activation could have been lost in isolated cases of Rhizomastix libera, Mastigamoeba abducta and Endolimax sp. The MIS (Fe-S) assembly system was duplicated in the common ancestor of Mastigamoebidae and Pelomyxidae, and one of the copies took over Fe-S assembly in their MRO. In Entamoebidae and Rhizomastixidae, we hypothesize that Fe-S cluster assembly in both compartments may be facilitated by dual localization of the single system. We could not find evidence for changes in metabolic functions of the MRO in response to changes in habitat; it appears that such environmental drivers do not strongly affect MRO reduction in this group of eukaryotes.

Morphology, morphogenesis, and molecular characterization of Castula specialis sp. nov. (Ciliophora, Armophorea, Metopida).

The morphology, morphogenesis, and molecular phylogeny of a new metopid ciliate, Castula specialis sp. nov., comprising three strains from geographically distant (China, Mexico, Czech Republic) anoxic freshwater habitats, were studied based on microscopic observation of live and protargol-stained specimens as well as SSU rRNA gene sequence data. The new species is characterized as follows: size in vivo 105-220 × 25-70 µm, body oblong to elongated ellipsoidal and asymmetrical; preoral dome distinctly projecting beyond the body; 32-46 adoral membranelles; 31-52 somatic kineties; and 4-7 setae. This study brings the first morphogenetic investigation of a member of the genus Castula. The morphogenesis of the type population (China) of the new species proceeds as in Metopus spp. comprising drastic changes in body shape and a pleurotelokinetal stomatogenesis; however, the main difference is the origin of the opisthe's paroral membrane that derives from all perizonal rows and some adjacent dome kineties. Phylogenetically, the genus Castula is paraphyletic.

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

Inteins are self-splicing protein elements found in viruses and all three domains of life. How the DNA encoding these selfish elements spreads within and between genomes is poorly understood, particularly in eukaryotes where inteins are scarce. Here, we show that the nuclear genomes of three strains of Anaeramoeba encode between 45 and 103 inteins, in stark contrast to four found in the most intein-rich eukaryotic genome described previously. The Anaeramoeba inteins reside in a wide range of proteins, only some of which correspond to intein-containing proteins in other eukaryotes, prokaryotes, and viruses. Our data also suggest that viruses have contributed to the spread of inteins in Anaeramoeba and the colonization of new alleles. The persistence of Anaeramoeba inteins might be partly explained by intragenomic movement of intein-encoding regions from gene to gene. Our intein dataset greatly expands the spectrum of intein-containing proteins and provides insights into the evolution of inteins in eukaryotes.

Phylogenetic and morphological diversity of free-living diplomonads.

Diplomonadida is a lineage of anaerobic protists belonging to Fornicata, Metamonada. Most diplomonads are endobiotic or parasitic, such as Giardia intestinalis, which is a famous human pathogen, but several free-living species exist as well. Although it has been proposed that the free-living diplomonads are descendants of endobiotic organisms and thus interesting from the evolutionary point of view, they have been largely neglected. We obtained 58 cultures of free-living diplomonads belonging to four genera (Hexamita, Trepomonas, Gyromonas, and Trimitus) and six strains of endobiotic diplomonads and analyzed their SSU rRNA gene sequences. We also studied light-microscopic morphology of selected strains and the ultrastructure of Trepomonas rotans for the first time. Our phylogenetic analysis showed that the genus Hexamita, and, possibly, also the genus Trepomonas, are polyphyletic. Trepomonas rotans, which may represent a novel genus, is unique among Diplomonadida by having the cell covered in scales. Our results suggest that the evolution of the endobiotic life style and cell organization in diplomonads is more complicated than previously thought.

Hydrogenotrophic methanogenesis is the key process in the obligately syntrophic consortium of the anaerobic ameba Pelomyxa schiedti.

Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti, which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria) and Candidatus Vesiculincola pelomyxae (class Clostridia), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca. Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca. Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca. Vesiculincola pelomyxae, while Ca. Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca. Methanoregula pelomyxae, which consumes H2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.

Host specificity of passerine Lankesterella (Apicomplexa: Coccidia).

Lankesterella parasites are blood coccidians that have recently gained attention as their records in common passerine species emerge. To date, their occurrence has been molecularly confirmed in several passerine genera, mainly among members of the families Paridae and Acrocephalidae. Despite their relatively high prevalence in some host populations, their life cycles remain unclear, mosquitoes or mites being the proposed vectors. The aim of this study was to reveal Lankesterella host specificity, focusing mainly on parasites of tit and warbler species (families Paridae and Acrocephalidae). We have determined the 18S rRNA gene sequences of Lankesterella from 35 individuals belonging to eight different host species. Phylogenetic analysis revealed that passerine Lankesterella are host-specific, with specificity at the host genus or species level. Besides Lankesterella, Isospora sequences were obtained from avian blood as well, pointing out the need for barcoding.

Anaerocyclidiidae fam. nov. (Oligohymenophorea, Scuticociliatia): A newly recognized major lineage of anaerobic ciliates hosting prokaryotic symbionts.

The research on anaerobic ciliates, to date, has mainly been focused on representatives of obligately anaerobic classes such as Armophorea or Plagiopylea. In this study, we focus on the anaerobic representatives of the subclass Scuticociliatia, members of the class Oligohymenophorea, which is mainly composed of aerobic ciliates. Until now, only a single anaerobic species, Cyclidium porcatum (here transferred to the genus Anaerocyclidium gen. nov.), has been described both molecularly and morphologically. Our broad sampling of anoxic sediments together with cultivation and single cell sequencing approaches have shown that scuticociliates are common and diversified in anoxic environments. Our results show that anaerobic scuticociliates represent a distinctive evolutionary lineage not closely related to the family Cyclidiidae (order Pleuronematida), as previously suggested. However, the phylogenetic position of the newly recognized lineage within the subclass Scuticociliatia remains unresolved. Based on molecular and morphological data, we establish the family Anaerocyclidiidae fam. nov. to accommodate members of this clade. We further provide detailed morphological descriptions and 18S rRNA gene sequences for six new Anaerocyclidium species and significantly broaden the described diversity of anaerobic scuticociliates.

Rediscovery of Remarkably Rare Anaerobic Tentaculiferous Ciliate Genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea).

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

Morphology and phylogenetic position of three anaerobic ciliates from the classes Odontostomatea and Muranotrichea (Ciliophora).

The diversity of the classes Odontostomatea and Muranotrichea, which contain solely obligate anaerobes, is poorly understood. We studied two populations of Mylestoma sp., one of Saprodinium dentatum (Odontostomatea), two of Muranothrix felix sp. nov., and one of Muranothrix sp. (Muranotrichea) employing live observation, protargol impregnation, scanning electron microscopy, and 18S rRNA gene sequencing. Conspecificity of Mylestoma sp., described here, with a previously described species of this genus cannot be excluded since no species have been studied with modern methods. Phylogenetically, the genus Mylestoma is closely related to the odontostomatid Discomorphella pedroeneasi, although the phylogenetic position of class Odontostomatea itself remains unresolved. The newly described muranotrichean species, Muranothrix felix sp. nov., is morphologically similar to M. gubernata but can be distinguished by its fewer macronuclear nodules and fewer adoral membranelles; moreover, it is clearly distinguished from M. gubernata by its 18S rRNA gene sequence. Another population, designated here as Muranothrix sp., most likely represents a separate species.

Blood parasites (Trypanosoma, Leucocytozoon, Haemoproteus) in the Eurasian sparrowhawk (Accipiter nisus): diversity, incidence and persistence of infection at the individual level.

BACKGROUND: A high prevalence of parasites may result from life-long persistence of infection or from high reinfection rates. We have studied blood parasites in a breeding population of the accipitrid raptor, Eurasian sparrowhawk (Accipiter nisus), to determine parasite diversity and turnover. METHODS: During this 7-year study, 210 adult Eurasian sparrowhawks breeding in the city of Prague were checked for parasites using several diagnostic methods. RESULTS: In both female and male raptors, parasites of the genus Leucocytozoon were the most prevalent (92% and 85%, respectively) followed in decreasing order of prevalence by those of genus Trypanosoma (74% and 68%, respectively) and genus Haemoproteus (46% and 16%, respectively). The prevalence of all parasites increased with age in both sexes, with the females at each respective age having the higher prevalence. There was a positive association between Haemoproteus and Leucocytozoon infections. Persistence at the individual level was higher than incidence for Trypanosoma and Haemoproteus. In the case of Leucocytozoon and Trypanosoma, most individuals probably become infected in their first year of life or even before dispersal from the nest. The detected parasites belonged to Trypanosoma avium sensu stricto, Leucocytozoon sp. (haplotypes ACNI1 and ACNI3) and Leucocytozoon mathisi (haplotype ACNI4) and two new lineages of the Haemoproteus elani complex (ACCNIS6 and ACCNIS7). Detailed analysis of parasite lineages in individuals that were repeatedly sampled revealed lineage turnover that would otherwise remain hidden. Phylogenetic analysis revealed that the detected Haemoproteus belongs to a phylogenetically distant group whose taxonomic position requires further analysis. CONCLUSIONS: All three genera of blood parasites persist in infected individuals, thus enabling sustainability of vector transmission cycles. Prevalence increases with age; however, there is a high turnover of Leucocytozoon lineages. No clear evidence of parasite-induced mortality was found, and most of the individuals were infected early in life, particularly in the case of Leucocytozoon.

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates.

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

Free-living Trichomonads are Unexpectedly Diverse.

The vast majority of the more than 450 described species of Parabasalia are intestinal symbionts or parasites of animals. This endobiotic life-history is presumably ancestral although the root of Parabasalia still needs to be robustly established. The half-dozen putatively free-living species thus far described are likely independently derived from endobiotic ancestors and represent the most neglected ecological group of parabasalids. Thus, we isolated and cultivated 45 free-living strains of Parabasalia obtained from a wide variety of anoxic sediments to conduct detailed morphological and SSU rRNA gene phylogenetic analyses. Sixteen species of trichomonads were recovered. Among them, we described seven new species, three new genera, two new families, and one new order. Most of the newly described species were more or less closely related to members of already described genera. However, we uncovered a new deep-branching lineage without affinity to any currently known group of Parabasalia. The newly discovered free-living parabasalids will be key taxa in comparative analyses aimed at rooting the entire lineage and deciphering the evolutionary innovations involved in transitioning between endobiotic and free-living habitats.