Single-Cell Genomics Reveals the Divergent Mitochondrial Genomes of Retaria (Foraminifera and Radiolaria).

Mitochondria originated from an ancient bacterial endosymbiont that underwent reductive evolution by gene loss and endosymbiont gene transfer to the nuclear genome. The diversity of mitochondrial genomes published to date has revealed that gene loss and transfer processes are ongoing in many lineages. Most well-studied eukaryotic lineages are represented in mitochondrial genome databases, except for the superphylum Retaria-the lineage comprising Foraminifera and Radiolaria. Using single-cell approaches, we determined two complete mitochondrial genomes of Foraminifera and two nearly complete mitochondrial genomes of radiolarians. We report the complete coding content of an additional 14 foram species. We show that foraminiferan and radiolarian mitochondrial genomes contain a nearly fully overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. In contrast to animals and fungi, many protists encode a diverse set of proteins on their mitochondrial genomes, including several ribosomal genes; however, some aerobic eukaryotic lineages (euglenids, myzozoans, and chlamydomonas-like algae) have reduced mitochondrial gene content and lack all ribosomal genes. Similar to these reduced outliers, we show that retarian mitochondrial genomes lack ribosomal protein and tRNA genes, contain truncated and divergent small and large rRNA genes, and contain only 14 or 15 protein-coding genes, including nad1, -3, -4, -4L, -5, and -7, cob, cox1, -2, and -3, and atp1, -6, and -9, with forams and radiolarians additionally carrying nad2 and nad6, respectively. In radiolarian mitogenomes, a noncanonical genetic code was identified in which all three stop codons encode amino acids. Collectively, these results add to our understanding of mitochondrial genome evolution and fill in one of the last major gaps in mitochondrial sequence databases. IMPORTANCE We present the reduced mitochondrial genomes of Retaria, the rhizarian lineage comprising the phyla Foraminifera and Radiolaria. By applying single-cell genomic approaches, we found that foraminiferan and radiolarian mitochondrial genomes contain an overlapping but reduced mitochondrial gene complement compared to other sequenced rhizarians. An alternative genetic code was identified in radiolarian mitogenomes in which all three stop codons encode amino acids. Collectively, these results shed light on the divergent nature of the mitochondrial genomes from an ecologically important group, warranting further questions into the biological underpinnings of gene content variability and genetic code variation between mitochondrial genomes.

Evolution: Divergent trajectories predate the origins of animals and fungi.

Animals, fungi, and their closest protist relatives comprise the clade Opisthokonta. Although they are comparatively closely related, animals and fungi have diverged greatly from one another. A new study demonstrates that the genomic features that are characteristic of animals and fungi arose even before the origin of these two kingdoms.

Redescription of Paradiplozoon opsariichthydis (Jiang, Wu et Wang 1984) Jiang, Wu et Wang, 1989 (Monogenea, Diplozoidae).

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis.

A Study on the Prevalence and Subtype Diversity of the Intestinal Protist Blastocystis sp. in a Gut-Healthy Human Population in the Czech Republic.

Blastocystis sp. is a common intestinal protist colonizing the human intestine the prevalence of which varies across non-industrialized and industrialized countries. Its role in the human gut ecosystem remains unclear due to persisting gaps in knowledge of epidemiology and factors affecting gut colonization. Here, we aimed to expand the knowledge of the epidemiology of Blastocystis sp. in the gut-healthy humans in one of the industrialized European countries, including the distribution of its subtypes, the correlation between its occurrence and several factors such as lifestyle, contact with animals, age, and sex. A total of 288 stool samples were obtained from asymptomatic individuals over the entire age-range and 136 samples from animals with which the volunteers were in frequent contact. All samples were examined in parallel by PCR and xenic in vitro culture. Blastocystis sp. was detected in samples from both human and non-human hosts. In humans, the overall prevalence was 24% and eight subtypes were found; in animals, the prevalence was 10%, and only five subtypes were detected. A higher incidence of Blastocystis sp. was observed in individuals (i) traveling outside Europe, (ii) in frequent contact with livestock, and (iii) over 50 years of age. We found no effect on gender on Blastocystis sp. colonization. Summary: This study provides data on the prevalence and diversity of the gut protist Blastocystis sp. and its subtypes in a gut-healthy human population with emphasis on several factors such as contact with animals, lifestyle, age, and gender.

Author Correction: From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

Cryptic genetic diversity and erroneous morphological species determination represent frequent problems in biodiversity research. Here, examination of 138 specimens of Synodontis (Mochokidae, Siluriformes) from the Nile River and Lake Turkana revealed the presence of both S. schall-like and S. frontosus-like morphotypes, with a phenotypic gradient between them. We concluded phylogenetic and population genetic analyses based on two mitochondrial and one nuclear marker including 131 coxI (565 bp), 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences from the Nile-Turkana population, plus additional GenBank data of Synodontis spp. Whilst nuclear data were inconclusive, mitochondrial sequences suggested that both morphotypes and intermediate forms are conspecific. The results imply probable synonymy of S. frontosus with S. schall. Conversely, a strong biogeographical signal was revealed among widely distributed and supposedly conspecific S. schall-like catfish of the Nilo-Sudanian ichthyological province. Synodontis schall sensu stricto (=Eastern clade), as defined by type locality in the Nile, is apparently restricted to the eastern part of the Nilo-Sudanian ichthyological province (e.g. Nile, Turkana, Chad). Synodontis schall Western clade (Senegambia, Niger, Chad) most probably represents a cryptic taxon, unrecognized thus far due to the absence of distinctive morphological differences.

Morphology, Ultrastructure, and Mitochondrial Genome of the Marine Non-Photosynthetic Bicosoecid Cafileria marina Gen. et sp. nov.

In this paper, we describe a novel bacteriophagous biflagellate, Cafileria marina with two smooth flagellae, isolated from material collected from a rock surface in the Kvernesfjorden (Norway). This flagellate was characterized by scanning and transmission electron microscopy, fluorescence, and light microscopy. The sequence of the small subunit ribosomal RNA gene (18S) was used as a molecular marker for determining the phylogenetic position of this organism. Apart from the nuclear ribosomal gene, the whole mitochondrial genome was sequenced, assembled, and annotated. Morphological observations show that the newly described flagellate shares key ultrastructural characters with representatives of the family Bicosoecida (Heterokonta). Intriguingly, mitochondria of C. marina frequently associate with its nucleus through an electron-dense disc at the boundary of the two compartments. The function of this association remains unclear. Phylogenetic analyses corroborate the morphological data and place C. marina with other sequence data of representatives from the family Bicosoecida. We describe C. marina as a new species from a new genus in this family.

Massive mitochondrial DNA content in diplonemid and kinetoplastid protists.

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

Redescription of Paradiplozoon hemiculteri (Monogenea, Diplozoidae) from the type host Hemiculter leucisculus, with neotype designation.

Paradiplozoon hemiculteri (Ling, 1973), a member of the Diplozoidae, parasitizes the gills of Asian fish. Not only is the type material unavailable for this species, the original description was poor and somewhat conflicting, and adequate molecular data were not available. What is more, the available morphological and molecular data are inconsistent and fluctuate significantly. Here, we present a redescription of P. hemiculteri based on morphological and molecular data from new isolates collected from the type host, the sharpbelly Hemiculter leucisculus (Basilewsky, 1855), captured at the neotype locality (Shaoguan, Guangdong Province, southern China); a neotype for P. hemiculteri was designated from this collection. The length and width of the body, buccal suckers, pharynx, attachment clamps, sickle and the central hook handle were all measured and the shape of the anterior and posterior part of the median plate and anterior and posterior joining sclerites accurately documented. Phylogenetic analyses based on the sequences of the second rDNA internal transcribed spacer (ITS2) indicated that all new samples clustered together and differed clearly from sequences attributed to P. hemiculteri, which are deposited in GenBank. Our results confirm that P. hemiculteri is the only diplozoid that has demonstrably been found on the gills of H. leucisculus to date.

Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid.

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.

Discordant population histories of host and its parasite: A role for ecological permeability of extreme environment?

Biogeographical and ecological barriers strongly affect the course of micro-evolutionary processes in free living organisms. Here we assess the impact of a recently emerged barrier on populations of limnic fauna. Genetic diversity and population structure in a host-parasite system (Wenyonia virilis tapeworm, Synodontis schall catfish) are analyzed in the recently divided Turkana and Nile basins. The two basins, were repeatedly connected during the Holocene wet/dry climatic oscillations, following late Pleistocene dessication of the Turkana basin. Mitochondrial DNA sequences for cytochrome oxidase I gene (cox I) and a whole genome scanning method-amplified fragment length polymorphism (AFLP) were employed. A total of 347 cox I sequences (representing 209 haplotypes) and 716 AFLP fragments, as well as 120 cox I sequences (20 haplotypes) and 532 AFLP fragments were obtained from parasites and hosts, respectively. Although results indicate that host and parasite populations share some formative traits (bottlenecks, Nilotic origin), their population histories/patterns differ markedly. Mitochondrial analysis revealed that parasite populations evolve significantly faster and show remarkably higher genetic variability. Analyses of both markers confirmed that the parasites undergo lineage fission, forming new clusters specific for either freshwater or saline parts of Lake Turkana. In congruence with the geological history, these clusters apparently indicate multiple colonisations of Lake Turkana from the Nile. In contrast, the host population pattern indicates fusion of different colonisation waves. Although fish host populations remain connected, saline habitats in Lake Turkana (absent in the Nile), apparently pose a barrier to the gene flow in the parasite, possibly due to its multihost lifecycle, which involves freshwater annelids. Despite partially corroborating mitochondrial results, AFLP data was not sufficiently informative for analyzing populations with recently mixed biogeographic histories.

Diversity and host specificity of coccidia (Apicomplexa: Eimeriidae) in native and introduced squirrel species.

Introduction of alien species into new areas can have detrimental effects on native ecosystems and impact the native species. The present study aims to identify coccidia infecting native and introduced squirrels in Italy, to gain insight into possible transmission patterns and role of monoxenous coccidia in mediating the competition between alien and native hosts. We collected 540 faecal samples of native red squirrels, Sciurus vulgaris, invasive alien grey squirrels, S. carolinensis, and introduced Pallas's squirrels, Callosciurus erythraeus. Total prevalence of Eimeria spp. was 95.6% in S. vulgaris, 95.7% in S. carolinensis and only 4.1% in C. erythraeus. Morphological examination revealed 3 Eimeria morphotypes. Phylogenetic analyses of Eimeria DNA based on 18S, ITS, cox I markers displayed fairly distinct monophyletic clades in the microscopically indistinguishable E2 morphotype, proving indisputable distinction between the isolates from red and grey squirrels. Grey squirrels successfully introduced E. lancasterensis from their native range, but this species does not spill over to native red squirrels. Similarly, there is no evidence for the transmission of E. sciurorum from red to grey squirrels. The possible transmission and the potential role of monoxenous coccidia in mediating the competition between native and invasive squirrels in Italy were not confirmed.

Cytauxzoon Infections in Wild Felids from Carpathian-Danubian-Pontic Space: Further Evidence for a Different Cytauxzoon Species in European Felids.

Parasitic protists of the genus Cytauxzoon are detected in a wide range of wild and domestic felids. Bobcats are a confirmed reservoir of Cytauxzoon felis in North America while domestic cats are susceptible hosts suffering from severe or fatal illness. Cytauxzoon infections are mainly reported from American felids and, recently, several sub-clinical and clinical findings were reported from European, Asian, and African felids. In 2014, the collection of organs of 4 Eurasian lynx and 12 wild cats from 11 Romanian localities was carried out to determine the prevalence and genetic diversity of Cytauxzoon spp. We detected an overall high prevalence of 62.5% in both species of wild felids; 50% in wild cats and 100% in Eurasian lynx. The phylogenetic analysis indicates 2 distinct clades of Cytauxzoon in felids, with all of our sequences clustering with sequences of Cytauxzoon sp./Cytauxzoon manul from Palaearctic felids. Further studies, development of new genetic markers, and experimental transmission studies are required for clarifying the taxonomy and life cycle of feline Cytauxzoon in the Old World.

A tsetse and tabanid fly survey of African great apes habitats reveals the presence of a novel trypanosome lineage but the absence of Trypanosoma brucei.

Tsetse and tabanid flies transmit several Trypanosoma species, some of which are human and livestock pathogens of major medical and socioeconomic impact in Africa. Recent advances in molecular techniques and phylogenetic analyses have revealed a growing diversity of previously unidentified tsetse-transmitted trypanosomes potentially pathogenic to livestock and/or other domestic animals as well as wildlife, including African great apes. To map the distribution, prevalence and co-occurrence of known and novel trypanosome species, we analyzed tsetse and tabanid flies collected in the primary forested part of the Dzanga-Sangha Protected Areas, Central African Republic, which hosts a broad spectrum of wildlife including primates and is virtually devoid of domestic animals. Altogether, 564 tsetse flies and 81 tabanid flies were individually screened for the presence of trypanosomes using 18S rRNA-specific nested PCR. Herein, we demonstrate that wildlife animals are parasitized by a surprisingly wide range of trypanosome species that in some cases may circulate via these insect vectors. While one-third of the examined tsetse flies harbored trypanosomes either from the Trypanosoma theileri, Trypanosoma congolense or Trypanosoma simiae complex, or one of the three new members of the genus Trypanosoma (strains 'Bai', 'Ngbanda' and 'Didon'), more than half of the tabanid flies exclusively carried T. theileri. To establish the putative vertebrate hosts of the novel trypanosome species, we further analyzed the provenance of blood meals of tsetse flies. DNA individually isolated from 1033 specimens of Glossina spp. and subjected to high-throughput library-based screening proved that most of the examined tsetse flies engorged on wild ruminants (buffalo, sitatunga, bongo), humans and suids. Moreover, they also fed (albeit more rarely) on other vertebrates, thus providing indirect but convincing evidence that trypanosomes can be transmitted via these vectors among a wide range of warm- and cold-blooded hosts.

Integrated morphological and molecular identification of cat fleas (Ctenocephalides felis) and dog fleas (Ctenocephalides canis) vectoring Rickettsia felis in central Europe.

Fleas of the genus Ctenocephalides are the most common ectoparasites infesting dogs and cats world-wide. The species Ctenocephalides felis and Ctenocephalides canis are competent vectors for zoonotic pathogens such as Rickettsia felis and Bartonella spp. Improved knowledge on the diversity and phylogenetics of fleas is important for understanding flea-borne pathogen transmission cycles. Fleas infesting privately owned dogs and cats from the Czech Republic (n=97) and Romania (n=66) were subjected to morphological and molecular identification and phylogenetic analysis. There were a total of 59 (60.82%) cat fleas (Ctenocephalides felis felis), 30 (30.93%) dog fleas (Ctenocephalides canis), 7 (7.22%) European chicken fleas (Ceratophyllus gallinae) and 1 (1.03%) northern rat flea (Nosopsyllus fasciatus) collected in the Czech Republic. Both C. canis and C. felis felis were identified in Romania. Mitochondrial DNA sequencing at the cox1 gene on a cohort of 40 fleas revealed the cosmopolitan C. felis felis clade represented by cox1 haplotype 1 is present in the Czech Republic. A new C. felis felis clade from both the Czech Republic and Romania is also reported. A high proportion of C. canis was observed from dogs and cats in the current study and phylogeny revealed that C. canis forms a sister clade to the oriental cat flea Ctenocephalides orientis (syn. C. felis orientis). Out of 33 fleas tested, representing C. felis felis, C. canis and Ce. gallinae, 7 (21.2%) were positive for R. felis using diagnostic real-time PCR targeting the gltA gene and a conventional PCR targeting the ompB gene. No samples tested positive for Bartonella spp. using a diagnostic real-time PCR assay targeting ssrA gene. This study confirms high genetic diversity of C. felis felis globally and serves as a foundation to understand the implication for zoonotic disease carriage and transmission by the flea genus Ctenocephalides.

Bothriocephalidean tapeworms (Cestoda) from the blackfish, Centrolophus niger (Perciformes: Centrolophidae).

Bothriocephalidean tapeworms parasitic in the blackfish, Centrolophus niger (Gmelin) (Perciformes: Centrolophidae), are redescribed on the basis of the evaluation of freshly collected specimens and museum material. This evaluation enabled us to supplement species diagnoses by new morphological characters of potential use for phylogenetic analyses, including the data from scanning electron microscopical observations, and to provide a key to identification of the following four species occurring in this fish: Amphicotyle heteropleura (Diesing, 1850); Milanella familiaris Kuchta et Scholz, 2008 (both Triaenophoridae); Bothriocotyle solinosomum Ariola, 1900; and Echinophallus wageneri (Monticelli, 1890) (both Echinophallidae). Large spiniform microtriches were observed on the surface of the posterodorsal margin of segments of B. solinosomum, E. wageneri and M. familiaris. The invalidity of Atelemerus Guiart, 1935, first proposed by Bray et al. (1994), is supported by the present data and its type species, A. acanthodes Guiart, 1935, is newly synonymised with E. wageneri.