A study on the non-monophyletic genera Australothrix and Holostichides based on multigene and morphological analyses with a reexamination of type materials (Protozoa: Ciliophora).

In a study of ciliate diversity, we collected two new species of the genera Australothrix and Holostichides. Based on nuclear ribosomal genes, our study shows that these two genera are genetically non-monophyletic. To clarify the issues of the non-monophyly, we reexamined type material of H. heterotypicus, H. terrae, and Birojimia soyaensis. Based on multigene and morphometric analyses, Australothrix and Holostichides are clearly non-monophyletic even in mitochondrial CO1 gene trees. The multigene analyses show a clade composed of A. lineae sp. nov., H. terrae, and B. soyaensis, suggesting that a cytopharynx with argyrophilic structures might be their synapomorphy. A list of species with this type of cytopharynx is provided for further studies to either accept or reject this hypothesis. Based on the reexamination, we discriminate the morphologically nearly identical (cryptic/sibling) species H. obliquocirratus sp. nov. from H. heterotypicus and they show significant genetic dissimilarities in the multigene trees. They showed only few morphological (non-quantitative) differences and thus distinguishing them morphologically needs careful investigation.

Morphology and Phylogeny of the Soil Ciliate Parabistichella multilineae sp. nov. (Protozoa: Ciliophora: Hypotricha).

The morphology and molecular phylogeny of Parabistichella multilineae sp. nov., which was discovered in soil from Gagye Beach, South Korea, were investigated. The new species is characterized as follows: body size about 200 × 47 µm in vivo, slenderly lanceolate and twisted; two ellipsoidal macronuclei and usually two globular micronuclei; two types of cortical granules; four or five frontoventral rows, of which rows 1 and 2 are conspicuously developed and rows 3 and 4 are fully extended; and caudal cirri are lacking. Phylogenetic analyses based on the SSU rDNA sequences showed that the new species clusters in a soft polytomy with P. variabilis and Uroleptoides magnigranulosus.

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora, Metopida), with Identification of the Intracellular Bacteria.

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18-21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21-29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion-resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).

Morphology and molecular phylogeny of two hypotrichous ciliates (Ciliophora, Spirotrichea) from South Korea, including Hemiurosomoida koreana n. sp.

The living morphology, infraciliature, and molecular phylogeny of a new soil ciliate, Hemiurosomoida koreana n. sp., discovered in a sample collected from a mountain in the northeast of South Korea, were investigated. The new species possesses the characteristics of the genus Hemiurosomoida, i.e., a reduced number of frontal-ventral-transverse cirri, three dorsal kineties of which kineties 1 and 2 each bears a caudal cirrus, and a single dorsomarginal kinety. It is distinguishable from congeners and other similar species by at least one distinct qualitative or quantitative character including the body size, the presence and arrangement of cortical granules, the number of adoral membranelles, marginal cirri, and dorsal dikinetids, or by the arrangement of transverse cirri. Phylogenetic analyses based on 18S rRNA gene sequences also support the assignment of the new species to the non-monophyletic genus Hemiurosomoida. In addition, the living morphology, infraciliature, and the 18S rRNA gene sequence of a Korean population of Nothoholosticha flava were studied.

Morphology, morphogenesis, and molecular phylogeny of Aspidisca koreana n. sp. (Ciliophora, Euplotida) from South Korea.

The morphology, morphogenesis, and molecular phylogeny of a new ciliate, Aspidisca koreana n. sp., discovered in the eastern coast of South Korea, were investigated. The morphological description is based on the observation of living cells, 4'-6-diamidino-2-phenylindole (DAPI) and silver-stained specimens (e.g., protargol, silver nitrate), and scanning electron micrographs. The new species is characterized by having a small body size (17-25 × 15-18 µm in vivo), a distinct peristomial spur on the posterior portion of left margin, seven frontoventral cirri in "polystyla-arrangement", and the arrangement of the anterior portion of adoral zone of membranelles, i.e., anteriormost membranelle is distinctly separated from the other three membranelles. The morphogenesis follows the typical pattern of this genus. Phylogenetic analyses, using the 18S rDNA sequence, also support the establishment of a new species.

Redescription and molecular phylogeny of Trochilia sigmoides Dujardin, 1841 (Ciliophora, Cyrtophoria) collected from South Korea.

During a survey of Korean marine ciliates, Trochilia sigmoides, the type species of the genus Trochilia, was collected and examined using in vivo observation and protargol impregnation. Moreover, scanning electron microscopy and 18S rRNA gene sequencing have been applied for the first time to study this species. Morphologically, T. sigmoides is characterized by the small body size, the oval body outline, and the spiral dorsal ridges. The Korean population of T. sigmoides shows only minute differences to other populations reported in the literature, mainly in body size and the number of dorsal ridges. Phylogenetic analyses based on 18S rRNA gene sequences show that T. sigmoides and T. petrani are placed together with two members of the family Kyaroikeidae, causing the family Dysteriidae to be non-monophyletic. The present new data increase the knowledge about the morphology and phylogeny of the genus Trochilia and would assist in understanding the phylogenetic relationship between the free-living Dysteriidae and the parasitic Kyaroikeidae.

Polymorphism, gigantism, and cannibalism, one stylonychid ciliate (Ciliophora, Hypotricha) to rule them all.

The morphology, ontogenesis, and molecular phylogeny of the polymorphic and cannibalistic giant forming Tetmemena polymorpha n. sp., found in a brackish water sample in South Korea, were investigated. The present species has long been misidentified as "Oxytricha bifaria." The new investigation shows that the species produces three morphologically different morphs. The small morph is bacterivorous and characterized by its small body size and slim body and it is found only in the stationary and decline phases of the culture. The large morph has a wide body, larger oral apparatus, and feeds on small eukaryotes such as yeast cells and small ciliates. It divides very quickly and produces the other two morphs and found in the exponential phase of the cultures. The giant morph is characterized by its huge body and oral apparatus. It feeds on the small morph cells of the same species and other ciliates, and occurs together with the small morph. Phylogenetic analyses based on the 18S rRNA gene sequences show that the new species is placed in a sister subclade to that containing other Tetmemena sequences. Moreover, Tetmemena indica Bharti et al., 2019 nov. stat. is raised to species level based on the differences in the cyst morphology and the dorsal ciliature to the authoritative Tetmemena pustulata population.

Neotypification and ontogenesis of Leptopharynx costatus costatus Mermod, 1914.

Using standard methods, we studied the morphology and ontogenesis of a German Leptopharynx costatus costatus. This population makes two morphs: microstomes with a size of 40 × 25 µm, about 190 basal bodies, and 5 µm wide oral basket; and macrostomes with a size of 55 × 40 µm, about 264 basal bodies, and 15 µm wide oral basket. Because the identity is threatened, this population is designated as the neotype of L. costatus costatus. Ontogenesis is complex due to the preoral kineties and the postoral complex produced by kineties 9 and 10. Stomatogenesis is mixokinetal: the opisthe membranelles 1 and 2 are formed by the oral primordium, whereas membranelle 3 is produced by the posterior portion of somatic kinety 1. The nasse kinetosomes are generated by the anterior portion of the oral primordium. Preoral kineties 1 and 3 develop de novo, while kinety 2 originates by intrakinetal proliferation of kinety 8; preoral kinety 4 is produced by the postoral complex, thus being a somatic kinety. Kinety 6 has two anterior kinetids in line with kinety 7. These observations require changes in the descriptive morphology, support the classification of Leptopharynx into the Microthoracidae, and sustain the nonmonophyly of the Nassophorea.

Morphology and Molecular Phylogeny of Two New Trachelophyllid Ciliates, Monolamellophrya terricola gen. nov., sp. nov. and Trachelophyllum parapiculatum sp. nov. (Litostomatea, Haptoria), From South Korea.

The morphology and molecular phylogeny of two new ciliates, Monolamellophrya terricola gen. nov., sp. nov. and Trachelophyllum parapiculatum sp. nov., discovered in South Korea, were investigated. The two species belong to the suborder Trachelophyllina, which is characterized by the presence of a mucilaginous layer containing lepidosomes covering the cortex. Monolamellophrya terricola gen. nov., sp. nov. is characterized by the presence of a single layer of type II lepidosomes, representing a new genus. Trachelophyllum parapiculatum sp. nov. has only type I lepidosomes covering the cortex, a generic character of the genus Trachelophyllum, and is distinguished from other congeners by a combination of morphological features, including the 15-24 µm long rod-shaped extrusomes, the 9-13 ciliary rows, the 7-11 and 17-25 dikinetids in brush rows 1 and 2, respectively, and the bipolar brush row 3. Furthermore, the 18S rRNA gene sequences of the two new species were provided. The phylogenetic analyses show that the sequence of M. terricola gen. nov., sp. nov. clusters with two other trachelophyllid sequences, and the sequence of T. parapiculatum sp. nov. is placed at the base of these three sequences with full support. Furthermore, the four trachelophyllid sequences that are available so far form a monophyletic clade.

A new "flagship" ciliate, Pseudostylonychia obliquocaudata n. gen., n. sp. (Ciliophora, Hypotricha), from South Korea.

The morphology, ontogenesis, and molecular phylogeny of a new ciliate, Pseudostylonychia obliquocaudata n. gen., n. sp., discovered in a semiterrestrial habitat in South Korea, were investigated. The new species is characterized by an obovate or triangular body with pointed posterior end. The caudal area is distinctly obliquely truncated rightwards. Pseudostylonychia n. gen. is established and characterized by the obovate or triangular body, Stylonychia-like infraciliature and macronuclear pattern, and undulating membranes in Laurentiella pattern. It is also characterized by the involvement of cirri III/2 and IV/3 in primordia formation. SEM investigations show that the resting cyst has high, spirally arranged ridges. Phylogenetic analyses based on SSU rRNA gene sequences show that the new species is placed as a sister to the clade containing sequences of Stylonychia mytilus complex, Stylonychia (Metastylonychia), and Coniculostomum. Furthermore, Stylonychia koreana Kumar et al., 2016 nov. stat. is raised to species level based on the differences in the cyst morphology between the two subspecies of S. ammermanni. Also, Metastylonychia Kumar and Foissner, 2017 nov. stat. is raised to genus rank based on the new molecular data and the morphological and ontogenetic differences to Coniculostomum and Stylonychia.

Diophrys pauciciliata n. sp. (Ciliophora, Euplotida), a new marine spirotrich ciliate with highly static morphometrics.

A new marine ciliate was discovered from intertidal mudflat from South Korea. Based on the observation of living cells, protargol-impregnated specimens and SEM, we describe it as new species belonging to the genus Diophrys Dujardin, 1841, namely Diophrys pauciciliata n. sp. Its nuclear small subunit ribosomal RNA gene also supports the taxonomic assignment. Diophrys pauciciliata n. sp. is one of the smallest species in the genus. Besides, the bipartite adoral zone and oligomerized dorsal bristles easily discriminate the new species from other congeners. Interestingly, D. pauciciliata n. sp. showed highly static states in all morphometrics (<8.5 % of coefficient of variation) and in particular, the number of frontal adoral membranelles and the counts of dorsal ciliature (e.g., number of dorsal kineties and numbers of dikinetids within each kinety) are invariable. Considering morphometric data of previous studies in the genus Diophrys, the narrow range of character states is unexpected because cell length is usually positively correlated with the numbers of adoral membranelles and dorsal dikinetids.

Molecular phylogeny of a new gonostomatid ciliate revealing a discrepancy between interphasic and cell divisional patterns (Ciliophora, Hypotricha).

The morphology, ontogenesis, and molecular phylogeny of a new ciliate, Gonostomum koreanum n. sp., discovered in a terrestrial moss sample from South Korea, were investigated. Morphologically, it is characterized by a gonostomatid oral apparatus, two macronuclear nodules, six frontoventral rows, the two rightmost of which (frontoventral rows V and VI) extend posteriorly to near pretransverse and transverse cirri, and three dorsal kineties each with a single caudal cirrus posteriorly. The new species is easily confused with members of the genus Metagonostomum because of the long frontoterminal cirral row but differs mainly in the de novo (vs. intrakinetal) origin of anlage VI, a character found only in Gonostomum and Paragonostomum. To solve the discrepancy between the interphasic and ontogenetic patterns, we additionally performed morphological and multigene analyses on three gonostomatid species, namely Gonostomum koreanum n. sp. and its morphologically (M. gonostomoida) and ontogenetically (G. kuehnelti) most similar species. The multigene analyses show that the new species is closely related to G. kuehnelti and the core gonostomatids consists of five groups based on the origin of the frontoventral rows.

Tunicothrix halophila n. sp., a Secondarily Oligomerized Parabirojimid Hypotrich (Ciliophora, Spirotrichea) From Hypersaline Costal Water in Korea.

Tunicothrix halophila n. sp. was discovered in a hypersaline marine sample from Jeju Island, Korea. It is characterized by the highly reduced number of dorsal bristles. In addition, the main character of the genus Tunicothrix (e.g., alveolar layer) is absent/indistinct. To figure out its identity and phylogenetic relationship, we examined the new species based on modern morphological methods and molecular phylogenetic analyses. Since the parabirojimids are of basal position to core hypotrichs and a smaller data set could show incorrect phylogenetic relationships among the hypotrichs, we used a huge data set composed of 1,460 DNA sequences to infer the phylogenetic tree. The reduction of dorsal bristles is very likely a secondarily evolved character in hypotrichs, resulting in the independent phenotypic adaptation in the hypersaline ecosystems as shown in other hypersaline hypotrichs. Furthermore, the so-called right marginal row 1 in other congeners is found to produce a pretransverse and transverse cirrus and thus we recommend using the term frontoventral row. Based on our data, we can justify Tunicothrix halophila n. sp. as a new species; however, despite the phenotypic distinctiveness, we refrain to establish a new genus because of the missing data and the non-monophyly of Tunicothrix.

Anteholosticha foissneri n. sp., a marine hypotrich ciliate (Ciliophora: Spirotrichea) from Vietnam: Morphology, morphogenesis, and molecular phylogeny.

In a study of marine ciliate diversity, we collected an Anteholosticha monilata-like population from Vietnam. To identify this population, we analyzed its morphology, some morphogenetic stages, and molecular phylogeny. Based on these data, we conclude that the Vietnamese population is new to science. Anteholosticha foissneri n. sp. resembles Anteholosticha monilata-like species considering (1) the number and arrangement of macronuclear nodules and micronuclei; (2) the presence of cortical granules; and (3) the saline habitat. However, the new species can be easily distinguished from these species by the arrangement, color, and shape of the cortical granules. The divisional morphogenesis commences with the de novo proliferation of basal bodies as a single longitudinal patch left of the posteriormost midventral cirral pair. This character state has not been reported before in Anteholosticha (based on check of the available data) and probably reflects a distinct clade within the nuclear small subunit ribosomal RNA gene tree.

Morphology and Molecular Phylogeny of the Soil Ciliate Cyrtohymena seorakensis sp. n. (Ciliophora, Hypotricha, Oxytrichidae).

The morphology and infraciliature of a new ciliate, Cyrtohymena seorakensis sp. n., discovered in a moss sample from South Korea, were investigated. The new species is distinguished from congeners by having rather fewer, randomly scattered, yellowish cortical granules, 23-30 adoral membranelles, invariably four transverse cirri in morphostatic specimens, and a total of 62 dorsal bristles on average. Phylogenetic analysis based on 18S rDNA sequences showed that the new species clusters with an Indian population of C. citrina.

Immunosuppressive therapy in children with steroid-resistant, frequently-relapsing, and steroid-dependent idiopathic nephrotic syndrome: a single center experience.

INTRODUCTION: Immunosuppressive agents are recommended for the management of children with steroid-resistant, frequently-relapsing, and steroid-dependent idiopathic nephrotic syndrome, i.e., SRNS, FRNS, and SDNS. This study evaluated the efficacy of immunosuppressive agents in these cases. METHODS: This is a retrospective analysis of the records of 130 pediatric cases recruited from a tertiary-care center over a period of two years. They were divided into two groups, i.e., 51 patients with SRNS (Group I) and 79 cases with SDNS and FRNS (Group II). They were treated with immunosuppressive agents in addition to steroids, either as double- or triple-combination therapy. Complete or partial remission was considered a good response. RESULTS: In group I, the proportions of good response to cyclophosphamide, cyclosporine A, and mycophenolate mofetil were 48.6, 60, and 80%, respectively (p = 0.162). In group II, the resistance rate was significantly higher with levamisole than with cyclophosphamide and azathioprine (p = 0.046). Leukopenia was reported infrequently after the administration of cyclophosphamide or azathioprine. The most serious adverse reaction was to cyclosporine A, which induced nephrotoxicity (6.4%), while no adverse effects were reported to be related to levamisole. Histopathological diagnoses were available in only 39 patients. CONCLUSION: The high potency of cyclosporine with steroids makes it useful in patients with idiopathic SRNS with a normal glomerular filtration rate. Its efficacy is augmented when combined with mycophenolate mofetil. Cyclophosphamide, orally or as intravenous boluses, together with alternate-day steroids, could be a good option outside the peripubertal age. The outcomes with FRNS and SDNS could be improved by encouraging compliance with the use of levamisole.

Iron chelation monotherapy in transfusion-dependent beta-thalassemia major patients: a comparative study of deferasirox and deferoxamine.

INTRODUCTION: Iron overload is the primary cause of mortality and morbidity in thalassemia major (TM) despite advances in chelation therapy. The aim of this study was to compare the effectiveness and safety of deferasirox (DFX) and deferoxamine (DFO) as iron-chelating agents in patients with transfusion-dependent ß-thalassemia major. METHODS: This prospective randomized study included 60 patients with transfusion-dependent ß-TM during the period from September 2014 to September 2015. Their ages were ≥ 6 years, and they had serum ferritin above 1500 µg/L and were on irregular DFO therapy. Patients had regular packed red cell transfusion in a dose of 10 mL/kg/session. They were randomized to receive DFX (single oral daily dose of 20-40 mg/kg/day) or DFO (20-50 mg/kg/day via subcutaneous infusion over 8-10 hours, 5 days a week). Iron overload was determined by serum ferritin level. The primary endpoint was decrease of serum ferritin level below 1500 µg/L. The secondary endpoint was drug safety. RESULTS: Both drugs significantly reduced serum ferritin (p < 0.001). At the end of follow-up, there were no significant differences between the two groups in serum ferritin levels (p = 0.673) and in percent reduction of ferritin (p = 0.315). There were no significant differences between the two groups in the total amount of blood transfusion (p = 0.166) and average iron intake (p = 0.227). There were no mortalities or any serious adverse effects, neutropenia, arthropathy, or pulmonary toxicity. Gastrointestinal upset and skin rash occurred more frequently with DFX than with DFO (p = 0.254 and 0.095, respectively). CONCLUSION: With appropriate dosing and compliance with drugs, both DFX and DFO are generally well tolerated, safe, and effective in reducing serum ferritin levels in iron-overloaded, regularly-transfused thalassemia major patients. Therefore, oral DFX is recommended for more convenience and adherence to the treatment regimen.

Description of two new Drepanomonas taxa and an account on features defining species in Drepanomonas Fresenius, 1858 (Ciliophora, Microthoracida).

Using standard methods, we describe two new Drepanomonas taxa: Drepanomonas hymenofera (Horváth 1956) nov. comb., which is composed of two (biogeographical?) subspecies, viz., D. hymenofera venezuelensis nov. subspec. and D. hymenofera hymenofera (Horváth 1956), was discovered in soil from Venezuela and Iceland, respectively. Both are comparatively large-sized (50×20µm and 40×18 µm in vivo), differing in the cortex pattern and the structure of kineties 3 and 4. We agree with Corliss (1979) and Chardez (1990) that the genus Pseudocristigera, which was established by Horváth (1956) for Drepanomonas hymenofera, is a junior synonym of Drepanomonas. Drepanomonas vasta nov. spec., which was discovered in the mud of a tree hole in Austria, is a middle-sized species (35×18 µm) with thick body, wide left side ridges, a single anterior dikinetid in kinety 4, and an average of 99 basal bodies; it is unique in having the dorsal side much more flattened than the ventral side, thus being cuneate in transverse view. Ontogenetic data show that the ciliary pattern of Drepanomonas is homologous to that of Leptopharynx, specifically, the structure and origin of the postoral complex. Main features for distinguishing Drepanomonas species are discussed.

Description of Leptopharynx brasiliensis nov. spec. and Leptopharynx costatus gonohymen nov. subspec. (Ciliophora, Microthoracida).

Using standard morphological methods, we describe one new Leptopharynx species and a new subspecies of L. costatus, both from soil of the neotropic region. Further, we studied two populations of L. costatus costatus. Leptopharynx brasiliensis nov. spec., which was discovered in the Mato Grosso, Brazil, is a large member (60µm) of the genus with an enormous oral basket. It differs from similar congeners in having six monokinetids in kinety 6, widely spaced kinetids in kinety 1, and an average of 294 kinetids. Leptopharynx costatus gonohymen nov. subspec., which was discovered in southern Florida, makes a small (35µm) and a large morph (55µm) both with narrow oral basket. The small morph is inseparable from the small morph of L. costatus costatus, while the large morph has right-angled adoral membranelles and widely (vs. narrowly) spaced kinetids in kinety 1. The small morphs of a Brazilian and an Austrian L. costatus match Mexican and other European populations, all having on average 181-187 kinetids. As yet, we know four morphs of L. costatus that differ by body size (small vs. large), the oral basket (narrow vs. wide), membranelle 1 (present vs. absent), and the arrangement of the membranelles (flat vs. angled).