SSU rRNA Gene Research Articles

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae).

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 μm (4.18-3.03 μm, n = 200) in length and 1.91 ± 0.23 μm (2.98-1.66 μm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 μm (3.72-2.41 μm, n = 150) in length and 1.76 ± 0.23 μm (2.16-1.25 μm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

Impact of Temperature, Nutrients and Heavy Metals on Bacterial Diversity and Ecosystem Functioning Studied by Freshwater Microcosms and High-Throughput DNA Sequencing.

Microbial communities are fundamental components in freshwater, and community shifts in ecosystem structure are indicative of changing environmental conditions. This study aimed at investigating the influence of key environmental parameters on bacterial diversity and ecosystem functioning (i.e. organic matter breakdown) in laboratory freshwater microcosms. The effects of varying temperatures (5, 20 and 35°C), nutrients (representing low, medium and high urbanization) and heavy metals Copper (Cu) and Zinc (Zn) on bacterial diversity and organic matter (OM) breakdown were studied by using leaf bags and capsules filled with polycaprolactonediol-2000 (PCP-2000), respectively. The leaf-associated bacterial diversity was determined by next-generation sequencing of SSU rRNA gene amplicons. The results showed that bacterial diversity increased at high temperature (35°C) with more operational taxonomic units (OTUs) as compared to medium (20°C) or low (5°C) temperatures, whereas nutrient variation had fewer effects on the bacterial community structure. In contrast, the presence of heavy metals, especially high concentrations (100μM) of Cu, reduced the number of OTUs in the leaf-associated bacterial community. The higher temperatures and nutrient levels accelerated PCP-2000 breakdown rate, but this was impeded by a high concentration (100μM) of Cu in the short term, though no effect of Zn on breakdown rate was observed. The overall results indicate that temperature and variated heavy metals are among the key factors that affect bacterial diversity and ecosystem functioning in freshwater systems.

First Report of Blastocystis Infection in Pigs from Large Farms in Xinjiang, China.

Blastocystis is a common intestinal protozoan in humans and various animals worldwide. A few studies have reported the genetic characterization of Blastocystis in pigs in China, but no epidemiological data are available from the Xinjiang Uygur Autonomous Region. In this study, 801 fecal samples were collected from seven scale pig farms in Xinjiang and tested by polymerase chain reaction and sequence analysis of the partial SSU rRNA gene. The average infection rate of Blastocystis was 21.7% (174/801), with 7.1% in preweaning piglets, 10.0% in postweaning piglets, 31.8% in fattening pigs, and 41.9% in sows (χ2 =104.89; P<0.01). Blastocystis subtypes ST1 (7/174), ST3 (2/174), and ST5 (165/174) were identified, with subtype ST5 being predominant in each of the pig farms and in each of the age groups. ST3 and ST5 were identified in preweaning piglets, and ST1, ST3, and ST5 were identified in postweaning piglets. In contrast, only the subtype ST5 was observed in fattening pigs and sows. Genetic polymorphisms were observed at the intrasubtype level, including two variations of ST1 (ST1A, ST1B), and seven of ST5 (ST5A to ST5G), by sequence alignment analysis and phylogenetic analysis. More studies are needed to elucidate the transmission and public health significance of Blastocystis in pigs in various areas.

Taxonomy and Phylogeny of a New Marine Planktonic Ciliate, Strombidium pseudorapulum sp. n. (Protozoa, Ciliophora, Oligotrichia)

As one of the main groups of marine planktonic protozoa, oligotrich ciliates have shown a high biodiversity. Recently, a new aloricate oligotrich ciliate, Strombidium pseudorapulum sp. n. was isolated from coastal waters of southeast China. The living morphology and infraciliature of this species were studied using live cell observation and protargol impregnation. Strombidium pseudorapulum sp. n. differs from its congeners by having a radish-like body with a bumped anterior end and an elongated tail-like posterior end, possessing a red ‘eyespot’ in apical protrusion of cell, adoral zone of membranelles composed of 12–16 collar and 9–12 buccal membranelles, one spindle-shaped macronucleus, girdle kinety in anterior 2/5 of cell with 45–60 densely spaced dikinetids, and ventral kinety occupying about the posterior 1/3 of the cell with 16–19 dikinetids. Strombidium pseudorapulum sp. n. clusters together with those species having ‘eyespot’ in apical protrusion in the phylogenetic tree based on the small subunit rRNA gene sequence. It is a sister species of S. cuneiforme with 31 unmatched nucleotides between their SSU rRNA gene sequences.

Zoonotic Transmission of Cryptosporidium and Microsporidia in Individuals of the Kurdistan Province, West of Iran.

Cryptosporidium species and microsporidia, which can cause zoonotic intestinal infections in humans, have become an emerging public health concern. It seems that the identification and genotyping of these parasites are necessary for the prevention, control, and establishment of appropriate treatment. This study aimed to evaluate the distribution and zoonotic transmission routes of Cryptosporidium species and microsporidia to humans referred to medical laboratories of Kurdistan Province, Iran. A total of 1,383 stool samples were collected and investigated. Cryptosporidium spp. and microsporidia were detected using microscopic methods (i.e., formol-ether concentration, Ziehl-Neelsen staining, and modified trichrome staining methods). DNA was extracted from positive samples, and specific fragments of the Cryptosporidium GP60 gene and microsporidia SSU rRNA gene were amplified. Furthermore, positive samples were sequenced for genotype identification and bioinformatics analysis. Based on the microscopic analysis of 1,383 stool samples, 5 (0.36%) and 6 (0.43%) samples were considered positive for Cryptosporidium oocysts and microsporidia spores, respectively. Molecular analysis of positive samples identified the isolates as Cryptosporidium parvum and Enterocytozoon bieneusi. According to comparative phylogenetics, cryptosporidiosis and microsporidiosis may occur via zoonotic transmission in this region. Therefore, proper control and health education are strongly recommended to prevent zoonotic diseases.

Molecular detection of protists associated with the Manila clam Ruditapes philippinarum inhabiting the Wajiro tidal flat in Hakata Bay, Fukuoka Prefecture

Tidal flats are ecologically important as they support a large community of animals (e.g., crabs, mollusks, and polychaetes) and restore water quality. However, information regarding microbial eukaryotes, most of which are protists, associated with animals inhabiting these environments is limited. In the present study, utilizing polymerase chain reaction techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene of protists from the gill tissues of the Manila clam Ruditapes philippinarum in the Wajiro tidal flat, Hakata Bay, Japan. Based on the phylogeny of the SSU rRNA gene, the detected organisms were shown to belong to not only Perkinsozoa but also to Ciliophora, Kinetoplastida, and Haplosporidia. For the protist affiliated with Perkinsozoa, the internal transcribed spacer (ITS) of the rRNA genes was retrieved from the same gill tissue samples for precise identification at the species level. The ITS sequence obtained was identical to that of Perkinsus olseni, which is highly prevalent in Manila clam populations in Japan. Our findings suggest that protists potentially parasitic on this representative mollusk in tidal flats are phylogenetically diverse, and that the organisms detected may affect the population dynamics of the host clams.

Molecular prevalence and subtypes of Blastocystis sp. in primates in northern China.

Blastocystis sp. is a common enteric protist that colonizes humans and a wide range of animals. Although some studies have reported incidences of Blastocystis sp. in humans and animals in China, there are limited data available concerning the prevalence among people and non-human primates. The aims of the present study were to determine the prevalence, subtype distribution and genetic characteristics of Blastocystis sp. in primates, and to investigate the potential for zoonotic transmission between human and non-human primates. A total of 185 faecal samples from non-human primates and 1,118 samples from human volunteers were collected in Hebei province. The overall prevalence of Blastocystis sp. in non-human primates was 32.97% (61/185) based on polymerase chain reaction (PCR) amplification of the barcode region of the SSU rRNA gene. Rates of prevalence were highest among Cercopithecus neglectus (100%, 5/5) and were absent in Cebus apella and Colobus guereza. The prevalence of Blastocystis sp. in humans was 34.88% (390/1,118), and the highest rates were 41.24% in children three years of age. There was a higher detection rate in humans with diarrhoea (53.68%). Five potentially zoonotic subtypes (ST1, ST2, ST3, ST5 and ST9) were identified; among these, ST1 and ST2 were more prevalent than others in non-human primates. Similarly, two subtypes (ST2 and ST5) were detected in humans and ST2 was also the most prevalent. To the best of our knowledge, this is the first report of the prevalence and subtype composition of Blastocystis sp. in Hebei province, and the first study concerning the relationship of Blastocystis sp. among primates in China. The findings of the study will improve our understanding of the genetic diversity and public health potential of Blastocystis sp. enteric infections in addition to providing a profile of subtype characteristics of Blastocystis sp. in primates of northern China.

The first report of Cryptosporidium testudinis in Chinese alligators (Alligator sinensis) in China.

Several Cryptosporidium species that infect reptiles, especially squamates, are well described, but there is limited data about Cryptosporidium species infecting crocodilians. In this study, we assess the occurrence of intestinal parasites using traditional microscopic examination and describe the prevalence and Cryptosporidium species in the captive-bred Chinese alligators (Alligator sinensis) in eastern China using molecular methods. The results of microscopic examination showed that no intestinal parasites were detected among the 491 fecal samples examined from the Chinese alligators. The overall prevalence for Cryptosporidium was 0.41% (2/491) by PCR detection using the SSU rRNA locus. Sequence and phylogenetic analysis of the SSU rRNA, COWP, and actin genes revealed the presence of Cryptosporidium testudinis, which has been isolated primarily from chelonians. This is the first detection of the specific DNA of C. testudinis in the feces of the Chinese alligator. This study expands our knowledge of the Cryptosporidium species involved in crocodiles, and more extensive studies are necessary to confirm the validity of C. testudinis in crocodiles.

Thecamoeba foliovenanda n. sp. (Amoebozoa, Discosea, Thecamoebida) – One more case of sibling species among amoebae of the genus Thecamoeba

Until recently it seemed that amoebae of the genus Thecamoeba can be reliably identified using light-microscopic characters, like the size and shape of the cell, a characteristic pattern of folds and ridges and structure of the nucleus. However, recent molecular studies show the presence of sibling species that can be reliably distinguished based on the gene sequence data only. Here we describe a new terrestrial species, Thecamoeba foliovenanda n. sp. This species is almost identical with Thecamoeba similis in light-microscopy, which has minor differences in the ultrastructure but considerable differences in the SSU rRNA gene sequence. We investigated the light-microscopic data, as well as transmission electron-microscopic images and videorecords on the type strain of T. similis 1583/8 and performed its comparison with the new species. This study further supports the conclusion that species of the genus Thecamoeba nowadays require gene sequencing for reliable identification and species distinction.

Redescription of Rigidohymena inquieta (Stokes, 1887) Berger, 2011 as Metahymena inquieta gen. nov., comb. nov. (Ciliophora, Hypotricha) Based on Morphology, Morphogenesis, and Molecular Phylogeny.

The morphology and morphogenesis of Rigidohymena inquieta (Stokes, 1887) Berger, 2011, isolated from a lawn soil in the campus of the University of Ulsan, Korea, was studied, using live observation and protargol impregnation. The molecular phylogeny was studied based on the SSU rRNA gene sequences. The morphology of the Korean population of R.inquieta matches the previously known populations; however, the morphogenetic pattern shows differences to the species R.candens in the involvement of cirrus V/3 in the anlagen formation. A novel genus namely Metahymena gen. nov. has been erected for the present species based on the ontogenetic difference, and the new combination Metahymena inquieta gen. nov., comb. nov. is proposed. The morphology, morphogenesis, distribution, and phylogeny of M.inquieta are presented. The morphologic and morphogenetic data corroborate the phylogenetic analyses as M.inquieta clusters among the stylonychid ciliates in a clade distant from Rigidohymena candens.

QPCR-based assessment of microfaunal indicators of oil for monitoring benthos around oil and gas platforms

Today's benthic offshore biological monitoring of oil & gas (O&G) activities relies on macrofauna taxa enumeration. For the future, analysis of DNA isolated directly from sediments holds great potential for multi-trophic biodiversity surveys and the monitoring of a larger spectrum of benthic taxa, including micro-fauna. Here, we evaluate more specifically the potential of microfauna-specific gene quantification in relation to both petroleum-related discharge compounds and other seafloor environmental properties. We carried out this evaluation using sediment samples collected at drilling Region III on the Norwegian continental shelf where DNA metabarcoding of eukaryotic diversity was already performed. Generally, the quantification of microfauna indicator taxa related well to the gradient of contamination on the seafloor. Contrary to eukaryotic Euplotida, metabarcoding data and qPCR numbers for indicative prokaryotic taxa showed the same relationship to offshore contaminants (both showed positive relationship). We found absolute numbers of SSU rRNA gene copies of (1) Dinophyceae, Bacillariophyceae and Alcanivorax were correlated with the level of petroleum-related compounds but not with other environmental variables, (2) bacteria closely related to Shewanella were correlated with the concentration of Ba, PAH, as well to percent of gravel, (3) Desulfobacteriales correlated with petroleum-related contaminants, but as well with percent of gravel and grain size.Findings from our study suggest that biomonitoring surveys of O&G activities on benthos could benefit from quantification of specific micro-fauna indicators that is simpler and faster than the methods currently used for impact assessment of benthos.

Closely related dinoflagellate species in vastly different habitats – an example of a marine–freshwater transition

ABSTRACT Phytoplankton are present in a large variety of aquatic environments, ranging from small freshwater ponds to the oceans. Typically, freshwater and marine species are not closely related, indicating an ancient divergence and that salinity poses a strong dispersal barrier. Here we reveal a common recent origin of two dinoflagellates that are well adapted to different habitats. Gymnodinium baicalense inhabits the geologically old freshwater Lake Baikal, which is located in the middle of the Eurasian continent. Gymnodinium corollarium, on the other hand, is a brackish water species from the Baltic Sea. Both species form blooms under ice during the spring. We generated 10 DNA sequences from these species. The partial 28S rRNA gene from G. baicalense and the partial 18S rRNA gene and internal transcribed spacer-2 (ITS2) from G. corollarium were sequenced for the first time. A more detailed description of G. baicalense, which was previously known only from light microscopy observations, is also provided. In the laboratory we demonstrated that G. baicalense is strictly adapted to fresh water, while G. corollarium had a wide salinity tolerance. However, the two species have almost identical morphology, identical SSU rRNA gene sequences, and only small differences in the LSU rRNA gene and ITS2. We infer a common ancestor, which was a species from the Arctic region with a tolerance to a range of salinities. Our data support the scenario that the under-ice phytoplankton community in Lake Baikal has been formed recently, even though the lake is the oldest in the world.

Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs

A new species of clevelandellid ciliate, Nyctotheroides grimi n. sp., is described from the frog Fejervarya limnocharis. Light and scanning electron microscopy were used for the morphological studies, and the DNA encoding the SSU rRNA gene (SSU rDNA) and the ITS1-5.8S subunit rRNA-ITS2 region (ITS) were sequenced for genetic comparisons and phylogenetic analysis. The main distinctive morphological feature is a knob-like projection in the left-posterior end; other differential characters are the cell size, the length of the oral groove and the shape of the infundibulum. Nyctotheroides grimi possess an apical suture line in the left and right side of the anterior end and in the left side of the caudal end. In the phylogenetic analyses, the new species engroups with other Nyctotheroides species forming a monophyletic group. The high similarity in the SSU rDNA and ITS sequences between Nyctotheroides species suggests a relative recent divergence. The genetic data and the different host range support the separation of Nyctotheroides and Nyctotherus; however the morphological criterion based on the presence (in Nyctotheroides)/absence (in Nyctothterus) of an apical kinetal suture line should be modified to consider the presence of kinetal suture lines in the apical and/or the caudal left side in Nyctotheroides.

New Intranuclear Symbiotic Bacteria from Macronucleus of Paramecium putrinum—“Candidatus Gortzia Yakutica”

Holospora-like bacteria (HLB) are obligate intracellular Alphaproteobacteria, inhabiting nuclei of Paramecium and other ciliates such as “Candidatus Hafkinia” is in Frontonia. The HLB clade is comprised of four genera, Holospora, Preeria, “Candidatus Gortzia”, and “Candidatus Hafkinia”. These bacteria have a peculiar life cycle with two morphological forms and some degree of specificity to the host species and the type of nucleus they inhabit. Here we describe a novel species of HLB—“Candidatus Gortzia yakutica” sp. nov.—a symbiont from the macronucleus of Paramecium putrinum, the first described HLB for this Paramecium species. The new endosymbiont shows morphological similarities with other HLB. The phylogenetic analysis of the SSU rRNA gene places it into the “Candidatus Gortzia” clade.

The red fox (Vulpes vulpes) as a potential natural reservoir of human cryptosporidiosis by Cryptosporidium hominis in Northwest Spain.

Giardia duodenalis and Cryptosporidium spp. are ubiquitous intestinal protozoa that parasitize domestic and wild animals, as well as human beings. Due to their zoonotic potential, the objective of the present study was to determine the presence of these pathogens in the fox population (Vulpes vulpes) located in Northwest Spain. A total of 197 faecal samples from legally hunted foxes were collected in the autonomous region of Galicia. The presence of G. duodenalis and Cryptosporidium spp. was investigated by PCR-based methods amplifying the small subunit ribosomal RNA (ssu rRNA) gene of the parasites. Attempts to genotype obtained positive samples were subsequently conducted at the glutamate dehydrogenase (gdh) and β-giardin (bg) genes of G. duodenalis, and the 60kDa glycoprotein (gp60) gene of Cryptosporidium. Giardia duodenalis and Cryptosporidium spp. were identified in 19 (9.6%) and 12 (6.1%) of the investigated samples, respectively. However, five Cryptosporidium species were detected at the ssu rRNA locus: C. hominis (33.4%, 4/12), C. canis (25.0%, 3/12), C. parvum (16.7%, 2/12), C. ubiquitum (8.3%, 1/12) and C. suis (8.3%, 1/12). An additional Cryptosporidium-positive sample was identified at the genus level only. Typing and subtyping of Giardia- and Cryptosporidium-positive samples were unsuccessful. The detection of C. hominis in wild foxes indicates the probable overlapping of sylvatic and domestic cycles of this parasite in rural settings. Besides, this finding raises the question of whether red foxes may act as natural reservoirs of C. hominis. The detection of C. parvum and C. suis is suggestive of active transmission events between farm and wild animals, opening up the possibility of transmission to human beings.

The first case of microsporidiosis in Paramecium.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 μm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Assignment of Vairimorpha leptinotarsae comb. nov. on the basis of molecular characterization of Nosema leptinotarsae Lipa, 1968 (Microsporidia: Nosematidae).

Nosema leptinotarsae Lipa, 1968 is a microsporidian pathogen of the Colorado potato beetle, Leptinotarsa decemlineata Say. (Coleoptera: Chrysomelidae). To determine the phylogenetic status of N. leptinotarsae, the 16S SSU rRNA gene was sequenced (GenBank Accession No. MN841279) and compared phylogenetically against 21 microsporidian 16S SSU rRNA sequences using neighbour-joining and maximum-parsimony methods. The per cent identities of the N. leptinotarsae and other members of the Nosema-Vairimorpha clade ranged from 78.1 to 98.5%. Pairwise phylogenetic distances between the N. leptinotarsae and other species ranged from 0.009 to 0.320. Phylogenetic analysis shows clearly that N. leptinotarsae is a member of the Vairimorpha clade rather than the Nosema clade. The sequence divergence and morphological traits separated the N. leptinotarsae from other species in the Vairimorpha complex. As a result, a new assignment of Vairimorpha leptinotarsae comb. nov. has been implemented for N. leptinotarsae according to the phylogenetical positioning in the present study.

Molecular characterization of Giardia spp. and Cryptosporidium spp. from dogs and coyotes in an urban landscape suggests infrequent occurrence of zoonotic genotypes

Giardia spp. and Cryptosporidium spp. are common gastrointestinal parasites with the potential for zoonotic transmission. This study aimed to (1) determine the genotypes occurring in dogs and coyotes occupying a similar urban area; (2) determine if these hosts were infected with potentially zoonotic genotypes; (3) provide baseline molecular data. In August and September 2012, 860 dog owners living in neighborhoods bordering six urban parks in Calgary, Alberta, Canada, provided faecal samples from their dogs. From March 2012 through July 2013, 193 coyote faeces were also collected from five of six of the same parks. Direct immunofluorescence microscopy (DFA) indicated that Giardia spp. and Cryptosporidium spp. infected a total of 64 (7.4%) and 21 (2.4%) dogs, as well as 15 (7.8%) and three (1.6%) coyotes, respectively. Semi-nested, polymerase chain reactions targeting the 16S small-subunit ribosomal ribonucleic acid (SSU rRNA) and 18S SSU rRNA genes of Giardia spp. and Cryptosporidium spp., respectively, were conducted on samples that screened positive by DFA, and products were sequenced and genotyped. Dogs were infected with Giardia intestinalis canid-associated assemblages C (n = 14), D (n = 13), and Cryptosporidium canis (n = 3). Similarly, G. intestinalis assemblages C (n = 1), D (n = 1) and C. canis (n = 1), were detected in coyotes, as well as G. intestinalis assemblage A (n = 1) and Cryptosporidium vole genotype (n = 1). Dogs and coyotes were predominantly infected with host-specific genotypes and few potentially zoonotic genotypes, suggesting that they may not represent a significant risk for zoonotic transmission of these parasites in urban areas where these hosts are sympatric.

Reclassification of Gonyaulax verior (Gonyaulacales, Dinophyceae) as Sourniaea diacantha gen. et comb. nov.

ABSTRACT Gonyaulax verior was initially described as Amylax diacantha from Belgian coastal waters a century ago but its detailed morphology needed restudy. Here, we established nine strains of G. verior by germinating cysts or isolating cells from localities from the European Atlantic to the Caspian Sea and the Pacific Ocean. Both cyst and thecal morphology were examined by light and scanning electron microscopy. SSU, LSU and/or ITS-5.8S rRNA gene sequences were obtained from all strains. Cells of G. verior have a plate formula of Po, 4′, 2a, 6-7′′, 6C, 6S, 6′′′, 1p, 1′′′′ with an L-type ventral organisation, characterised by two either straight or curved antapical horns of variable length. Cysts of G. verior are oval, smooth and contain one or two yellow accumulation bodies. The maximum-likelihood and Bayesian inference analyses based on SSU and LSU rRNA gene sequences revealed two clades of G. verior, referred to as ribotypes A and B. Genetic distances based on ITS-5.8S rRNA gene sequences within the same ribotype were less than 0.06, but greater than 0.32 between ribotypes. G. verior is reclassified as Sourniaea diacantha gen. et comb. nov., which is attributed to Lingulodiniaceae together with Pyxidinopsis, Lingulodinium and Amylax. Our results suggest that Lingulodiniaceae can be separated from Protoceratiaceae and Gonyaulacaceae based on ventral organisation, apical complex, ventral pore and number of anterior intercalary plates. One strain of S. diacantha was examined for yessotoxin production by LC-MS/MS but did not produce toxin.

Description of a new species of Tetmemena (Ciliophora, Oxytrichidae) using classical and molecular markers

Tetmemena saprai n. sp. was isolated from fresh-water samples collected from Okhla Bird Sanctuary, Delhi, India and described based on its morphology, morphogenesis and molecular markers, namely the small-subunit (SSU) rRNA gene and internal transcribed spacers (ITS1-5.8S-ITS2). The morphological features of T. saprai n. sp. are as follows: colourless; rigid body with no cortical granules; water expelling vesicle about 15 µm in size, located below the adoral zone of membranelles near the left body margin; body size about 125–140 × 50–60 μm in live and about 100 × 45 μm in protargol preparations, with body length : width ratio should be 2:1; undulating membranes in a Stylonychia-pattern; adoral zone about 44 μm in length with (on average) 45 adoral membranelles; 18 frontal-ventral-transverse cirri; one right and one left marginal row; six dorsal rows including two dorsomarginals, of which the fourth row is shortened anteriorly; three caudal cirri which are not equidistant; two macronuclear nodules; 2–4 micronuclei. A detailed morphogenetic study revealed the oral primordium to originate near the leftmost transverse cirrus, and the involvement of five parental cirri (three frontals and two ventrals) in the formation of streaks I–VI for the proter and opisthe. The genetic distance between T. saprai n. sp. and its congeners T. pustulata and T. vorax (S. vorax) varied from between 1% and 2% in the SSU rRNA gene and 1% and 6% in the ITS1-5.8S-ITS2 sequence. The secondary structures of the ITS1 and ITS2 RNA transcripts of four different species of genus Tetmemena (T. saprai n. sp., T. pustulata, T. vorax and T. bifaria) were also compared, revealing that the ITS1 region was more variable than ITS2. The morphological and morphogenetic characterisation, and phylogenetic analyses based on the molecular markers, confirm that the present species is a distinct species of the genus Tetmemena belonging to the subfamily Stylonychinae. This study also reveals that the macronuclear ITS region can be a suitable candidate for species identification.