SSrRNA Gene Sequences Research Articles

De novo transcriptome assembly of a facultative parasitic nematode Pelodera (syn. Rhabditis) strongyloides

Pelodera strongyloides is a generally free-living gonochoristic facultative nematode. The whole genomic sequence of P. strongyloides remains unknown but 4 small subunit ribosomal RNA (ssrRNA) gene sequences are available. This project launched a de novo transcriptome assembly with 100 bp paired-end RNA-seq reads from normal, starved and wet-plate cultured animals. Trinity assembly tool generated 104,634 transcript contigs with N50 contig being 2195 bp and average contig length at 1103 bp. Transcriptome BLASTX matching results of five nematodes (C. elegans, Strongyloides stercoralis, Necator americanus, Trichuris trichiura, and Pristionchus pacificus) were consistent with their evolutionary relationships. Sixteen genes were identified to be homologous to key elements of the C. elegans RNA interference system, such as Dicer, Argonaute, RNA-dependent RNA polymerase and double strand RNA transport proteins. In starved samples, we observed up-regulation of cuticle related genes and 3 dauer formation genes. Dauer morphology was captured with enlarged phasmid under light microscopy, and dauer and normal larvae counts in clumps had a Pearson's product-moment correlation of 0.805 with P-value = 0.0088. Our results demonstrate that P. strongyloides could be used for studying nematode-related human or pet parasitic diseases. The sequenced assembled transcriptome reported here may be useful to understand the evolution of parasitism in Nematoda.

Morphological and molecular characterization of Thelohanellus macrovacuolaris n. sp. (Myxosporea: Bivalvulida) infecting the palate in the mouth of common carp Cyprinus carpio L. in China

Thelohanellus macrovacuolaris n. sp. is described during a survey on myxozoan diversity of common carp Cyprinus carpio L. in China. It is characterized by the presence of round or ellipsoidal plasmodium in the palate in the mouth of host. Mature spores were pyriform in frontal view, lemon shaped in lateral view, measuring 21.6±0.9 (19.3–23.8) long, 12.5±0.7 (10.3–13.6) wide, and 10.2±0.4 (9.8–11.8) thick. Most spores were surrounded by the membrane sheath. Single polar capsule was round with an apophysis at its top end presented close to apex of spore, measuring 9.1±0.6 (8.0–10.0) in length, 8.6±0.5 (7.8–9.6) in width. Polar filaments coiled, with 7 to 8 turns. A large, round iodinophilous vacuole was present, with 5.8–7.5 in diameter. The present species is morphologically distinct from all other Thelohanellus species. The BLAST search indicated that the newly obtained small subunit ribosomal RNA (ssrRNA) gene sequence of T. macrovacuolaris n. sp. did not match any available sequences in GenBank. Phylogenetically, T. macrovacuolaris n. sp. was placed sister to Thelohanellus wangi in the Thelohanellus clade. Both morphology and ssrRNA gene sequence data revealed that the present parasite is a new species of genus Thelohanellus.

Taxonomy and molecular phylogeny of four Strombidium species, including description of S. pseudostylifer sp. nov. (Ciliophora, Oligotrichia)

Oligotrich ciliates are important components of marine microplankton and have a high biodiversity. In the present paper, the morphology and small subunit rRNA (SSrRNA) gene sequence of four Strombidium species are recorded, including a new species, S. pseudostylifer sp. nov. that was sampled from a mangrove wetland. This new species is characterized by its prominent apical protrusion, long constant tail and large number of ventral dikinetids. In addition, the morphology of two poorly known species, S. chlorophilum Montagnes et al., 1988 and S. oculatum Gruber, 1884 are redescribed based on the Chinese populations and their diagnoses are improved. The morphology of three populations of S. stylifer Levander, 1894 collected from three sampling sites in China are also compared, revealing that the only difference between them are the sizes of their cells and macronuclei. Finally, the phylogenetic analyses of the above four Strombidium species, as inferred from their SSrRNA gene sequence data, show that S. oculatum, S. stylifer and S. pseudostylifer cluster within the main Strombidium branch while S. chlorophilum clusters with S. conicum and branches from the main Strombidium group.http://zoobank.org/urn:lsid:zoobank.org:pub:E869E485-8F29-4B2B-8BB2-D3A039AA6403

Myxidium ceccarellii n. sp. (Myxosporea) from the gallbladder of Leporinus elongatus (Anastomidae) from the São Francisco River, Brazil.

During a survey of myxozoan parasites of freshwater fish from the São Francisco River in Minas Gerais State, Brazil, plasmodia of Myxidium ceccarellii n. sp. were found in gallbladders of two out of six specimens (22%) of Leporinus elongatus (Anastomidae). Parasite plasmodia were translucent and greenish, with disporic sporoblasts that develop asynchronously. Mature myxospores were ellipsoidal in frontal and lateral views, with slightly pointed ends. The surfaces of each valve had four to six longitudinal grooves. Spores dimensions were as follows: length 17.7 ± 0.5 μm (17.1-18.1), width 10.4 ± 0.47 μm (9.8-11.3), and thickness 10.1 ± 0.27 μm (9.6-10.4). Two polar capsules, one at either end of the spore, had the length of 6.3 ± 0.5 μm (5.7-7.0) and width of 6.4 ± 0.44 μm (5.7-6.9), with four to five polar filament turns. Some aberrant spores had one or three polar capsules. Partial sequencing of M. ceccarellii n. sp. small subunit ribosomal RNA (ssrRNA) gene resulted in 1,845 bp. This is the first molecular study of a Myxidium species that parasitizes a South American freshwater fish. Phylogenetic reconstruction using ssrRNA gene sequences showed that M. ceccarellii n. sp. was positioned basally in a recognized clade of myxozoans that infect the biliary systems of nonfish vertebrates.

Thelohanellus testudineus n. sp. (Myxosporea: Bivalvulida) infecting the skin of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) in China

A Thelohanellus species was encountered during a survey on Thelohanellus diversity of Carassius auratus gibelio (Bloch) in China. The infection is characterized by the presence of large cysts of 1.4-3.2 cm in diameter in the skin of host. Mature spores were ampullaceous in frontal view and testudinate in lateral view, measuring 19.7 ± 0.7 (18.6-20.8) μm long, 7.6 ± 0.4 (6.6-8.4) μm wide and 7.3 ± 0.5 (6.6-8.8) μm thick. The single polar capsule was elongated pyriform, with 11.1 ± 0.5 (10.0-11.9) μm long and 5.3 ± 0.3 (4.3-5.8) μm wide. Polar filaments coiled with 7-8 turns. Scanning electron microscopy revealed a smooth spore surface with flat side and convex side. The sutural line was straight or 'S' like, running near the middle of the valves. Histologically, the large cysts consisting of numerous small plasmodia developed in the dermis of the skin. The BLAST search indicated that the newly obtained ssrRNA gene sequences did not match any available sequences in GenBank and phylogenetic analysis placed it in the Thelohanellus clade. Based on morphology and molecular differences with reported Thelohanellus spp., this parasite was described as a new species of genus Thelohanellus.

Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene

Coccidian parasites in the genus Sarcocystis have a two-host life cycle, and have traditionally been identified on the basis of morphological features of the sarcocyst stage in their intermediate hosts. Additional molecular species identification, delimitation and phylogeny of Sarcocystis spp. have been based mainly on the nuclear ssrRNA gene. This gene is well suited for discrimination between more distant species but less so for closely related species. The objective of this study was therefore to establish the mitochondrial cytochrome c oxidase subunit I gene (cox1) as a novel genetic marker for Sarcocystis spp. and assess its utility for species identification and delimitation. New primers were developed and 1,020–1,095bp long cox1 sequences were obtained from 155 isolates of 22 Sarcocystis spp. from cattle, sheep, red deer, reindeer, roe deer and moose, and used for phylogenetic reconstructions. For 18 species, the intraspecific and interspecific sequence identities were 98.5–100% and 58–92%, respectively. The four other species had previously been regarded as two species (Sarcocystis rangiferi, Sarcocystis tarandi), each infecting both reindeer and red deer. From cox1 data, each of those appeared to be two separate species, with S. rangiferi and S. tarandi being restricted to reindeer. Thus, cox1 sequences seem to perform better than ssrRNA gene sequences for delimitation of closely related species. The 22 species were distributed in three major clades according to their definitive hosts as in phylogenetic trees obtained from the ssrRNA gene. There were only minor differences in the branching order of different taxa between the trees obtained from either gene. This study has successfully established cox1 as a novel genetic marker for future research on Sarcocystis spp. It has also provided the first published molecular identification of Sarcocystis gigantea and Sarcocystis tenella in Norwegian sheep, and of Sarcocystis hirsuta and Sarcocystis sinensis in Argentinean cattle.

Supplemental Description ofMyxobolus squamalis(Myxozoa)

Myxobolus squamalis is a myxozoan skin parasite first reported from rainbow trout (Oncorhynchus mykiss). Identification of the parasite based on myxospore morphology is unreliable because M. squamalis is similar to several other myxobolids that share host species and geographic ranges. The only ssrRNA gene sequence available for M. squamalis is from Chinook salmon, Oncorhynchus tschawytscha, but these data are not linked with any information on spore morphology. Here we provide a supplemental description of M. squamalis from its type host, O. mykiss, using myxospore morphology, morphometry, and ssrRNA gene sequence data. Our ssrRNA sequence data were only 78% similar to the GenBank M. squamalis sequence from Chinook salmon, which raises the possibility of misidentification. We suggest that Chinook salmon are not a host of the parasite, as no infections were found in current stocks or in >30 yr of historical data from hatchery fish held in waters that harbored M. squamalis in rainbow trout, and we could find no well-identified report of M. squamalis from Chinook salmon. Our complementary morphological and molecular data sets will facilitate unambiguous future identification of M. squamalis.

Plastic parasites: Extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia

In this paper, we combine field observations of highly statistically significant co-occurrence with histopathological, ultrastructural and molecular phylogenetic analyses, to provide evidence for extreme morphological plasticity in a microsporidium parasite infecting the musculature of marine crabs. The parasite appears to alternate between lineages that culminate in production of either bizarre needle-like spores in the peripheral sarcoplasm of heart and skeletal muscle fibres (reminiscent of Nadelspora canceri infecting Cancer magister) or alternatively, Ameson-like spores with pronounced surface projections, in the skeletal muscles (as for Ameson pulvis, previously described infecting Carcinus maenas). Both lineages occur in direct contact with the cytoplasm of host muscle cells and can exist simultaneously within the same cell. Pathological data appears to reveal a remarkable shift in morphology during pathogenic remodelling of host tissues. Sequence analysis of multiple clones derived from amplification of the ssrRNA gene from infected regions of the heart and skeletal muscles appear to confirm the genetic identity of the two lineages. Furthermore, derived ssrRNA gene sequences are more similar (>99%) to N. canceri than to the coparasite Ameson michaelis infecting Callinectes sapidus (93%). Although molecular phylogenetic data support transfer of A. pulvis into the genus Nadelspora, the expansion in the generic description required to include such widely divergent characteristics is so significant as to be unfeasible within the current taxonomic framework of the phylum Microsporidia. At present, it is preferable to propose that the parasite infecting C. maenas forms a clade with other morphologically diverse but phylogenetically and ecologically similar muscle-infecting microsporidians from marine crustacean hosts. Given the strong evidence for significant plasticity in morphology amongst members of the phylum Microsporidia, novel approaches to phylogeny, based predominantly upon the informed use of molecular sequence data, are now deemed a necessity.

Taxonomy, morphology and molecular systematics of a new oligotrich ciliate, Williophrya maedai gen. nov., sp. nov., with redescriptions of Strombidium basimorphum and Pseudotontonia simplicidens (Protozoa, Ciliophora, Oligotrichia)

The morphology and small subunit rRNA (SSrRNA) gene sequence of a marine oligotrich ciliate, Williophrya maedai gen. nov., sp. nov., are reported. The new genus Williophrya is characterized by the adoral zone with no differentiation of membranelles, and the reduced somatic ciliature which comprises a bipartite girdle kinety only. In addition, the in vivo morphologies of two other oligotrichs, namely Strombidium basimorphum Martin & Montagnes, 1993 and Pseudotontonia simplicidens (Lynn & Gilron, 1993) Agatha, 2004, are reported for the first time based on Chinese populations. Improved diagnoses of both species are supplied. The phylogenetic position of Williophrya maedai is investigated based on SSrRNA gene sequence data. These show that: (1) Williophrya maedai is most closely related to Strombidium purpureum and S. apolatum; (2) Williophrya is assigned to the family Strombidiidae although it has some unique morphological features regarding its oral and somatic ciliatures.

Insights into the phylogeny of sporadotrichid ciliates (Protozoa, Ciliophora: Hypotricha) based on genealogical analyses of multiple molecular markers

The sporadotrichid ciliates are an especially diverse group. A number of investigators have studied the morphological, morphogenetic, and molecular relationships among members of this group. Despite this, a consistent classification is still lacking and several important questions about the phylogenetic relationships within this group remain unsolved. To improve our understanding of these relationships, we constructed phylogenetic trees using the nucleotide sequences of the small-subunit rRNA (SSrRNA) gene and amino acid sequences of actin I and α-tubulin. Analyses of SSrRNA gene sequences indicated that: 1) the Sporadotrichida sensu Lynn (2008) and the Oxytrichidae are polyphyletic; 2) the Uroleptus species, which are classified to urostylids, formed a sister group with the oxytrichids; 3) Halteria grandinella, which is grouped morphologically with oligotrich species, clustered within the oxytrichids. These results are congruent with previous studies based on SSrRNA gene sequences. However, the amino acid sequences of actin I and α-tubulin yielded different topologies. The main results are: 1) in all phylogenetic trees, the genus Oxytricha was paraphyletic; 2) Uroleptus was sister to a subset of Urostyla and Holosticha, albeit with low supporting values; 3) Halteria grandinella was separated distantly from the Oxytrichidae in trees inferred from actin I amino acid sequences but clustered with oligotrichids in the α-tubulin analysis. The inconsistency among the trees inferred from these different molecular markers may be caused by rapidly accumulated genetic characterizations of ciliates. Further studies with additional molecular markers and sampling of more taxa are expected to better address the relationships among sporadotrichids.

Comparison of microscopical examination and semi-nested multiplex polymerase chain reaction in diagnosis of Plasmodium Falciparm and P. vivax

We compared light microscopy examination and a semi-nested multiplex PCR (SnM-PCR) assay in endemic areas of the Islamic Republic of Iran. A total of 68 individuals with malaria-positive and suspected malaria symptoms were included in the study. Giemsa-stained thick blood films were examined under a light microscope for malaria parasites in 100 and 200 fields. DNA was extracted from blood samples and SnM-PCR based on the amplification of the small sub-unit ribosomal RNA (ssrRNA) gene sequences was applied. Microscopical examination showed that 48.5% (33.8% P. vivax and 14.7% P.falciparum) and 50% (35.3% P. vivax and 14.7% P. falciparum) of the samples were positive in 100 and 200 fields respectively. SnM-PCR showed the same results as the 200 field microscopy.

Re‐Evaluation on the Diversity of the Polyphyletic Genus Metaurostylopsis (Ciliophora, Hypotricha): Ontogenetic, Morphologic, and Molecular Data Suggest the Establishment of a New Genus Apourostylopsis n. g.

The urostylid genus Metaurostylopsis Song et al., 2001 was considered to be a well-outlined taxon. Nevertheless, recent evidence, including morphological, ontogenetic, and molecular information, have consistently revealed conflicts among congeners, regarding their systematic relationships, ciliature patterns, and origins of ciliary organelles. In the present work, the morphogenetic and morphogenetic features were re-checked and compared, and the phylogeny of nominal species was analysed based on information inferred from the small subunit ribosomal RNA (SS rRNA) gene sequence. In addition, the binary divisional process in a new isolate of Metaurostylopsis struederkypkeae Shao et al., 2008 is described. All results obtained reveal that the genus is a polyphyletic assemblage whose nominal congeners fall into three clades within the core Urostylida, based on SS rRNA gene sequences. These three clades not match the groups inferred from morphological/morphogenetical evidences. Some conflicting data from molecular and ontogenetic studies also indicate that single-gene information might not be consistently reliable in detecting the phylogenetic relationships among closely related groups and comprehensive multi-gene analyses are necessary to give a more exact evaluation for this divergent assemblage. According to our new understandings, five forms are confirmed to be true Metaurostylopsis. The morphotype Metaurostylopsis sinica Shao et al., 2008 should be excluded from the genus and represents a distinct type, and, thus, a new genus Apourostylopsis n. g. with it as the type specie, i.e. Apourostylopsis sinica (Shao et al., 2008) n. comb.

On four closely related hypotrichous ciliates (Protozoa, Ciliophora, Spirotrichea): molecular characters, interspecific relationships and phylogeny defined with multigene sequence information

In order to clarify the phylogeny and relationships of the most confused hypotrichous ciliates, Holosticha-complex, four closely related holostichids (five populations), Holosticha bradburyae, H. diademata, Anteholosticha sp., and A. manca, were compared and analyzed using ITS2 secondary structures, ITS1-5.8S-ITS2 region and SSrRNA gene sequences. The ITS1-5.8S-ITS2 region sequences of these four species were first sequenced, and they shared sequence identities ranging from 68.0% to 90.1%, while two populations of Anteholosticha sp. differed in three nucleotides (sequence identity 99.8%). There were several minor differences among ITS2 secondary structures of these species, while two populations of Anteholosticha sp. had the identical secondary structure. Phylogenetic trees inferred from the ITS1-5.8S-ITS2 region sequences of stichotrichs using multiple algorithms (Neighbor-Joining, Maximum Parsimony and Bayesian) revealed similar topologies. The results show that: (1) Holosticha bradburyae and H. diademata firmly clustered together with strong bootstrap supports, forming a sister clade with Anteholosticha sp., (2) Anteholosticha appeared to be a paraphyletic assemblage, in which the morphotype A. manca was more closely related to Diaxonella trimarginata than to its congener Anteholosticha sp. Phylogenetic analyses based on the SSrRNA gene and the combined sequences of SSrRNA gene and ITS1-5.8S-ITS2 region revealed the similar relationships between Holosticha and Anteholosticha, nevertheless their positions within the subclass Stichotrichia differed from each other inferred from different genes.

Phylogeny of subclass Scuticociliatia (Protozoa, Ciliophora) using combined data inferred from genetic, morphological, and morphogenetic evidence

Gene sequence-based genealogies of scuticociliates are different from those produced by morphological analyses. For this reason, 11 representative scuticociliates and two ambiguously related genera were chosen to test the ability of combined phylogenetic analyses using both gene sequences and morphological/morphogenetic characteristics. Analyses of both the SSrRNA gene sequences and the combined datasets revealed a consistent branching pattern. While the terminal branches and the order level relationships were generally well resolved, the family level relationships remain unresolved. However, two other trees based on ITS1-5.8S-ITS2 region sequences and morphological/morphogenetic characters showed limited information, due to a lack of informative sites in these two datasets. Our data suggest, however, that the combined analysis of morphological/morphogenetic characters and gene sequences did produce some changes to the phylogenetic estimates of this group.

Molecular phylogeny of oligotrich genera Omegastrombidium and Novistrombidium (Protozoa, Ciliophora) for the systematical relationships within Family Strombidiidae

The phylogeny of the oligotrich ciliates is currently a hot debate despite the availability of both morphological and molecular data. In the present paper, further small subunit rRNA (SS rRNA) genes were analyzed from the Genera Omegastrombidium and Novistrombidium, as well as from Strombidium, and combined with three new SS rRNA sequences from Strombidium basimorphum, S. sulcatum population QD-1, and Novistrombidium testaceum population GD. The phylogenetic positions of these organisms were inferred using Bayesian inference, Maximum Likelihood, and Maximum Parsimony methods. The main results are: (1) the SS rRNA gene sequence analyses match the recent findings about the molecular evolution of oligotrichs, indicating that the family Strombidiidae is paraphyletic; (2) the Genus Omegastrombidium is separated from the Genus Strombidium, as shown in recent cladistic analyses; (3) morphospecies in Genus Novistrombidium, based on similarity of somatic ciliature, are separated from each other in all topological trees, indicating that this genus could be a paraphyletic group; (4) the molecular data indicate a possibility of paraphyly for the genus Strombidium; and (5) the similarities of the SS rRNA gene of specimens identified as S. sulcatum and S. inclinatum are 99.8%-100%. However, present knowledge on the oligotrichs sensu stricto is still insufficient and further studies based on both molecular and other technologies are required.

Morphological and molecular characterization of Euplotes encysticus (Protozoa: Ciliophora: Euplotida)

The ciliate Euplotes encysticus Yonezawa, 1985 was insufficiently described so far regarding its living morphology, infraciliature and silverline system. Recently, a China population was isolated from brackish water (salinity 6‰) near a coastal sandy beach (114°32′E 22°43′N) of the Daya Bay, South China Sea and subsequent investigations were performed on it in detail. Based on data available, the morphology of this species was summarized and a new diagnosis is given which is characterized by: medium-sized body with consistently 9 frontoventral, 2 caudal and 2 left marginal cirri; 30–40 adoral membranelles; 7 dorsal kineties; dorsal silverline system consisting of complex type with irregular network; capable of encystment. Both the morphology and SSr RNA gene sequence show, with high support, a close relationship of E. encysticus and Euplotes muscorum.

Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence

Spores of the myxozoan parasite Myxobolus turpisrotundus Zhang 2009 were observed for the first time bearing caudal appendages. Most spores had the typical Myxobolus spp. morphology, but approximately 10% of spores possessed a spore body that was slightly elongated with a short tail projecting from the spore valve. In other spores, the tail was much more clearly visible and elongate. The spore body of these unusual spores is consistent in morphology and dimension to the normal spores of M. turpisrotundus. Both spore types were found within individual cysts, and the small subunit ribosomal RNA (ssrRNA) gene sequence from parasite cysts of this type was nearly identical to the previously published sequence of M. turpisrotundus from allogynogenetic gibel carp Carassius auratus gibelio (Bloch). The phenomenon of Myxobolus spores with caudal appendages provides additional evidence that the use of this character to separate Myxobolus and Henneguya into distinct genera is not reflective of an evolutionarily accurate classification scheme. Phylogenetic analysis of ssrDNA sequence from Myxobolus and Henneguya species showed clustering of species in some locations of the tree, but ultimately these genera are intermixed. The use of a single character to delineate species in the two most species-rich myxozoan genera has been consistently challenged where DNA analyses are used. The present finding of a single species bearing both Myxobolus-type and Henneguya-type spores emphasizes the inadequacy of this classification scheme, and highlights the need for careful consideration of these variable characteristics when describing myxozoan species.

Molecular phylogeny and species separation of five morphologically similar Holosticha-complex ciliates (Protozoa, Ciliophora) using ARDRA riboprinting and multigene sequence data

To separate and redefine the ambiguous Holosticha-complex, a confusing group of hypotrichous ciliates, six strains belonging to five morphospecies of three genera, Holosticha heterofoissneri, Anteholosticha sp. pop1, Anteholosticha sp. pop2, A. manca, A. gracilis and Nothoholosticha fasciola, were analyzed using 12 restriction enzymes on the basis of amplified ribosomal DNA restriction analysis. Nine of the 12 enzymes could digest the DNA products, four (Hinf I, Hind III, Msp I, Taq I) yielded species-specific restriction patterns, and Hind III and Taq I produced different patterns for two Anteholosticha sp. populations. Distinctly different restriction digestion haplotypes and similarity indices can be used to separate the species. The secondary structures of the five species were predicted based on the ITS2 transcripts and there were several minor differences among species, while two Anteholosticha sp. populations were identical. In addition, phylogenies based on the SSrRNA gene sequences were reconstructed using multiple algorithms, which grouped them generally into four clades, and exhibited that the genus Anteholosticha should be a convergent assemblage. The fact that Holosticha species clustered with the oligotrichs and choreotrichs, though with very low support values, indicated that the topology may be very divergent and unreliable when the number of sequence data used in the analyses is too low.

Reevaluation of cortical developmental patterns in Euplotes (s. l.), including a morphogenetic redescription of E. charon (Protozoa, Ciliophora, Euplotida)

We documented the pattern of cell development in Euplotes charon. The ontogenesis of this species was similar to many of its congeners, except for the formation of the caudal cirri. In E. Charon, a caudal cirrus is formed posterior to each of the rightmost two or three dorsal kinety anlage in the proter, and the second rightmost dorsal kinety in the opisthe. In addition, two caudal cirri are formed posterior to the rightmost dorsal kinety in the opisthe. This pattern of development represents a completely new type. Based on our evaluation, and in comparison with previous studies, we also conclude that the pattern of cell development is variable among species in the Euplotes genera. The variation is particularly evident during the formation of frontoventral and caudal cirri. Based on the segmentation pattern of frontal-midventral transverse cirral anlagen, cirri reduction, and migration of frontoventral cirri, we identified five types: the affinis-type, the eurystomus-type, the charon-type, the raikovi-type and orientalis-type. Euplotes (s. l.) can also be divided into three types based on the formation of caudal cirri: focardii-type, vannus-type and charon-type. Indeed, we conclude that the number (one or two) of marginal cirri should be given as much consideration as the genetic separation. Given this, we reassessed the validity of using genetic separation to classify the group. Generally, the morphogenetic data disagreed with the molecular data (SSrRNA gene sequences). Given these discrepancies, it is too early to draw conclusions on the systematic arrangement of this species-rich taxon.

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.