Gi-Sik Min

Taxonomic Redescriptions of Two Ciliates, Protogastrostyla pulchra n. g., n. comb. and Hemigastrostyla enigmatica (Ciliophora: Spirotrichea, Stichotrichia), with Phylogenetic Analyses Based on 18S and 28S rRNA Gene Sequences

ABSTRACT. The morphology and infraciliature of two stichotrichid ciliates, Gastrostyla pulchra (Perejaslawzewa 1886) Kahl, 1932 and Hemigastrostyla enigmatica (Dragesco and Dragesco‐Kernéis 1986) Song & Wilbert, 1997 , collected from marine and brackish sediments, were investigated by using living observations and protargol impregnations. Both 18S and 28S rRNA genes of these two species were sequenced. The 18S rDNA show high similarities (98.4%–99.7%) among populations of each species. There is about 94% similarity in 18S rDNA genes between G. pulchra and Gastrostyla steinii, the type species of the genus, which has been confirmed to be an oxytrichid by previous studies. In the phylogenetic trees of 18S, 28S, and combined 18S and 28S rDNA, both G. pulchra and H. enigmatica are consistently placed outside the well‐established oxytrichid clade. Based on our analyses and previous ontogenetic data, we conclude that these two species may represent some lower groups in the subclass Stichotrichia, and that G. pulchra should represent a new genus, Protogastrostyla n. g. This new genus, which is morphologically similar to Gastrostyla, differs in its morphogenesis: the apical part of the old AZM is retained combining with the newly built membranelles that develop from the proters oral primordium; the primary primordia of the dorsal kinety; and marginal primordia commence de novo without a definite contribution from the old structure.

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

BackgroundThe orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.ResultsFor this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomiasiamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.ConclusionThe phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

An approach to analyses of periphytic ciliate colonization for monitoring water quality using a modified artificial substrate in Korean coastal waters

Structural and functional parameters of periphytic ciliate communities were studied for monitoring water quality in Korean coastal waters during April 2007. The PFES (polyurethane foam enveloped slide) system, a modified glass slide method, was used to analyze periphytic ciliate colonization in marine ecosystems. A total of 27 ciliate species were identified using living observation and silver impregnation method with this system. Although the ciliate colonizations had similar species composition, they represented considerable differences in both structural and functional parameters between the PFES system and the conventional slide system. The species diversity, evenness and the colonization rate (G) were distinctly higher, but the time for reaching 90% equilibrium species number (T(90%)) was shorter in the PFES system than those on the naked slides. Results suggest that the PFES system is more effective than the conventional slide method for periphytic ciliate colonization with high species diversity, evenness, and colonization rate in marine ecosystems.

An approach to analyses of periphytic ciliate communities for monitoring water quality using a modified artificial substrate in Korean coastal waters

Structural parameters of periphytic ciliate communities on a modified substrate were studied in Korean coastal waters during the period August-November 2007. In order to reduce the strong disturbances from tidal current and circulation in marine ecosystems, a modified slide method, named the polyurethane foam enveloped slide (PFES) system, was used to host ciliate communities. A total of 37 ciliate species, about half of which belong to the orders Hypotrichida and Cyrtophorida, were identified using living observation and silver impregnation method with this system. The sessile ciliates belonged to the orders Peritrichida and Suctorida, while the motile forms were represented primarily by the species of the orders Hypotrichida, Cyrtophorida and Pleurostomatida. The species diversity and evenness were significantly higher in the PFES system than those on the conventional slides (paired t-test: t = 2.384, 2.415; P < 0.05). Multivariate analysis revealed that the ciliate communities from both sampling systems had similar species composition, but represented significant differences in species distribution and temporal dynamics mainly due to the most dominant peritrich Zoothaminium duplicatum, which overly colonized the conventional slides. Results suggest that the PFES system is more effective than the conventional slide method for periphytic ciliate colonization with high species diversity, evenness and sensitive temporal dynamics mainly due to the reduction of disturbances from tidal current and circulation in marine ecosystems.

Comparative analysis of complete mitochondrial genome sequences confirms independent origins of plant-parasitic nematodes

BackgroundThe nematode infraorder Tylenchomorpha (Class Chromadorea) includes plant parasites that are of agricultural and economic importance, as well as insect-associates and fungal feeding species. Among tylenchomorph plant parasites, members of the superfamily Tylenchoidea, such as root-knot nematodes, have great impact on agriculture. Of the five superfamilies within Tylenchomorpha, one (Aphelenchoidea) includes mainly fungal-feeding species, but also some damaging plant pathogens, including certain Bursaphelenchus spp. The evolutionary relationships of tylenchoid and aphelenchoid nematodes have been disputed based on classical morphological features and molecular data. For example, similarities in the structure of the stomatostylet suggested a common evolutionary origin. In contrast, phylogenetic hypotheses based on nuclear SSU ribosomal DNA sequences have revealed paraphyly of Aphelenchoidea, with, for example, fungal-feeding Aphelenchus spp. within Tylenchomorpha, but Bursaphelenchus and Aphelenchoides spp. more closely related to infraorder Panagrolaimomorpha. We investigated phylogenetic relationships of plant-parasitic tylenchoid and aphelenchoid species in the context of other chromadorean nematodes based on comparative analysis of complete mitochondrial genome data, including two newly sequenced genomes from Bursaphelenchus xylophilus (Aphelenchoidea) and Pratylenchus vulnus (Tylenchoidea).ResultsThe complete mitochondrial genomes of B. xylophilus and P. vulnus are 14,778 bp and 21,656 bp, respectively, and identical to all other chromadorean nematode mtDNAs in that they contain 36 genes (lacking atp8) encoded in the same direction. Their mitochondrial protein-coding genes are biased toward use of amino acids encoded by T-rich codons, resulting in high A+T richness. Phylogenetic analyses of both nucleotide and amino acid sequence datasets using maximum likelihood and Bayesian methods did not support B. xylophilus as most closely related to Tylenchomorpha (Tylenchoidea). Instead, B. xylophilus, was nested within a strongly supported clade consisting of species from infraorders Rhabditomorpha, Panagrolaimomorpha, Diplogasteromorpha, and Ascaridomorpha. The clade containing sampled Tylenchoidea (P. vulnus, H. glycines, and R. similis) was sister to all analyzed chromadoreans. Comparison of gene arrangement data was also consistent with the phylogenetic relationships as inferred from sequence data. Alternative tree topologies depicting a monophyletic grouping of B. xylophilus (Aphelenchoidea) plus Tylenchoidea, Tylenchoidea plus Diplogasteromorpha (Pristionchus pacificus), or B. xylophilus plus Diplogasteromorpha were significantly worse interpretations of the mtDNA data.ConclusionsPhylogenetic trees inferred from nucleotide and amino acid sequences of mtDNA coding genes are in agreement that B. xylophilus (the single representative of Aphelenchoidea) is not closely related to Tylenchoidea, indicating that these two groups of plant parasites do not share an exclusive most recent common ancestor, and that certain morphological similarities between these stylet-bearing nematodes must result from convergent evolution. In addition, the exceptionally large mtDNA genome size of P. vulnus, which is the largest among chromadorean nematode mtDNAs sequenced to date, results from lengthy repeated segments in non-coding regions.

The susceptibility of Anopheles lesteri to infection with Korean strain of Plasmodium vivax.

BackgroundFollowing its recent re-emergence, malaria has gained renewed attention as a serious infectious disease in Korea. Three species of the Hyrcanusgroup, Anopheles lesteri, Anopheles sinensis and Anopheles pullus, have long been suspected malaria vectors. However, opinions about their vector ability are controversial. The present study was designed with the aim of determining the susceptibility of these mosquitoes to a Korean isolate of Plasmodium vivax. Also, An. sinensis is primarily suspected to be vector of malaria in Korea, but in Thailand, the same species is described to have less medical importance. Therefore, comparative susceptibility of Thai and Korean strains of An. sinensis with Thai strain of P. vivax may be helpful to understand whether these geographically different strains exhibit differences in their susceptibility or not.MethodsThe comparative susceptibility of An. lesteri, An. sinensis and An. pullus was studied by feeding laboratory-reared mosquitoes on blood from patients carrying gametocytes from Korea and Thailand.ResultsIn experimental feeding with Korean strain of P. vivax, oocysts developed in An. lesteri, An. sinensis and An. pullus. Salivary gland sporozoites were detected only in An. lesteri and An. sinensis but not in An. pullus. Large differences were found in the number of sporozoites in the salivary glands, with An. lesteri carrying much higher densities, up to 2,105 sporozoites in a single microscope field of 750 × 560 μM, whereas a maximum of 14 sporozoites were found in any individual salivary gland of An. sinensis. Similar results were obtained from a susceptibility test of two different strains of An. sinensis to Thai isolate of P. vivax, and differences in vector susceptibility according to geographical variation were not detected.ConclusionThe high sporozoite rate and sporozoite loads of An. lesteri indicate that this species is highly susceptible to infection with P. vivax. Anopheles sinensis appears to have a markedly reduced ability to develop salivary gland infection, whilst in An. pullus, no sporozoites were found in the salivary glands. Provided that the survival rate of An. lesteri is sufficiently high in the field, it would be a highly competent vector of vivax malaria.

Eurotatorian paraphyly: Revisiting phylogenetic relationships based on the complete mitochondrial genome sequence of Rotaria rotatoria (Bdelloidea: Rotifera: Syndermata)

BackgroundThe Syndermata (Rotifera+Acanthocephala) is one of the best model systems for studying the evolutionary origins and persistence of different life styles because it contains a series of lineage-specific life histories: Monogononta (cyclic parthenogenetic and free-living), Bdelloidea (entirely parthenogenetic and mostly benthic dweller), Seisonidea (exclusively bisexual and epizoic or ectoparasitic), and Acanthocephala (sexual and obligatory endoparasitic). Providing phylogenetic resolution to the question of Eurotatoria (Monogononta and Bdelloidea) monophyly versus paraphyly is a key factor for better understanding the evolution of different life styles, yet this matter is not clearly resolved. In this study, we revisited this issue based on comparative analysis of complete mitochondrial genome information for major groups of the Syndermata.ResultsWe determined the first complete mitochondrial genome sequences (15,319 bp) of a bdelloid rotifer, Rotaria rotatoria. In order to examine the validity of Eurotatoria (Monogononta and Bdelloidea) monophyly/paraphyly, we performed phylogenetic analysis of amino acid sequences for eleven protein-coding genes sampled from a wide variety of bilaterian representatives. The resulting mitochondrial genome trees, inferred using different algorithms, consistently failed to recover Monogononta and Bdelloidea as monophyletic, but instead identified them as a paraphyletic assemblage. Bdelloidea (as represented by R. rotatoria) shares most common ancestry with Acanthocephala (as represented by L. thecatus) rather than with monogonont B. plicatilis, the other representative of Eurotatoria.ConclusionComparisons of inferred amino acid sequence and gene arrangement patterns with those of other metazoan mtDNAs (including those of acanthocephalan L. thecatus and monogonont B. plicatilis) support the hypothesis that Bdelloidea shares most common ancestry with Acanthocephala rather than with Monogononta. From this finding, we suggest that the obligatory asexuality of bdelloideans may have secondarily derived from some other preexisting condition in earlier lineage of rotifers. Providing a more complete assessment of phylogenetic relationships and inferring patterns of evolution of different types of life styles among Syndermata awaits comparisons requiring mitochondrial genome sequencing of Seisonidea.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae).

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Multiplex assay to identify Korean vectors of malaria

Following the recent emergence of malaria in South Korea, vector control has been an important task. For this, vector identification is very important. Earlier, two PCR‐based assays have been described. But, poor species resolution and their ability to include only 4–5 species limit their use. Thus, it has now become important to revise the assay identifying these members. In this study, a new assay based on internal transcribed spacer 2 and 28S of ribosomal DNA has been described. The assay successfully identified all the Korean malaria vector mosquitoes. Therefore, it is an indispensable tool to study ecology, abundance and biology of these species.