Phylogeny and Genetic/Morphological Variation of Strombidinopsis minima-like Species (Ciliophora: Choreotrichia).

Six isolates of mineral-enveloped Strombidinopsis minima-like species were collected from the coastal waters across several regions in Korea. Morphological observations and molecular analyses were performed. The ribosomal DNA sequences (including small subunit ribosomal DNA, internal transcriber spacer 1-5.8S ribosomal DNA-internal transcriber spacer 2; and part of large subunit ribosomal DNA) of these six isolates were compared. Their morphological characteristics were also compared with those of S. minima populations reported. The marked genetic differences (with a similarity range of 96.85-98.48%) in SSU rDNA among these S. minima-like entities suggest the existence of multiple species. This finding is also supported by morphological variations detected in this study and reported in the literature (e.g. 15-32 collar membranelles in different populations). In addition, S. minima-like species are clustered with S. batos and S. sinicum, and therefore, our SSU rDNA results support previous results suggesting that the genus Strombidinopsis is not monophyletic in origin. Further collection of morphological and molecular data may facilitate the determination of a new genus carrying mineral-enveloped Strombidinopsis species.

Morphological and ribosomal DNA-based characterization of six Antarctic ciliate morphospecies from the Amundsen Sea with phylogenetic analyses.

We characterized six tintinnid ciliates from Antarctic waters using molecular markers and morphological traits: Amphorellopsis quinquealata, Codonellopsis gaussi, Cymatocylis convallaria, Cy. calyciformis, Cy. drygalskii, and Laackmanniella prolongata. The 100% similarity in SSU-ITS1-5.8S rDNA-ITS2-partial LSU rDNA sequences among Cy. convallaria, Cy. calyciformis, and Cy. drygalskii is supportive of synonymy. Codonellopsis gaussi and L. prolongata also showed high levels of similarity in SSU rDNA (99.83%) and the D2 domain of LSU rDNA (95.77%), suggesting that they are closely related. Phylogenetic analysis placed Cymatocylis in the Rhabdonellidae, Amphorellopsis in the Tintinnidae and L. prolongata/Co. gaussi within the Dictyocystidae.

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

Facile control of C2H5OH sensing characteristics by decorating discrete Ag nanoclusters on SnO2 nanowire networks.

The effect of Ag decoration on the gas sensing characteristics of SnO(2) nanowire (NW) networks was investigated. The Ag layers with thicknesses of 5-50 nm were uniformly coated on the surface of SnO(2) NWs via e-beam evaporation, which were converted into isolated or continuous configurations of Ag islands by heat treatment at 450 °C for 2 h. The SnO(2) NWs decorated by isolated Ag nano-islands displayed a 3.7-fold enhancement in gas response to 100 ppm C(2)H(5)OH at 450 °C compared to pristine SnO(2) NWs. In contrast, as the Ag decoration layers became continuous, the response to C(2)H(5)OH decreased significantly. The enhancement and deterioration of the C(2)H(5)OH sensing characteristics by the introduction of the Ag decoration layer were strongly governed by the morphological configurations of the Ag catalysts on SnO(2) NWs and their sensitization mechanism.

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with phylogenetic analyses based on small subunit rRNA gene sequences.

The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.

New observations on the choreotrich ciliate Strombidinopsis acuminata Fauré-Fremiet 1924, and comparison with Strombidinopsis jeokjo Jeong et al., 2004.

A choreotrich ciliate collected in waters off Geoje Island, on the eastern coast of the South Sea of the southern coastal water of Korea was morphologically and biometrically investigated through observations of permanent preparations after protargol impregnations. Morphological data and ciliary patterns indicated that the ciliate should be identified as Strombidinopsis acuminata based on the number of somatic kineties. The vertical distribution of the population abundance was also examined in relation to environmental conditions: higher abundances occurred in surface water with higher temperature (22-23 degrees C), lower salinity (32 psu), and higher concentration (ca 10 microg/L) of chlorophyll a. The 18S rRNA gene of specimens from clonal cultures isolated from natural assemblages was sequenced. While this isolate of S. acuminata is closer to Strombidinopsis jeokjo (99.6-99.8%) than other isolates of S. acuminata (<99.0%), monophyly of S. acuminata was not rejected by the AU test. We discuss the discordance between morphological and molecular data for Strombidinopsis. In order to confirm the species level of the Strombidinopsis specimens, additional information is required based on genealogical analyses of multiple loci.

Study of harmful algal blooms in a eutrophic pond, Bangladesh.

The purpose of this research was to analyze the underlying mechanisms and contributing factors related to the seasonal dynamic of harmful algal blooms in a shallow eutrophic pond, Bangladesh during September 2005-July 2006. Two conspicuous events were noted simultaneously throughout the study period: high concentration of phosphate-phosphorus (>3.03; SD 1.29 mg l(-1)) and permanent cyanobacterial blooms {>3,981.88×10(3) cells l(-1) (SD 508.73)}. Cyanobacterial blooms were characterized by three abundance phases, each of which was associated with different ecological processes. High nitrate-nitrogen (>2.35; SD 0.83 mg l(-1)), for example, was associated with high cyanobacterial abundance, while low nitrate-nitrogen (0.36; SD 0.2 mg l(-1)) was recorded during moderate abundance phase. Extremely low NO3-N/PO4-P ratio (>3.55, SD 2.31) was recorded, and all blooming taxa were negatively correlated with this ratio. Cyanobacterial blooms were positively correlated with temperature (r=0.345) and pH (0.833; p=0.05) and negatively correlated with transparency (r=-0.956; p=0.01). Although Anabaena showed similar relationship with water quality parameters as cyanobacteria, the co-dominant Microcystis exhibited negative relationship with temperature (r=-0.386) and nitrate-nitrogen (r=-0.172). This was attributed to excessive growth of Anabaena that suppressed Microcystis's growth. Planktothrix was the third most dominant taxa, while Euglena was regarded as opportunistic.

Community structure and seasonal dynamics of planktonic ciliates along salinity gradients.

The ciliate community structure and seasonal dynamics in a solar saltern of the Yellow Sea were studied based on 4 sampling dates and 8 stations with salinities from 27.7 per thousand to 311.0 per thousand. The effects of the type and concentration of the fixative used (Lugol's and Bouin's) were tested at the first sampling date. Fixative type and fixative concentration had significant effects on ciliate abundance and biovolume, with 1% Lugol's giving the best results. A detailed investigation using live observations and protargol staining techniques revealed a total of 98 morphospecies from 8 sampling stations. There was obvious seasonal variation in species composition at most of the stations, but this tended to be less distinct with increasing salinity, as the dominant ciliate group shifted from oligotrichs to heterotrichs. Ciliate abundance varied from 4.40 x 10(1) to 2.11 x 10(5) cellsl(-1) and biomass ranged between 2.39 and 9.87 x 10(3)microg Cl(-1) (at a salinity of 147.6 per thousand). Both abundance and biomass decreased abruptly when salinity exceeded 100-150 per thousand. Statistical analyses suggested that the dynamics of ciliate abundance and biomass were regulated by both salinity and by season, but those of diversity and species richness were mainly controlled by salinity and both significantly decreased with increasing salinity.

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.

Morphology and molecular phylogeny of Trimyema koreanum n. sp., a ciliate from the hypersaline water of a solar saltern.

A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293 per thousand) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 x 13 microm in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).

Kiitricha minuta n. sp., a peculiar hypotrichous ciliate (Ciliophora, Spirotrichea) from the Yellow Sea.

The morphology and infraciliature of a new ciliate, Kiitricha minuta n. sp., isolated from the Yellow Sea, were investigated using live observation and protargol impregnation. Kiitricha minuta represents a third member of the rarely known order Kiitrichida. It is unique in the subclass Hypotrichia in having many rows of small uniform cirri along the right side of the body and the dorsal kineties composed of dikinetids, most of which bear two cilia. Kiitricha minuta n. sp. is ovoid and measures about 60 x 45 microm in vivo. It has a huge buccal cavity occupying about 80% of the body length, numerous body extrusomes, one macronucleus and two micronuclei, 27-27 adoral membranelles, 9-12 frontoventral cirral rows, a submarginal row of 7-9 cirri, 6 or 7 transverse cirri, and roughly 7-9 dorsal kineties. This new species differs distinctly from its only congener Kiitricha marina by its smaller size (60 microm vs. 80-150 microm), the presence of body extrusomes (vs. absent), the different macronuclear pattern (one vs. two nodules), and the lower number of frontoventral cirral rows (9-12 vs. 21-26), which terminate at the anterior two-thirds of body (vs. extend to the posterior). The new term "submarginal cirral row" is introduced to distinguish from the marginal cirral row in typical hypotrichs sensu lato. Based on our new observations and the literature, an improved diagnosis for the genus Kiitricha is provided and its phylogenetic importance is discussed.

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.

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 proter's oral primordium; the primary primordia of the dorsal kinety; and marginal primordia commence de novo without a definite contribution from the old structure.

Morphological descriptions of new and little-known benthic ciliates from Ganghwa tidal flat, Korea.

Ciliates are highly diverse in the benthos where there are rare species to be unrevealed and described. By isolating species during successive and diversified cultivations, we discovered several new and interesting taxa from the top layer sediment of a muddy site in the Ganghwa tidal flat. These include three new species Spirodysteria ganghwaensis n. sp., Uronemella parafilificum n. sp., Zosterodasys minuta n. sp., and one poorly known form Loxophyllum chaetonotumBorror 1965. The morphology of live cells and infraciliature of these four species are described based on living observations, protargol impregnations, and morphometrics. Diagnoses and improved definitions are also provided. The newly established genus Spirodysteria n. g. differs from Dysteria mainly in its spirally twisted body shape. Spirodysteria kahli (Tucolesco 1962) n. comb. (formerly Dysteria kahliTucolesco 1962) has been included in this new genus.

Tunicothrix rostrata n. g., n. sp., a new urostylid ciliate (Ciliophora, Stichotrichia) from the Yellow Sea.

The morphology and infraciliature of a new ciliate, Tunicothrix rostrata n. g., n. sp., isolated from the Yellow Sea, are investigated using live observation and protargol impregnation. Tunicothrix rostrata measures about 160 x 40 mum in vivo, and has a frontal beak-like protrusion, a conspicuous cortical alveolar layer, two right marginal rows, and usually three distinct midventral pairs. The discovery of T. rostrata enables us to reconsider the classification of Erniella wilberti, a curious ciliate with obscure midventral pairs. Both species are highly similar in overall appearance and nuclear and ciliary pattern. Thus, they are united in a new genus of the family Urostylidae, Tunicothrix, and E. wilberti is transferred to Tunicothrix: Tunicothrix wilbertiLin and Song, 2004 n. comb. By contrast, Erniella filiformis, type species of Erniella, has several ventral rows and does not belong to the urostylids. Tunicothrix rostrata is easily distinguished from T. wilberti by its beak-like anterior protrusion and by the distinctly elongated right marginal row 2, which curves anteriorly on the dorsal side of the cells. Tunicothrix is closely related to Parabirojimia, differing by the invariably two (vs. five-eight) right marginal rows and the conspicuous (vs. ordinary) alveolar layer, a unique feature in urostylid ciliates.

Holosticha hamulata n. sp. and Holosticha heterofoissneri Hu and Song, 2001, two urostylid ciliates (protozoa, ciliophora) from intertidal sediments of the yellow sea.

Two marine urostylid ciliates, Holosticha hamulata n. sp. and Holosticha heterofoissneri Hu and Song, 2001, were investigated using live observation and protargol impregnation. Both species were isolated from Korean intertidal sediments of the Yellow Sea. Holosticha hamulata measures about 150 x 25 mum in vivo, and is characterized by a tripartite body shape with a narrow head, an inflated trunk, and a tail that distally projects ventrally forming a hook-like structure. It is the characteristic body shape that distinguishes H. hamulata distinctly from congeners. Holosticha hamulata differs from H. heterofoissneri, possibly the nearest relative, also by the location of the contractile vacuole (ahead of mid-body versus near posterior body third) and the configuration of the macronucleus (on average, 33 scattered nodules assuming a Y-shape versus 17 nodules that may form a U shape). The average number of the macronuclear nodules is a pronounced feature showing great consistency in populations of each species. However, their arrangement is variable in H. heterofoissneri where the nodules are basically scattered or connected by fine fibers forming an elongate U-shape. The location of the contractile vacuole as a taxonomic feature is discussed and a dichotomous key to the species of Holosticha sensu stricto is provided.

Morphology and infraciliature of three species of Metaurostylopsis (Ciliophora, Stichotrichia): M. songi n. sp., M. salina n. sp., and M. marina (Kahl 1932) from sediments, saline ponds, and coastal waters.

Two new urostylid ciliates, Metaurostylopsis songi n. sp. and Metaurostylopsis salina n. sp. and Metaurostylopsis marina (Kahl 1932) are investigated using live observation and protargol impregnation. These species were isolated in Korea from intertidal sediments, saline ponds, and coastal waters. Metaurostylopsis songi is in vivo about 120 microm x 25 microm, has a slenderly ellipsoidal body, colorless cortical granules in rows on ventral and dorsal body sides, about 54 macronuclear nodules, 28-47 adoral membranelles, five frontal, two or three frontoterminal and six or seven transverse cirri, and 9-12 midventral cirral pairs followed posteriorly by 1-3 single cirri. In vivo M. salina is about 60 microm x 25 microm, has a pyriform body, colorless cortical granules irregularly arranged, about 45 macronuclear nodules, 18-23 adoral membranelles, three frontal, three to five frontoterminal and two to five transverse cirri, and four or five midventral cirral pairs followed posteriorly by five to seven single cirri. Both species have three marginal cirral rows on each body side and 3 long dorsal kineties. The Korean specimens of M. marina match the Chinese population in all main features. Metaurostylopsis songi differs from M. marina by the more slender body, the number of frontal cirri (invariably five vs. four), and the arrangement of cortical granules (in rows on dorsal and ventral cortex vs. only along dorsal kineties and anterior body margin). Metaurostylopsis salina differs from its congeners by the distinctly smaller size, the pyriform body shape, the scattered cortical granules (vs. in rows), and number of frontal cirri. It differs from M. marina also by the number of midventral cirral pairs (four or five vs. seven to 11).

Synergistic effect of sophorolipid and loess combination in harmful algal blooms mitigation.

The inhibition effect of sophorolipid and removal efficiency of loess on Cochlodinium polykrikoides and Alexandrium tamarense was investigated separately in the laboratory. Based on this, the combination of sophorolipid and loess for harmful algal bloom mitigation was proposed. Algal sedimentation tests in the laboratory and in the field revealed that the combination of sophorolipid and loess showed synergistic effects both on the removal efficiencies and on the mitigation cost. The concentration of 1 g/l loess and 5 mg/l sophorolipid was determined as the optimum ratio for C. polykrikoides mitigation. In the field test, the effective concentration of loess and sophorolipid in the combination group was reduced to 10% and 25%, respectively, compared to the non-combination group, and the cost decreased more than 60%. The combination of loess and sophorolipid was considered as a promising novel method in harmful algal bloom mitigation.

Screening of surfactants for harmful algal blooms mitigation.

Screening experiments were conducted in order to find promising synthetic surfactants for harmful algal blooms (HABs) mitigation. The chemically synthesized surfactant cocamidopropyl betaine (CAPB) showed characteristics of relatively high inhibition efficiency, high biodegradability and low cost. The motility inhibition ratios of 10 mg/L CAPB on Cochlodinium polykrikoides and Alexandrium tamarense were about 60% after 5 min. The biodegradation test indicated that the half-life of CAPB in seawater was shorter than one day and 90% was biodegraded after five days under the initial concentration of 100 mg/L at 25 degrees C. Further cell lysis experiments revealed the selective lysis effect of CAPB on different HAB organisms. More than 90% of C. polykrikoides lysed at the concentration of 10 mg/L CAPB after 24 h and at 15 mg/L CAPB after 4 h, whereas the lysis effect of CAPB on A. tamarense was slight, no more than 10% after 2 h interaction with 50 mg/L CAPB. This research provided preliminary data for CAPB as a candidate in harmful algal blooms mitigation and pointed out unresolved problems for its practical application in the meantime.

Morphology and infraciliature of a new marine ciliate Euplotidium smalli n. sp. with description of a new genus, Paraeuplotidium n. g. (Ciliophora, Euplotida).

The morphology and infraciliature of a new hypotrichous ciliate, Euplotidium smalli n. sp., isolated from eutrophic coastal water in Korea, were observed in living cells and investigated using the protargol impregnation technique. This new ciliate bears 13-14 frontoventral cirri, 7 transverse cirri, and 5-6 dorsal kineties. Neither left marginal cirrus nor caudal cirrus is present. The new species differs from the related species, Euplotidium agitatum Noland, 1937 in the different number of frontoventral and transverse cirri and different body shape. With the exception of Euplotidium agitatum, the known species of the genus Euplotidium Noland, 1937 with the presence of left marginal cirrus are assigned to a new genus, Paraeuplotidium n. g. Diagnosis of Paraeuplotidium is: Gastrocirrhidae with funnel-shaped buccal cavity; with frontoventral and transverse cirri; left marginal cirrus present. Paraeuplotidium itoi (Ito, 1958) n. comb. is designated here the type species. Four additional species are included: Paraeuplotidium psammophilus (Vacelet, 1961) n. comb., Paraeuplotidium arenarium (Magagnini & Nobili, 1964) n. comb., Paraeuplotidium helgae (Hartwig, 1980) n. comb., and Paraeuplotidium prosaltans (Tuffrau, 1985) n. comb. An improved generic diagnosis of Euplotidium is suggested based on morphology and infraciliature characters: marine hypotrichs with a funnel-shaped buccal cavity; with frontoventral and transverse cirri; neither left marginal cirrus nor caudal cirri present.

Biomonitoring of coastal pollution status using protozoan communities with a modified PFU method.

Structural and functional parameters of protozoan communities were assessed as indicators of water quality in Korean coastal waters in the summer of 2000. A modified polyurethane foam unit (PFU) method, named the bottled PFU (BPFU) system, was used in order to carry out the bioassessment. Both parameters suggested that biomonitoring using the BPFU system was more effective than the conventional PFU method in offshore areas. The species number collected by the BPFU system generally decreased as pollution intensity increased at three main stations and was greater than that collected using the PFU method (paired t-test, t = 4.83, p < 0.0001). The Margalef diversity index coincided well with the water conditions. The diversity index values calculated from the BPFU system were also significantly higher than those from the PFU method (paired t-test, t = 5.37, p < 0.0001). Furthermore, the functional parameters, i.e. S(eq),G and T90%, correlated with the pollution status and could thus clearly discriminate the different classes of water quality.