Cellular Identification of a Novel Uncultured Marine Stramenopile (MAST-12 Clade) Small-Subunit rRNA Gene Sequence from a Norwegian Estuary by Use of Fluorescence In Situ Hybridization-Scanning Electron Microscopy

Revealing the cellular identity of organisms behind environmental eukaryote rRNA gene sequences is a major objective in microbial diversity research. We sampled an estuarine oxygen-depleted microbial mat in southwestern Norway and retrieved an 18S rRNA gene signature that branches in the MAST-12 clade, an environmental marine stramenopile clade. Detailed phylogenetic analyses revealed that MAST-12 branches among the heterotrophic stramenopiles as a sister of the free-living Bicosoecida and the parasitic genus Blastocystis. Specific sequence signatures confirmed a relationship to these two groups while excluding direct assignment. We designed a specific oligonucleotide probe for the target sequence and detected the corresponding organism in incubation samples using fluorescence in situ hybridization (FISH). Using the combined FISH-scanning electron microscopy approach (T. Stoeck, W. H. Fowle, and S. S. Epstein, Appl. Environ. Microbiol. 69:6856-6863, 2003), we determined the morphotype of the target organism among the very diverse possible morphologies of the heterotrophic stramenopiles. The unpigmented cell is spherical and about 5 μm in diameter and possesses a short flagellum and a long flagellum, both emanating anteriorly. The long flagellum bears mastigonemes in a characteristic arrangement, and its length (30 μm) distinguishes the target organism from other recognized heterotrophic stramenopiles. The short flagellum is naked and often directed posteriorly. The organism possesses neither a lorica nor a stalk. The morphological characteristics that we discovered should help isolate a representative of a novel stramenopile group, possibly at a high taxonomic level, in order to study its ultrastructure, physiological capabilities, and ecological role in the environment.     

Distribution of the uncultured protist MAST-4 in the Indian Ocean, Drake Passage and Mediterranean Sea assessed by real-time quantitative PCR

Molecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2–3 μm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal–offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml⁻¹) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importance.     

Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae sedis)

A new heterotrophic flagellate Wobblia lunata gen. et sp. nov. is described. This organism usually attaches to the substratum showing a wobbling motion, and sometimes glides on the substratum or swims freely in the medium. W. lunata has various features characteristic of the stramenopiles. These include a hairy flagellum with tripartite tubular hairs, a mitochondrion with tubular cristae, arrangement of flagellar apparatus components and a double helix in the flagellar transition zone. W. lunata shares a double helix with heterotrophic stramenopiles, including Developayella elegans, oomycetes, hyphochytrids, opalinids and proteromonads, and could be placed in the phylum Bigyra Cavalier-Smith. However, from 18S rDNA tree analysis, these organisms form two distantly-related clades in the stramenopiles, and Wobblia appears at the base of the stramenopiles. Evaluation of morphological features and comparison of 18S rDNA sequences indicate that W. lunata is a member of the stramenopiles, but it is distinct from any other stramenopiles so far described. Its phylogenetic position within the stramenopiles is uncertain and therefore W. lunata is described as a stramenopile incertae sedis.     

Biogeography of the uncultured marine picoeukaryote MAST-4: temperature-driven distribution patterns

The MAST-4 (marine stramenopile group 4) is a widespread uncultured picoeukaryote that makes up an important fraction of marine heterotrophic flagellates. This group has low genetic divergence and is composed of a small number of putative species. We combined ARISA (automated ribosomal intergenic spacer analysis) and ITS (Internal Transcribed Spacer) clone libraries to study the biogeography of this marine protist, examining both spatial and temporal trends in MAST-4 assemblages and associated environmental factors. The most represented MAST-4 clades appeared adapted to different temperature ranges, and their distributions did not suggest clear geographical barriers for dispersal. Distant samples sharing the same temperature had very similar assemblages, especially in cold temperatures, where only one clade, E1, dominated. The most highly represented clades, A and E1, showed very little differentiation between populations from distant geographical regions. Within a single site, temporal variation also followed patterns governed by temperature. Our results contribute to the general discussion on microbial biogeography by showing strong environmental selection for some picoeukaryotes in the marine environment.     

Distribution and abundance of uncultured heterotrophic flagellates in the world oceans

Heterotrophic flagellates play fundamental roles in marine ecosystems as picoplankton grazers. This recognized importance contrasts with our ignorance of the taxonomic composition of this functional group, which remains mostly unidentified by microscopical and culturing approaches. Recent molecular marine surveys based on 18S rDNA genes have retrieved many sequences unrelated to cultured organisms and marine stramenopiles were among the first reported uncultured eukaryotes. However, little is known about the organisms corresponding to these sequences. Here we determine the abundance of several marine stramenopile lineages in surface marine waters using molecular probes and fluorescent in situ hybridization. We show that these protists are free-living bacterivorous heterotrophic flagellates. They were widely distributed, occurring in the five world oceans, and accounted for a significant fraction (up to 35%) of heterotrophic flagellates in diverse geographic regions. A single group, MAST-4, represented 9% of cells within this functional assemblage, with the intriguing exception of polar waters where it was absent. MAST-4 cells likely contribute substantially to picoplankton grazing and nutrient re-mineralization in vast areas of the oceans and represent a key eukaryotic group in marine food webs.     

Environmental selection of marine stramenopile clades in the Arctic Ocean and coastal waters

Single-celled bacterial grazers are key components of marine ecosystems, and at times represent the dominant eukaryotes in the Arctic Ocean. Among these are small, phylogenetically diverse stramenopiles, known almost exclusively from their 18S rRNA gene sequences. Marine stramenopiles (MAST) have been found in the upper waters of the world ocean and in all Arctic seas. In particular, three sub-clades of MAST-1, MAST-1A, 1B and 1C co-occur in Arctic waters, but ecological and distributional details are lacking. Here, we have updated phylogenies of the MAST-1 clade to test whether there are Arctic-specific MAST-1 ecotypes. In addition, taking advantage of samples collected over 2 years as part of several Arctic cruises leading up to and during the International Polar Year, we applied sub-clade-specific probes and fluorescent in situ hybridization to describe the distribution of the three sub-clades in the Canadian Arctic. The three groups were found principally in the euphotic zone; however, evidence of Arctic ecotypes remains elusive. Some environment-specific separation among sub-clades was detected, with MAST-1C reaching significantly greater concentrations near the marginal ice zone, while MAST-1A and MAST-1B were associated with ice-covered and open-water stations, respectively. MAST-1B appeared to be able to persist at greater depths. Changing ice cover and mixing regimes are therefore expected to have an impact on the concentrations and distribution of these sub-clades, with possible downstream consequences on the marine food webs.     

Distribution patterns and phylogeny of marine stramenopiles in the north pacific ocean

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml(-1), respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Homologs of the sexually induced gene 1 (sig1) product constitute the stramenopile mastigonemes

The tripartite tubular mastigoneme on the anterior flagellum is a morphological feature that characterizes the stramenopiles. Mastigonemes are significant and potentially informative structures not only from the viewpoint of systematics, but also of cell biology. Nevertheless, few biochemical studies have been reported on stramenopile mastigonemes. The flagella of Scytosiphon lomentaria (Phaeophyceae) were successfully isolated and analyzed using SDS-PAGE followed by protein sequencing. The partial amino acid sequence of one flagellar protein (115kDa) showed high similarity with the sexually induced gene 1 (sig1) product of centric diatoms. A polyclonal antibody against the 115-kDa protein reacted not only to the shaft of mastigonemes in Scytosiphon lomentaria, but also another distinctly different stramenopile flagellate, Sulcochrysis biplastida (Dictyochophyceae). Therefore, we propose that the 115-kDa protein (i.e. Sig1 homologs) is a constituent of the tubular shaft of the mastigoneme.     

Phylogeny of Heterokonta: Incisomonas marina,a uniciliate gliding opalozoan related to Solenicola (Nanomonadea), and evidence that Actinophryida evolved from raphidophytes

Environmental rDNA sequencing has revealed many novel heterokont clades of unknown morphology. We describe a new marine heterotrophic heterokont flagellate, Incisomonas marina, which most unusually lacks an anterior cilium. It glides and swims with its cilium trailing behind, but is predominantly sedentary on the substratum, with or without a cilium. 18S rDNA sequence phylogeny groups Incisomonas strongly within clade MAST-3; with others it forms a robust sister clade to Solenicola, here grouped with it as new order Uniciliatida, placed within new class Nanomonadea encompassing MAST-3. Our comprehensive maximum likelihood heterokont phylogeny shows Nanomonadea as sister to MAST-12 plus Opalinata within Opalozoa, and that Actinophryida are not Opalozoa (previously suggested by distance trees), but highly modified raphidomonads, arguably related to Heliorapha (formerly Ciliophrys) azurina gen., comb. n. We discuss evolution of Actinophryida from photosynthetic raphidophytes. Clades MAST-4,6-11 form one early-branching bigyran clade. Olisthodiscus weakly groups with Hypogyristea not Raphidomonadea. Phylogenetic analysis shows that MAST-13 is all Bicosoeca. Some gliding uniciliates similar to Incisomonas marina seem to have been misclassified: therefore we establish Incisomonas devorata comb. n. for Rigidomastix devoratum, revise the genus Rigidomastix, transfer Clautriavia parva to Kiitoksia. We make 17 new familes (13 heterokont (three algal), two cercozoan, two amoebozoan).     

Mesoscale distribution and functional diversity of picoeukaryotes in the first-year sea ice of the Canadian Arctic

Sea ice, a characteristic feature of polar waters, is home to diverse microbial communities. Sea-ice picoeukaryotes (unicellular eukaryotes with cell size <3 μm) have received little attention compared with diatoms that dominate the spring bloom in Arctic first-year sea ice. Here, we investigated the abundance of all picoeukaryotes, and of 11 groups (chlorophytes, cryptophytes, bolidophytes, haptophytes, Pavlovaphyceae, Phaeocystis spp., pedinellales, stramenopiles groups MAST-1, MAST-2 and MAST-6 and Syndiniales Group II) at 13 first-year sea-ice stations localized in Barrow Strait and in the vicinity of Cornwallis Island, Canadian Arctic Archipelago. We applied Catalyzed Reporter Deposition–Fluorescence In Situ Hybridization to identify selected groups at a single cell level. Pavlovaphyceae and stramenopiles from groups MAST-2 and MAST-6 were for the first time reported from sea ice. Total numbers of picoeukaryotes were significantly higher in the vicinity of Cornwallis Island than in Barrow Strait. Similar trend was observed for all the groups except for haptophytes. Chlorophytes and cryptophytes were the dominant plastidic, and MAST-2 most numerous aplastidic of all the groups investigated. Numbers of total picoeukaryotes, chlorophytes and MAST-2 stramenopiles were positively correlated with the thickness of snow cover. All studied algal and MAST groups fed on bacteria. Presence of picoeukaryotes from various trophic groups (mixotrophs, phagotrophic and parasitic heterotrophs) indicates the diverse ecological roles picoeukaryotes have in sea ice. Yet, >50% of total sea-ice picoeukaryote cells remained unidentified, highlighting the need for further study of functional and phylogenetic sea-ice diversity, to elucidate the risks posed by ongoing Arctic changes.     

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300‰ salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30–35°C. The isolated HNF grew optimally at 150‰ salinity and tolerated up to 363‰ salinity, but it failed to grow below 75‰ salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.     

Unveiling the Organisms behind Novel Eukaryotic Ribosomal DNA Sequences from the Ocean

Despite the fact that the smallest eukaryotes (cells less than 5 μm in diameter) play key roles in marine food webs, particularly in open oligotrophic areas, the study of their in situ diversity started just one year ago. Perhaps the most remarkable finding of the most recent studies has been the discovery of completely new phylogenetic lineages, such as novel clades belonging to the stramenopile and alveolate phyla. The two new groups account for a significant fraction of clones in genetic libraries from North Atlantic, equatorial Pacific, Antarctic, and Mediterranean Sea waters. However, the identities and ecological relevance of these organisms remain unknown. Here we investigate the phylogenetic relationships, morphology, in situ abundance, and ecological role of novel stramenopiles. They form at least eight independent clades within the stramenopile basal branches, indicating a large phylogenetic diversity within the group. Two lineages were visualized and enumerated in field samples and enrichments by fluorescent in situ hybridization using specific rRNA-targeted oligonucleotide probes. The targeted organisms were 2- to 3-μm-diameter, round-shaped, nonpigmented flagellates. Further, they were found to be bacterivorous. One lineage accounted for up to 46% (average during an annual cycle, 19%) of heterotrophic flagellates in a coastal environment, providing evidence that novel stramenopiles are important and unrecognized components of the total stock of bacterial grazers.     

Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean

Despite the fact that the smallest eukaryotes (cells less than 5 micro m in diameter) play key roles in marine food webs, particularly in open oligotrophic areas, the study of their in situ diversity started just one year ago. Perhaps the most remarkable finding of the most recent studies has been the discovery of completely new phylogenetic lineages, such as novel clades belonging to the stramenopile and alveolate phyla. The two new groups account for a significant fraction of clones in genetic libraries from North Atlantic, equatorial Pacific, Antarctic, and Mediterranean Sea waters. However, the identities and ecological relevance of these organisms remain unknown. Here we investigate the phylogenetic relationships, morphology, in situ abundance, and ecological role of novel stramenopiles. They form at least eight independent clades within the stramenopile basal branches, indicating a large phylogenetic diversity within the group. Two lineages were visualized and enumerated in field samples and enrichments by fluorescent in situ hybridization using specific rRNA-targeted oligonucleotide probes. The targeted organisms were 2- to 3- micro m-diameter, round-shaped, nonpigmented flagellates. Further, they were found to be bacterivorous. One lineage accounted for up to 46% (average during an annual cycle, 19%) of heterotrophic flagellates in a coastal environment, providing evidence that novel stramenopiles are important and unrecognized components of the total stock of bacterial grazers.     

Protist diversity in suboxic and sulfidic waters of the Black Sea

The oxic-anoxic transition zone of the Black Sea comprises a large suboxic zone as well as anoxic and sulfidic waters. While the prokaryotes and biogeochemical cycles that characterize this zone have been frequently studied, little is known about the diversity or ecology of its microbial eukaryotes. Here, we present the first broad qualitative report of the protist species composition in the Black Sea redoxcline using molecular tools. Fingerprint analysis from the whole redoxcline revealed a complex community structure of metabolically active protists with distinct shifts along the redox gradient. Additionally, 18S rRNA gene clone libraries were used to compare protist species composition of suboxic and sulfidic water layers. Among the ciliates, sequences related to Pleuronema and Strombidium were dominant in both water layers whereas sequences affiliated with anaerobic plagiopylids and Cyclidium were detected only in the sulfidic zone. Among the flagellates, mainly stramenopiles (mostly bicosoecids and chrysophytes) occurred throughout the redoxcline. In the sulfidic zone we found stramenopile sequences but also euglenozoans, jakobids and choanoflagellates that were related to clonal sequences from other anoxic marine habitats, thus indicating the existence of globally distributed groups of anoxic flagellates. Higher species diversity in the sulfidic zone and about twice as many novel sequence types of ciliates and stramenopiles compared with the suboxic layer emphasizes the importance of anoxic, sulfidic waters as habitat for high protist diversity although the function of these organisms is yet unknown.     

Low evolutionary diversification in a widespread and abundant uncultured protist (MAST-4)

Recent culture-independent studies of marine planktonic protists have unveiled a large diversity at all phylogenetic scales and the existence of novel groups. MAST-4 represents one of these novel uncultured lineages, and it is composed of small (~2 μm) bacterivorous eukaryotes that are widely distributed in marine systems. MAST-4 accounts for a significant fraction of the marine heterotrophic flagellates at the global level, playing key roles in the marine ecological network. In this study, we investigated the diversity of MAST-4, aiming to assess its limits and structure. Using ribosomal DNA (rDNA) sequences obtained in this study (both pyrosequencing reads and clones with large rDNA operon coverage), complemented with GenBank sequences, we show that MAST-4 is composed of only five main clades, which are well supported by small subunit and large subunit phylogenies. The differences in the conserved regions of the internal transcribed spacers 1 and 2 (ITS1 and ITS2) secondary structures strongly suggest that these five clades are different biological species. Based on intraclade divergence, ITS secondary structures and comparisons of ITS1 and ITS2 trees, we did not find evidence of more than one species within clade A, whereas as many as three species might be present within other clades. Overall, the genetic divergence of MAST-4 was surprisingly low for an organism with a global population size estimated to be around 10(24), indicating a very low evolutionary diversification within the group.     

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.     

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Ultrastructure and taxonomy of a marine photosynthetic stramenopile Phaeomonas parva gen. et sp. nov. (Pinguiophyceae) with emphasis on the flagellar apparatus architecture

Phaeomonas parva gen. et sp. nov., a marine photosynthetic stramenopile from oceanic water near the Caroline Islands, is described. Cells are naked and spherical to ovoid. The alga is motile with two laterally inserted flagella during the light period, whereas during the dark period, it absorbs the flagella and rounds up. The anterior (immature, No. 2) long flagellum possesses tubular tripartite mastigonemes. The posterior (mature, No. 1) short flagellum is smooth and has autofluorescence at the base. The cupshaped, yellowish‐brown chloroplast occupies the posterior half of the cell, and a pyrenoid occurs in the inner cavity of the cup‐shaped chloroplast. The flagellar apparatus has several unusual features. Two basal plates and a two‐gyred proximal helix in the flagellar transitional region may suggest that P. parva is related to the Pelagophyceae, Dictyochophyceae and Sulcochrysis biplastida, a photosynthetic stramenopile of uncertain taxonomic position. The R3 and R4 roots form a loop that resembles phagotrophic chrysophytes. However, this resemblance is superficial because Phaeomonas is not phagotrophic, its R3 root has a different number of microtubules and its R3 root does not split to form a food‐uptake mouth. Phaeomonas has a ‘bypassing root’, which is found only with the Phaeophyceae, Giraudyopsis stellifera (Chrysomerophyceae), and Ankylochrysis lutea (probably a member of the Pelagophyceae). The taxonomic position of P. parva could not be determined solely from ultrastructural features. However, molecular phylogeny and biochemical analyses (published separately) strongly supported a relationship between P. parva and four other monotypic strameno‐piles, Glossomastix, Pinguiochrysis, Pinguiococcus and Polypodochrysis. Although these algae are morphologically distinct, they have unusually high percentages of polyunsaturated fatty acids, especially eicosapentoic acid. This unusual assemblage of stramenopiles is classified in a new class, the Pinguiophyceae (published separately), and P. parva is its only biflagellate member.