Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection.

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3-V4 region to investigate whether the RC-PCR reveals more of the 'unseen' diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3-V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.

Monitoring of biofilm development and physico-chemical changes of floating microplastics at the air-water interface.

Microplastics in the aquatic environment serve as a habitat for microbial life, on which they can form biofilms. However, how the development of the biofilm alters the properties of floating microplastics that are at the air-water interface and, therefore, not fully submerged, is not well understood. In this context, an aging experiment was conducted to monitor biofilm formation and changes in physico-chemical properties of low-density polyethylene (floating) microplastics over time. The growth of the biofilm followed the typical bacterial/biofilm growth phases and reached about 30% of the total mass of the microplastics, while the concentration of extracellular polymeric substances within the biofilm remained stable. Presence of chlorophyll a and urease activity indicated presence of photosynthetic microrganisms within the biofilm which was also confirmed by analysis of the biofilm composition. Chemical characterization by FTIR showed the formation of additional functional groups attributed to the formed biofilm, and SEM imaging showed cracks on the surface of the aged microplastics, indicating incipient degradation of the polyethylene. Moreover, the adsorption capacity of the aged particles for metals (Pb(II)) was 52% higher compared to the pristine ones. Aging increased the density and size of the particles; however, it did not lead to the submersion of the aged particles even after 12 weeks of aging, suggesting that additional environmental processes may influence the transport of microplastics from the air-water interface into the water body.

Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts.

Paramecium bursaria is a mixotrophic ciliate species, which is common in stagnant and slow-flowing, nutrient-rich waters. It is usually found living in symbiosis with zoochlorellae (green algae) of the genera Chlorella or Micractinium. We investigated P. bursaria isolates from around the world, some of which have already been extensively studied in various laboratories, but whose morphological and genetic identity has not yet been completely clarified. Phylogenetic analyses of the SSU and ITS rDNA sequences revealed five highly supported lineages, which corresponded to the syngen and most likely to the biological species assignment. These syngens R1-R5 could also be distinguished by unique synapomorphies in the secondary structures of the SSU and the ITS. Considering these synapomorphies, we could clearly assign the existing GenBank entries of P. bursaria to specific syngens. In addition, we discovered synapomorphies at amino acids of the COI gene for the identification of the syngens. Using the metadata of these entries, most syngens showed a worldwide distribution, however, the syngens R1 and R5 were only found in Europe. From morphology, the syngens did not show any significant deviations. The investigated strains had either Chlorella variabilis, Chlorella vulgaris or Micractinium conductrix as endosymbionts.

Historical Review of Studies on Cyrtophorian Ciliates (Ciliophora, Cyrtophoria) from China.

The subclass Cyrtophoria are a group of morphologically specialized ciliates which mainly inhabit soil, freshwater, brackish water, and marine environments. In this study, we revise more than 50 publications on the taxonomy, phylogeny, and ecology of cyrtophorian ciliates in China since the first publication in 1925, most of which were carried out in coastal areas. The research history can be divided into three periods: the early stage, the Tibet stage, and the molecular stage. To date, 103 morpho-species (147 isolates) have been formally recorded in China, with ciliature patterns described for 82 of them. A species checklist and an illustrated identification key to the genera are provided. A total of 100 small subunit rDNA sequences have been obtained for 74 taxonomic hits (lowest taxonomic rank to species or genus). These sequences are used for the study of molecular phylogeny. Based on these morphological data and molecular phylogeny analyses, we synthesize the understanding of the phylogeny of cyrtophorian ciliates. We hypothesize that the key evolutionary event of cyrtophorian ciliates lies in the separation of the stomatogenesis zone (postoral kineties) from the left kineties, namely, the formation of an independent "sexual organelle". We, furthermore, briefly summarize the ecological features of cyrtophorian ciliates and provide a comprehensive bibliography of related research from China. Finally, we give an outlook on the future research directions of these taxa.

The ER protein Ema19 facilitates the degradation of nonimported mitochondrial precursor proteins.

For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of nonproductive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homologue is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting.

Molecular Data Reveal a Cryptic Diversity in the Genus Urotricha (Alveolata, Ciliophora, Prostomatida), a Key Player in Freshwater Lakes, With Remarks on Morphology, Food Preferences, and Distribution.

Species of the ciliate genus Urotricha are key players in freshwater plankton communities. In the pelagial of lakes, about 20 urotrich species occur throughout an annual cycle, some of which play a pivotal role in aquatic food webs. For example, during the phytoplankton spring bloom, they consume a remarkable proportion of the algal production. In ecological studies, urotrich ciliates are usually merely identified to genus rank and grouped into size classes. This is unsatisfying considering the distinct autecological properties of individual species and their specific spatial and temporal distribution patterns. As a basis for future research, we characterized in detail four common urotrich morphotypes, i.e., specimens identified as U. furcata and tentatively as U. agilis, U. pseudofurcata, and U. castalia, using state-of-the-art methods. We used an integrative polyphasic approach, in which morphological studies (in vivo observation, silver staining methods, scanning electron microscopy) were linked with a molecular approach exploiting four different gene fragments as taxonomic DNA barcodes with different resolution potential (SSU rDNA, ITS-1, ITS-2, hypervariable V4 and V9 regions of the SSU rDNA). We shed light on the diversity of urotrich ciliates as well as on their global distribution patterns, and annual cycles. Additionally, we coupled individual species occurrences and environmental parameters, and subsequently modeled the distribution and occurrence, using logistic regressions. Furthermore, for one strain putatively identified as U. castalia, we ascertained the optimal cultivation media and food preferences. Thereby, our comprehensive view on these important freshwater ciliates that frequently occur in environmental high throughput sequencing datasets worldwide will allow future studies to better exploit protistan plankton data from lakes.

Bathymetric gradient shapes the community composition rather than the species richness of deep-sea benthic ciliates.

The bathymetric gradient is one of the most important factors that regulate the distribution of life. However, community variations of benthic ciliates along bathymetric gradients in the deep sea remain rather unexplored. In this study, we hypothesize that in the deep sea, the bathymetric gradient shapes the benthic ciliate community composition rather than the species richness. Here, we evaluated the distribution patterns and drivers of benthic ciliate communities of an abyssal plain, a seamount, and a trench with water depths ranging from 800 m down to 6600 m by high throughput eDNA sequencing and statistical analyses. We observed no significant correlation between ciliate operated taxonomic unit (OTU) richness and water depth. A meta-analysis, which combined our previously published data from the neritic habitats, supports the notion that water depth exceeding 800 m has little effect on the richness of benthic ciliate species. In contrast, the composition of deep-sea ciliate communities was significantly distinct in different habitats along the bathymetric gradients. A SourceTracker analysis revealed extremely low connectivity among ciliate communities along the bathymetric gradients. More than 95% of the community dissimilarity in the deep-sea floor was attributed to species replacement, which might be caused by environmental sorting or historical constraints. Furthermore, the observed community variations could be ascribed more to water depth than to geographic distance. The findings imply that the strong force of environmental sorting along the bathymetric gradients and the low connectivity among the ciliate communities might lead to an isolated evolution. This could shape the community composition rather than the species richness, which is mainly determined by the limited nutrient availability and the extreme environmental conditions in the deep sea.

Morphological, Phylogenetic and Ecophysiological Characterization of a New Ciliate, Platynematum rossellomorai n. sp. (Oligohymenophorea, Scuticociliatia), Detected in a Hypersaline Pond on Mallorca, Spain.

With highly specialized morphology and unexplored functional capacities, ciliates from extreme habitats are drawing increasing attention. During a microbial investigation of a solar saltern pond (salinity 240‰) on Mallorca, Spain, a previously unknown scuticociliate, Platynematum rossellomorai n. sp. was isolated, cultured and studied using a tripartite approach consisting of a morphological description, a molecular analysis and an ecophysiological characterization. The ciliate has distinct morphological characteristics and its main diagnostic features include a large anteriorly positioned oral area (occupying almost half of the body length), two caudal cilia and a small number of somatic kineties. However, due to the most important generic feature of Cinetochilidae, the consistency of the arrangement of the adoral membranes, the ciliate is classified as a new member of the genus Platynematum. Its 18S rRNA gene sequence shows a sequence similarity of 91.0% to the closest deposited relative, Platynematum salinarum, and a phylogenetic analysis reveals a close relationship to other members of the family Cinetochilidae Perty, 1852. Growth experiments identify the ciliate as a borderline halophile, with a tolerance range between 180 and 280‰ salinity. The ciliate apparently accumulates the compatible solutes glycine betaine and ectoine to counterbalance osmotic stress, however, other osmoregulatory mechanisms are not excluded.

Perspectives from Ten Years of Protist Studies by High-Throughput Metabarcoding.

During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding.

Spatio-temporal patterns of zooplankton in a main-stem dam affected tributary: a case study in the Xiangxi River of the Three Gorges Reservoir, China.

As the ecologically important recipient channels for riverine ecosystems, tributaries provide unique microhabitats for microorganisms, among which zooplankton constitutes the most important heterotrophic organisms. In particular, the reduced water velocity caused by dams is more favorable for zooplankton development; therefore, dammed rivers are expected to support extremely diverse and abundant zooplankton communities and notably different spatiotemporal distribution patterns. So far, however, only very few molecular studies support these assumptions. Using high-throughput sequencing, a high number of 350 operational taxonomic units (OTUs; 97% cutoff) were retrieved from 30 samples collected in the Xiangxi River, the nearest large tributary upstream of the Three Gorges Dam. Zooplankton did not show significant spatial distribution in the channel. Instead, the community structures varied significantly over sampling dates, corroborating the seasonal patterns found in lakes and ponds in the subtropical zone. As expected, the community compositions were deterministically governed by environmental filtering processes (phylogenetic clustering), in which water velocity appeared to be much less important than other investigated environmental factors. Moreover, most of the detected phylotypes (OTUs) had a relatively high (>90%) sequence similarity to previously deposited sequences, suggesting a mediocre degree of genetic novelty within the zooplankton communities in the Xiangxi River.

Improving eDNA-based protist diversity assessments using networks of amplicon sequence variants.

Effective and precise grouping of highly similar sequences remains a major bottleneck in the evaluation of high-throughput sequencing datasets. Amplicon sequence variants (ASVs) offer a promising alternative that may supersede the widely used operational taxonomic units (OTUs) in environmental sequencing studies. We compared the performance of a recently developed pipeline based on the algorithm DADA2 for obtaining ASVs against a pipeline based on the algorithm SWARM for obtaining OTUs. Illumina-sequencing of 29 individual ciliate species resulted in up to 11 ASVs per species, while SWARM produced up to 19 OTUs per species. To improve the congruency between species diversity and molecular diversity, we applied sequence similarity networks (SSNs) for second-level sequence grouping into network sequence clusters (NSCs). At 100% sequence similarity in SWARM-SSNs, NSC numbers decreased from 7.9-fold overestimation without abundance filter, to 4.5-fold overestimation when an abundance filter was applied. For the DADA2-SSN approach, NSC numbers decreased from 3.5-fold to 3-fold overestimation. Rand index cluster analyses predicted best binning results between 97% and 94% sequence similarity for both DADA2-SSNs and SWARM-SSNs. Depending on the ecological questions addressed in an environmental sequencing study with protists we recommend ASVs as replacement for OTUs, best in combination with SSNs.

Redescription of Dexiotricha colpidiopsis (Kahl, 1926) Jankowski, 1964 (Ciliophora, Oligohymenophorea) from a Hot Spring in Iceland with Identification Key for Dexiotricha species.

We isolated an encysted ciliate from a geothermal field in Iceland. The morphological features of this isolate fit the descriptions of Dexiotricha colpidiopsis Kahl, 1926) Jankowski, 1964 very well. These comprise body shape and size in vivo, the number of somatic kineties, and the positions of macronucleus and contractile vacuole. Using state-of-the-art taxonomic methods, the species is redescribed, including phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) gene as molecular marker. In the phylogenetic analyses, D. colpidiopsis clusters with the three available SSU rRNA gene sequences of congeners, suggesting a monophyly of the genus Dexiotricha. Its closest relative in phylogenetic analyses is D. elliptica, which also shows a high morphological similarity. This is the first record of a Dexiotricha species from a hot spring, indicating a wide temperature tolerance of this species at least in the encysted state. The new findings on D. colpidiopsis are included in a briefly revision of the scuticociliate genus Dexiotricha and an identification key to the species.

Morphology, Morphogenesis and Molecular Phylogeny of a New Obligate Halophile Ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov. (Ciliophora, Hypotrichia) Isolated from a Volcanic Crater on Sal (Cape Verde Islands).

A new hypotrichous ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov., was isolated from a solar saltern on the island of Sal, Cape Verde. The possession of only one short dorsal kinety clearly distinguishes S. ultrahalophila from other known hypotrichous genera and species. Further diagnostic characters include: a flexible and slender body, an average size of 85 × 15 µm in vivo; a bipartite adoral zone with two hypertrophied frontal adoral membranelles and nine to twelve ventral adoral membranelles; three frontal, one parabuccal, two frontoventral, two or three postoral ventral, and two or three frontoterminal cirri; and marginal cirral rows variable in number, usually one on each side. Ontogenetic data indicate the following: the frontal-ventral cirri originate from six or five anlagen; the proter inherits the parental adoral zone; the frontal and ventral cirri originate from five or six anlagen; and the marginal cirral rows and the dorsal kinety tend to originate intrakinetally. Additional marginal rows are rarely derived from de novo anlagen. Based on its morphology, morphogenesis and its SSU rRNA phylogenetic placement, the new species should be assigned to the order Sporadotrichida Fauré-Fremiet, 1961. Due to low taxon sampling, however, its exact position in this order remains enigmatic.

A fundamental difference between macrobiota and microbial eukaryotes: protistan plankton has a species maximum in the freshwater-marine transition zone of the Baltic Sea.

Remane's Artenminimum at the horohalinicum is a fundamental concept in ecology to describe and explain the distribution of organisms along salinity gradients. However, a recent metadata analysis challenged this concept for protists, proposing a species maximum in brackish waters. Due to data bias, this literature-based investigation was highly discussed. Reliable data verifying or rejecting the species minimum for protists in brackish waters were critically lacking. Here, we sampled a pronounced salinity gradient along a west-east transect in the Baltic Sea and analysed protistan plankton communities using high-throughput eDNA metabarcoding. A strong salinity barrier at the upper limit of the horohalinicum and 10 psu appeared to select for significant shifts in protistan community structures, with dinoflagellates being dominant at lower salinities, and dictyochophytes and diatoms being keyplayers at higher salinities. Also in vertical water column gradients in deeper basins (Kiel Bight, Arkona and Bornholm Basin) appeared salinity as significant environmental determinant influencing alpha- and beta-diversity patterns. Importantly, alpha-diversity indices revealed species maxima in brackish waters, that is, indeed contrasting Remane's Artenminimum concept. Statistical analyses confirmed salinity as the major driving force for protistan community structuring with high significance. This suggests that macrobiota and microbial eukaryotes follow fundamentally different rules regarding diversity patterns in the transition zone from freshwater to marine waters.

Correction to: Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

The original version of this article unfortunately contained mistakes in the author affiliation, the references given in two tables and in a figure legend.

Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

One decisive factor controlling the distribution of organisms in their natural habitats is the cellular response to environmental factors. Compared to prokaryotes, our knowledge about salt adaptation strategies of microbial eukaryotes is very limited. We, here, used a recently introduced approach (implementing proton nuclear magnetic resonance spectroscopy) to investigate the presence of compatible solutes in halophilic, heterotrophic ciliates. Therefore, we isolated four ciliates from solar salterns, which were identified as Cyclidium glaucoma, Euplotes sp., Fabrea salina, and Pseudocohnilembus persalinus based on their 18S rRNA gene signatures and electron microscopy. The results of 1H-NMR spectroscopy revealed that all four ciliates employ the "low-salt-in" strategy by accumulating glycine betaine and ectoine as main osmoprotectants. We recorded a linear increase of these compatible solutes with increasing salinity of the external medium. Ectoine in particular stands out as its use as compatible solute was thought to be exclusive to prokaryotes. However, our findings and those recently made on two other heterotroph species call for a re-evaluation of this notion. The observation of varying relative proportions of compatible solutes within the four ciliates points to slight differences in haloadaptive strategies by regulatory action of the ciliates. Based on this finding, we provide an explanatory hypothesis for the distribution of protistan diversity along salinity gradients.

A Comparison of Different Ciliate Metabarcode Genes as Bioindicators for Environmental Impact Assessments of Salmon Aquaculture.

Ciliates are powerful indicators for monitoring the impact of aquaculture and other industrial activities in the marine environment. Here, we tested the efficiency of four different genetic markers (V4 and V9 regions of the SSU rRNA gene, D1 and D2 regions of the LSU rRNA gene, obtained from environmental (e)DNA and environmental (e)RNA) of benthic ciliate communities for environmental monitoring. We obtained these genetic metabarcodes from sediment samples collected along a transect extending from below salmon cages toward the open sea. These data were compared to benchmark data from traditional macrofauna surveys of the same samples. In beta diversity analyses of ciliate community structures, the V4 and V9 markers had a higher resolution power for sampling sites with different degrees of organic enrichment compared to the D1 and D2 markers. The eDNA and eRNA V4 markers had a higher discriminatory power than the V9 markers. However, results obtained with the eDNA V9 marker corroborated better with the traditional macrofauna monitoring. This allows for a more direct comparison of ciliate metabarcoding with the traditional monitoring. We conclude that the ciliate eDNA V9 marker is the best choice for implementation in routine monitoring programs in marine aquaculture.

Characterization of protistan plankton diversity in ancient salt evaporation ponds located in a volcanic crater on the island Sal, Cape Verde.

Salinity is an important factor when exploring the limits known for life. Therefore, hypersaline systems have attracted much attention in recent years. In this study, we investigated the protistan diversity and community composition in two natural salt evaporation ponds (27-30% salinity) located in an ancient volcanic crater on the Cape Verde island Sal using high-throughput DNA sequencing. Our study revealed a broad range of protistan taxa and a high taxonomic diversity within the Ciliophora, Dinophyceae, and Chlorophyta. We detected a total of 23 Dinophyceae families, although Dinophyceae were generally considered to be only this diverse in aquatic environments of less than 10% salinity. Moreover, we uncovered a high degree of genetic novelty in this habitat. The mean similarity of all detected OTUs to previously described sequences was only 93.6%. These findings strongly dispute the traditional view that extreme hypersaline environments generally maintain low protistan diversity. A meta-analysis covering our and previously published data from other inland and coastal salt ponds clearly showed that our samples clustered according to salinity and not biogeography. This result further supports the claim that salinity is a major transition boundary for protistan communities, regardless of their biogeographic origin.