Changes in community structure of active protistan assemblages from the lower Pearl River to coastal Waters of the South China Sea

Protists make up an important component of aquatic ecosystems, playing crucial roles in biogeochemical processes on local and global scales. To reveal the changes of diversity and community structure of protists along the salinity gradients, community compositions of active protistan assemblages were characterized along a transect from the lower Pearl River estuary to the open waters of the South China Sea (SCS), using high-throughput sequencing of the hyper-variable V9 regions of 18S rRNA. This study showed that the alpha diversity of protists, both in the freshwater and in the coastal SCS stations was higher than that in the estuary. The protist community structure also changed along the salinity gradient. The relative sequence abundance of Stramenopiles was highest at stations with lower salinity and decreased with the increasing of salinity. By contrast, the contributions of Alveolata, Hacrobia and Rhizaria to the protistan communities generally increased with the increasing of salinity. The composition of the active protistan community was strongly correlated with salinity, indicating that salinity was the dominant factor among measured environmental parameters affecting protistan community composition and structure.     

Seasonal diversity of planktonic protists in Southwestern Alberta rivers over a 1-year period as revealed by terminal restriction fragment length polymorphism and 18S rRNA gene library analyses

The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.     

Microbial eukaryotes in the hypersaline anoxic L'Atalante deep-sea basin

The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l⁻¹ NaCl) anoxic L'Atalante deep-sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea , Acantharea and Euglenozoa , all of which are typically found in non-hypersaline deep-sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

Impact of protists on the activity and structure of the bacterial community in a rice field soil

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a gamma-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G + C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH4+ was lower and the level of extractable SO4(2-) was higher without protists, indicating that nitrogen mineralization and SO4(2-) reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O2 was a major control, and O2 availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.     

An optimised PCR/T-RFLP fingerprinting approach for the investigation of protistan communities in groundwater environments

Due to the scarcity or complete absence of higher organisms, protists may represent an important higher trophic level (above Prokaryotes) in the food webs of groundwater habitats. Nevertheless, the importance of aquifer protists, especially in contaminated groundwater environments, is poorly understood. Partly, this may be due to a lack of adequate PCR and fingerprinting approaches for protists in aquifers, which can be considered low in protistan or high in non-target rRNA gene copy numbers. Therefore, we have validated the suitability of distinct eukaryote-targeted primer pairs and restriction endonucleases for T-RFLP fingerprinting of protistan communities. By in silico predictions, and by fingerprinting, cloning and sequencing of microeukaryote amplicons from hydrocarbon-contaminated aquifer sediment DNA, we show that the Euk20f/Euk516r primer set in combination with Bsh1236I digestion is best suited for the recovery of diverse protistan 18S rRNA lineages. In contrast to other tested primer sets, a preferred recovery of fungal and archaeal non-target amplicons was not observed. In summary, we present an optimised microeukaryote-targeted PCR/T-RFLP fingerprinting approach which may be of value for the characterisation of protistan communities in groundwater and other habitats.     

土壤原生生物多样性及其生态功能研究进展

原生生物广泛分布在土壤和不同生境中, 其数量庞大、种类繁多, 在生态系统物质循环和能量流动以及维持土壤和植物健康中起着举足轻重的作用。与土壤其他生物类群相比, 原生生物分类体系和生态类型复杂, 分类鉴定困难且分子检测技术不够成熟, 目前对原生生物的认识相对不足。本文对当前原生生物的相关研究进展进行了总结和梳理, 系统阐述了原生生物的分类系统和营养功能群特征、土壤原生生物的多样性分布特征及影响因子, 重点介绍了原生生物群落在参与土壤养分循环、维持土壤和植物健康等中的功能作用, 并对未来的研究方向与应用前景进行了展望。对土壤原生生物的研究有助于我们深入认识土壤生物多样性资源, 并进行保护性地开发和利用, 维护土壤和生态系统健康。     

Fertilization alters protistan consumers and parasites in crop-associated microbiomes

Crop plants carry an enormous diversity of microbiota that provide massive benefits to hosts. Protists, as the main microbial consumers and a pivotal driver of biogeochemical cycling processes, remain largely understudied in the plant microbiome. Here, we characterized the diversity and composition of protists in sorghum leaf phyllosphere, and rhizosphere and bulk soils, collected from an 8-year field experiment with multiple fertilization regimes. Phyllosphere was an important habitat for protists, dominated by Rhizaria, Alveolata and Amoebozoa. Rhizosphere and bulk soils had a significantly higher diversity of protists than the phyllosphere, and the protistan community structure significantly differed among the three plant–soil compartments. Fertilization significantly altered specific functional groups of protistan consumers and parasites. Variation partitioning models revealed that soil properties, bacteria and fungi predicted a significant proportion of the variation in the protistan communities. Changes in protists may in turn significantly alter the compositions of bacterial and fungal communities from the top-down control in food webs. Altogether, we provide novel evidence that fertilization significantly affects the functional groups of protistan consumers and parasites in crop-associated microbiomes, which have implications for the potential changes in their ecological functions under intensive agricultural managements.     

Spatial distribution of protists in the presence of macroaggregates in a marine system

The spatial distribution of marine protistan communities in the presence of organic macroaggregates, formed from natural seawater, was studied in several microcosm experiments. The presence of macroaggregates had two main effects. First, the size of the communities of bacteria, flagellates and ciliates increased, as these communities were three orders of magnitude higher in the aggregates than in the microcosm water. In addition, it brought the diversification on the niches accessible to planktonic microorganisms, as three phases were formed: water, aggregates and aggregate-water interphase. Some of the detected protistan taxa were only found in the water, and therefore they can be considered as truly free-swimming protists. Others quickly colonised the aggregates, and finally, some of them showed a preference for the aggregate-water interphase. We discuss this spatial structuring of the protistan community on the basis of their feeding strategies and structural and behavioural characteristics.     

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a γ-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G+C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH₄⁺ was lower and the level of extractable SO₄²⁻ was higher without protists, indicating that nitrogen mineralization and SO₄²⁻ reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O₂ was a major control, and O₂ availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.     

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.     

Histones in protistan evolution

The potential of comparative studies on histones for use in protistan evolution is discussed, using algal histones as specific examples. A basic premise for the importance of histones in protistan evolution is the observation that these proteins are completely absent in prokaryotes (and cytoplasmic organelles), but with few exceptions, the same five major histone types are found in all higher plants and animals. Since the histone content of the algae and other protists is not constant, some of these organisms may represent transition forms between the prokaryotic and eukaryotic modes of packaging the genetic material. Comparative studies of protistan histones may thus be of help in determining evolutionary relationships. However, several problems are encounter with protistan histones, including difficulties in isolating nuclei, proteolytic degradation, anomalous gel migration of histones, and difficulties in histone identification. Because of the above problems, and the observed variability in protistan histones, it is suggested that several criteria be employed for histone identification in protists.     

Comparisons of the Fungal and Protistan Communities among Different Marine Sponge Holobionts by Pyrosequencing

To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.     

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Unveiling trophic functions of uncultured protist taxa by incubation experiments in the brackish baltic sea

Background

Our knowledge of the phylogeny and diversity of aquatic protists is rapidly increasing due to molecular surveys and next-generation sequencing approaches. This has led to a considerable discrepancy between the taxa known from cultures and those known from environmental 18S rRNA gene sequences. Hence, it is generally difficult to assign ecological functions to new taxa detected by culture-independent molecular approaches.

Methodology/Principal Findings

A combination of unamended dark incubations and 18S rRNA sequencing was chosen to link molecular diversity data of uncultured protists with heterotrophic, presumably bacterivorous, growth. The incubations, conducted with Baltic Sea brackish water, resulted in a consistent shift from a protistan community dominated by phototrophs to one in which heterotrophs predominated. This was determined on the basis of cell abundance and 18S rRNA sequences derived from fingerprint analysis and clone libraries. The bulk of enriched phylotypes after incubation were related to hitherto uncultured marine taxa within chrysophytes, ochrophytes, choanoflagellates, cercozoans, and picobiliphytes, mostly represented in recently established or here defined environmental clades. Their growth in the dark, together with coinciding results from studies with a similar objective, provides evidence that these uncultured taxa represent heterotrophic or mixotrophic species.

Conclusions/Significance

These findings shed some light into the trophic role of diverse uncultured protists especially within functionally heterogeneous groups (e.g., chrysophytes, ochrophytes) and groups that appear to be puzzling with regard to their nutrition (picobiliphytes). Additionally, our results indicate that the heterotrophic flagellate community in the southwestern Baltic Sea is dominated by species of marine origin. The combination of unamended incubations with molecular diversity analysis is thus confirmed as a promising approach to explore the trophic mode of environmentally relevant protist taxa for which only sequence data are currently available.     

Dynamics and trophic roles of heterotrophic protists in the plankton of a freshwater tidal estuary

Freshwater tidal estuaries comprise the most upstream reaches of estuaries and are often characterised by the presence of dense bacterial and algal populations which provide a large food source for bacterivorous and algivorous protists. In 1996, the protistan community in the freshwater tidal reaches of the Schelde estuary was monitored to evaluate whether these high food levels are reflected in a similarly high heterotrophic protistan biomass. Protistan distribution patterns were compared to those of metazoan zooplankton to evaluate the possible role of top-down regulation of protists by metazoans. Apart from the algivorous sarcodine Asterocaelum, which reached high densities in summer, heterotrophic protistan biomass was dominated by ciliates and, second in importance, heterotrophic nanoflagellates (HNAN). HNAN abundance was low (annual average 2490 cells ml^–1) and did not display large seasonal variation. It is hypothesised that HNAN were top-down controlled by oligotrich ciliates throughout the year and by rotifers in summer. Ciliate abundance was generally relatively high (annual average 65 cells ml^–1) and peaked in winter (maximum 450 cells ml^–1). The decline of ciliate populations in summer was ascribed to grazing by rotifers, which developed dense populations in that season. In winter, ciliate populations were probably regulated `internally' by carnivorous ciliates (haptorids and Suctoria). Our observations suggest that, in this type of productive ecosystems, the microbial food web is mainly top-down controlled rather than regulated by food availability.