Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing

Flagellated heterotrophic microeukaryotes have key roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. Still, we know very little on the diversity patterns of most groups constituting this evolutionary heterogeneous assemblage. Here, we investigate 11 groups of uncultured flagellates known as MArine STramenopiles (MASTs). MASTs are ecologically very important and branch at the base of stramenopiles. We explored the diversity patterns of MASTs using pyrosequencing (18S rDNA) in coastal European waters. We found that MAST groups range from highly to lowly diversified. Pyrosequencing (hereafter ‘454'') allowed us to approach to the limits of taxonomic diversity for all MAST groups, which varied in one order of magnitude (tens to hundreds) in terms of operational taxonomic units (98% similarity). We did not evidence large differences in activity, as indicated by ratios of DNA:RNA-reads. Most groups were strictly planktonic, although we found some groups that were active in sediments and even in anoxic waters. The proportion of reads per size fraction indicated that most groups were composed of very small cells (∼2–5 μm). In addition, phylogenetically different assemblages appeared to be present in different size fractions, depths and geographic zones. Thus, MAST diversity seems to be highly partitioned in spatial scales. Altogether, our results shed light on these ecologically very important but poorly known groups of uncultured marine flagellates.     

Distribution patterns of heterotrophic flagellates and bacteria in acidic and neutral Karelian lakes

The abundance and taxonomic and trophic structures of the community of planktonic heterotrophic nanoflagellates, as well as the quantitative distribution of bacteria as their main object of food, have been studied in six lakes of Karelia with differing pH values, concentrations and compositions of organic substances, and degrees of anthropogenic load. Thirty-eight species and forms of colorless flagellates from seven orders and groups of uncertain taxonomic statuses have been recorded. Most of them belong to the trophic group of bacteriodetritophages. The minimal abundance and the lowest diversity of flagellates have been documented in clear- and brown-water lakes with low pH values, and maximal values have been observed in the polluted Kondopoga Bay in Lake Onega.     

The structure of extrusive organelles in alveolate and other heterotrophic flagellates

The structure of ejective organelles (extrusomes) in 12 species of heterotrophic flagellates belonging to 8 taxonomic groups is considered. Trichocysts of various structures have been found in colpodellids, Spumella and Metromonas. Kinetocysts of amoeboid flagellates, thaumatomonads, contain a cylindrical element. These kinetocysts are attached to bacteria after discharge. When being discharged, trichocysts of colpodellids produce filaments which have transversal striation. Discobolocysts have been found in excavate flagellate Reclinomonas, and ejectisomes--in Goniomonas. Toxicysts have been marked in carnivorous alveolate flagellate Colponema. The most of studied extrusomes lie inside the cytoplasm, while exstrusomes in chrysomonad Spumella are also found inside the nucleus. Trichocysts of colpodellids and Metromonas as well as ejectisomes of Goniomonas are located near the branches of Golgi apparatus. The comparison of the data obtained confirms the hypothesis about the correlation of taxonomic position of flagellates and the structure of their extrusomes that allows in some cases using these features as phylogenetic markers.     

Species diversity of heterotrophic flagellates in White Sea littoral sites

The species diversity and distribution of benthic heterotrophic flagellates in sediment samples from along the salinity gradient in the Chernaya River Estuary and from Velikaya Salma Strait (Kandalaksha Bay, the White Sea) were investigated during August 2004. One hundred and six taxa have been identified by means of phase and interference contrast light microscopy and transmission electron microscopy. The majority of observed flagellates were bacterivores. The species diversity of the following groups: choanoflagellates, euglenids, kinetoplastids, bicosoecids, chrysomonads, thaumatomonads and flagellates Incertae sedis was the highest. Ancyromonas sigmoides and Petalomonas pusilla were the most common species. The species richness was lowest in the brackish water estuarine part with salinity levels between 5 per thousand and 8 per thousand. The distribution of heterotrophic flagellates conforms to the so-called "rule of critical salinity", possessing, apparently, the same universal character for organisms of different size levels. Heterotrophic flagellate communities in these littoral sites were highly heterogeneous. The curve of "cumulative species number vs. sampling effort" is well fitted by equation S=21.17N(0.50) and unsaturated, which indicates that more intensive investigations of the heterotrophic flagellates in the White Sea should be expected to reveal more species.     

自由生活的异养鞭毛虫多样性及生态功能

随着对水生态系统结构与功能的深入研究,自由生活的异养鞭毛虫在水生态系统中的作用成为当前生态学领域的热点之一.已有的研究结果表明,异养鞭毛虫物种丰富,摄食方式多样,周转迅速,是微食物环的重要成分,在水生态系统中起着十分重要的作用.异养鞭毛虫物种多样性及生态学方面的系统研究将有助于对水生态系统结构、功能和过程深入了解.综述了异养鞭毛虫多样性、群落结构、摄食生态学以及在氮、磷循环中的作用,分析了其在生态系统中的功能.     

Ecology of planktonic heterotrophic flagellates. A review.

In aquatic environments heterotrophic flagellates are an important component within the microbial loop and the food web, owing to their involvement in the energy transfer and flux and as an intermediate link between bacteria and primary producers, and greater organisms, such as other protists and metazoan consumers. In the microbial loop heterotrophic flagellates highly contribute to fast biomass and nutrient recycling and to the production in aquatic environments. In fact, these protists consume efficiently viruses, bacteria, cyanobacteria and picophytoplankton, and are grazed mainly by other protists, rotifers and small crustaceans. In this paper the knowledge about these unicellular organisms is reviewed, taking into particular account their ecological relationships and trophic role within the plankton community of marine and freshwater environments.     

Hydrological connectivity determining metacommunity structure of planktonic heterotrophic flagellates

Dispersal potential of organisms is directly related to the hydrological connectivity among habitats. We investigated if the relative role of the environmental and spatial components in structuring the heterotrophic flagellates depends on the degree of hydrological connectivity. Samplings were performed in aquatic environments of the Paraná River floodplain, which differ in their degree of connectivity: lotic environments, connected lakes and isolated lakes (temporarily connected to the main river only during floods). We expect that communities in isolated lakes would be more subject to dispersal limitation, while in connected lakes and lotic environments the communities would be regulated mainly by environmental variables (species sorting). We sampled in the pelagic region of 23 environments during the low water period in 2014. The results of pRDA revealed that the contributions of environmental and spatial factors differed between the types of environments. The greater contribution of the environmental variables in structuring the flagellates metacommunity, regardless of the type of environment, may be related to the elevated dispersal capacity of microorganisms. The spatial component was also significant in the isolated lakes. Our results support the idea that microorganism communities are mainly structured by environmental factors, although the spatial component seems important when lakes are isolated.     

Bacterivory by heterotrophic flagellates: community structure and feeding strategies

Heterotrophic flagellates (HF) are known as most important grazers of bacteria in many aquatic ecosystem. HF cannot be treated as a black box since HF generally contain a diverse community of species significantly differing in their feeding behaviour and other ecological properties. Today it seems that the dominant taxonomic groups among heterotrophic nano- and microflagellate communities within different marine, brackish and limnetic pelagic communities (heterokont taxa, dinoflagellates, choanoflagellates, kathablepharids) and benthic communities (euglenids, bodonids, thaumatomonads, apusomonads, cercomonads) are relatively similar. HF among protista incertae sedis, often neglected in ecological studies, are abundant bacterivores in all investigated habitats. Recent studies of flagellate feeding processes indicated that there are significant species-specific differences and individual variability regarding the food uptake and food selection of bacterivorous flagellates: Variability of bacterivory is discussed regarding the prevailing feeding modes, the energy budgets, the considerable importance of slight deviations in the time budgets of feeding phases, the ingestion rates and the feeding microhabitat, respectively. The significant flexibility of the grazing impact of bacterivorous flagellate communities creates a complex top-down pressure on bacteria which should have lead to the evolution of efficient predator avoidance mechanisms in bacteria and should be at least partly responsible for the diversity of present bacteria. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution of heterotrophic flagellates at the littoral of estuary of Chernaya River (Kandalaksha Bay, White Sea)

Species composition, distribution, and the character of structural changes in the heterotrophic flagellate community were studied along environmental gradients in the Chernaya River estuary. There were 99 species and forms of heterotrophic flagellates, subdivided into three groups: prevalently marine species and euryhaline species preferring biotopes either of higher or decreased salinity. The heterotrophic flagellate community of the estuary was continuously divided into two distinct variants: (1) cenosis of halophilic species, prevalently of sea forms and euryhaline species preferring biotopes of increased salinity; (2) cenosis of halophobic species with prevalence of euryhaline forms gravitating to fresh biotopes. The arbitrary and indistinct boundary between the variants of the community ran at a salinity of 9–10‰. The response of estuarine communities of heterotrophic flagellates and infusorians to variation of abiotic factors was similar and differed from response of communities of microphyto-, meiozoo-and macrozoobenthos; this implied similarity of the response mechanism to environmental factors in organisms of one level of organization.     

Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary

The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.     

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.     

Indirectly fluorescently labelled flagellates (IFLF): a tool to estimate the predation on free-living heterotrophic flagellates

A procedure was developed to estimate the direct grazing impacton free-living heterotrophic nanoflagellates (HNF). Culturedflagellates were labelled by feeding on brightly fluorescingbacteria (FLB) and then offered as indirectly fluorescentlylabelled flagellates (IFLF) to potential predators of HNF. Thenumber of FLB in the predators' food vacuoles could be convertedinto IFLF uptake and consumption of HNF. This new techniquewas used to study the HNF-ciliate relationship in the pelagiczone of Lake Constance. Three groups of ciliates were detectedas HNF grazers: small representatives of the genus Strobilidium.a small Haltena-like ciliate (probably Halteria grandinella)and a Codonella sp. Tintinnidium sp. group The ingestion ofHNF by these groups of ciliates ranged between 3 and 15, 3 and39, and 3 and 7 HNF ciliate^–1 h^–1; respectively.The IFLF method allows the direct determination of ingestedflagellate prey in the food vacuoles of their predators. Becauseindigenous living prey organisms were used, tracer discriminationcan be reduced.     

Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates

Heterotrophic flagellates are key components of all ecosystems. Understanding the patterns of biodiversity of these organisms is thus particularly important. Here we analyzed the intraspecific diversity of 10 morphospecies of heterotrophic flagellates comprising representatives of the Apusozoa (2 morphospecies) and Kinetoplastea (8 morphospecies), all belonging to the most common flagellates with worldwide distribution. Most morphospecies showed a mixing of lineages isolated from diverse habitats, indicating that some lineages of these morphospecies had been able to colonize different habitats several times. Furthermore, our results revealed remarkable levels of genetic divergence within most of the morphospecies studied, underlining the difficulty of correctly determining species by means of morphology alone. Many cryptic or pseudocryptic species seem to occur. Our results revealed clear divergence between marine and freshwater lineages of the morphospecies Ancyromonas sigmoides, showing that freshwater lineages have not been able to colonize marine environments and marine lineages have not been able to colonize freshwater environments for a long time.     

Taxonomic composition and biomass of heterotrophic flagellates in relation to lake trophy and season

1. Taxonomic composition and abundance of heterotrophic flagellates (HF) were studied in 55 lakes with different trophy in northern Germany using a live-counting technique.
2. Mean abundances and biomasses of HF ranged from 169 cells L⁻¹ and 22 μg L⁻¹ in mesotrophic lakes to 2439 cells mL^–1 and 475 μg L⁻¹ in hypertrophic lakes, respectively. Highest values were generally observed in spring, but mesotrophic lakes showed maximum values in early summer.
3. The taxonomic composition of HF was not significantly influenced by lake trophy and season. The major fraction of HF consisted of chrysomonads and Protista incertae sedis; other important groups were choanoflagellates and bicosoecids. The size distribution of HF changed with lake trophy and season, with a higher proportion of large HF (> 10 μm) in hypertrophic lakes and in spring, respectively.
4. Correlation analyses revealed a strong negative impact of cladocerans on total HF biomass and especially on large HF. Ciliates and large bacteria (> 10 μm) were strongly positively correlated with HF biomass; small bacteria (< 2 μm) showed a weak positive correlation.
5. Analyses at the level of species and genera revealed distinct distribution patterns of some taxa. Paraphysomonas , Aulacomonas and Quadricilia as large-bodied HF showed highest abundance in hypertrophic lakes and in spring. Attached taxa (e.g. Monosiga , Salpingoeca amphoridium ) were highly abundant in late summer and autumn, whereas Spumella and Kathablepharis occurred frequently in most samples.     

Global abundance of planktonic heterotrophic protists in the deep ocean

The dark ocean is one of the largest biomes on Earth, with critical roles in organic matter remineralization and global carbon sequestration. Despite its recognized importance, little is known about some key microbial players, such as the community of heterotrophic protists (HP), which are likely the main consumers of prokaryotic biomass. To investigate this microbial component at a global scale, we determined their abundance and biomass in deepwater column samples from the Malaspina 2010 circumnavigation using a combination of epifluorescence microscopy and flow cytometry. HP were ubiquitously found at all depths investigated down to 4000 m. HP abundances decreased with depth, from an average of 72±19 cells ml⁻¹ in mesopelagic waters down to 11±1 cells ml⁻¹ in bathypelagic waters, whereas their total biomass decreased from 280±46 to 50±14 pg C ml⁻¹. The parameters that better explained the variance of HP abundance were depth and prokaryote abundance, and to lesser extent oxygen concentration. The generally good correlation with prokaryotic abundance suggested active grazing of HP on prokaryotes. On a finer scale, the prokaryote:HP abundance ratio varied at a regional scale, and sites with the highest ratios exhibited a larger contribution of fungi molecular signal. Our study is a step forward towards determining the relationship between HP and their environment, unveiling their importance as players in the dark ocean''s microbial food web.     

Salinity tolerances of 62 strains of Pflesteria and Pfiesteria-like heterotrophic flagellates (Dinophyceae)

The salinity tolerance of 62 strains of Pfiesteria and Pfiesteria‐like heterotrophic dinoflagellates was measured. All strains were acclimated at 12 psu for at least 1 year before experimentation. Strains isolated from the Chesapeake Bay and Neuse River systems tolerated lower salinities than strains isolated from the Wilmington River system (P< 0.005). Swimming cells were still observed after 5 days at 0.5 psu for one strain, and at 1 psu for most other Chesapeake Bay and Neuse River strains. Swimming cells for the Wilmington River were still observed after 5 days at 3–5 psu, but no swimming cells were observed at ≤ 2 psu. With regard to the upper salinity tolerance, the Wilmington River strains tolerated higher salinities than the Chesapeake Bay and Neuse River systems (P< 0.005). Most Wilmington River strains were swimming after 5 days at salinities ≥ 50 psu, whereas the Chesapeake Bay and Neuse River system strains rarely had swimming cells at salinities exceeding 35–45 psu. For all three water systems and for both lower and higher salinities, cells apparently encysted in many instances. However, when salinities were returned to 12 psu, swimming cells often re‐appeared. Statistically significant geographic differences in salinity tolerance suggest a geographic adaptation has occurred and that salinity tolerance is under genetic control. The results also suggest there is diversity among the strains.     

Patterns of the quantitative distribution of plankton heterotrophic flagellates in the high-mountain Lake Sevan (Armenia)

Patterns of the quantitative development and spatial distribution of heterotrophic flagellates have been studied in Lake Sevan (Armenia). The abundance and biomass of flagellates is lower in summer than in autumn, but on the whole these parameters correspond to the quantitative parameters in oligotrophic lakes. Differences in the vertical and horizontal distribution of flagellates have been found during summer stratification and autumn homothermy.