Capturing diversity of marine heterotrophic protists: one cell at a time

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.     

High diversity and isolated distribution of aquatic heterotrophic protists in salars of the Atacama Desert at different salinities

The species richness of eukaryotes in the hypersaline environment is generally thought to be low. However, recent studies showed a high degree of phylogenetic novelty at these extreme conditions with variable chemical parameters. These findings call for a more thorough look into the species richness of hypersaline environments. In this study, various hypersaline lakes (salars, 1–348 PSU) as well as further aquatic ecosystems of northern Chile were investigated regarding diversity of heterotrophic protists by metabarcoding studies of surface water samples. Investigations of genotypes of 18S rRNA genes showed a unique community composition in nearly each salar and even among different microhabitats within one salar. The genotype distribution showed no clear connection to the composition of main ions at the sampling sites, but protist communities from similar salinity ranges (either hypersaline, hyposaline or mesosaline) clustered together regarding their OTU composition. Salars appeared to be fairly isolated systems with only little exchange of protist communities where evolutionary lineages could separately evolve.     

First glimpse into Lower Jurassic deep-sea biodiversity: in situ diversification and resilience against extinction

Owing to the assumed lack of deep-sea macrofossils older than the Late Cretaceous, very little is known about the geological history of deep-sea communities, and most inference-based hypotheses argue for repeated recolonizations of the deep sea from shelf habitats following major palaeoceanographic perturbations. We present a fossil deep-sea assemblage of echinoderms, gastropods, brachiopods and ostracods, from the Early Jurassic of the Glasenbach Gorge, Austria, which includes the oldest known representatives of a number of extant deep-sea groups, and thus implies that in situ diversification, in contrast to immigration from shelf habitats, played a much greater role in shaping modern deep-sea biodiversity than previously thought. A comparison with coeval shelf assemblages reveals that, at least in some of the analysed groups, significantly more extant families/superfamilies have endured in the deep sea since the Early Jurassic than in the shelf seas, which suggests that deep-sea biota are more resilient against extinction than shallow-water ones. In addition, a number of extant deep-sea families/superfamilies found in the Glasenbach assemblage lack post-Jurassic shelf occurrences, implying that if there was a complete extinction of the deep-sea fauna followed by replacement from the shelf, it must have happened before the Late Jurassic.     

A glimpse of the diversity of complex polysaccharide-degrading culturable bacteria from Kongsfjorden,Arctic Ocean

Cold-adapted, complex polysaccharide-degrading marine bacteria have important implications in biogeochemical processes and biotechnological applications. Bacteria capable of degrading complex polysaccharide substrates, mainly starch, have been isolated from various cold environments, such as sea ice, glaciers, subglacial lakes, and marine sediments. However, the total diversity of polysaccharide-degrading culturable bacteria in Kongsfjorden, Arctic Ocean, remains unexplored. In the study reported here, we tested 215 cold-adapted heterotrophic bacterial cultures (incubated at 4 and 20 °C, respectively) isolated from Kongsfjorden, for the production of cold-active extracellular polysaccharide-degrading enzymes, including amylase, pectinase, alginase, xylanase, and carboxymethyl (CM)-cellulase. Our results show that 52 and 41% of the bacterial isolates tested positive for extracellular enzyme activities at 4 and 20 °C, respectively. A large fraction of the bacterial isolates (37% of the positive isolates) showed multiple extracellular enzyme activities. Alginase and pectinase were the most predominantly active enzymes, followed by amylase, xylanase, and CM-cellulase. All isolates which tested positive for extracellular enzyme activities were affiliated to microbial class Gammaproteobacteria. The four genera with the highest number of isolates were Pseudomonas, followed by Psychrobacter, Pseudoalteromonas, and Shewanella. The prevalence of complex polysaccharide-degrading enzymes among the isolates indicates the availability of complex polysaccharide substrates in the Kongsfjorden, likely as a result of glacial melting and/or macroalgal load. In addition, the observed high functional/phenotypic diversity in terms of extracellular enzyme activities within the bacterial genera indicates a role in regulating carbon/carbohydrate turnover in the Kongsfjorden, especially by reducing recalcitrance.     

The diversity and distribution of heterotrophic nannoflagellates in the eutrophic Lake Nero

The fauna and abundance of heterotrophic nannoflagellates (HNF) and the quantitative distribution of their main food items (bacteria) have been studied in the highly eutrophic Lake Nero (Yaroslavskaya oblast). A total of 70 HNF species and forms were found, with representatives of the order Choanoflagellida dominant. Abundances of HNF and bacteria were high, reaching levels typical for productive waters. The pattern of HNF seasonal dynamics was characterized by two peaks in June and September and one low in August. The minimal levels of HNF coincided with the peak of bacterioplankton abundance.     

The candidate phylum 'Termite Group 1' of bacteria: phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists

The candidate phylum 'Termite Group 1' (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.     

A glimpse into the epigenetic landscape of gene regulation

Post-translational modifications to histone proteins and methylation of DNA comprise the epigenome of a cell. The epigenome, which changes through development, controls access to our genes. Recent advances in DNA sequencing technology has led to genome-wide distribution data for a limited number of histone modifications in mammalian stem cells and some differentiated lineages. These studies reveal predictive correlations between histone modifications, different classes of gene and chromosomal features. Moreover, this glimpse into our epigenome challenges current ideas about regulation of gene expression. Many genes in stem cells are poised for expression with initiated RNA polymerase II at the promoter. This state is maintained by an epigenetic mark through multiple lineages until the gene is expressed.     

Last of the human protists: the phylogeny and genetic diversity of Iodamoeba

Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.     

On the allelochemical potency of the marine dinoflagellate Alexandrium ostenfeldii against heterotrophic and autotrophic protists

Three strains of the marine dinoflagellate Alexandrium ostenfeldiiof different geographic origin were tested for their short-termdeleterious effects on a diversity of marine protists. All A.ostenfeldii strains were capable of eliciting an apparent allelochemicalresponse, but the various protistan target species were differentiallyaffected. Protists that were negatively affected by exposureto cells of A. ostenfeldii and associated extracellular metabolitescomprised both autotrophs (Rhodomonas sp., Dunaliella salina,Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina,Amphidinium crassum, Rimostrombidium caudatum). Observed effectsincluded immobilisation (e.g. of O. marina), morphological changes(e.g. in D. salina) and/or aberrant behaviour (e.g. of R. caudatum),mainly as preliminary stages of cell lysis. Immobilization andlytic effects against O. marina were strongly dependent on A.ostenfeldii cell concentrations. Effects also differed substantiallyamong strains and different batch cultures of the same strain.Values of EC₅₀, defined as the A. ostenfeldii cell concentrationcausing lysis of 50% of O. marina cells, ranged from 0.3 to1.9 x 10³ mL^–1, depending on the A. ostenfeldii strain.The autotrophic dinoflagellate Scrippsiella trochoidea reactedto exposure to A. ostenfeldii cells by formation of temporary(ecdysal) cysts, whereas, in contrast, the flagellates Emilianiahuxleyi and Prymnesium parvum and the ciliate Strombidium sp.were relatively refractory or even unaffected. As long as cellsdid not lyse, the fluorescence yield of target autotrophs, estimatedby pulse-amplitude modulation fluorometry, did not significantlychange during the first 3 h of incubation, suggesting that allelochemicalsproduced by A. ostenfeldii caused no short-term negative effectson the photosynthetic apparatus. Overall, the allelochemicalresponses of target species showed no obvious relationship tocell quota or extracellular concentrations of either toxic macrocyclicimines (spirolides) or tetrahydropurine neurotoxins (saxitoxinand analogues) produced by various strains of A. ostenfeldii.Instead, the potency of A. ostenfeldii, eliciting immobilizationand lytic species-specific responses in potential predatorsand competitors, is consistent with the existence of an allelochemicalmechanism unrelated to the bioactivity of known phycotoxinsof the genus Alexandrium.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Cow behaviour on a new grooved floor in comparison with a slatted floor, taking claw health and floor properties into account

The objective of this study was to compare the behaviour of cows on a grooved floor with that of cows kept on a slatted floor. The trial was carried out with two groups of 12 Holstein-Friesian cows kept in a cowshed with two symmetrical halves, identical except for the floor. One floor was grooved longitudinally to the feeding fence (width of grooves 35mm) and the other was slatted (gaps 35mm wide) perpendicular to the feeding fence. Both floors had scrapers to remove manure. After 3 weeks of being kept on these two floors, cows were switched between floors for 3 weeks. In the third week of each 3-week-period, behavioural observations of cows related to their time budget over 24h, relocation on each floor indicated by index of movement and specific behaviours (aggression, self maintenance) performed on the floors were executed. The health of claws was examined before the trial and 6 weeks later, after the trial. The grooved floor influenced the cows' daily time budget: cows kept on the grooved floor stood less (P<0.05) with four legs inside the cubicles (group 1: 36min, group 2: 39min) than cows kept on the slatted floor (group 1: 57min, group 2: 60min). Neither the specific behaviours of cows nor their movement performed on both floors were different. After switching from the grooved floor to the slatted floor, cows lay for 669min a day (in comparison to 746min a day while kept on the grooved floor, P<0.05) and they stood parallel to the feeding fence for 174min a day (in comparison to 126min a day while kept on the grooved floor, P<0.05). Given that both groups of cows on the grooved floor and the group that began on the slatted floor had a similar daily time budget, it is possible that the different time budget of the remaining group, which started off on the grooved floor, was a reaction (pleasure or disappointment) induced by returning to the familiar floor. The grooved floor was more fouled with faeces (P<0.05) than the slatted floor. The grooved floor can be evaluated as being equal to the slatted floor with a scraper in terms of the behaviour performed on it. There were hardly any slip incidents on it (during 64h of observations, two slip incidents on the grooved floor, four slip incidents on the slatted floor). However, the occurrence of stumble incidents involving the manure scraper (66 cases on the grooved floor and 48 on the slatted floor during 64h of observations) and the occurrence of foot lesions (probably of traumatic origin) suggests that the functioning of the manure scraper, which is indispensable on grooved floors, needs to be optimised.     

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.     

Distribution and diversity of aquatic protists: an evolutionary and ecological perspective

Assessment of the distribution and diversity of free-living protists is currently hampered by a limited taxonomic resolution of major phyla and by neglecting the significance of spatial and temporal scaling for speciation. There is a tremendous physiological and ecological diversity that is hidden at the morphological level and not apparent at the level of conserved genes. A conceptual framework linking the various levels of diversity is lacking. Neutral genetic markers are useful indicators of population structure and gene flow between populations, but do not explain adaptation to local habitat conditions. The correspondence between protein-coding genes, ecophysiological performance, and fitness needs to be explored under natural conditions. The area and the associated typical temporal dimension of active cells (their ‘home range’) are much smaller, respectively shorter, than the area and time period potentially covered during passive dispersal of protist resting stages. The assumptions that dispersal rates are generally high in free-living protists and that extinction of local populations is, therefore, infinitesimally small wait rigorous testing. Gene flow may be uncoupled largely from dispersal, because local adaptation and numerical effects of residents may strongly reduce or even prevent successful invasion (immigration). The significance of clonal selection depends on the as yet unknown frequency and timing of sexual reproduction, and on the stability of the environment. The extent of local adaptation and the fitness-related ecophysiological divergence are critical for the speciation process and, hence, for defining protist species. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.