Assessing morphological congruence in Dinobryon species and their stomatocysts, including a newly established Dinobryon pediforme-stomatocyst connection.

The chrysophyte genus Dinobryon Ehrenberg consists of 44 taxa, which occur in freshwaters, rarely marine waters, mostly in temperate regions of the world. The taxa of Dinobryon produce characteristic solitary or dendroid colonies and resting stages called stomatocysts. Only 20 Dinobryon taxa have information on produced stomatocysts and only four stomatocysts are reliably linked with vegetative stages using modern identification standards employing scanning electron microscopy (SEM) analyses. In this study, an encysted material of Dinobryon pediforme (Lemmermann) Steinecke was collected in two lakes in contrasting regions of Poland. Light microscopy (LM) and scanning electron microscopy (SEM) analyses revealed that Dinobryon pediforme produces stomatocyst #61, Piatek J. that is described here as new morphotype following the International Statospore Working Group (ISWG) guidelines. This raises to five the number of reliable links between vegetative stages of Dinobryon species and corresponding stomatocysts. Phenotypic similarities between Dinobryon species and their stomatocysts, analysed for five reliably established links, showed no relationships in size and shape between loricas and stomatocysts belonging to the same species. The morphological characters of loricas and stomatocysts mapped onto the phylogenetic tree of the five Dinobryon species revealed only little congruence between their morphology and phylogenetic relationships.

Speciation in protists: Spatial and ecological divergence processes cause rapid species diversification in a freshwater chrysophyte.

Although eukaryotic microorganisms are extremely numerous, diverse and essential to global ecosystem functioning, they are largely understudied by evolutionary biologists compared to multicellular macroscopic organisms. In particular, very little is known about the speciation mechanisms which may give rise to the diversity of microscopic eukaryotes. It was postulated that the enormous population sizes and ubiquitous distribution of these organisms could lead to a lack of population differentiation and therefore very low speciation rates. However, such assumptions have traditionally been based on morphospecies, which may not accurately reflect the true diversity, missing cryptic taxa. In this study, we aim to articulate the major diversification mechanisms leading to the contemporary molecular diversity by using a colonial freshwater flagellate, Synura sphagnicola, as an example. Phylogenetic analysis of five sequenced loci showed that S. sphagnicola differentiated into two morphologically distinct lineages approximately 15.4 million years ago, which further diverged into several evolutionarily recent haplotypes during the late Pleistocene. The most recent haplotypes are ecologically and biogeographically much more differentiated than the old lineages, presumably because of their persistent differentiation after the allopatric speciation events. Our study shows that in microbial eukaryotes, species diversification via the colonization of new geographical regions or ecological resources occurs much more readily than was previously thought. Consequently, divergence times of microorganisms in some lineages may be equivalent to the estimated times of speciation in plants and animals.

Change of volatile components in six microalgae with different growth phases.

BACKGROUND: Head space solid-phase microextraction-gas chromatography-mass spectrometry has been applied to analyze the volatile components of six marine microalgae (Thalassiosira weissflogii, Nitzschia closterium, Chaetoceros calcitrans, Platymonas helgolandica, Nannochloropsis spp. and Dicrateria inornata) from Bacillariophyta, Chlorophyta and Chrysophyta, respectively, in different growth phases. RESULTS: All volatile compounds were identified by database searching in the NIST08 Mass Spectral Library and analyzed by principal component analysis with SIMCA-P software (Umetrics, Umea, Sweden). The results clearly revealed that the volatile components of the six microalgae were significantly different in the exponential, stationary and declining phases. Aldehydes, alkanes, some esters and dimethyl sulfide significantly changed in different growth phases. CONCLUSION: This is the first report on the comprehensive characteristics of volatile components in different microalgae and in different growth phases. The results may provide reference data for studies on the flavor of cultivated aquatic organism, odor formation in nature water, choice of feeding period and microalgae species selection for the artificial rearing of marine organisms. © 2016 Society of Chemical Industry.

Effect of high pH on growth of Synechocystis sp. PCC 6803 cultures and their contamination by golden algae (Poterioochromonas sp.).

Culturing cyanobacteria in a highly alkaline environment is a possible strategy for controlling contamination by other organisms. Synechocystis PCC 6803 cells were grown in continuous cultures to assess their growth performance at different pH values. Light conversion efficiency linearly decreased with the increase in pH and ranged between 12.5 % (PAR) at pH 7.5 (optimal) and decreased to 8.9 % at pH 11.0. Photosynthetic activity, assessed by measuring both chlorophyll fluorescence and photosynthesis rate, was not much affected going from pH 7.5 to 11.0, while productivity, growth yield, and biomass yield on light energy declined by 32, 28, and 26 % respectively at pH 11.0. Biochemical composition of the biomass did not change much within pH 7 and 10, while when grown at pH 11.0, carbohydrate content increased by 33 % while lipid content decreased by about the same amount. Protein content remained almost constant (average 65.8 % of dry weight). Cultures maintained at pH above 11.0 could grow free of contaminants (protozoa and other competing microalgae belonging to the species of Poterioochromonas).

Iron uptake mechanism in the chrysophyte microalga Dinobryon.

The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.

Negative consequences of glacial turbidity for the survival of freshwater planktonic heterotrophic flagellates.

Heterotrophic (phagotrophic) flagellates are key components of planktonic food webs in freshwater and marine ecosystems because they are the main consumers of bacteria. Although they are ubiquitous in aquatic ecosystems, they were numerically undetectable in turbid glacier-fed lakes. Here we show that glacial particles had negative effects on the survival and growth of heterotrophic flagellates. The effect of glacial particles was concentration-dependent and was caused by their interference with bacterial uptake rather than by physical damage. These results are the first to reveal why establishment of heterotrophic flagellates populations is hindered in very turbid glacial lakes. Because glaciers are vanishing around the world, recently formed turbid meltwater lakes represent an excellent opportunity to understand the environmental conditions that probably shaped the establishment of lake communities at the end of the last glaciation.

Applicability of hydrogen peroxide in brown tide control - culture and microcosm studies.

Brown tide algal blooms, caused by the excessive growth of Aureococcus anophagefferens, recur in several northeastern US coastal bays. Direct bloom control could alleviate the ecological and economic damage associated with bloom outbreak. This paper explored the effectiveness and safety of natural chemical biocide hydrogen peroxide (H(2)O(2)) for brown tide bloom control. Culture studies showed that H(2)O(2) at 1.6 mg L(-1) effectively eradicated high density A. anophagefferens within 24-hr, but caused no significant growth inhibition in the diatoms, prymnesiophytes, green algae and dinoflagellates of >2-3 µm cell sizes among 12 phytoplankton species tested over 1-week observation. When applied to brown tide bloom prone natural seawater in a microcosm study, this treatment effectively removed the developing brown tide bloom, while the rest of phytoplankton assemblage (quantified via HPLC based marker pigment analyses), particularly the diatoms and green algae, experienced only transient suppression then recovered with total chlorophyll a exceeding that in the controls within 72-hr; cyanobacteria was not eradicated but was still reduced about 50% at 72-hr, as compared to the controls. The action of H(2)O(2) against phytoplankton as a function of cell size and cell wall structure, and a realistic scenario of H(2)O(2) application were discussed.

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing.

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator-prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.

[Phytoplankton assemblages in Yangtze River Estuary in the first sluice discharge duration of Three Gorges Dam in late spring].

In June 15-25, 2003, the first sluice discharge duration of the Three Gorges Dam, water samples were taken from a grid of survey stations around the Yangtze River Estuary and its adjacent waters to analyze the characteristics of phytoplankton assemblages. In the survey area, the major phytoplankton groups were diatoms and dinoflagellates, and a few species belonging to Chrysophyceae and Chlorophyceae were observed. The cell abundance of the assemblages ranged from 0.2 to 1504.2 cells ml(-1), with an average of 72.7 cells ml(-1), and the dominant species were Skeletonema spp., Prorocentrum dentatum, and Scrippsiella trochoidea. The highest cell abundance was appeared in north diluted waters and southwest inshore waters. The cell abundance was the maximum in mid (10 m) water layer, and the minimum in bottom layer. In diluted waters, Skeletonema spp. was the dominant species, and mainly presented in surface water layer; while in the waters with the diluted water of Yangtze River and the mixed water of Taiwan Warmer Current and Yellow Sea, Prorocentrum dentatum dominated, and mainly distributed in surface and mid water layers.

Utilizing the effective xanthophyll cycle for blooming of Ochromonas smithii and O. itoi (Chrysophyceae) on the snow surface.

Snow algae inhabit unique environments such as alpine and high latitudes, and can grow and bloom with visualizing on snow or glacier during spring-summer. The chrysophytes Ochromonas smithii and Ochromonas itoi are dominant in yellow-colored snow patches in mountainous heavy snow areas from late May to early June. It is considered to be effective utilizing the xanthophyll cycle and holding sunscreen pigments as protective system for snow algae blooming in the vulnerable environment such as low temperature and nutrients, and strong light, however the study on the photoprotection of chrysophytes snow algae has not been shown. To dissolve how the chrysophytes snow algae can grow and bloom under such an extreme environment, we studied with the object of light which is one point of significance to this problem. We collected the yellow snows and measured photosynthetically active radiation at Mt. Gassan in May 2008 when the bloom occurred, then tried to establish unialgal cultures of O. smithii and O. itoi, and examined their photosynthetic properties by a PAM chlorophyll fluorometer and analyzed the pigment compositions before and after illumination with high-light intensities to investigate the working xanthophyll cycle. This experimental study using unialgal cultures revealed that both O. smithii and O. itoi utilize only the efficient violaxanthin cycle for photoprotection as a dissipation system of surplus energy under prolonged high-light stress, although they possess chlorophyll c with diadinoxanthin.

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy.

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.

Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains.

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica, on strains of marine Synechococcus. Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas, 9 for Pteridomonas) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.

Feeding characteristics of a golden alga (Poterioochromonas sp.) grazing on toxic cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa has quickly risen in infamy as one of the most universal and toxic bloom-forming cyanobacteria. Here we presented a species of golden alga (Poterioochromonas sp. strain ZX1), which can feed on toxic M. aeruginosa without any adverse effects from the cyanotoxins. Using flow cytometry, the ingestion and maximal digestion rates were estimated to be 0.2 approximately 1.2 and 0.2 M. aeruginosa cells (ZX1 cell)(-1)h(-1), respectively. M. aeruginosa in densities below 10(7)cells mL(-1) could be grazed down by ZX1, but no significant decrease was observed when the initial density was 3.2 x 10(7)cells mL(-1). ZX1 grazing was a little influenced by the light intensity (0.5 approximately 2500l x) and initial pH of the medium (pH=5.0 approximately 9.5). ZX1 could not survive in continuous darkness for longer than 10 days. The pH value was adjusted to 8 by ZX1 while to 10 by M. aeruginosa. This study may shed light on understanding the ecological interactions between M. aeruginosa and mixotrophic Poterioochromonas sp. in aquatic ecosystems.

Reduced models of algae growth.

The simulation of biological systems is often plagued by a high level of noise in the data, as well as by models containing a large number of correlated parameters. As a result, the parameters are poorly identified by the data, and the reliability of the model predictions may be questionable. Bayesian sampling methods provide an avenue for proper statistical analysis in such situations. Nevertheless, simulations should employ models that, on the one hand, are reduced as much as possible, and, on the other hand, are still able to capture the essential features of the phenomena studied. Here, in the case of algae growth modeling, we show how a systematic model reduction can be done. The simplified model is analyzed from both theoretical and statistical points of view.

Effects of temperature on growth rate and gross growth efficiency of an Antarctic bacterivorous protist.

The effects of temperature on the growth rate and gross growth efficiency (GGE) of the heterotrophic nanoflagellate, Paraphysomonas imperforata, cultured from the Ross Sea, Antarctica were investigated using five experimental temperatures (range=0-20 degrees C). This bacterivorous protist exhibited measurable growth over the temperature range examined, although temperature exerted a significant effect on its growth rate. There was no evidence for an effect of temperature on GGE. The growth rates and GGE of our Antarctic P. imperforata isolate were compared to values reported for other cultures of species from this genus. A wide range of growth efficiencies have been reported for different strains of Paraphysomonas spp., but our estimates were comparable to mean/median values reported in the literature. The growth rates of our Antarctic P. imperforata were similar to rates obtained for an Arctic conspecific at low temperatures (0-5 degrees C), among the highest reported rates for any Paraphysomonas species at intermediate temperatures (10-15 degrees C) and similar to rates reported for temperate congeners and conspecifics at 20 degrees C. Q(10) values of 15, 2.2, 3.6 and 0.93 were calculated for growth rates at 5 degrees C intervals between 0 and 20 degrees C, respectively. Results indicated that our Antarctic P. imperforata grew at rates comparable to other polar isolates at ambient polar temperatures, but these low temperatures may be outside the physiological optimum for the isolate.

Effect of temperature and prey type on nutrient regeneration by an antarctic bacterivorous protist.

Experimental studies were carried out on an Antarctic isolate of the heterotrophic nanoflagellate Paraphysomonas imperforata to examine the efficiency of incorporation and remineralization of nitrogen and phosphorus from bacterial prey. Experiments were carried out over a temperature range from ambient Antarctic temperature (0 degrees C) to 10 degrees C. Temperature had a marked effect on the maximal growth rate of the phagotrophic nanoflagellate. Growth rate in the presence of high prey abundance ranged from 0.6 day(-1) at 0 degrees C to 2.6 day(-1) at 10 degrees C. In contrast, temperature had no discernable effect on the efficiencies of incorporation and remineralization of major nutrients by P. imperforata. The efficiencies of phosphorus and nitrogen incorporation from prey biomass averaged over the temperature range examined were 58 and 39%, respectively, for the two elements. Ammonium and phosphate were the dominant forms of dissolved nitrogen and phosphorus appearing in the culture medium during the grazing phase of the experiments. Overall, dissolved organic nitrogen and phosphorus constituted minor components of these elements released by the grazing activities of the protist. The results of this study indicated that incorporation/remineralization of nitrogen and phosphorus contained in prey was relatively unaffected by culture temperature in this heterotrophic nanoflagellate, although low temperature significantly depressed its growth rate. This finding has important implications for energy utilization and elemental cycling in perennially cold ecosystems and is at odds with conclusions that have been reached in some previous studies regarding the growth efficiency of phagotrophic Antarctic protists.

Differential thermal adaptation of clonal strains of a protist morphospecies originating from different climatic zones.

Eco-physiological variation and local adaptation are key issues in microbial ecology. Here, we investigated the thermal adaptation of 19 strains of the same Spumella morphospecies (Chrysophyceae, Heterokonta). In order to test for local adaptation and the existence of specific ecotypes we analysed growth rates of these strains, which originated from different climate regions. We applied temperature-adaptation as an eco-physiological marker and analysed growth rates of the different Spumella strains at temperatures between 0 degrees C and 35 degrees C. The temperatures allowing for maximal growth of strains from temperate and warm climatic zones ranged between 19.9 degrees C and 33.4 degrees C. Phylogenetically, most of these 'warm'-adapted strains fall into two different previously defined 18S rDNA Spumella clusters, one of them consisting of mostly soil organisms and the other one being a freshwater cluster. As a rule, the 'warm'-adapted strains of the soil cluster grew slower than the 'warm'-adapted isolates within the freshwater cluster. This difference most probably reflect different strategies, i.e. the formation of cysts at the expense of lower growth rates in soil organisms. In contrast, as expected, all isolates from Antarctica were cold-adapted and grew already around melting point of freshwater. Surprisingly, optimum temperature for these strains was between 11.8 degrees C and 17.7 degrees C and maximum temperature tolerated was between 14.6 degrees C and 23.5 degrees C. Our data indicate that despite the relatively high optimal temperature of most Antarctic strains, they may have a relative advantage below 5-10 degrees C only. Based on the thermal adaptation of the flagellate strains the Antarctic strains were clearly separated from the other investigated strains. This may indicate a limited dispersal of flagellates to and from Antarctica. Even if the latter assumption needs support from more data, we argue that the high levels of eco-physiological and molecular microdiversity indicate that the current species concepts do not sufficiently reflect protist eco-physiological differentiation.

Evidence for geographic isolation and signs of endemism within a protistan morphospecies.

The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of "Spumella-like" flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.

Energy--dependent bacterivory in Ochromonas minima--a strategy promoting the use of substitutable resources and survival at insufficient light supply.

Phagotrophy and competitive ability of the mixotrophic Ochromonas minima were investigated in a three-factorial experiment where light intensity (low: 1.0 micromol m(-2)s(-1) and high: 60 micromol m(-2)s(-1) PPFD), nutrient concentration (ambient: 7.0 micromolNl(-1), 0.11 micromol P l(-1) and enriched: 88 micromol N l(-1), 6.3 micro mol P l(-1)) and DOC supply (without and with enrichment, 250 micromol C l(-1)) were manipulated. Ochromonas minima and bacterial abundance were monitored for 12 days. We found significant and interacting effects of light and nutrients on Ochromonas minima growth rate and abundance. At high light intensity, nutrient enrichment resulted in increased growth rates and population sizes. In contrast, reduced growth rates and population sizes were observed for nutrient enrichment when light intensity was low. Although, Ochromonas minima was able to ingest bacteria under both high and low light conditions, it grew only when light intensity was high. At high light intensity, Ochromonas minima grew exponentially under nutrient conditions that would have been limiting for photoautotrophic microalgae. In non-enriched low light treatments, Ochromonas minima populations survived, probably by using background DOC as an energy source, indicating that this ability can be of relevance for natural systems even when DOC concentrations are relatively low. When competing with photoautotrophic microalgae, the ability to grow under severe nutrient limitation and to survive under light limitation should be advantageous for Ochromonas minima.