Tracking a century of changes in microbial eukaryotic diversity in lakes driven by nutrient enrichment and climate warming.

High-throughput sequencing of sedimentary DNA (sed-DNA) was utilized to reconstruct the temporal dynamics of microbial eukaryotic communities (MECs) at a centennial scale in two re-oligotrophicated lakes that were exposed to different levels of phosphorus enrichment. The temporal changes within the MECs were expressed in terms of richness, composition and community structure to investigate their relationships with two key forcing factors (i.e., nutrient enrichment and climate warming). Various groups, including Apicomplexa, Cercozoa, Chrysophyceae, Ciliophora, Chlorophyceae and Dinophyceae, responded to phosphorus enrichment levels with either positive or negative impacts on their richness and relative abundance. For both lakes, statistical modelling demonstrated that phosphorus concentration ([P]) was a dominant contributor to MECs modifications before the 1980s; after the mid-80s, the contribution of air temperature changes increased and potentially surpassed the contribution of [P]. Co-occurrence network analysis revealed that some clusters of taxa (i.e., modules) composed mainly of Dinophyceae and unclassified Alveolata were strongly correlated to air temperature in both lakes. Overall, our data showed that sed-DNA constitutes a precious archive of information on past biodiversity changes, allowing the study of the dynamics of numerous eukaryotic groups that were not traditionally considered in paleo-reconstructions.

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.

Temperature-dependent phagotrophy and phototrophy in a mixotrophic chrysophyte.

The roles of temperature and light on grazing and photosynthesis were examined for Dinobryon sociale, a common freshwater mixotrophic alga. Photosynthetic rate was determined for D. sociale adapted to temperatures of 8, 12, 16, and 20°C under photosynthetically active radiation light irradiances of 25, 66, and 130 µmol photons · m(-2)  · s(-1) , with concurrent measurement of bacterial ingestion at all temperatures under medium and high light (66 and 130 µmol photons · m(-2)  · s(-1) ). Rates of ingestion and photosynthesis increased with temperature to a maximum at 16°C under the two higher light regimes, and declined at 20°C. Although both light and temperature had a marked effect on photosynthesis, there was no significant difference in bacterivory at medium and high irradiances at any given temperature. At the lowest light condition (25 µmol photons · m(-2)  · s(-1) ), photosynthesis remained low and relatively stable at all temperatures. D. sociale acquired the majority of carbon from photosynthesis, although the low photosynthetic rate without a concurrent decline in feeding rate at 8°C suggested 20%-30% of the carbon budget could be attributed to bacterivory at low temperatures. Grazing experiments in nutrient-modified media revealed that this mixotroph had increased ingestion rates when either dissolved nitrogen or phosphorus was decreased. This work increases our understanding of environmental effects on mixotrophic nutrition. Although the influence of abiotic factors on phagotrophy and phototrophy in pure heterotrophs and phototrophs has been well studied, much less is known for mixotrophic organisms.

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.

Characterization and antioxidant activities of degraded polysaccharides from two marine Chrysophyta.

Water-soluble polysaccharides from Pavlova viridis and Sarcinochrysis marina Geitler (P0 and S0, respectively) and their degradation fragments (P1, P2, S1, S2 and S3) were screened for their antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl-radical (OH) scavenging, lipid peroxidation (LPO) inhibition and the mouse red blood cells (RBCs) hemolysis assay. The physicochemical properties of the polysaccharides were also determined. Chemical analysis showed the presence of sulfate groups and uronic acids. Degradation increased the sulfate group content, but also, in part, damaged the uronic acids. FTIR spectroscopy showed that P0 and S0 had ß-pyranose and α-pyranose configurations, respectively. The low molecular weight fragments after degradation exhibited higher antioxidant capacities, of which P2 and S3 showed the strongest antioxidant activity in the given assay system. The half-maximal inhibitory concentration (IC50) values of P2 on DPPH, OH, LPO and RBCs hemolysis assays were 0.45, 0.42, 0.88, and 1.51 mg/ml, respectively, and the corresponding IC50 values of S3 were 0.41, 0.41, 0.79, and 1.04 mg/ml, respectively. All the polysaccharide fragments evoked a significant dose dependent inhibitory effect or scavenging ability. Altogether, these results suggest that the polysaccharide of two marine Chrysophyta could be considered as a potential antioxidant.

Impact of protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea pelagic oxic/anoxic interfaces.

Barrier zones between oxic and anoxic water masses (redoxclines) host highly active prokaryotic communities with important roles in biogeochemical cycling. In Baltic Sea pelagic redoxclines, Epsilonproteobacteria of the genus Sulfurimonas (subgroup GD17) have been shown to dominate chemoautotrophic denitrification. However, little is known on the loss processes affecting this prokaryotic group. In the present study, the protist grazing impact on the Sulfurimonas subgroup GD17 was determined for suboxic and oxygen/hydrogen sulphide interface depths of Baltic Sea redoxclines, using predator exclusion assays and bacterial amendment with the cultured representative 'Sulfurimonas gotlandica' strain GD1. Additionally, the principal bacterivores were identified by RNA-Stable Isotope Probing (RNA-SIP). The natural Sulfurimonas subgroup GD17 population grew strongly under oxygen/hydrogen sulphide interface conditions (doubling time: 1-1.5 days), but protist grazing could consume the complete new cell production per day. In suboxic samples, little or no growth of Sulfurimonas subgroup GD17 was observed. RNA-SIP identified five active grazers, belonging to typical redoxcline ciliates (Oligohymenophorea, Prostomatea) and globally widespread marine flagellate groups (MAST-4, Chrysophyta, Cercozoa). Overall, we demonstrate for the first time that protist grazing can control the growth, and potentially the vertical distribution, of a chemolithoautotrophic key-player of oxic/anoxic interfaces.

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.

Evidence for the occurrence of photorespiration in synurophyte algae.

The fluxes of CO(2) and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO(2,) only a slow drawdown to compensation concentrations of 26 µM for T. volvocina and 18 µM for M. papillosa, when O(2) evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO(2), indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO(2) was observed similar to the post-illumination burst characteristic of C(3) higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO(2) burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O(2). This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: photosynthetic O(2) evolution rates in all four algae, measured by O(2) electrode, were significantly higher (40-50%) in media at low O(2) (4%) than in air-equilibrated (21% O(2)) media, indicating an O(2) inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species.

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.

Intracellular uptake: a possible mechanism for silver engineered nanoparticle toxicity to a freshwater alga Ochromonas danica.

The behavior and toxicity of silver engineered nanoparticles (Ag-ENs) to the mixotrophic freshwater alga Ochromonas danica were examined in the present study to determine whether any other mechanisms are involved in their algal toxicity besides Ag(+) liberation outside the cells. Despite their good dispersability, the Ag-ENs were found to continuously aggregate and dissolve rapidly. When the initial nanoparticle concentration was lower than 10 µM, the total dissolved Ag(+) concentration ([Ag(+)](T)) in the suspending media reached its maximum after 1 d and then decreased suggesting that Ag(+) release might be limited by the nanoparticle surface area under these conditions. Furthermore, Ag-EN dissolution extent remarkably increased in the presence of glutathione. In the Ag-EN toxicity experiment, glutathione was also used to eliminate the indirect effects of Ag(+) that was released. However, remarkable toxicity was still observed although the free Ag(+) concentration in the media was orders of magnitude lower than the non-observed effect concentration of Ag(+) itself. Such inhibitive effects were mitigated when more glutathione was added, but could never be completely eliminated. Most importantly, we demonstrate, for the first time, that Ag-ENs can be taken in and accumulated inside the algal cells, where they exerted their toxic effects. Therefore, nanoparticle internalization may be an alternative pathway through which algal growth can be influenced.

Life history responses of Daphnia magna feeding on toxic Microcystis aeruginosa alone and mixed with a mixotrophic Poterioochromonas species.

The toxic effects of a mixotrophic golden alga (Poterioochromonas sp. strain ZX1) and a cyanobacterium Microcystis aeruginosa grazed by Poterioochromonas to a cladoceran were investigated through life history experiments using Daphnia magna. Poterioochromonas cultured in two ways (fed M. aeruginosa in an inorganic medium or grown in an organic medium) both induced starvation-like effects on D. magna, indicating that Poterioochromonas is neither acutely toxic nor a good food for D. magna. Despite a microcystin-LR content of 10(-8)microgcell(-1) in M. aeruginosa, no toxins were accumulated in Poterioochromonas fed the cyanobacterium. The toxic effect of M. aeruginosa to D. magna was significantly reduced in the presence of Poterioochromonas, which may be performed in two ways: decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; and decrease the toxicity of the medium by degrading the toxins released by M. aeruginosa. This study provides new information on the interactions between a cyanobacterium and its grazer under laboratory conditions and may increase our understanding of the ecological significance of such interactions in the aquatic food webs.

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.

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.

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.

Heavy metal toxicity and bioavailability of dissolved nutrients to a bacterivorous flagellate are linked to suspended particle physical properties.

Many dissolved substances attach easily to sediment particles. In the presence of suspended sediments bioavailability of dissolved substances is therefore, usually reduced and clays are even applied to "wash" natural waters upon pollution. In organisms which feed on food organisms in the size range of these suspended sediment particles, however, bioavailability of such substances may even increase. For microorganisms the interaction with dissolved substances and suspended sediment particles so far has hardly been investigated. We specifically tested: (1) the importance of suspended particles as an uptake route for dissolved substances; and (2) the significance of particle surface properties, i.e. surface load and mineralogy. As a model system we used an axenically cultured strain of a widespread and often abundant flagellate ("Spumella-like" flagellate strain JBM10). We tested the toxicity of cadmium (II) and mercury (II) as well as availability of dissolved organic matter (DOM) in the absence as well as in the presence of different natural clays, i.e. a kaolinite, a montmorillonite, and a mixed clay, and of artificial silicate particles of different surface charge. When applied separately the presence of the heavy metals cadmium and mercury as well as of suspended particles negatively affected the investigated flagellate but nutritive organics supported growth of the investigated flagellate. Toxic stress response comprises behavioral changes including enhanced swimming activity and stress egestion of ingested particles and was generally similar for a variety of different flagellate species. In combination with suspended particles, the respective effect of trace metals and nutritive substances decreased. Regarding the particle quality, cadmium toxicity increased with increasingly negative surface charge, i.e. increasing surface density of silanol groups (Pearson's product moment, P = 0.005). For mercury particle mineralogy still had a significant effect (P < 0.001) but surface load seems to play a minor role and for nutritive organics no significant effect of the investigated particle properties was found. We conclude that: (i) flagellates are as sensitive as higher animals to heavy metal pollution; (ii) suspended particles decrease bioavailability of dissolved substances and ingestion of suspended particles probably play a minor role as uptake route for dissolved substances; and (iii) suspended sediment particle properties, i.e. surface charge and mineralogy, are key factors for the interaction between microorganisms and dissolved substances in the presence of suspended sediments.

Cyanobacterial blooms and the presence of cyanotoxins in small high altitude tropical headwater reservoirs in Kenya.

The phytoplankton community in three small (0.065-0.249 km2) reservoirs in the stepped plateau landscape in the Kinangop area above the Rift Valley floor in Kenya were studied between 1998 and 2000. Approximately 70 species of phytoplankton were identified. The community was dominated by chlorophytes, cyanobacteria and chrysophytes. Diatoms were rare. The phytoplankton assemblage was frequently dominated by cyanobacteria in the dry season. The phytoplankton assemblage transformed to a mixture of cyanobacteria, chlorophytes and chrysophytes at the onset of the long rains, and mixture of cyanobacteria and chlorophytes after the long rains. Thereafter the phytoplankton assemblage consisted mainly of a mix of cyanobacteria and chrysophytes until the onset of the short rains when cyanobacterial dominance re-emerged. The most common phytoplankton species included Microcystis spp., Botryococcus braunii, Ceratium hirundinella, Anabaena spp. and Euglena viridis. The dry season cyanobacterial blooms produced cyanotoxins that included microcystin and endotoxins. The concentrations were well above the recommended safe limits for drinking water. The patterns of cyanotoxin production showed that the growth of the toxin-producing cyanobacteria was regulated by water temperature, pH and nutrients. The appearance of cyanotoxins in the small reservoirs is a serious public health issue in rural Kenya because such reservoirs are key sources of water for humans, livestock and wildlife.

Community structure and photosynthetic activity of epilithon from a highly acidic (pH < or = 2) mountain stream in Patagonia, Argentina.

We explored a benthic community living on stones in an acidic (pH< or =2) stream of active volcanic origin from Patagonia, Argentina, by combining in situ measurements (temperature, pH, conductivity, dissolved oxygen), photosynthesis of intact biofilms (measured with microsensors by the light-dark shift method), pure-culture experiments on isolated algae, and confocal laser scanning microscopy on the biofilms. The epilithon of the Agrio River was dominated (99% of total biomass) by one species: Gloeochrysis (Chrysophyceae). This species was observed as brown, mucilaginous, 200-microm-thick films on stones, growing in clumps in a dense matrix of fungal hyphae, bacteria, and inorganic particles held together by extracellular polymeric substances. Gloeochrysis was isolated and cultivated. The photosynthetic rate measured at saturation irradiance was 120 micromol oxygen (mg chlorophyll a)(-1) h(-1) under laboratory conditions, and the saturation rate of photosynthesis by carbon dioxide was 90 micromol oxygen (mg chlorophyll a)(-1) h(-1) for oxygen evolution. Photosynthetic activity of the biofilm was light-dependent and saturated above 200 micromol photons m(-2) s(-1). In the dark, the stone surface became anoxic. Our data suggest that primary production in the Agrio River was not limited by light, carbon, or phosphorus but instead, nitrogen-limited.