Exploration of an odorous aldehydes and ketones produced by Uroglena americana using high resolution mass spectrometry, GC-Olfactometry, and multivariate analysis.

Off-flavor events in tap water have been reported from various regions of Japan. Fishy smell is the second most common off-flavor in Japan and Uroglena americana (U. americana) is known to be a major contributor to the smell. However, the causative compound of the smell it produces still remains unrevealed to the best of our knowledge. In this study, an exploration of odorous aldehydes and ketones originating from U. americana was performed with a view to discovering a possible candidate substance of causative compounds. Environmental samples containing U. americana colony and cultured media with U. americana were analyzed with two high resolution mass spectrometers, one of them is coupled with liquid chromatography (LC-HRMS), and the other is with gas chromatography and a sniffing port (GC-O-HRMS). Multivariate analyses (MVA) were utilized to explore a compound that is likely to be odorous aldehydes or ketones with a reduced time of exploration. A combination of LC-HRMS and MVA resulted in the selection of one candidate substance and its formula was determined to be C13H20O3 on the basis of its accurate mass and natural isotopic pattern. The candidate substance underwent GC-O-HRMS analyses and milk-like smell was detected at around its retention time. Although the detected smell was different from fishy smell, it is expected that the fishy smell is caused by multiple compounds to which the candidate substance belongs. First generation product ion spectra of the candidate substance suggested that it contains a hydroxyl group, a cyclohexene ring, and a ketone moiety.

Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 µm × 19.2 µm × 4.2 µm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 µm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.

Factors Affecting the Mixotrophic Flagellate Poterioochromonas malhamensis Grazing on Chlorella Cells.

Grazing behaviour between protozoa and phytoplankton exists widely in planktonic ecosystems. Poterioochromonas malhamensis is a well-known and widespread mixotrophic flagellate, which is recognized to play an important role within marine and freshwater planktonic ecosystems and regarded as the greatest contamination threat for mass algal cultures of Chlorella. In this study, a comprehensive range of factors, including morphological characters, biochemical compositions, and specific growth rate of ten species or strains of Chlorella, were evaluated for their effect on the feeding ability of P. malhamensis, which was assessed by two parameters: the clearance rate of P. malhamensis on Chlorella spp. and the specific growth rate of P. malhamensis. The results showed that the clearance rate of P. malhamensis was negatively correlated with cell wall thickness and specific growth rate of Chlorella spp., while the specific growth rate of P. malhamensis was positively correlated with carbohydrate percentage and C/N ratio and negatively correlated with protein, lipid percentage, and nitrogen mass. In conclusion, the factors influencing feeding selectivity include not only the morphological character and chemical composition of Chlorella, but also its population dynamics. Our study provides useful insights into the key factors that affect the feeding selectivity of P. malhamensis and provides basic and constructive data to help in screening for grazing-resistant microalgae.

Control of Waterborne Human Viruses by Indigenous Bacteria and Protists Is Influenced by Temperature, Virus Type, and Microbial Species.

Human viruses are ubiquitous contaminants in surface waters, where they can persist over extended periods of time. Among the factors governing their environmental persistence, the control (removal or inactivation) by microorganisms remains poorly understood. Here, we determined the contribution of indigenous bacteria and protists to the decay of human viruses in surface waters. Incubation of echovirus 11 (E11) in freshwater from Lake Geneva and seawater from the Mediterranean Sea led to a 2.5-log10 reduction in the infectious virus concentration within 48 h at 22°C, whereas E11 was stable in sterile controls. The observed virus reduction was attributed to the action of both bacteria and protists in the biologically active matrices. The effect of microorganisms on viruses was temperature dependent, with a complete inhibition of microbial virus control in lake water at temperatures of ≤16°C. Among three protist isolates tested (Paraphysomonas sp., Uronema marinum, and Caecitellus paraparvulus), Caecitellus paraparvulus was particularly efficient at controlling E11 (2.1-log10 reduction over 4 days with an initial protist concentration of 103 cells ml-1). In addition, other viruses (human adenovirus type 2 and bacteriophage H6) exhibited different grazing kinetics than E11, indicating that the efficacy of antiviral action also depended on the type of virus. In conclusion, indigenous bacteria and protists in lake water and seawater can modulate the persistence of E11. These results pave the way for further research to understand how microorganisms control human viral pathogens in aquatic ecosystems and to exploit this process as a treatment solution to enhance microbial water safety.IMPORTANCE Waterborne human viruses can persist in the environment, causing a risk to human health over long periods of time. In this work, we demonstrate that in both freshwater and seawater environments, indigenous bacteria and protists can graze on waterborne viruses and thereby reduce their persistence. We furthermore demonstrate that the efficiency of the grazing process depends on temperature, virus type, and protist species. These findings may facilitate the design of biological methods for the disinfection of water and wastewater.

The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction.

Planktonic cyanobacteria belonging to the genus Synechococcus are ubiquitously distributed in marine and fresh waters, substantially contributing to total carbon fixation on a global scale. While their ecological relevance is acknowledged, increasing resolution in molecular techniques allows disentangling cyanobacteria's role at the micro-scale, where complex microbial interactions may drive the overall community assembly. The interplay between phylogenetically different Synechococcus clades and their associated bacterial communities can affect their ecological fate and susceptibility to protistan predation. In this study, we experimentally promoted different levels of ecological interaction by mixing two Synechococcus ribotypes (MW101C3 and LL) and their associated bacteria, with and without a nanoflagellate grazer (Poterioochromonas sp.) in laboratory cultures. The beta-diversity of the Synechococcus-associated microbiome in laboratory cultures indicated that the presence of the LL ribotype was the main factor determining community composition changes (41% of total variance), and prevailed over the effect of protistan predation (18% of total variance). Our outcomes also showed that species coexistence and predation may promote microbial diversity, thus highlighting the underrated ecological relevance of such micro-scale factors.

Some Mixotrophic Flagellate Species Selectively Graze on Archaea.

Many phototrophic flagellates ingest prokaryotes. This mixotrophic trait becomes a critical aspect of the microbial loop in planktonic food webs because of the typical high abundance of these flagellates. Our knowledge of their selective feeding upon different groups of prokaryotes, particularly under field conditions, is still quite limited. In this study, we investigated the feeding behavior of three species (Rhodomonas sp., Cryptomonas ovata, and Dinobryon cylindricum) via their food vacuole content in field populations of a high mountain lake. We used the catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol with probes specific for the domain Archaea and three groups of Eubacteria: Betaproteobacteria, Actinobacteria, and Cytophaga-Flavobacteria of Bacteroidetes Our results provide field evidence that contrasting selective feeding exists between coexisting mixotrophic flagellates under the same environmental conditions and that some prokaryotic groups may be preferentially impacted by phagotrophic pressure in aquatic microbial food webs. In our study, Archaea were the preferred prey, chiefly in the case of Rhodomonas sp., which rarely fed on any other prokaryotic group. In general, prey selection did not relate to prey size among the grazed groups. However, Actinobacteria, which were clearly avoided, mostly showed a size of <0.5 µm, markedly smaller than cells from the other groups. IMPORTANCE: That mixotrophic flagellates are not randomly feeding in the main prokaryotic groups under field conditions is a pioneer finding in species-specific behavior that paves the way for future studies according to this new paradigm. The particular case that Archaea were preferentially affected in the situation studied shows that phagotrophic pressure cannot be disregarded when considering the distribution of this group in freshwater oligotrophic systems.

Heterosides--compatible solutes occurring in prokaryotic and eukaryotic phototrophs.

The acclimation to osmotic and/or salt stress conditions induces an integrated response at different cellular levels. One acclimation strategy relies on the massive accumulation of low molecular mass compounds, so-called compatible solutes, to balance osmotic gradients and to directly protect critical macromolecules. Heterosides are compounds composed of a sugar and a polyol moiety that represent one chemical class of compatible solutes with interesting features. Well-investigated examples are glucosylglycerol, which is found in many cyanobacteria, and galactosylglycerols (floridoside and isofloridoside), which are accumulated by eukaryotic algae under salt stress conditions. Here, we review knowledge on physiology, biochemistry and genetics of heteroside accumulation in pro- and eukaryotic photoautotrophic organisms.

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.

A global perspective on marine photosynthetic picoeukaryote community structure.

A central goal in ecology is to understand the factors affecting the temporal dynamics and spatial distribution of microorganisms and the underlying processes causing differences in community structure and composition. However, little is known in this respect for photosynthetic picoeukaryotes (PPEs), algae that are now recognised as major players in marine CO2 fixation. Here, we analysed dot blot hybridisation and cloning-sequencing data, using the plastid-encoded 16S rRNA gene, from seven research cruises that encompassed all four ocean biomes. We provide insights into global abundance, α- and ß-diversity distribution and the environmental factors shaping PPE community structure and composition. At the class level, the most commonly encountered PPEs were Prymnesiophyceae and Chrysophyceae. These taxa displayed complementary distribution patterns, with peak abundances of Prymnesiophyceae and Chrysophyceae in waters of high (25:1) or low (12:1) nitrogen:phosphorus (N:P) ratio, respectively. Significant differences in phylogenetic composition of PPEs were demonstrated for higher taxonomic levels between ocean basins, using Unifrac analyses of clone library sequence data. Differences in composition were generally greater between basins (interbasins) than within a basin (intrabasin). These differences were primarily linked to taxonomic variation in the composition of Prymnesiophyceae and Prasinophyceae whereas Chrysophyceae were phylogenetically similar in all libraries. These data provide better knowledge of PPE community structure across the world ocean and are crucial in assessing their evolution and contribution to CO2 fixation, especially in the context of global climate change.

[Seasonal dynamics of algae species composition and biomass in the coastal ice of Kandalaksha Bay, the White Sea].

Ice algae were investigated in January - April in Velikaya Salma Sound (1997), Kandalaksha Inlet (2002), and Chupa Inlet (2003) of Kandalaksha Bay, the White Sea. In total, 146 taxa were found. By species number, diatoms predominated. The highest species richness was observed in Velikaya Salma Sound (108 taxa), the lowest--in Kandalarsha Inlet (51 taxa). Between the three regions, algae species composition differed significantly from each other (with similarity measured by Shimkevich-Simpson index being on the level of 0.61-0.63). Total ice algae biomass varied substantially over time and space. In all the regions it increased during ice season: in Velikaya Salma Sound from 0.08 to 4.10 mg C/m2 in Kandalaksha Inlet from 0.38 to 89 mg C/m2, in Chupa Inlet from 1.72 to 64.70 mg C/m2. Species composition of those algae contributing to biomass value to the greatest extent varied over time and space, within each region, and between the regions. Among dominating species not only ice-neritic algae (Nitzschia frigida, Pauliella taeniata, Entomoneis kjellmanii, E. paludosa, and others), but also neritic (Thalassiosira gravida, T. nordenskioeldii) and littoral ones (Amphora laevis and other) were registered. In Chupa Inlet, at sites affected by river discharge, freshwater cyanobacteria Gomphosphaeria lacustris and chlorophyte Ulothrix implexa dominated. In one case the dominating species was found to be snow algae Rhaphidonema nivale.

Life history response of Daphnia magna to a mixotrophic golden alga, Poterioochromonas sp., at different food levels.

The toxicity of Poterioochromonas to Daphnia magna was investigated at different food (Scenedesmus acutus) levels. Poterioochromonas alone of 0.4-20 mg C L(-1) was not acutely toxic to D. magna, but did not support D. magna growth, either. When fed mixed diets (2 mg C L(-1) in total), D. magna's survival and reproduction were significantly depressed when Poterioochromonas comprised above 50%, likely due to the inhibition of food ingestion. Large juveniles were less sensitive to poor quality food than neonates. Therefore, Poterioochromonas may affect D. magna living to various extents depending on its concentration, age structure of D. magna populations and availability of other food.

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.

Scent of danger: floc formation by a freshwater bacterium is induced by supernatants from a predator-prey coculture.

We investigated predator-prey interactions in a model system consisting of the bacterivorous flagellate Poterioochromonas sp. strain DS and the freshwater bacterium Sphingobium sp. strain Z007. This bacterial strain tends to form a subpopulation of grazing-resistant microscopic flocs, presumably by aggregation. Enhanced formation of such flocs could be demonstrated in static batch culture experiments in the presence of the predator. The ratio of aggregates to single cells reached >0.1 after 120 h of incubation in an oligotrophic growth medium. The inoculation of bacteria into supernatants from cocultures of bacteria and flagellates (grown in oligotrophic or in rich media) also resulted in a substantially higher level of floc formation than that in supernatants from bacterial monocultures only. After separation of supernatants on a C(18) cartridge, the aggregate-inducing activity could be assigned to the 50% aqueous methanolic fraction, and further separation of this bioactive fraction could be achieved by high-pressure liquid chromatography. These results strongly suggest the involvement of one or several chemical factors in the induction of floc formation by Sphingobium sp. strain Z007 that are possibly released into the surrounding medium by flagellate grazing.

Prymnesins: toxic metabolites of the golden alga, Prymnesium parvum Carter (Haptophyta).

Increasingly over the past century, seasonal fish kills associated with toxic blooms of Prymnesium parvum have devastated aquaculture and native fish, shellfish, and mollusk populations worldwide. Protracted blooms of P. parvum can result in major disturbances to the local ecology and extensive monetary losses. Toxicity of this alga is attributed to a collection of compounds known as prymnesins, which exhibit potent cytotoxic, hemolytic, neurotoxic and ichthyotoxic effects. These secondary metabolites are especially damaging to gill-breathing organisms and they are believed to interact directly with plasma membranes, compromising integrity by permitting ion leakage. Several factors appear to function in the activation and potency of prymnesins including salinity, pH, ion availability, and growth phase. Prymnesins may function as defense compounds to prevent herbivory and some investigations suggest that they have allelopathic roles. Since the last extensive review was published, two prymnesins have been chemically characterized and ongoing investigations are aimed at the purification and analysis of numerous other toxic metabolites from this alga. More information is needed to unravel the mechanisms of prymnesin synthesis and the significance of these metabolites. Such work should greatly improve our limited understanding of the physiology and biochemistry of P. parvum and how to mitigate its blooms.

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.

Direct effects of UV-B radiation on the freshwater heterotrophic nanoflagellate Paraphysomonas sp.

The formation of DNA photoproducts in organisms exposed to ambient levels of UV-B radiation can lead to death and/or reduced population growth in aquatic systems. Dependence on photoenzymatic repair to reverse DNA damage caused by UV-B radiation is demonstrated for Paraphysomonas sp., a member of a widely distributed genus of heterotrophic nanoflagellates. At 20 degrees C, Paraphysomonas sp. was exposed to a range of UV-B intensities encountered in natural systems. Populations of the flagellate survived and grew in a dose-dependent manner, but only when simultaneously exposed to photorepair radiation (PRR). In contrast, flagellates exposed to UV-B at 15 degrees C suffered 100% mortality except at the lowest UV-B level (with PRR) tested, which suggested a photorepair temperature optimum above 15 degrees C. After acute UV-B exposures, DNA damage (measured as the formation of pyrimidine dimers) was reduced only in organisms that underwent subsequent exposure to PRR. Populations kept in the dark after UV-B exposure maintained the initial levels of pyrimidine dimers. These results are the first to demonstrate the reliance of a heterotrophic flagellate on photoenzymatic DNA repair for survival from UV-B exposure.

Isolation of a Poterioochromonas capable of feeding on Microcystis aeruginosa and degrading microcystin-LR.

Algal blooms have become a worldwide issue recently, especially those comprised of toxic cyanobacteria. Grazers' predation of bloom-forming algae plays an important role in water clearing. In this study, a species of golden alga (strain ZX1), capable of feeding on the toxic cyanobacteria Microcystis aeruginosa, was isolated and identified as Poterioochromonas sp. (GenBank accession: EU586184) on the basis of morphological characteristics and 18s rRNA gene sequencing. Feeding experiments showed that ZX1 could clear high densities of M. aeruginosa (7.3 x 10(5)-4.3 x 10(6) cells mL(-1)) in a short time (40 h), with inhibition ratios higher than 99.9%. ZX1 grew during the feeding processes and achieved a maximum density of 10-20% of the initial M. aeruginosa density. Furthermore, this study is the first to report that ZX1 was able to degrade microcystin-LR (MC-LR) in cells of M. aeruginosa while digesting the whole cells, and that the degradation process was determined to be carried out inside the ZX1 cell. For a total MC-LR (intra- and extracellular) concentration of up to 114 microg L(-1), 82.7% was removed in 40 h. This study sheds light on the importance of golden alga in aquatic microbial ecosystems and in the natural transportation/transformation of MC-LR.

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.