In situ associations between marine photosynthetic picoeukaryotes and potential parasites - a role for fungi?

Photosynthetic picoeukaryotes (PPEs) are important components of the marine picophytoplankton community playing a critical role in CO2 fixation but also as bacterivores, particularly in the oligotrophic gyres. Despite an increased interest in these organisms and an improved understanding of the genetic diversity of this group, we still know little of the environmental factors controlling the abundance of these organisms. Here, we investigated the quantitative importance of eukaryotic parasites in the free-living fraction as well as in associations with PPEs along a transect in the South Atlantic. Using tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH), we provide quantitative evidence of the occurrence of free-living fungi in open ocean marine systems, while the Perkinsozoa and Syndiniales parasites were not abundant in these waters. Using flow cytometric cell sorting of different PPE populations followed by a dual-labelled TSA-FISH approach, we also demonstrate fungal associations, potentially parasitic, occurring with both pico-Prymnesiophyceae and pico-Chrysophyceae. These data highlight the necessity for further work investigating the specific role of marine fungi as parasites of phytoplankton to improve understanding of carbon flow in marine ecosystems.

Resilience of SAR11 bacteria to rapid acidification in the high-latitude open ocean.

Ubiquitous SAR11 Alphaproteobacteria numerically dominate marine planktonic communities. Because they are excruciatingly difficult to cultivate, there is comparatively little known about their physiology and metabolic responses to long- and short-term environmental changes. As surface oceans take up anthropogenic, atmospheric CO2, the consequential process of ocean acidification could affect the global biogeochemical significance of SAR11. Shipping accidents or inadvertent release of chemicals from industrial plants can have strong short-term local effects on oceanic SAR11. This study investigated the effect of 2.5-fold acidification of seawater on the metabolism of SAR11 and other heterotrophic bacterioplankton along a natural temperature gradient crossing the North Atlantic Ocean, Norwegian and Greenland Seas. Uptake rates of the amino acid leucine by SAR11 cells as well as other bacterioplankton remained similar to controls despite an instant ∼50% increase in leucine bioavailability upon acidification. This high physiological resilience to acidification even without acclimation, suggests that open ocean dominant bacterioplankton are able to cope even with sudden and therefore more likely with long-term acidification effects.

Dominant oceanic bacteria secure phosphate using a large extracellular buffer.

The ubiquitous SAR11 and Prochlorococcus bacteria manage to maintain a sufficient supply of phosphate in phosphate-poor surface waters of the North Atlantic subtropical gyre. Furthermore, it seems that their phosphate uptake may counter-intuitively be lower in more productive tropical waters, as if their cellular demand for phosphate decreases there. By flow sorting (33)P-phosphate-pulsed (32)P-phosphate-chased cells, we demonstrate that both Prochlorococcus and SAR11 cells exploit an extracellular buffer of labile phosphate up to 5-40 times larger than the amount of phosphate required to replicate their chromosomes. Mathematical modelling is shown to support this conclusion. The fuller the buffer the slower the cellular uptake of phosphate, to the point that in phosphate-replete tropical waters, cells can saturate their buffer and their phosphate uptake becomes marginal. Hence, buffer stocking is a generic, growth-securing adaptation for SAR11 and Prochlorococcus bacteria, which lack internal reserves to reduce their dependency on bioavailable ambient phosphate.

Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition.

To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition.

Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean.

Nearly half of the Earth's surface is covered by the ocean populated by the most abundant photosynthetic organisms on the planet--Prochlorococcus cyanobacteria. However, in the oligotrophic open ocean, the majority of their cells in the top half of the photic layer have levels of photosynthetic pigmentation barely detectable by flow cytometry, suggesting low efficiency of CO2 fixation compared with other phytoplankton living in the same waters. To test the latter assumption, CO2 fixation rates of flow cytometrically sorted (14)C-labelled phytoplankton cells were directly compared in surface waters of the open Atlantic Ocean (30°S to 30°N). CO2 fixation rates of Prochlorococcus are at least 1.5-2.0 times higher than CO2 fixation rates of the smallest plastidic protists and Synechococcus cyanobacteria when normalised to photosynthetic pigmentation assessed using cellular red autofluorescence. Therefore, our data indicate that in oligotrophic oceanic surface waters, pigment minimisation allows Prochlorococcus cells to harvest plentiful sunlight more effectively than other phytoplankton.

In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy.

Heterotrophic bacterioplankton, cyanobacteria and phototrophic picoeukaryotes (< 5 µm in size) numerically dominate planktonic oceanic communities. While feeding on bacterioplankton is often attributed to aplastidic protists, recent evidence suggests that phototrophic picoeukaryotes could be important bacterivores. Here, we present direct visual evidence from the surface mixed layer of the Atlantic Ocean that bacterioplankton are internalized by phototrophic picoeukaryotes. In situ interactions of phototrophic picoeukaryotes and bacterioplankton (specifically Prochlorococcus cyanobacteria and the SAR11 clade) were investigated using a combination of flow cytometric cell sorting and dual tyramide signal amplification fluorescence in situ hybridization. Using this method, we observed plastidic Prymnesiophyceae and Chrysophyceae cells containing Prochlorococcus, and to a lesser extent SAR11 cells. These microscopic observations of in situ microbial trophic interactions demonstrate the frequency and likely selectivity of phototrophic picoeukaryote bacterivory in the surface mixed layer of both the North and South Atlantic subtropical gyres and adjacent equatorial region, broadening our views on the ecological role of the smallest oceanic plastidic protists.

Comparable light stimulation of organic nutrient uptake by SAR11 and Prochlorococcus in the North Atlantic subtropical gyre.

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic, that is, potentially enhance uptake of scarce organic molecules using the available solar radiation to energise appropriate transport systems. Here, we assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions using (33)P-ATP, (3)H-ATP and (35)S-methionine tracers. Light-stimulated uptake of these substrates was assessed in two dominant bacterioplankton groups discriminated by flow cytometric sorting of tracer-labelled cells and identified using catalysed reporter deposition fluorescence in situ hybridisation. One group of cells, encompassing 48% of all bacterioplankton, were identified as members of the SAR11 clade, whereas the other group (24% of all bacterioplankton) was Prochlorococcus. When exposed to light, SAR11 cells took 31% more ATP and 32% more methionine, whereas the Prochlorococcus cells took 33% more ATP and 34% more methionine. Other bacterioplankton did not demonstrate light stimulation. Thus, the SAR11 and Prochlorococcus groups, with distinctly different light-harvesting mechanisms, used light equally to enhance, by approximately one-third, the uptake of different types of organic molecules. Our findings indicate the significance of light-driven uptake of essential organic nutrients by the dominant bacterioplankton groups in the surface waters of one of the less productive, vast regions of the world's oceans-the oligotrophic North Atlantic subtropical gyre.

Mixotrophic basis of Atlantic oligotrophic ecosystems.

Oligotrophic subtropical gyres are the largest oceanic ecosystems, covering >40% of the Earth's surface. Unicellular cyanobacteria and the smallest algae (plastidic protists) dominate CO(2) fixation in these ecosystems, competing for dissolved inorganic nutrients. Here we present direct evidence from the surface mixed layer of the subtropical gyres and adjacent equatorial and temperate regions of the Atlantic Ocean, collected on three Atlantic Meridional Transect cruises on consecutive years, that bacterioplankton are fed on by plastidic and aplastidic protists at comparable rates. Rates of bacterivory were similar in the light and dark. Furthermore, because of their higher abundance, it is the plastidic protists, rather than the aplastidic forms, that control bacterivory in these waters. These findings change our basic understanding of food web function in the open ocean, because plastidic protists should now be considered as the main bacterivores as well as the main CO(2) fixers in the oligotrophic gyres.

Recovery of as-yet-uncultured soil acidobacteria on dilute solid media.

A growing number of Acidobacteria strains have been isolated from environments worldwide, with most isolates derived from acidic samples and affiliated with subdivision 1. We recovered 18 Acidobacteria strains from an alkaline soil, among which 11 belonged to the previously uncultured subdivision 6. Various medium formulations were tested for their effects on Acidobacteria growth.

Invariable biomass-specific primary production of taxonomically discrete picoeukaryote groups across the Atlantic Ocean.

Oceanic photosynthetic picoeukaryotes (< 3 µm) are responsible for > 40% of total primary production at low latitudes such as the North-Eastern tropical Atlantic. In the world ocean, warmed by climate changes, the expected gradual shift towards smaller primary producers could render the role of photosynthetic picoeukaryotes even more important than they are today. Little is still known, however, about how the taxonomic composition of this highly diverse group affects primary production at the basin scale. Here, we combined flow cytometric cell sorting, NaH¹4CO3 radiotracer incubations and class-specific fluorescence in situ hybridization (FISH) probes to determine cell- and biomass-specific inorganic carbon fixation rates and taxonomic composition of two major photosynthetic picoeukaryote groups on a ∼7500-km-long latitudinal transect across the Atlantic Ocean (Atlantic Meridional Transect, AMT19). We show that even though larger cells have, on average, cell-specific CO2 uptake rates ∼5 times higher than the smaller ones, the average biomass-specific uptake is statistically similar for both groups. On the other hand, even at a high taxonomic level, i.e. class, the contributions to both groups by Prymnesiophyceae, Chrysophyceae and Pelagophyceae are significantly different (P < 0.001 in all cases). We therefore conclude that these group's carbon fixation rates are independent of the taxonomic composition of photosynthetic picoeukaryotes across the Atlantic Ocean. Because the above applies across different oceanic regions the diversity changes seem to be a secondary factor determining primary production.

Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre.

The smallest phototrophic protists (<3 µm) are important primary producers in oligotrophic subtropical gyres - the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4-29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2-3 µm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition - predation on phosphorus-rich bacterioplankton cells.

Assessing amino acid uptake by phototrophic nanoflagellates in nonaxenic cultures using flow cytometric sorting.

Biologically available concentrations of individual dissolved amino acids in the open ocean are generally <1 nM. Despite this, the microbial turnover of amino acids is usually measured in hours indicating high demand. It is thought that the majority of uptake is due to bacterioplankton, although protists, particularly phototrophic protists, are also expected to take up amino acids. In order to assess the ability of protists to compete with prokaryotes for amino acids at subnanomolar concentrations, we examined the direct uptake of (3)H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to (3)H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of (3)H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved (3)H-leucine at natural oceanic concentrations. More practically, the flow-sorting technique allowed rapid and unequivocal differentiation of organic nitrogen uptake between prokaryotic cells and eukaryotic cells in mixed microbial populations, reducing the need to establish and maintain axenic algal cultures.

Changes in soil Acidobacteria communities after 2,4,6-trinitrotoluene contamination.

Despite their widespread occurrence in soils, the ecology of Acidobacteria and their response to environmental perturbations due to human activities remain very poorly documented. This study was aimed at assessing changes in the diversity and abundance of Acidobacteria in soils contaminated with 2,4,6-trinitrotoluene (TNT) compared with nonpolluted soils. The analysis of Acidobacteria communities at two sites with long-term and short-term contamination revealed that TNT has a drastic impact on the relative abundance of Acidobacteria in soil bacterial 16S rRNA gene libraries. The disappearance of most Acidobacteria from these soils was concomitant with a shift in Acidobacteria community composition and a loss of diversity, although the extent of diversity erosion depended on the sampling site.