Small pigmented eukaryote assemblages of the western tropical North Atlantic around the Amazon River plume during spring discharge.

Small pigmented eukaryotes (⩽ 5 µm) are an important, but overlooked component of global marine phytoplankton. The Amazon River plume delivers nutrients into the oligotrophic western tropical North Atlantic, shades the deeper waters, and drives the structure of microphytoplankton (> 20 µm) communities. For small pigmented eukaryotes, however, diversity and distribution in the region remain unknown, despite their significant contribution to open ocean primary production and other biogeochemical processes. To investigate how habitats created by the Amazon river plume shape small pigmented eukaryote communities, we used high-throughput sequencing of the 18S ribosomal RNA genes from up to five distinct small pigmented eukaryote cell populations, identified and sorted by flow cytometry. Small pigmented eukaryotes dominated small phytoplankton biomass across all habitat types, but the population abundances varied among stations resulting in a random distribution. Small pigmented eukaryote communities were consistently dominated by Chloropicophyceae (0.8-2 µm) and Bacillariophyceae (0.8-3.5 µm), accompanied by MOCH-5 at the surface or by Dinophyceae at the chlorophyll maximum. Taxonomic composition only displayed differences in the old plume core and at one of the plume margin stations. Such results reflect the dynamic interactions of the plume and offshore oceanic waters and suggest that the resident small pigmented eukaryote diversity was not strongly affected by habitat types at this time of the year.

Experimental identification and in silico prediction of bacterivory in green algae.

While algal phago-mixotrophs play a major role in aquatic microbial food webs, their diversity remains poorly understood. Recent studies have indicated several species of prasinophytes, early diverging green algae, to be able to consume bacteria for nutrition. To further explore the occurrence of phago-mixotrophy in green algae, we conducted feeding experiments with live fluorescently labeled bacteria stained with CellTracker Green CMFDA, heat-killed bacteria stained with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), and magnetic beads. Feeding was detected via microscopy and/or flow cytometry in five strains of prasinophytes when provided with live bacteria: Pterosperma cristatum NIES626, Pyramimonas parkeae CCMP726, Pyramimonas parkeae NIES254, Nephroselmis pyriformis RCC618, and Dolichomastix tenuilepis CCMP3274. No feeding was detected when heat-killed bacteria or magnetic beads were provided, suggesting a strong preference for live prey in the strains tested. In parallel to experimental assays, green algal bacterivory was investigated using a gene-based prediction model. The predictions agreed with the experimental results and suggested bacterivory potential in additional green algae. Our observations underline the likelihood of widespread occurrence of phago-mixotrophy among green algae, while additionally highlighting potential biases introduced when using prey proxy to evaluate bacterial ingestion by algal cells.

AFISsys - An autonomous instrument for the preservation of brackish water samples for microbial metatranscriptome analysis.

Microbial communities are the main drivers of biogeochemical cycling of multiple elements sustaining life in the ocean. The rapidity of their response to stressors and abrupt environmental changes implies that even fast and infrequent events can affect local transformations of organic matter and nutrients. Modern molecular techniques now allow for monitoring of microbial activities and functions in the environment through the analysis of genes and expressed genes contained in natural microbial assemblages. However, messenger RNA turnover in cells can be as short as 30 seconds and stability varies greatly between transcripts. Sampling of in situ communities involves an inevitable delay between the collection of seawater and the extraction of its RNA, leaving the bacterial communities plenty of time to alter their gene expression. The characteristics of microbial RNA turnover make time-series very difficult because samples need to be processed immediately to limit alterations to the metatranscriptomes. To address these challenges we designed an autonomous in situ fixation multi-sampler (AFISsys) for the reliable sampling of microbial metatranscriptomes at frequent intervals, for refined temporal resolution. To advance the development of this instrument, we examined the minimal seawater volume necessary for adequate coverage of community gene expression, and the suitability of phenol/ethanol fixation for immediate and long-term preservation of transcripts from a microbial community. We then evaluated the field eligibility of the instrument itself, with two case studies in a brackish system. AFISsys is able to collect, fix, and store water samples independently at a predefined temporal resolution. Phenol/ethanol fixation can conserve metatranscriptomes directly in the environment for up to a week, for later analysis in the laboratory. Thus, the AFISsys constitutes an invaluable tool for the integration of molecular functional analyses in environmental monitoring in brackish waters and in aquatic environments in general.

Mixotrophy in Chlorophytes and Haptophytes-Effect of Irradiance, Macronutrient, Micronutrient and Vitamin Limitation.

Chlorophytes and haptophytes are key contributors to global phytoplankton biomass and productivity. Mixotrophic bacterivory has been detected for both groups, but a shortage of studies with cultured representatives hinders a consistent picture of the ecological relevance and regulation of this trophic strategy. Here, the growth, primary production, fraction of feeding cells (acidotropic probes) and bacterivory rates (surrogate prey) are tested for two species of the chlorophyte genus Nephroselmis and the haptophyte Isochrysis galbana under contrasting regimes of light (high vs. low) and nutrients (non-limited and macronutrient-, micronutrient- and vitamin-limited), at low bacterial concentrations (<107 bacteria mL-1). All three species were obligate phototrophs, unable to compensate for low light conditions through feeding. Under nutrient limitation, N. rotunda and I. galbana fed, but growth ceased or was significantly lower than in the control. Thus, mixotrophic bacterivory could be a survival rather than a growth strategy for certain species. In contrast, nutrient-limited N. pyriformis achieved growth rates equivalent to the control through feeding. This strikingly differs with the classical view of chlorophytes as primarily non-feeders and indicates mixotrophic bacterivory can be a significant trophic strategy for green algae, even at the low bacterial concentrations found in oligotrophic open oceans.

Diversity of Pico- to Mesoplankton along the 2000 km Salinity Gradient of the Baltic Sea.

Microbial plankton form the productive base of both marine and freshwater ecosystems and are key drivers of global biogeochemical cycles of carbon and nutrients. Plankton diversity is immense with representations from all major phyla within the three domains of life. So far, plankton monitoring has mainly been based on microscopic identification, which has limited sensitivity and reproducibility, not least because of the numerical majority of plankton being unidentifiable under the light microscope. High-throughput sequencing of taxonomic marker genes offers a means to identify taxa inaccessible by traditional methods; thus, recent studies have unveiled an extensive previously unknown diversity of plankton. Here, we conducted ultra-deep Illumina sequencing (average 10(5) sequences/sample) of rRNA gene amplicons of surface water eukaryotic and bacterial plankton communities sampled in summer along a 2000 km transect following the salinity gradient of the Baltic Sea. Community composition was strongly correlated with salinity for both bacterial and eukaryotic plankton assemblages, highlighting the importance of salinity for structuring the biodiversity within this ecosystem. In contrast, no clear trends in alpha-diversity for bacterial or eukaryotic communities could be detected along the transect. The distribution of major planktonic taxa followed expected patterns as observed in monitoring programs, but groups novel to the Baltic Sea were also identified, such as relatives to the coccolithophore Emiliana huxleyi detected in the northern Baltic Sea. This study provides the first ultra-deep sequencing-based survey on eukaryotic and bacterial plankton biogeography in the Baltic Sea.

Late winter under ice pelagic microbial communities in the high Arctic Ocean and the impact of short-term exposure to elevated CO2 levels.

Polar Oceans are natural CO2 sinks because of the enhanced solubility of CO2 in cold water. The Arctic Ocean is at additional risk of accelerated ocean acidification (OA) because of freshwater inputs from sea ice and rivers, which influence the carbonate system. Winter conditions in the Arctic are of interest because of both cold temperatures and limited CO2 venting to the atmosphere when sea ice is present. Earlier OA experiments on Arctic microbial communities conducted in the absence of ice cover, hinted at shifts in taxa dominance and diversity under lowered pH. The Catlin Arctic Survey provided an opportunity to conduct in situ, under-ice, OA experiments during late Arctic winter. Seawater was collected from under the sea ice off Ellef Ringnes Island, and communities were exposed to three CO2 levels for 6 days. Phylogenetic diversity was greater in the attached fraction compared to the free-living fraction in situ, in the controls and in the treatments. The dominant taxa in all cases were Gammaproteobacteria but acidification had little effect compared to the effects of containment. Phylogenetic net relatedness indices suggested that acidification may have decreased the diversity within some bacterial orders, but overall there was no clear trend. Within the experimental communities, alkalinity best explained the variance among samples and replicates, suggesting subtle changes in the carbonate system need to be considered in such experiments. We conclude that under ice communities have the capacity to respond either by selection or phenotypic plasticity to heightened CO2 levels over the short term.

Effects of light and prey availability on Arctic freshwater protist communities examined by high-throughput DNA and RNA sequencing.

Protists in high-latitude lakes are constrained by cold temperatures, low inorganic nutrient supply and low light availability for much of the year due to ice cover and polar darkness. The lengthening ice-free periods in these freshwater ecosystems due to a warming climate results in increased light availability, but the overall impacts on phytoplankton and other protists are unknown. We experimentally investigated protist community responses to changes in light and prey availability in a dilution series in Ward Hunt Lake (latitude 83°05'N), in the Canadian High Arctic. The communities at the end of the experiment were characterized using high-throughput pyrosequencing of the V4 region of the 18S rRNA gene as a measure of taxonomic presence, and of 18S rRNA (from RNA converted to cDNA) as a taxon-specific indicator of community response. At the end of the experiment under low irradiance, cDNA reads were dominated by photosynthetic dinoflagellate genera, except at the greatest dilution where Cercozoa were most abundant. In contrast, the cDNA reads in the high light treatments were dominated by chrysophytes. Given the known trophic differences among dinoflagellates, cercozoans and chrysophytes, this apparent environmental selection implies that the rise in underwater irradiance associated with increasing ice-free conditions may affect microbial food web structure and function in polar lakes.

Pyrosequencing analysis of the protist communities in a High Arctic meromictic lake: DNA preservation and change.

High Arctic meromictic lakes are extreme environments characterized by cold temperatures, low nutrient inputs from their polar desert catchments and prolonged periods of low irradiance and darkness. These lakes are permanently stratified with an oxygenated freshwater layer (mixolimnion) overlying a saline, anoxic water column (monimolimnion). The physical and chemical properties of the deepest known lake of this type in the circumpolar Arctic, Lake A, on the far northern coast of Ellesmere Island, Canada, have been studied over the last 15 years, but little is known about the lake's biological communities. We applied high-throughput sequencing of the V4 region of the 18S ribosomal RNA gene to investigate the protist communities down the water column at three sampling times: under the ice at the end of winter in 2008, during an unusual period of warming and ice-out the same year, and again under the ice in mid-summer 2009. Sequences of many protist taxa occurred throughout the water column at all sampling times, including in the deep anoxic layer where growth is highly unlikely. Furthermore, there were sequences for taxonomic groups including diatoms and marine taxa, which have never been observed in Lake A by microscopic analysis. However, the sequences of other taxa such as ciliates, chrysophytes, Cercozoa, and Telonema varied with depth, between years and during the transition to ice-free conditions. These seasonally active taxa in the surface waters of the lake are thus sensitive to depth and change with time. DNA from these taxa is superimposed upon background DNA from multiple internal and external sources that is preserved in the deep, cold, largely anoxic water column.

Prevalence of partnerships between bacteria and ciliates in oxygen-depleted marine water columns.

Symbioses between Bacteria, Archaea, and Eukarya in deep-sea marine environments represent a means for eukaryotes to exploit otherwise inhospitable habitats. Such symbioses are abundant in many low-oxygen benthic marine environments, where the majority of microbial eukaryotes contain prokaryotic symbionts. Here, we present evidence suggesting that in certain oxygen-depleted marine water-column habitats, the majority of microbial eukaryotes are also associated with prokaryotic cells. Ciliates (protists) associated with bacteria were found to be the dominant eukaryotic morphotype in the haloclines of two different deep-sea hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea. These findings are compared to associations between ciliates and bacteria documented from the permanently anoxic waters of the Cariaco Basin (Caribbean Sea). The dominance of ciliates exhibiting epibiotic bacteria across three different oxygen-depleted marine water column habitats suggests that such partnerships confer a fitness advantage for ciliates in these environments.

Virgibacillus arcticus sp. nov., a moderately halophilic, endospore-forming bacterium from permafrost in the Canadian high Arctic.

A novel, moderately halophilic, endospore-forming bacterial strain, designated Hal 1T, was isolated from a permafrost core collected from the Canadian high Arctic. The temperature for growth of strain Hal 1T was 0-30 degrees C with no growth observed at either -5 or 37 degrees C (optimum growth at about 25 degrees C). Strain Hal 1T was able to grow at NaCl concentrations of 0-20% (w/v) and did not have an absolute NaCl requirement for growth; optimal growth was at 5% (w/v) NaCl. The level of 16S rRNA gene sequence similarity between strain Hal 1T and the type strains of Virgibacillus carmonensis and Virgibacillus necropolis was 98.2%; values with respect to the type strains of other recognized Virgibacillus species were below 96.0%. The DNA G+C content of strain Hal 1T was 38.2 mol%. Levels of DNA-DNA relatedness between strain Hal 1T and the type strains of V. carmonensis and V. necropolis were 14.0 and 21.0%, respectively. The major fatty acid of strain Hal 1T was anteiso-C15:0, consistent with species of the genus Virgibacillus. The cell-wall peptidoglycan of strain Hal 1T was type A1alpha and the major respiratory quinone was MK-7. On the basis of genotypic and physiological results, strain Hal 1T (=DSM 19574T=JCM 14839T) is proposed as the type strain of a novel species of the genus Virgibacillus, namely Virgibacillus arcticus sp. nov.