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Predicting outcomes of marine disease outbreaks presents a challenge in the face of both global and local stressors. Host-associated microbiomes may play important roles in disease dynamics but remain understudied in marine ecosystems. Host-pathogen-microbiome interactions can vary across host ranges, gradients of disease, and temperature; studying these relationships may aid our ability to forecast disease dynamics. Eelgrass, Zostera marina , is impacted by outbreaks of wasting disease caused by the opportunistic pathogen Labyrinthula zosterae . We investigated how Z. marina phyllosphere microbial communities vary with rising wasting disease lesion prevalence and severity relative to plant and meadow characteristics like shoot density, longest leaf length, and temperature across 23° latitude in the Northeastern Pacific. We detected effects of geography (11%) and smaller, but distinct, effects of temperature (30-day max sea surface temperature, 4%) and disease (lesion prevalence, 3%) on microbiome composition. Declines in alpha diversity on asymptomatic tissue occurred with rising wasting disease prevalence within meadows. However, no change in microbiome variability (dispersion) was detected between asymptomatic and symptomatic tissues. Further, we identified members of Cellvibrionaceae, Colwelliaceae, and Granulosicoccaceae on asymptomatic tissue that are predictive of wasting disease prevalence across the geographic range (3,100 kilometers). Functional roles of Colwelliaceae and Granulosicoccaceae are not known. Cellvibrionaceae, degraders of plant cellulose, were also enriched in lesions and adjacent green tissue relative to nonlesioned leaves. Cellvibrionaceae may play important roles in disease progression by degrading host tissues or overwhelming plant immune responses. Thus, inclusion of microbiomes in wasting disease studies may improve our ability to understand variable rates of infection, disease progression, and plant survival. IMPORTANCE The roles of marine microbiomes in disease remain poorly understood due, in part, to the challenging nature of sampling at appropriate spatiotemporal scales and across natural gradients of disease throughout host ranges. This is especially true for marine vascular plants like eelgrass ( Zostera marina ) that are vital for ecosystem function and biodiversity but are susceptible to rapid decline and die-off from pathogens like eukaryotic slime-mold Labyrinthula zosterae (wasting disease). We link bacterial members of phyllosphere tissues to the prevalence of wasting disease across the broadest geographic range to date for a marine plant microbiome-disease study (3,100 km). We identify Cellvibrionaceae, plant cell wall degraders, enriched (up to 61% relative abundance) within lesion tissue, which suggests this group may be playing important roles in disease progression. These findings suggest inclusion of microbiomes in marine disease studies will improve our ability to predict ecological outcomes of infection across variable landscapes spanning thousands of kilometers.

A barrier to realizing Nannochloropsis oceanica's potential for omega-3 eicosapentaenoic acid (EPA) production is the disparity between conditions that are optimal for growth and those that are optimal for EPA biomass content. A case in point is temperature: higher content of polyunsaturated fatty acid, and especially EPA, is observed in low-temperature (LT) environments, where growth rates are often inhibited. We hypothesized that mutant strains of N. oceanica resistant to the singlet-oxygen photosensitizer Rose Bengal (RB) would withstand the oxidative stress conditions that prevail in the combined stressful environment of high light (HL; 250 μmol photons m-2 s-1) and LT (18°C). This growth environment caused the wild-type (WT) strain to experience a spike in lipid peroxidation and an inability to proliferate, whereas growth and homeostatic reactive oxygen species levels were observed in the mutant strains. We suggest that the mutant strains' success in this environment can be attributed to their truncated photosystem II antennas and their increased ability to diffuse energy in those antennas as heat (non-photosynthetic quenching). As a result, the mutant strains produced upward of four times more EPA than the WT strain in this HL-LT environment. The major plastidial lipid monogalactosyldiacylglycerol was a likely target for oxidative damage, contributing to the photosynthetic inhibition of the WT strain. A mutation in the NO10G01010.1 gene, causing a subunit of the 2-oxoisovalerate dehydrogenase E1 protein to become non-functional, was determined to be the likely source of tolerance in the RB113 mutant strain., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The microalgal genus Nannochloropsis has broad applicability to produce biofuels, animal feed supplements and other value-added products including proteins, carotenoids and lipids. This study investigated a potential role of N. oceanica in the reversal of metabolic syndrome. Male Wistar rats ( n = 48) were divided into four groups in a 16-week protocol. Two groups were fed either corn starch or high-carbohydrate, high-fat diets (C and H, respectively) for the full 16 weeks. The other two groups received C and H diets for eight weeks and then received 5% freeze-dried N. oceanica in these diets for the final eight weeks (CN and HN, respectively) of the protocol. The H diet was high in fructose and sucrose, together with increased saturated and trans fats. H rats developed obesity, hypertension, dyslipidaemia, fatty liver disease and left ventricular fibrosis. N. oceanica increased lean mass in CN and HN rats, possibly due to the increased protein intake, and decreased fat mass in HN rats. Intervention with N. oceanica did not change cardiovascular, liver and metabolic parameters or gut structure. The relative abundance of Oxyphotobacteria in the gut microbiota was increased. N. oceanica may be an effective functional food against metabolic syndrome as a sustainable protein source.

Iron-containing proteins, including iron-sulfur (Fe-S) proteins, are essential for numerous electron transfer and metabolic reactions. They are present in most subcellular compartments. In plastids, in addition to sustaining the linear and cyclic photosynthetic electron transfer chains, Fe-S proteins participate in carbon, nitrogen, and sulfur assimilation, tetrapyrrole and isoprenoid metabolism, and lipoic acid and thiamine synthesis. The synthesis of Fe-S clusters, their trafficking, and their insertion into chloroplastic proteins necessitate the so-called sulfur mobilization (SUF) protein machinery. In the first part, we describe the molecular mechanisms that allow Fe-S cluster synthesis and insertion into acceptor proteins by the SUF machinery and analyze the occurrence of the SUF components in microalgae, focusing in particular on the green alga Chlamydomonas reinhardtii . In the second part, we describe chloroplastic Fe-S protein-dependent pathways that are specific to Chlamydomonas or for which Chlamydomonas presents specificities compared to terrestrial plants, putting notable emphasis on the contribution of Fe-S proteins to chlorophyll synthesis in the dark and to the fermentative metabolism. The occurrence and evolutionary conservation of these enzymes and pathways have been analyzed in all supergroups of microalgae performing oxygenic photosynthesis.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Here, we analyzed patterns of taxon richness and endemism of freshwater protists in Europe. Even though the significance of physicochemical parameters but also of geographic constraints for protist distribution is documented, it remains unclear where regional areas of high protist diversity are located and whether areas of high taxon richness harbor a high proportion of endemics. Further, patterns may be universal for protists or deviate between taxonomic groups. Based on amplicon sequencing campaigns targeting the SSU and ITS region of the rDNA we address these patterns at two different levels of phylogenetic resolution. Our analyses demonstrate that protists have restricted geographical distribution areas. For many taxonomic groups the regions of high taxon richness deviate from those having a high proportion of putative endemics. In particular, the diversity of high mountain lakes as azonal habitats deviated from surrounding lowlands, i.e. many taxa were found exclusively in high mountain lakes and several putatively endemic taxa occurred in mountain regions like the Alps, the Pyrenees or the Massif Central. Beyond that, taxonomic groups showed a pronounced accumulation of putative endemics in distinct regions, e.g. Dinophyceae along the Baltic Sea coastline, and Chrysophyceae in Scandinavia. Many other groups did not have pronounced areas of increased endemism but geographically restricted taxa were found across Europe.

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-log 10 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-log 10 reduction over 4 days with an initial protist concentration of 10 3 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., (Copyright © 2020 American Society for Microbiology.)

The species of the genus Nannochloropsis are unique in their maintenance of a nucleus-plastid continuum throughout their cell cycle, non-motility and asexual reproduction. These characteristics should have been endorsed in their gene assemblages (genomes). Here we show that N. oceanica has a genome of 29.3 Mb consisting of 32 pseudochromosomes and containing 7,330 protein-coding genes; and the host nucleus may have been overthrown by an ancient red alga symbiont nucleus during speciation through secondary endosymbiosis. In addition, N. oceanica has lost its flagella and abilities to undergo meiosis and sexual reproduction, and adopted a genome reduction strategy during speciation. We propose that N. oceanica emerged through the active fusion of a host protist and a photosynthesizing ancient red alga and the symbiont nucleus became dominant over the host nucleus while the chloroplast was wrapped by two layers of endoplasmic reticulum. Our findings evidenced an alternative speciation pathway of eukaryotes., Competing Interests: Competing interestsThe authors declare no competing interests.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31 h that damped within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

In the marine environment, the abundance of Bacteria and Archaea is either controlled bottom-up via nutrient availability or top-down via grazing. Heterotrophic nanoflagellates (HNF) are mainly responsible for prokaryotic grazing losses besides viral lysis. However, the grazing specificity of HNF on specific bacterial and archaeal taxa is under debate. Bacteria and Archaea might have different nutritive values and surface properties affecting the growth rates of HNF. In this study, we offered different bacterial and archaeal strains with different morphologic and physiologic characteristics to Cafeteria roenbergensis, one of the most abundant and ubiquitous species of HNF in the ocean. Two Nitrosopumilus maritimus-related strains isolated from the northern Adriatic Sea (Nitrosopumilus adriaticus, Nitrosopumilus piranensis), two Nitrosococcus strains, and two fast growing marine Bacteria (Pseudoalteromonas sp. and Marinobacter sp.) were fed to Cafeteria cultures. Cafeteria roenbergensis exhibited high growth rates when feeding on Pseudoalteromonas sp., Marinobacter sp., and Nitrosopumilus adriaticus, while the addition of the other strains resulted in minimal growth. Taken together, our data suggest that the differences in growth of Cafeteria roenbergensis associated to grazing on different thaumarchaeal and bacterial strains are likely due to the subtle metabolic, cell size, and physiological differences between different bacterial and thaumarchaeal taxa. Moreover, Nitrosopumilus adriaticus experienced a similar grazing pressure by Cafeteria roenbergensis as compared to the other strains, suggesting that other HNF may also prey on Archaea which might have important consequences on the global biogeochemical cycles.

Ocean acidification (O.A.) influences the ecology of oceans and it may impact plant-animal interactions at various levels. Seagrass meadows located at acidified vents in the Bay of Naples (Italy) are considered an open window to forecast the effects of global-changes on aquatic communities. Epiphytic diatoms of the genus Cocconeis are abundant in seagrass meadows, including acidified environments, where they play key ecological roles. A still-unknown apoptogenic compound produced by Cocconeis triggers the suicide of the androgenic gland of Hippolyte inermis Leach 1816, a protandric hermaphroditic shrimp distributed in P. oceanica meadows located both at normal pH and in acidified vents. Feeding on Cocconeis sp. was proven important for the stability of the shrimp's natural populations. Since O.A. affects the physiology of diatoms, we investigated if, in future scenarios of O.A., Cocconeis scutellum parva will still produce an effect on shrimp's physiology. Cell densities of Cocconeis scutellum parva cultivated in custom-designed photobioreactors at two pH conditions (pH 7.7 and 8.2) were compared. In addition, we determined the effects of the ingestion of diatoms on the process of sex reversal of H. inermis and we calculated the % female on the total of mature individuals-1 (F/mat). We observed significant differences in cell densities of C. scutellum parva at the two pH conditions. In fact, the highest cell densities (148,808 ±13,935 cells. mm-2) was obtained at day 13 (pH 7.7) and it is higher than the highest cell densities (38,066 (±4,166) cells. mm-2, day 13) produced at pH 8.2. Diatoms cultured at acidified conditions changed their metabolism. In fact, diatoms grown in acidified conditions produced in H. inermis a proportion of females (F/mat 36.3 ±5.9%) significantly lower than diatoms produced at normal pH (68.5 ±2.8), and it was not significantly different from that elicited by negative controls (31.7 ±5.6%)., Competing Interests: The authors have declared that no competing interests exist.

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690 nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S 2 and hot S 1 states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Heterokont algae are significant contributors to marine primary productivity. These algae have a photosynthetic machinery that shares many common features with that of Viridiplantae (green algae and land plants). Here we demonstrate, however, that the photosynthetic machinery of heterokont algae responds to light fundamentally differently than that of Viridiplantae. While exposure to high light leads to electron accumulation within the photosynthetic electron transport chain in Viridiplantae, this is not the case in heterokont algae. We use this insight to manipulate the photosynthetic electron transport chain and demonstrate that heterokont algae can dynamically distribute excitation energy between the two types of photosystems. We suggest that the reported electron transport and excitation distribution features are adaptations to the marine light environment., Competing Interests: The authors have declared that no competing interests exist.

In humans, dietary polyunsaturated fatty acids (PUFAs) are involved in therapeutic processes such as prevention and treatment of cardiovascular diseases, neuropsychiatric disorders, and dementia. We examined the physiology, PUFA accumulation and glycerol lipid biosynthesis in the marine microalga Nannochloropsis salina in response to constant suboptimal temperature (<20 °C). As expected, N. salina exhibited significantly reduced growth rate and photosynthetic activity compared to optimal cultivation temperature. Total fatty acid contents were not significantly elevated at reduced temperatures. Cultures grown at 5 °C had the highest quantity of eicosapentanoic acid (EPA) (C20:5n3) and the lowest growth rate. Additionally, we monitored broadband lipid composition to model the occurrence of metabolic alteration and remodeling for various lipid pools. We focused on triacylglycerol (TAG) with elevated PUFA content. TAGs with EPA at all three acyl positions were higher at a cultivation temperature of 15 °C. Furthermore, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, which are polar lipids associated with chloroplast membranes, decreased with reduced cultivation temperatures. Moreover, gene expression analysis of key genes involved in Kennedy pathway for de novo TAG biosynthesis revealed bimodal variations in transcript level amongst the temperature treatments. Collectively, these results show that Nannochloropsis salina is a promising source of PUFA containing lipids.

Aurantiochytrium sp. PKU#SW7 is a thraustochytrid strain that was found to exhibit high potential for docosahexaenoic acid (DHA, C22:6n-3) production. In this work, the transcriptome of Aurantiochytrium sp. PKU#SW7 was analyzed for the study of genes involved in basic metabolic functions and especially in the mechanisms of DHA biosynthesis. Sequence annotation and functional analysis revealed that the strain contains components of fatty acid synthesis (FAS) and polyketide synthase (PKS) pathways. Fatty acid desaturases and elongases were identified as components of FAS pathway, whilst key components of PKS pathway were also found in the cDNA library. The relative contribution of the two pathways to the synthesis of DHA was unknown, as both pathways appeared to be lacking full complement of genes for standalone synthesis of DHA. Further analysis of two putative genes encoding the very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase and dehydrase/isomerase involved in FAS and PKS pathways, respectively, revealed that under various salinity conditions, their relative expression levels changed corresponding to the variation of DHA content in Aurantiochytrium sp. Independent knock outs of these genes in Aurantiochytrium sp. resulted in poor cell growth, probably due to little or no intracellular DHA accumulation. Hence, it can be speculated that both genes are engaged in DHA biosynthesis and DHA in Aurantiochytrium sp. could be produced by jointed actions of both FAS and PKS systems.

Eelgrass wasting disease, caused by the marine pathogen Labyrinthula zosterae, has the potential to devastate important eelgrass habitats worldwide. Although this host-pathogen interaction may increase under certain environmental conditions, little is known about how disease severity is impacted by multiple components of a changing environment. In this study, we investigated the effects of variation in 3 different L. zosterae isolates, pathogen dosage, temperature, and light on severity of infections. Severity of lesions on eelgrass varied among the 3 different isolates inoculated in laboratory trials. Our methods to control dosage of inoculum showed that disease severity increased with pathogen dosage from 104 to 106 cells ml-1. In a dosage-controlled light and temperature 2-way factorial experiment consisting of 2 light regimes (diel light cycle and complete darkness) and 2 temperatures (11 and 18°C), L. zosterae cell growth rate in vitro was higher at the warmer temperature. In a companion experiment that tested the effects of light and temperature in in vivo inoculations, disease severity was higher in dark treatments and temperature was marginally significant. We suggest that the much greater impact of light in the in vivo inoculation experiment indicates an important role for plant physiology and the need for photosynthesis in slowing severity of infections. Our work with controlled inoculation of distinct L. zosterae isolates shows that pathogen isolate, increasing dosage of inoculum, increasing temperature, and diminishing light increase disease severity, suggesting L. zosterae will cause increased damage to eelgrass beds with changing environmental conditions.

Marine heterotrophic protists of the Labyrinthulomycota are of interest for their biotechnological (e.g. thraustochytrid production of lipids) and ecological (e.g. wasting disease and rapid blight by pathogens of the genus Labyrinthula) applications; culture-based laboratory studies are a central technique of this research. However, maintaining such microorganism cultures can be labour- and cost-intensive, with a high risk of culture contamination and die-off over time. Deep-freeze storage, or cryopreservation, can be used to maintain culture back-ups, as well as to preserve the genetic and phenotypic properties of the microorganisms; however, this method has not been tested for the ubiquitous marine protists Labyrinthula spp. In this study, we trialled 12 cryopreservation protocols on 3 Labyrinthula sp. isolates of varying colony morphological traits. After 6 mo at -80°C storage, the DMSO and glycerol protocols were the most effective cryoprotectants compared to methanol (up to 90% success vs. 50% success, respectively). The addition of 30% horse serum to the cryoprotectant solution increased Labyrinthula sp. growth success by 20-30%. We expect that these protocols will provide extra security for culture-based studies, as well as opportunities for long-term research on key Labyrinthula sp. isolates.

Sacoglossan sea slugs offer fascinating systems to study the onset and persistence of algal-plastid symbioses. Elysia chlorotica is particularly noteworthy because it can survive for months, relying solely on energy produced by ingested plastids of the stramenopile alga Vaucheria litorea that are sequestered in cells lining its digestive diverticula. How this animal can maintain the actively photosynthesizing organelles without replenishment of proteins from the lost algal nucleus remains unknown. Here, we used RNA-Seq analysis to test the idea that plastid sequestration leaves a significant signature on host gene expression during E. chlorotica development. Our results support this hypothesis and show that upon exposure to and ingestion of V. litorea plastids, genes involved in microbe-associated molecular patterns and oxidative stress-response mechanisms are significantly up-regulated. Interestingly, our results with E. chlorotica mirror those found with corals that maintain dinoflagellates as intact cells in symbiosomes, suggesting parallels between these animal-algal symbiotic interactions.

Diacylglyceryl- N,N,N -trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica , DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica ., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

The White Sea is a unique marine environment combining features of temperate and Arctic seas. The composition and abundance of photosynthetic picoeukaryotes (PPEs) were investigated in the land-fast ice of the White Sea, Russia, in March 2013 and 2014. High-throughput tag sequencing (Illumina MiSeq system) of the V4 region of the 18S rRNA gene was used to reveal the diversity of PPE ice community. The integrated PPE abundance varied from 11 × 10 6 cells/m 2 to 364 × 10 6 cells/m 2 ; the integrated biomass ranged from 0.02 to 0.26 mg С/m 2 . The composition of sea-ice PPEs was represented by 16 algae genera belonging to eight classes and three super-groups. Chlorophyta, especially Mamiellophyceae, dominated among ice PPEs. The detailed analysis revealed the latent diversity of Micromonas and Mantоniella. Micromonas clade E2 revealed in the subarctic White Sea ice indicates that the area of distribution of this species is wider than previously thought. We suppose there exists a new Micromonas clade F. Micromonas clade C and Minutocellulus polymorphus were first discovered in the ice and extend the modern concept of sympagic communities' diversity generally and highlights the importance of further targeting subarctic sea ice for microbial study.

Molecular mechanisms of phosphorus (P) limitation are of great interest for understanding algal production in aquatic ecosystems. Previous studies point to P limitation-induced changes in lipid composition. As, in microalgae, the molecular mechanisms of this specific P stress adaptation remain unresolved, we reveal a detailed phospholipid-recycling scheme in Nannochloropsis oceanica and describe important P acquisition genes based on highly corresponding transcriptome and lipidome data. Initial responses to P limitation showed increased expression of genes involved in P uptake and an expansion of the P substrate spectrum based on purple acid phosphatases. Increase in P trafficking displayed a rearrangement between compartments by supplying P to the chloroplast and carbon to the cytosol for lipid synthesis. We propose a novel phospholipid-recycling scheme for algae that leads to the rapid reduction of phospholipids and synthesis of the P-free lipid classes. P mobilization through membrane lipid degradation is mediated mainly by two glycerophosphoryldiester phosphodiesterases and three patatin-like phospholipases A on the transcriptome level. To compensate for low phospholipids in exponential growth, N. oceanica synthesized sulfoquinovosyldiacylglycerol and diacylglyceroltrimethylhomoserine. In this study, it was shown that an N. oceanica strain has a unique repertoire of genes that facilitate P acquisition and the degradation of phospholipids compared with other stramenopiles. The novel phospholipid-recycling scheme opens new avenues for metabolic engineering of lipid composition in algae., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The development of mathematical models capable of accurate predictions of the photosynthetic productivity of microalgae under variable light conditions is paramount to the development of large-scale production systems. The process of photoacclimation is particularly important in outdoor cultivation systems, whereby seasonal variation of the light irradiance can greatly influence microalgae growth. This paper presents a dynamic model that captures the effect of photoacclimation on the photosynthetic production. It builds upon an existing semi-empirical model describing the processes of photoproduction, photoregulation and photoinhibition via the introduction of acclimation rules for key parameters. The model is calibrated against a dataset comprising pulsed amplitude modulation fluorescence, photosynthesis rate, and antenna size measurements for the microalga Nannochloropsis gaditana in several acclimation states. It is shown that the calibrated model is capable of accurate predictions of fluorescence and respirometry data, both in interpolation and in extrapolation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nannochloropsis spp. are algae with high potential for biotechnological applications due to their capacity to accumulate lipids. However, little is known about their photosynthetic apparatus and acclimation/photoprotective strategies. In this work, we studied the mechanisms of non-photochemical quenching (NPQ), the fast response to high light stress, in Nannochloropsis gaditana by "locking" the cells in six different states during quenching activation and relaxation. Combining biochemical analysis with time-resolved fluorescence spectroscopy, we correlated each NPQ state with the presence of two well-known NPQ components: de-epoxidized xanthophylls and stress-related antenna proteins (LHCXs). We demonstrated that after exposure to strong light, the rapid quenching that takes place in the antennas of both photosystems was associated with the presence of LHCXs. At later stages, quenching occurs mainly in the antennas of PSII and correlates with the amount of de-epoxidised xanthophylls. We also observed changes in the distribution of excitation energy between photosystems, which suggests redistribution of excitation between photosystems as part of the photo-protective strategy. A multistep model for NPQ induction and relaxation in N. gaditana is discussed., (Copyright © 2017. Published by Elsevier B.V.)

Aquatic photosynthetic organisms cope with low environmental CO 2 concentrations through the action of carbon-concentrating mechanisms (CCMs). Known eukaryotic CCMs consist of inorganic carbon transporters and carbonic anhydrases (and other supporting components) that culminate in elevated [CO 2 ] inside a chloroplastic Rubisco-containing structure called a pyrenoid. We set out to determine the molecular mechanisms underlying the CCM in the emerging model photosynthetic stramenopile, Nannochloropsis oceanica , a unicellular picoplanktonic alga that lacks a pyrenoid. We characterized CARBONIC ANHYDRASE 1 ( CAH1 ) as an essential component of the CCM in N. oceanica CCMP1779. We generated insertions in this gene by directed homologous recombination and found that the cah1 mutant has severe defects in growth and photosynthesis at ambient CO 2 We identified CAH1 as an α-type carbonic anhydrase, providing a biochemical role in CCM function. CAH1 was found to localize to the lumen of the epiplastid endoplasmic reticulum, with its expression regulated by the external inorganic carbon concentration at both the transcript and protein levels. Taken together, these findings show that CAH1 is an indispensable component of what may be a simple but effective and dynamic CCM in N. oceanica ., Competing Interests: The authors declare no conflict of interest.

The functional and numerical responses of the facultative anaerobic heterotrophic nanoflagellate, Suigetsumonas clinomigrationis NIES-3647 to prey density were examined under oxic and anoxic conditions. S. clinomigrationis grew at temperatures between 10 and 30°C and in the salinity range of 3.9-36.9 psu. The maximum specific growth and ingestion rates of S. clinomigrationis were lower under anoxic conditions than under oxic conditions. Half-saturation constants for the growth of S. clinomigrationis were within or greater than the range of bacterial densities in the water column of Lake Suigetsu, suggesting that its growth rate is limited by bacterial prey densities in natural environments.

While it is crucial to understand the factors that determine the biodiversity of primary producer communities, the relative importance of bottom-up and top-down control factors is still poorly understood. Using freshwater benthic algal communities in the laboratory as a model system, we find an unimodal relationship between nutrient availability and producer diversity, and that increasing number of consumer species increases producer diversity, but overall grazing decreases algal biodiversity. Interestingly, these two factors interact strongly in determining producer diversity, as an increase in nutrient supply diminishes the positive effect of consumer species richness on producer biodiversity. This novel and thus-far overlooked interaction of bottom-up and top-down control mechanisms of biodiversity may have a pronounced impact on ecosystem functioning and thus have repercussions for the fields of biodiversity conservation and restoration.

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

Objectives: To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems., Results: We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms., Conclusions: These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Reliable quantitative description of light-limited growth in microalgae is key to improving the design and operation of industrial production systems. This article shows how the capability to predict photosynthetic processes can benefit from a synergy between mathematical modelling and lab-scale experiments using systematic design of experiment techniques. A model of chlorophyll fluorescence developed by the authors [Nikolaou et al., J Biotechnol 194:91-99, 2015] is used as starting point, whereby the representation of non-photochemical-quenching (NPQ) process is refined for biological consistency. This model spans multiple time scales ranging from milliseconds to hours, thus calling for a combination of various experimental techniques in order to arrive at a sufficiently rich data set and determine statistically meaningful estimates for the model parameters. The methodology is demonstrated for the microalga Nannochloropsis gaditana by combining pulse amplitude modulation (PAM) fluorescence, photosynthesis rate and antenna size measurements. The results show that the calibrated model is capable of accurate quantitative predictions under a wide range of transient light conditions. Moreover, this work provides an experimental validation of the link between fluorescence and photosynthesis-irradiance (PI) curves which had been theoricized.

A continuous flow process is developed for recovery of the biomass of the marine microalga Nannochloropsis salina. Flocculation-sedimentation is used to recover the biomass from an algal suspension with an initial dry biomass concentration of 0.5 g L(-1), as would be typical of a raceway-based biomass production system. More than 85% of the biomass initially in suspension could be settled by gravity in a flocculation-sedimentation device with a total residence time of ∼148 min. Aluminum sulfate was used as an inexpensive, readily available and safe flocculant. The optimal flocculant dosage (as Al2(SO4)3) was 229 mg L(-1). Relative to a highly effective 62-min batch flocculation-sedimentation process for the same alga and flocculant, the continuous flow operation took longer and required nearly double the flocculant dose. The design of the flocculation-sedimentation system is explained., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nannochloropsis oceanica CCMP1779 is a marine unicellular stramenopile and an emerging reference species for basic research on oleogenic microalgae with biotechnological relevance. We investigated its physiology and transcriptome under light/dark cycles. We observed oscillations in lipid content and a predominance of cell division in the first half of the dark phase. Globally, more than 60% of the genes cycled in N. oceanica CCMP1779, with gene expression peaking at different times of the day. Interestingly, the phase of expression of genes involved in certain biological processes was conserved across photosynthetic lineages. Furthermore, in agreement with our physiological studies we found the processes of lipid metabolism and cell division enriched in cycling genes. For example, there was tight coordination of genes involved in the lower part of glycolysis, fatty acid synthesis and lipid production at dawn preceding lipid accumulation during the day. Our results suggest that diel lipid storage plays a key role for N. oceanica CCMP1779 growth under natural conditions making this alga a promising model to gain a basic mechanistic understanding of triacylglycerol production in photosynthetic cells. Our data will help the formulation of new hypotheses on the role of cyclic gene expression in cell growth and metabolism in Nannochloropsis., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Defining reference conditions is central to identifying environmental effects of anthropogenic activities. Using a watershed approach, we quantified reference conditions for benthic algal communities and their relations to physico-chemical conditions in rivers in the South Nahanni River watershed, NWT, Canada, in 2008 and 2009. We also compared the ability of three descriptors that vary in terms of analytical costs to define algal community structure based on relative abundances of (i) all algal taxa, (ii) only diatom taxa, and (iii) photosynthetic pigments. Ordination analyses showed that variance in algal community structure was strongly related to gradients in environmental variables describing water physico-chemistry, stream habitats, and sub-watershed structure. Water physico-chemistry and local watershed-scale descriptors differed significantly between algal communities from sites in the Selwyn Mountain ecoregion compared to sites in the Nahanni-Hyland ecoregions. Distinct differences in algal community types between ecoregions were apparent irrespective of whether algal community structure was defined using all algal taxa, diatom taxa, or photosynthetic pigments. Two algal community types were highly predictable using environmental variables, a core consideration in the development of Reference Condition Approach (RCA) models. These results suggest that assessments of environmental impacts could be completed using RCA models for each ecoregion. We suggest that use of algal pigments, a high through-put analysis, is a promising alternative compared to more labor-intensive and costly taxonomic approaches for defining algal community structure.

Vibrio species are an abundant and diverse group of bacteria that form associations with phytoplankton. Correlations between Vibrio and phytoplankton abundance have been noted, suggesting that growth is enhanced during algal blooms or that association with phytoplankton provides a refuge from predation. Here, we investigated relationships between particle-associated Vibrio spp. and phytoplankton in Delaware's inland bays (DIB). The relative abundances of particle-associated Vibrio spp. and algal classes that form blooms in DIB (dinoflagellates, diatoms, and raphidophytes) were determined using quantitative PCR. The results demonstrated a significant correlation between particle-associated Vibrio abundance and phytoplankton, with higher correlations to diatoms and raphidophytes than to dinoflagellates. Species-specific associations were examined during a mixed bloom of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) and indicated a significant positive correlation for particle-associated Vibrio abundance with H. akashiwo but a negative correlation with F. japonica. Changes in Vibrio assemblages during the bloom were evaluated using automated ribosomal intergenic spacer analysis (ARISA), which revealed significant differences between each size fraction but no significant change in Vibrio assemblages over the course of the bloom. Microzooplankton grazing experiments showed that losses of particle-associated Vibrio spp. may be offset by increased growth in the Vibrio population. Moreover, analysis of Vibrio assemblages by ARISA also indicated an increase in the relative abundance for specific members of the Vibrio community despite higher grazing pressure on the particle-associated population as a whole. The results of this investigation demonstrate links between phytoplankton and Vibrio that may lead to predictions of potential health risks and inform future management practices in this region., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A new relative of the chrysophyte genus Chrysopodocystis was found in Tenerife and termed Guanchochroma wildpretii. This unicellular alga was most noticeably discernible from Chrysopodocystis socialis (the only species of this genus) by the presence of a cyst-like stage with a multilayered lorica, which also functions as a dispersal unit and shows secondary wall growth. Secondary expansion of loricae (cell casings not involved in cell division, usually with a more or less pronounced opening) has never been observed previously and marks a unique feature of the new taxon. Plastids are non-randomly distributed within cells of G. wildpretii. 18S rRNA gene analyses identified the two species as sister lineages and placed them in a monophyletic group with the Synchromophyceae, a heterokont algal (Ochrophyta) class characterized by the presence of chloroplast complexes. Yet, neither Chrysopodocystis nor Guanchochroma showed this feature in ultrastructure analyses. Additionally, their 18S rRNA genes possessed distinct inserts, the highest GC-content known for Ochrophyta and exceptionally long branches on the Ochrophyta 18S rDNA phylogenetic tree, suggesting substantially increased substitution rates along their branch compared to Synchromophyceae. Plastid marker data (rbcL) recovered a monophyletic clade of Chrysopodocystis, Guanchochroma and Synchromophyceae as well, yet with lower supports for internal split order due to limited resolution of the marker. Evidence for the sequence of events leading to the formation of the plastid complex of Synchromophyceae still remains ambiguous because of the apparently short timeframe in which they occurred.

Objectives: To investigate the roles of fatty acid biosynthesis in carotenogenesis in the high-lipid accumulating aerial microalga Vischeria helvetica KGU-Y001, we cultured algal cells with fatty acid biosynthesis inhibitors., Results: Under nitrogen-deficient, high-light (200 µmol photons m(-2) s(-1)) conditions, the alga accumulated 6.2 mg carotenoids g(-1) dry weight cells (DWC) after 1 week of culture. The total fatty acid content increased gradually, and reached 290 mg g(-1) DWC after 9 weeks. When algal cells were cultured with a fatty acid biosynthesis inhibitor (molinate) under nitrogen-deficient, high-light conditions for 1 week, carotenoid accumulation was synergistically increased to 2.4 times that in algal cells cultured without the inhibitor in nitrogen-deficient, low-light conditions (40 µmol photons m(-2) s(-1)). The synergistic induction of carotenogenesis was suppressed by an inhibitor of c-jun N-terminal kinase, a mitogen-activated protein kinase-like protein., Conclusion: In a commercial context, carotenoid production could be increased by using fatty acid biosynthesis inhibitors to redirect metabolic flux to carotenoid biosynthesis instead of fatty acid synthesis.

The origin of phytohormones is poorly understood, and their physiological roles in microalgae remain elusive. Genome comparison of photosynthetic autotrophic eukaryotes has revealed that the biosynthetic pathways of abscisic acid (ABA) and cytokinins (CKs) emerged in unicellular algae. While ABA and CK degradation mechanisms emerged broadly in algal lineages, complete vascular plant-type conjugation pathways emerged prior to the rise of Streptophyta. In microalgae, a complete set of proteins from the canonical ABA and CK sensing and signaling pathways is not essential, but individual components are present, suggesting stepwise recruitment of phytohormone signaling components. In the oleaginous eustigmatophyte Nannochloropsis oceanica IMET1, UHPLC-MS/MS detected a wide array of plant hormones, despite a phytohormone profile that is very distinct from that of flowering plants. Time-series transcriptional analysis during nitrogen depletion revealed activation of the ABA biosynthetic pathway and antagonistic transcription of CK biosynthetic genes. Correspondingly, the ABA level increases while the dominant bioactive CK forms decrease. Moreover, exogenous CKs stimulate cell-cycle progression while exogenous ABA acts as both an algal growth repressor and a positive regulator in response to stresses. The presence of such functional flowering plant-like phytohormone signaling systems in Nannochloropsis sp. suggests a much earlier origin of phytohormone biosynthesis and degradation than previously believed, and supports the presence in microalgae of as yet unknown conjugation and sensing/signaling systems that may be exploited for microalgal feedstock development., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Algae frequently get a bad press. Pond slime is a problem in garden pools, algal blooms can produce toxins that incapacitate or kill animals and humans and even the term seaweed is pejorative - a weed being a plant growing in what humans consider to be the wrong place. Positive aspects of algae are generally less newsworthy - they are the basis of marine food webs, supporting fisheries and charismatic marine megafauna from albatrosses to whales, as well as consuming carbon dioxide and producing oxygen. Here we consider what algae are, their diversity in terms of evolutionary origin, size, shape and life cycles, and their role in the natural environment and in human affairs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Alga-derived biofuels are one of the best alternatives for economically replacing liquid fossil fuels with a fungible renewable energy source. Production of fuel from algae is technically feasible but not yet economically viable. Harvest of dilute algal biomass from the surrounding water remains one of the largest barriers to economic production of algal biofuel. We identified Bacillus sp. strain RP1137 in a previous study and showed that this strain can rapidly aggregate several biofuel-producing algae in a pH- and divalent-cation-dependent manner. In this study, we further characterized the mechanism of algal aggregation by RP1137. We show that aggregation of both algae and bacteria is optimal in the exponential phase of growth and that the density of ionizable residues on the RP1137 cell surface changes with growth stage. Aggregation likely occurs via charge neutralization with calcium ions at the cell surface of both algae and bacteria. We show that charge neutralization occurs at least in part through binding of calcium to negatively charged teichoic acid residues. The addition of calcium also renders both algae and bacteria more able to bind to hydrophobic beads, suggesting that aggregation may occur through hydrophobic interactions. Knowledge of the aggregation mechanism may enable engineering of RP1137 to obtain more efficient algal harvesting., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Generation of renewable energy is one of the grand challenges facing our society. We present a new bio-electric technology driven by chemical gradients generated by photosynthesis and respiration. The system does not require pure cultures nor particular species as it works with the core metabolic principles that define phototrophs and heterotrophs. The biology is interfaced with electrochemistry with an alkaline aluminum oxide cell design. In field trials we show the system is robust and can work with an undefined natural microbial community. Power generated is light and photosynthesis dependent. It achieved a peak power output of 33 watts/m(2) electrode. The design is simple, low cost and works with the biological processes driving the system by removing waste products that can impede growth. This system is a new class of bio-electric device and may have practical implications for algal biofuel production and powering remote sensing devices.

Many species of harmful algae transition between a motile, vegetative stage in the water column and a non-motile, resting stage in the sediments. Physiological and behavioral traits expressed during benthic-pelagic transition potentially regulate the timing, location and persistence of blooms. The roles of key physiological and behavioral traits involved in resting cell emergence and bloom formation were examined in two geographically distinct strains of the harmful alga, Heterosigma akashiwo. Physiological measures of cell viability, division and population growth, and cell fatty acid content were made using flow cytometry and gas chromatography - mass spectrometry techniques as cells transitioned between the benthic resting stage and the vegetative pelagic stage. Video-based tracking was used to quantify cell-level swimming behaviors. Data show increased temperature and light triggered rapid emergence from the resting stage and initiated cell swimming. Algal strains varied in important physiological and behavioral traits, including survivorship during life-stage transitions, population growth rates and swimming velocities. Collectively, these traits function as "population growth strategies" that can influence bloom formation. Many resting cells regained the up-swimming capacity necessary to cross an environmentally relevant halocline and the ability to aggregate in near-surface waters within hours after vegetative growth supporting conditions were restored. Using a heuristic model, we illustrate how strain-specific population growth strategies can govern the timescales over which H. akashiwo blooms form. Our findings highlight the need for identification and quantification of strain-specific physiological and behavioral traits to improve mechanistic understanding of bloom formation and successful bloom prediction.

Algal biofuels represent one of the most promising means of sustainably replacing liquid fuels. However, significant challenges remain before alga-based fuels become competitive with fossil fuels. One of the largest challenges is the ability to harvest the algae in an economical and low-energy manner. In this article, we describe the isolation of a bacterial strain, Bacillus sp. strain RP1137, which can rapidly aggregate several algae that are candidates for biofuel production, including a Nannochloropsis sp. This bacterium aggregates algae in a pH-dependent and reversible manner and retains its aggregation ability after paraformaldehyde fixation, opening the possibility for reuse of the cells. The optimal ratio of bacteria to algae is described, as is the robustness of aggregation at different salinities and temperatures. Aggregation is dependent on the presence of calcium or magnesium ions. The efficiency of aggregation of Nannochloropsis oceanica IMET1 is between 70 and 95% and is comparable to that obtained by other means of harvest; however, the rate of harvest is fast, with aggregates forming in 30 s.

The MAST-4 (marine stramenopile group 4) is a widespread uncultured picoeukaryote that makes up an important fraction of marine heterotrophic flagellates. This group has low genetic divergence and is composed of a small number of putative species. We combined ARISA (automated ribosomal intergenic spacer analysis) and ITS (Internal Transcribed Spacer) clone libraries to study the biogeography of this marine protist, examining both spatial and temporal trends in MAST-4 assemblages and associated environmental factors. The most represented MAST-4 clades appeared adapted to different temperature ranges, and their distributions did not suggest clear geographical barriers for dispersal. Distant samples sharing the same temperature had very similar assemblages, especially in cold temperatures, where only one clade, E1, dominated. The most highly represented clades, A and E1, showed very little differentiation between populations from distant geographical regions. Within a single site, temporal variation also followed patterns governed by temperature. Our results contribute to the general discussion on microbial biogeography by showing strong environmental selection for some picoeukaryotes in the marine environment.

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nM. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.

The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.

Protozoans of the family Opalinidae are intestinal commensals in amphibians. To test the hypothesis that these organisms are susceptible to the antiprotozoal antibiotic metronidazole, we randomly assigned 60 juvenile Woodhouse's toads ( Bufo woodhousii ) to receive a single oral dose of metronidazole or water. In pilot trials, the prevalence of opalinids in untreated members of this population was over 70%. One-third of the study population was dissected at each of 3 time points: 18 hr, 1 wk, and 2 wk post-treatment. An examiner blinded to the toad's treatment history determined the presence or absence of opalinids using a dissecting microscope. Opalinids were found in 3/10 toads in the treatment group and 9/10 in the control group after 18 hr (P < 0.02), in none of the treatment group and 8/10 in the control group after 1 wk (P < 0.001), and in none of the treatment group and 10/10 in the control group after 2 wk (P < 0.0001). These results suggest that a single-dose of metronidazole quickly and reliably clears opalinids from juvenile Woodhouse's toads with no evidence of short-term recurrence. The treatment was well tolerated, with no apparent morbidity and no mortality in either group. Future exploration of opalinid-related host fitness consequences may be facilitated by this simple method of developing a protozoan-free host population.

Seagrass beds are the foundation species of functionally important coastal ecosystems worldwide. The world's largest losses of the widespread seagrass Zostera marina (eelgrass) have been reported as a consequence of wasting disease, an infection with the endophytic protist Labyrinthula zosterae. During one of the most extended epidemics in the marine realm, ∼90% of East and Western Atlantic eelgrass beds died-off between 1932 and 1934. Today, small outbreaks continue to be reported, but the current extent of L. zosterae in European meadows is completely unknown. In this study we quantify the abundance and prevalence of the wasting disease pathogen among 19 Z. marina populations in northern European coastal waters, using quantitative PCR (QPCR) with primers targeting a species specific portion of the internally transcribed spacer (ITS1) of L. zosterae. Spatially, we found marked variation among sites with abundances varying between 0 and 126 cells mg(-1) Z. marina dry weight (mean: 5.7 L. zosterae cells mg(-1) Z. marina dry weight ±1.9 SE) and prevalences ranged from 0-88.9%. Temporarily, abundances varied between 0 and 271 cells mg(-1) Z. marina dry weight (mean: 8.5±2.6 SE), while prevalences ranged from zero in winter and early spring to 96% in summer. Field concentrations accessed via bulk DNA extraction and subsequent QPCR correlated well with prevalence data estimated via isolation and cultivation from live plant tissue. L. zosterae was not only detectable in black lesions, a sign of Labyrinthula-induced necrosis, but also occurred in green, apparently healthy tissue. We conclude that L. zosterae infection is common (84% infected populations) in (northern) European eelgrass populations with highest abundances during the summer months. In the light of global climate change and increasing rate of marine diseases our data provide a baseline for further studies on the causes of pathogenic outbreaks of L. zosterae.

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in the stramenopile alga, Vaucheria frigida. AUREO1 harbors a basic leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage-sensing (LOV) domain within the C-terminal region, and has been suggested to function as a light-regulated transcription factor. To understand the molecular mechanism of AUREO1, we have prepared three recombinant proteins: a full-length AUREO1 (FL), an N-terminal truncated construct containing bZIP and LOV (ZL) and a LOV-only (LOV) construct. The constructs showed the same absorption and fluorescent spectra in the dark state and underwent the characteristic cyclic reaction as previously observed in LOV domains upon BL excitation. FL and ZL bound to DNA in a sequence-specific manner. BL appeared to induce a shift of the α-helical structure of the LOV domain to a β-sheet structure, but did not alter the hydrodynamic radius (R(H)) of this domain. ZL formed a dimer possibly through disulfide linkages in the bZIP and the linker region between bZIP and LOV. BL induced an approximately 5% increase in the R(H) of ZL, although its secondary structure was unchanged. These results support a schema where BL-induced changes in the LOV domain may cause conformational changes in the bZIP and/or the linker of a dimeric ZL molecule. Since a 5% increase of the R(H) was also observed with the FL construct, BL may induce global conformational changes similar to those observed for ZL, and formation of the FL dimer may facilitate DNA binding.

Many organisms, including bacteria, fungi, animal, plants and algae, utilize blue light to adapt to a fluctuating light environment. Plants and algae, and photosynthetic stramenopiles in particular, require light energy for photosynthesis and have thus evolved a range of sophisticated light-sensing systems to utilize light information efficiently for growth, development and physiological responses. LOV (light, oxygen or voltage) domain photoreceptors are widely distributed among prokaryotic and eukaryotic organisms, and the number of specific LOV photoreceptors are increased in certain taxa. In this review, we summarize the molecular basis and physiological functions of three different families of LOV blue light receptors specific to green plants and photosynthetic stramenopiles: phototropin, ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LOV KELCH PROTEIN 2 (ZTL/FKF1/LKP2) and aureochrome.

Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochloropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001.