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The seasonal and geographical dynamics of phytoplankton have important implications for primary production, carbon sequestration, and predicting future ocean change. The distribution of phytoplankton is influenced by their environmental needs and preferences and can be examined at various levels, from strain or species to size class or broad taxonomic grouping. The first two chapters of this thesis focus on phytoplankton distribution by taxonomic group as determined by 16S and 18S rRNA gene amplicon sequencing. In chapter one, a time-series study of the seasonally oligotrophic northwestern Sargasso Sea underscores the importance of prasinophytes, a polyphyletic group of green algae. Prasinophytes (mostly Class II Mamiellophyceae) comprised approximately half the eukaryotic phytoplankton amplicons during the time of year where deep mixing brings nutrients to the surface. At the end of the deep mixing, the mixed layer shoaled quickly, which could lead to carbon export when the algae were trapped at depth. The focus of the second chapter is expanded to include other phytoplankton groups in addition to prasinophytes. This chapter explores the eukaryotic phytoplankton communities of various tropical habitats, from the green-algae-dominated salt ponds to the mangroves, reefs, and offshore habitats where stramenopiles become more relatively abundant. The Stramenopile group dictyochophytes were examined at high taxonomic resolution previously not reported for this region, with much of that group found to be comprised of uncultured environmental clades. The third chapter moves from the field to the laboratory, measuring the cell quotas of cultured representatives of Mamiellophyceae under nutrient replete, limiting, and starved conditions. Together, these chapters seek to increase the knowledge of prasinophytes across geographic areas and environmental parameters.

Phytoplankton are sensitive to temperature and other environmental conditions expected to change with warming over the next century. We quantified the capacity of an ecologically dominant Arctic phytoplankton species, Micromonas polaris, to adapt to changes in temperature, increased temperature and irradiance, and increased temperature and periodic nitrogen starvation, over several hundred generations. When originally isolated, this strain of Micromonas had its maximum growth rate at 6°C, and its growth rate declined above 10°C. We find an evolutionary increase in growth rate, with the largest increases associated with the elevated temperature treatments, especially when combined with repeated nitrate starvation. After several hundred generations of exposure, the growth rate of Micromonas under 13°C almost doubled and was higher than under 6°C. This increase in growth rate is consistent with the Arrhenius model of temperature effects on metabolism and suggests a general hypothesis for the evolutionary potential of phytoplankton to respond evolutionarily to temperature change.

Year-round reports of phytoplankton dynamics in the West Antarctic Peninsula are rare and mainly limited to microscopy and/or pigment-based studies. We analyzed the phytoplankton community from coastal waters of Fildes Bay in the West Antarctic Peninsula between January 2014 and 2015 using metabarcoding of the nuclear and plastidial 18/16S rRNA gene from both size-fractionated and flow cytometry sorted samples. Overall 14 classes of photosynthetic eukaryotes were present in our samples with the following dominating: Bacillariophyta (diatoms), Pelagophyceae and Dictyochophyceae for division Ochrophyta, Mamiellophyceae and Pyramimonadophyceae for division Chlorophyta, Haptophyta and Cryptophyta. Each metabarcoding approach yielded a different image of the phytoplankton community with for example Prymnesiophyceae more prevalent in plastidial metabarcodes and Mamiellophyceae in nuclear ones. Diatoms were dominant in the larger size fractions and during summer, while Prymnesiophyceae and Cryptophyceae were dominant in colder seasons. Pelagophyceae were particularly abundant towards the end of autumn (May). In addition of Micromonas polaris and Micromonas sp. clade B3, both previously reported in Arctic waters, we detected a new Micromonas 18S rRNA sequence signature, close to, but clearly distinct from M. polaris, which potentially represents a new clade specific of the Antarctic. These results highlight the need for complementary strategies as well as the importance of year-round monitoring for a comprehensive description of phytoplankton communities in Antarctic coastal waters. Ministry of Education (MOE) Published version This work was funded by INACH RG_31-15, INACH RT_34-17 and FONDECYT No. 1190879 grants. Collaboration between Chile and France was funded through ECOS-CONICYT No. C16B02 and CNRS International Research Network “Diversity, Evolution and Biotechnology of Marine Algae” (GDRI No. 0803). Catherine Géri-kas Ribeiro was supported by the FONDECYT Project No. 3190827. The authors thank Dr. Ernesto Molina for sampling during autumn and spring, as well the logistic support at the scientific station Professor Julio Escudero, INACH. Adriana Lopes dos Santos was supported by the Singapore Ministry of Education, Academic Research Fund Tier 1 (RG26/19). We thank the Roscoff ABIMS platform of the FR2424 (CNRS, Sorbonne Université) for bioinformatics resources.

Polar seas are under threat of enhanced UV-radiation as well as increasing shipping activities. Considering the ecological importance of marine viruses, it is timely to study the impact of UV-AB on Arctic phytoplankton host–virus interactions and also test the efficacy of ballast water (BW) UV-C treatment on virus infectivity. This study examined the effects of:(i) ecologically relevant doses of UV-AB radiation on Micromonas polaris RCC2258 and its virus MpoV-45T, and (ii) UV-C radiation (doses 25–800 mJ cm−2) on MpoV-45T and other temperate algal viruses. Total UV-AB exposure was 6, 12, 28 and 48 h (during the light periods, over 72 h total). Strongest reduction in algal growth and photosynthetic efficiency occurred for 28 and 48 h UV-AB treatments, and consequently the virus production rates and burst sizes were reduced by more than half (compared with PAR-only controls). For the shorter UV-AB exposed cultures, negative effects by UV (especially Fv/Fm) were overcomewithout impacting virus proliferation. To obtain the BW desired log −4 reduction in virus infectivity, a UV-C dose of at least 400 mJ cm−2 was needed for MpoV-45T and the temperate algal viruses. This is higher than the commonly used dose of 300 mJ cm−2 in BW treatment. View Full-Text

This survey is the first to explore the seasonal cycle of microbial eukaryote diversity (

Marine phytoplankton constitute the base of ocean food webs and are responsible for nearly half of the total annual carbon dioxide uptake on the planet acting as important players in biogeochemical cycles. Nitrogen is an essential nutrient that can limit phytoplankton growth. Eukaryotic phytoplankton within the prasinophytes are widespread in the ocean, found from polar to tropical regions under a wide range of environmental conditions, where nitrogen can be an essential nutrient limiting their growth. However, little is known about prasinophyte population dynamics, distributions, growth and abundance, as well as prasinophyte physiological strategies to cope with fluctuation in nitrogen availability in the ocean. Chapter 2 focuses primarily on phytoplankton population dynamics in offshore mesotrophic sites in the dynamic transition zone of the central California Current System. Based on hierarchical clustering of abiotic factors, two modalities were identified within the mesotrophic sites; each modality with distinct phytoplankton communities (determined by flow cytometry and high resolution 16S V1-V2 region rRNA gene amplicon sequencing) that appear to underlie the differences in phytoplankton growth and biomass production observed (determined by dilution experiments and 14C method). The highest biomass production (38.7 ± 8.1 µg C L-1 D-1 by 14C, 86% from photosynthetic eukaryotes) was found in a mesotrophic high modality (MHM) where nitrate was approximately 4-fold higher than in the MLM (mesotrophic low modality). The pico-prasinophyte Ostreococcus clade OI dominated the MHM, whereas in the MLM higher relative contributions of another prasinophyte (uncultured prasinophyte class VIII) and Pelagomonas (stramenopile) were found. Furthermore, the effects of nitrogen (N) depletion on growth, cellular parameters (Chapter 3) and whole-genome transcriptional responses (Chapter 4) of the pico-prasinophyte Micromonas, were investigated in laboratory experiments. This study showed common responses of three Micromonas species that come from three divergent and ecologically important clades. The main commonalities observed were that nitrogen depletion induced a pronounced decrease in growth rate compared to controls within 1 or 2 days and N-deplete treatments take longer to show signs of growth recovery than stationary phase control cells. One major difference was that M. pusilla under N-depletion exhibited a pronounced increase in mean FALS cell-1 (proxy of cell size). At a transcriptional level (RNA-seq), also several commonalities were observed for both two of the Micromonas species studied under N-depletion, such as a major decrease in the relative transcript abundance of genes related to the photosynthetic machinery, carbon transport and metabolism and fatty acid biosynthesis, and an increase in the relative transcript abundance of genes related to flagella and microtubule formation as well as an increase of a number of transcription factors (TFs). In addition, despite the slower growth recovery of N-deplete treatments after transferring to replete medium, a rapid transcriptional response was observed. The Micromonas species exhibited differences in the transcriptional patterns of genes related to nitrogen transport and assimilation. Under N-depletion, the majority of these genes increased in Micromonas commoda while results were more variable for homologous genes in Micromonas pusilla. Distinct transcriptional patterns were observed for ammonium transporter (AMT) homologous in each Micromonas species. In M. pusilla, one ammonium transporter (AMT1.3) appears to only increase under nitrogen depletion and may be a good marker of nutrient status in the field.

Context: Delayed, postoperative, spine infections are rare, most commonly occurring secondary to fastidious, less virulent pathogens. The etiology may involve a distant infectious focus, not related to the index operation. Patients may present months, or even years postoperatively with pain related to mechanical implant failure, often without additional signs of systemic infection. Findings: We present the case of a 59-year-old male who developed rapid disk degeneration and implant failure seven months following instrumented lumbar fusion surgery. The causal organism was found to be Micromonas micros, an anaerobic bacterium typically located in the oral cavity and associated with periodontal disease. The patient was found to have extensive oral caries, which were presumed to have occurred secondary to poor oral hygiene and his use of fentanyl lozenges for chronic back pain. The patient was treated with revision staged spinal surgery and long-term intravenous antibiotics. Conclusion/clinical relevance: This case highlights an unusual etiology of delayed postoperative spinal implant failure and provides evidence for periodontal disease as a source of hematogenous seeding in postoperative spinal infections. The orthopaedist should also be aware of the potential relationship between poor oral hygiene and the use of high sugar content fentanyl lozenges in treating chronic back pain in these patients.

Marine phytoplankton perform approximately half of global CO2 uptake. The phytoplankton communities that perform this photosynthesis are diverse and belong to different size classes. In many marine settings picophytoplankton (size) are abundant and among eukaryotic picoplankton the prasinophyte class Mamiellophyceae is particularly well-represented. Three Mamiellophyceae genera found in many marine settings are Micromonas, Ostreococcus and Bathycoccus. This thesis provides analyses of features shared by the Mamiellophyceae, features unique to an individual genus, and overall environmental distributions of the above genera. Specifically, Mamiellophyceae genome features were characterized quantitatively in Micromonas (Chapter 2, 3) and led to the detection of repetitive elements that can be used as tools for identifying Micromonas clades (Chapter 3). Application of these 'markers' to metagenomic data led to the discovery of Micromonas in the Southern Ocean and at salinities and depths it was previously not known to inhabit. In Ostreococcus and Bathycoccus different types of molecular markers were used to define distances between putative ecotypes within these genera (Chapter 4). This analysis showed two coastal Bathycoccus targeted metagenomes belong to the same clade as a cultured species, Bathycoccus prasinos, while sequences from a Tropical Atlantic Bathycoccus belong to a separate clade. Using this comprehension of Bathycoccus and Ostreococcus diversity, ecomarker genes were selected and used to analyze ecotypes of these genera in metatranscriptome and metagenome data. These analyses were combined with qPCR, flow cytometry and nutrient measurements (Chapter 4). The results provided in this thesis improve our knowledge of the diversity and biogeography of members of the Mamiellophyceae class. Collectively, these studies provide a baseline for future comparisons, as speciation and environmental adaptations are ongoing and may increase with global climate change.

International audience; In the Arctic Ocean, the small green alga Micromonas polaris dominates picophytoplankton during the summer months but is also present in winter. It has been previously hypothesized to be phago-mixotrophic (capable of bacteria ingestion) based on laboratory and field experiments. Prey uptake was analyzed in several M. polaris strains isolated from different regions and depths of the Arctic Ocean and in Ochromonas triangulata, a known phago-mixotroph used as a control. Measuring ingestion of either fluorescent beads or fluorescently labeled bacteria by flow cytometry, we found no evidence of phago-mixotrophy in any M. polaris strain while O. triangulata was ingesting both beads and bacteria. In addition, in silico predictions revealed that members of the genus Micromonas lack a genetic signature of phagocytotic capacity.

Global climate changes and anthropogenic activity greatly impact Arctic marine biodiversity including phytoplankton which contribute greatly to atmospheric oxygen production. Thus the study of microalgae has rising topicality. Class Mamiellophyceae is an important component of phototrophic picoplankton. To gain more knowledge about Mamiellophyceae distribution and diversity special studies were performed in such remote areas as the Russian Arctic seas. A metabarcoding of pico-sized Mamiellophyceae was undertaken by high-throughput sequencing of the 18S rRNA gene sequence V4 region from samples collected in July–September 2017 in the Barents, Kara and Laptev seas, and in the adjacent waters of the Norwegian Sea. Our study is the first to show that Mamiellophyceae among the summer picoplankton of Russian Arctic seas are diverse and represented by 16 algae species/phylotypes. We discovered a new candidate species of Bathycoccus assigned to a new Bathycoccus clade A—uncultured Bathycoccus Kara 2017. It was found that several Micromonas species can co-exist, with Micromonas polaris dominating north of 72°N. The presence of Ostreococcus tauri, Ostreococcus lucimarinus and Ostreococcus mediterraneus at high latitudes beyond 65°N was documented for the first time, similar to findings for some other taxa. Our results will be important for obtaining a global view of Mamiellophyceae community dynamics.

In the Arctic Ocean, the small green alga Micromonas polaris dominates pico-phytoplankton during the summer months. It has been previously hypothesized to be phago-mixotrophic (capable of bacteria ingestion) based on laboratory and field experiments. Prey uptake was analysed in several M. polaris strains isolated from different regions and depths of the Arctic Ocean. Using both fluorescent beads and fluorescently labelled bacteria as prey, we found no evidence of phago-mixotrophy in any M. polaris strain by flow cytometric measurement of prey ingestion. In addition, in silico predictions reveal that members of the genus Micromonas lack a genetic signature of phagocytotic capacity.

The White Sea is a unique basin combining features of temperate and arctic seas. The current state of its biocenoses can serve as a reference point in assessing the expected desalination of the ocean as a result of climate change. A metagenomic study of under-ice ice photosynthetic picoeukaryotes (PPEs) was undertaken by Illumina high-throughput sequencing of the 18S rDNA V4 region from probes collected in March 2013 and 2014. The PPE biomass in samples was 0.03&ndash, 0.17 &micro, g C&middot, L&minus, 1 and their abundance varied from 10 cells&middot, mL&minus, 1 to 140 cells&middot, 1. There were representatives of 16 algae genera from seven classes and three supergroups, but Chlorophyta, especially Mamiellophyceae, dominated. The most represented genera were Micromonas and Mantoniella. For the first time, the predominance of Mantoniella (in four samples) and Bolidophyceae (in one sample) was observed in under-ice water. It can be assumed that a change in environmental conditions will lead to a considerable change in the structure of arctic PPE communities.

Massive phytoplankton blooms develop at the Arctic ice edge, sometimes extending far under the pack ice. An extensive culturing effort was conducted before and during a phytoplankton bloom in Baffin Bay between April and July 2016. Different isolation strategies were applied, including flow cytometry cell sorting, manual single cell pipetting and serial dilution. Although all three techniques yielded the most common organisms, each technique retrieved specific taxa, highlighting the importance of using several methods to maximize the number and diversity of isolated strains. More than 1,000 cultures were obtained, characterized by 18S rRNA sequencing and optical microscopy and de-replicated to a subset of 276 strains presented in this work. Strains grouped into 57 genotypes defined by 100% 18S rRNA sequence similarity. These genotypes spread across five divisions: Heterokontophyta, Chlorophyta, Cryptophyta, Haptophyta and Dinophyta. Diatoms were the most abundant group (193 strains), mostly represented by the generaChaetocerosandAttheya. The generaRhodomonasandPyramimonaswere the most abundant non-diatom nanoplankton strains, whileMicromonas polarisdominated the picoplankton. Diversity at the class level was higher during the peak of the bloom. Potentially new species were isolated, in particular within the generaNavicula,Nitzschia,Coscinodiscus,Thalassiosira,Pyramimonas,MantoniellaandIsochrysis.Submitted to: Elementa: Science of the Anthropocene Date: May 17, 2019

Prasinophytes are widespread marine algae for which responses to nutrient limitation and viral infection are not well understood. We studied the picoprasinophyte, Micromonas pusilla, grown under phosphate-replete (0.65 ± 0.07 d-1 ) and 10-fold lower (low)-phosphate (0.11 ± 0.04 d-1 ) conditions, and infected by the phycodnavirus MpV-SP1. Expression of 17% of Micromonas genes in uninfected cells differed by >1.5-fold (q < 0.01) between nutrient conditions, with genes for P-metabolism and the uniquely-enriched Sel1-like repeat (SLR) family having higher relative transcript abundances, while phospholipid-synthesis genes were lower in low-P than P-replete. Approximately 70% (P-replete) and 30% (low-P) of cells were lysed 24 h post-infection, and expression of ≤5.8% of host genes changed relative to uninfected treatments. Host genes for CAZymes and glycolysis were activated by infection, supporting importance in viral production, which was significantly lower in slower growing (low-P) hosts. All MpV-SP1 genes were expressed, and our analyses suggest responses to differing host-phosphate backgrounds involve few viral genes, while the temporal program of infection involves many more, and is largely independent of host-phosphate background. Our study (i) identifies genes previously unassociated with nutrient acclimation or viral infection, (ii) provides insights into the temporal program of prasinovirus gene expression by hosts and (iii) establishes cell biological aspects of an ecologically important host-prasinovirus system that differ from other marine algal-virus systems.

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42–49%, 35–49% and 13–17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

International audience; The green picoalgal genus Micromonas is broadly distributed in estuaries, coastal marine habitats and open oceans, from the equator to the poles. Phylogenetic, ecological and genomic analyses of culture strains and natural populations have suggested that this cosmopolitan genus is composed of several cryptic species corresponding to genetic lineages. We performed a detailed analysis of variations in morphology, pigment content, and sequences of the nuclear-encoded small-subunit rRNA gene and the second internal transcribed spacer (ITS2) from strains isolated worldwide. A new morphological feature of the genus, the presence of tip hairs at the extremity of the hair point, was discovered and subtle differences in hair point length were detected between clades. Clear non-homoplasious synapomorphies were identified in the small-subunit rRNA gene and ITS2 spacer sequences of five genetic lineages. These findings lead us to provide emended descriptions of the genus Micromonas, of the type species M. pusilla, and of the recently described species M. commoda, as well as to describe 2 new species, M. bravo and M. polaris. By clarifying the status of the genetic lineages identified within Micromonas, these formal descriptions will facilitate further interpretations of large-scale analyses investigating ecological trends in time and space for this widespread picoplankter.

Viruses have recurrently been hypothesized as instrumental in driving microbial population diversity. Nonetheless, viral mediated co-existence of r/k-strategists, predicted in the Killing-the-Winner (KtW) hypothesis, remains controversial and demands empirical evidence. Therefore, we measured the life strategy parameters that characterize the relevant system Micromonas-Micromonas Virus (MicV). A large number of host and viral strains (37 and 17, respectively) were used in a total of 629 cross-infectivity tests. Algal and viral abundances were monitored by flow cytometry and used to calculate values of growth rate, resistance capacity, and viral production. Two main assumptions of the KtW model, namely (1) a resistance-associated cost on growth and (2) a negative correlation between resistance and viral production capacity, were mildly observed and lacked statistical significance. Micromonas strains infected by more MicV strains presented higher lysis and viral production rates as the number of infectious virus strains increased, suggesting a 'one-gate' regulation of infection in this system. MicV strains demonstrated a vast range of virion production capacity, which unexpectedly grew with increasing host-range. Overall, the significant trends observed in here demonstrate strong co-interactions at different levels between Micromonas and MicV populations, however, the role of viruses as major driving force in phytoplankton fitness wasn't explicitly observed.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, Ostreococcus , Bathycoccus and Micromonas , are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue ‘Single cell ecology’.

Micromonas commoda(previouslyMicromonas pusilla, a unicellular photosynthetic picoeukaryote globally dominant in marine ecosystems, has previously been qualified as being strongly phototactic. To date, no detailed quantitative or qualitative description of this behaviour has been reported, nor have thorough studies of its motility been undertaken. This primary producer has only been qualitatively described as utilizing run-and-tumble motion, but such motility strategy is incompatible with its morphology comprising only one propelling flagellum. Moreover, it is still unclear as to howMicromonas sp.detects a light direction due to the lack of a dedicated eyespot; the organism is essentially blind. Here we first perform population-scale phototactic experiments to show that this organism actively responds to a wide range of light wavelengths and intensities. These population responses are well accounted for within a simple drift-diffusion framework. Based on single-cell tracking experiments, we then detail thoroughlyMicromonas sp.’s motility which resembles run-and-reverse styles of motion commonly observed in marine prokaryotes and that we namestop-run or reverse. The associated peculiar microscopic changes upon photo-stimulation are finally described and integrating those into jump-diffusion simulations appears to produce phototactic drifts that are quantitatively compatible with those obtained experimentally at the population level.

The global phytoplankton community, comprised of aquatic photosynthetic organisms, is acknowledged for being responsible for half of the global oxygen production Prominent among these is the pico-eukaryoteMicromonas commoda(formallyMicromonas pusillaof the genusMicromonas), which can be found in marine and coastal environments across the globe. Cell death of phytoplankton has been identified as contributing to the largest carbon transfers on the planet moving 109tonnes of carbon in the oceans every day. During a cell death organic matter is released into the local environment which can act as both a food source and a warning signal for nearby organisms. Here we present a novel motility response to single cell death in populations ofMicromonas sp., where the death of a single cell releases a chemical patch triggers surrounding cells to escape the immediate affected area. These so-called “burst events” are then modelled and compared with a spherically symmetric diffusing patch which is found to faithfully reproduce the observed behaviour. Finally, laser ablation of single cells reproduces the observed avoidance response, confirming thatMicromonas sp. has evolved a specific motility response in order to escape harmful environments for example nearby predator-prey interactions or virus lysis induced cell death.

International audience; Seasonality in marine microorganisms has been classically observed in phytoplankton blooms, and more recently studied at the community level in prokaryotes, but rarely investigated at the scale of individual microbial taxa. Here we test if specific marine eukaryotic phytoplankton, bacterial and archaeal taxa display yearly rhythms at a coastal site impacted by irregular environmental perturbations. Our seven-year study in the Bay of Banyuls (North Western Mediterranean Sea) shows that despite some fluctuating environmental conditions, many microbial taxa displayed significant yearly rhythms. The robust rhythmicity was found in both autotrophs (picoeukaryotes and cyanobacteria) and heterotrophic prokaryotes. Sporadic meteorological events and irregular nutrient supplies did, however, trigger the appearance of less common non-rhythmic taxa. Among the environmental parameters that were measured, the main drivers of rhythmicity were temperature and day length. Seasonal autotrophs may thus be setting the pace for rhythmic heterotrophs. Similar environmental niches may be driving seasonality as well. The observed strong association between Micromonas and SAR11, which both need thiamine precursors for growth, could be a first indication that shared nutritional niches may explain some rhythmic patterns of co-occurrence.

International audience; Photosynthetic picoeukaryotes in the genus Micromonas show among the widest latitudinal distributions on Earth, experiencing large thermal gradients from poles to tropics. Micromonas comprises at least four different species often found in sympatry. While such ubiquity might suggest a wide thermal niche, the temperature response of the different strains is still unexplored, leaving many questions as for their ecological success over such diverse ecosystems. Using combined experiments and theory, we characterize the thermal response of eleven Micromonas strains belonging to four 1 species. We demonstrate that the variety of specific responses to temperature in the Micromonas genus makes this environmental factor an ideal marker to describe its global distribution and diversity. We then propose a diversity model for the genus Micromonas, which proves to be representative of the whole phytoplankton diversity. This prominent primary producer is therefore a sentinel organism of phytoplankton diversity at the global scale. We use the diversity within Micromonas to anticipate the potential impact of global warming on oceanic phytoplankton. We develop a dynamic, adaptive model and ran forecast simulations, exploring a range of adaptation time scales, to probe the likely responses to climate change. Results stress how biodiversity erosion depends on the ability of organisms to adapt rapidly to temperature increase.

Western boundary currents support high primary production and carbon export. Here, we performed a survey of photosynthetic picoeukaryotes in the North Pacific Ocean in four transects crossing the Kuroshio Front. Prasinophyte algae comprised 85% of 18S rRNA gene sequences for photosynthetic taxa in the

Expressed sequence tag analyses revealed that two marine Chlorophyceae green algae, Chlamydomonas sp. W80 and Chlamydomonas sp. HS5, contain genes coding for chloroplastic class IIA aldolase (fructose-1, 6-bisphosphate aldolase: FBA). These genes show robust monophyly with those of the marine Prasinophyceae algae genera Micromonas, Ostreococcus and Bathycoccus, indicating that the acquisition of this gene through horizontal gene transfer by an ancestor of the green algal lineage occurred prior to the divergence of the core chlorophytes (Chlorophyceae and Trebouxiophyceae) and the prasinophytes. The absence of this gene in some freshwater chlorophytes, such as Chlamydomonas reinhardtii, Volvox carteri, Chlorella vulgaris, Chlorella variabilis and Coccomyxa subellipsoidea, can therefore be explained by the loss of this gene somewhere in the evolutionary process. Our survey on the distribution of this gene in genomic and transcriptome databases suggests that this gene occurs almost exclusively in marine algae, with a few exceptions, and as such, we propose that chloroplastic class IIA FBA is a marine environment-adapted enzyme. This hypothesis was also experimentally tested using Chlamydomonas W80, for which we found that the transcript levels of this gene to be significantly lower under low-salt (that is, simulated terrestrial) conditions. Expression analyses of transcriptome data for two algae, Prymnesium parvum and Emiliania huxleyi, taken from the Sequence Read Archive database also indicated that the expression of this gene under terrestrial conditions (low NaCl and low sulfate) is significantly downregulated. Thus, these experimental and transcriptome data provide support for our hypothesis.

High-throughput sequencing of Prasinovirus DNA polymerase and host green algal (Mamiellophyceae) ribosomal RNA genes was used to analyse the diversity and distribution of these taxa over a ∼10 000 km latitudinal section of the Indian Ocean. New viral and host groups were identified among the different trophic conditions observed, and highlighted that although unknown prasinoviruses are diverse, the cosmopolitan algal genera Bathycoccus, Micromonas and Ostreococcus represent a large proportion of the host diversity. While Prasinovirus communities were correlated to both the geography and the environment, host communities were not, perhaps because the genetic marker used lacked sufficient resolution. Nevertheless, analysis of single environmental variables showed that eutrophic conditions strongly influence the distributions of both hosts and viruses. Moreover, these communities were not correlated, in their composition or specific richness. These observations could result from antagonistic dynamics, such as that illustrated in a prey–predator model, and/or because hosts might be under a complex set of selective pressures. Both of these reasons must be considered to interpret environmental surveys of viruses and hosts, because covariation does not always imply interaction.

Mixotrophic flagellates can comprise significant proportions of plankton biomass in marine ecosystems. Despite the growing recognition of the importance of this ecological strategy, and the identification of major environmental factors controlling phagotrophic behavior (light and nutrients), the physiological and molecular mechanisms underlying mixotrophic behavior are still unclear. In this study, we performed RNA-Seq transcriptomic analysis for two mixotrophic prasinophytes, Micromonas polaris and Pyramimonas tychotreta, under dissolved nutrient regimes that altered their ingestion of bacteria prey. Though the strains examined were polar isolates, both belong to genera with widespread distribution. Our aim was to characterize the transcriptomes of these two non-model phytoflagellates, identify transcripts consistent with phagotrophic activity and assess their differential expression in response to nutrient stress. De novo assembly of the transcriptomes yielded large numbers of novel coding transcripts with no known match within public databases. A summary of the transcripts by Gene Ontology terms showed many expected expression patterns, including genes involved in photosynthetic pathways and enzymes implicated in nutrient uptake pathways. Searches of KEGG databases identified several genes associated with intra-cellular digestive pathways actively transcribed in both prasinophytes. Differential expression analysis showed a larger response in P. tychotreta, where 23,373 genes were up-regulated and 1,752 were down-regulated in the low nutrient treatment when phagotrophy was enhanced. In contrast, in M. polaris, low nutrient treatments resulted in up-regulation of 314 transcripts while down-regulating 371. With respect to phagotrophic-related expression, 37 genes were co-expressed in both P. tychotreta and M. polaris, and although the response was less pronounced in M. polaris, it is consistent with differences in observed ingestion behavior. This study presents the first genomic data for Pyramimonas tychotreta, and also contributes to the limited available data for Micromonas polaris. Furthermore, it provides insight into the presence of genes associated with phagocytosis within the Prasinophyceae and contributes to the understanding of potential target genes required for the construction of a complete model of gene regulation of phagocytic behavior in algae.

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2 μm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2 μm filters, targeting the free-living fraction only. From the 0.27–0.34 Gb WGS library, 0.9 × 106–1.2 × 106 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0–19.6% Planktomarina (Family Rhodobacteraceae) and 13.7–17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5–6.1%), Flavobacterium (1.8–2.6%), Sphingobacterium (1.4–1.6%) and Cellulophaga (1.4–2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8–15.2%, 3.6–4.9% and 2.1–3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing.

Nitrogen stress is an important control on the growth of phytoplankton and varying responses to this common condition among taxa may affect their relative success within phytoplankton communities. We analyzed photosynthetic responses to nitrogen (N) stress in two classes of phytoplankton that often dominate their respective size ranges, diatoms and prasinophytes, selecting species of distinct niches within each class. Changes in photosynthetic structures appeared similar within each class during N stress, but photophysiological and growth responses were more species- or niche-specific. In the coastal diatom Thalassiosira pseudonana and the oceanic diatom T. weissflogii, N starvation induced large declines in photosynthetic pigments and Photosystem II (PSII) quantity and activity as well as increases in the effective absorption cross-section of PSII photochemistry (σʹPSII). These diatoms also increased photoprotection through energy-dependent non-photochemical quenching (NPQ) during N starvation. Resupply of N in diatoms caused rapid recovery of growth and relaxation of NPQ, while recovery of PSII photochemistry was slower. In contrast, the prasinophytes Micromonas sp., an Arctic Ocean species, and Ostreococcus tauri, a temperate coastal eutrophile, showed little change in photosynthetic pigments and structures and a decline or no change, respectively, in σʹPSII with N starvation. Growth and PSII function recovered quickly in Micromonas sp. after resupply of N while O. tauri failed to recover N-replete levels of electron transfer from PSII and growth, possibly due to their distinct photoprotective strategies. O. tauri induced energy-dependent NPQ for photoprotection that may suit its variable and nutrient-rich habitat. Micromonas sp. relies upon both energy-dependent NPQ and a sustained, energy-independent NPQ mechanism. A strategy in Micromonas sp. that permits photoprotection with little change in photosynthetic structures is consistent with its Arctic niche, where low temperatures and thus low biosynthetic rates create higher opportunity costs to rebuild photosynthetic structures.

Arctic marine ecosystems are currently undergoing rapid changes in temperature and light availability. Picophytoplankton, such as Micromonas polaris, are predicted to benefit from such changes. However, little is known about how these environmental changes affect the viruses that exert a strong mortality pressure on these small but omnipresent algae. Here we report on one-step infection experiments, combined with measurements of host physiology and viability, with 2 strains of M. polaris and the virus MpoV-45T under 3 light intensities (5, 60 and 160 &mu, mol quanta m&minus, 2 s&minus, 1), 2 light period regimes (16:8 and 24:0 h light:dark cycle) and 2 temperatures (3 and 7 &deg, C). Our results show that low light intensity (16:8 h light:dark) delayed the decline in photosynthetic efficiency and cell lysis, while decreasing burst size by 46%. In contrast, continuous light (24:0 h light:dark) shortened the latent period by 5 h for all light intensities, and even increased the maximum virus production rate and burst size under low light (by 157 and 69%, respectively). Higher temperature (7 &deg, C vs 3 &deg, C) led to earlier cell lysis and increased burst size (by 19%), except for the low light conditions. These findings demonstrate the ecological importance of light in combination with temperature as a controlling factor for Arctic phytoplankton host and virus dynamics seasonally, even more so in the light of global warming.

Micromonas strains of small prasinophyte green algae are found throughout the world's oceans, exploiting widely different niches. We grew arctic and temperate strains of Micromonas and compared their susceptibilities to photoinactivation of Photosystem II, their counteracting Photosystem II repair capacities, their Photosystem II content, and their induction and relaxation of non-photochemical quenching. In the arctic strain Micromonas NCMA 2099, the cellular content of active Photosystem II represents only about 50 % of total Photosystem II protein, as a slow rate constant for clearance of PsbA protein limits instantaneous repair. In contrast, the temperate strain NCMA 1646 shows a faster clearance of PsbA protein which allows it to maintain active Photosystem II content equivalent to total Photosystem II protein. Under growth at 2 °C, the arctic Micromonas maintains a constitutive induction of xanthophyll deepoxidation, shown by second-derivative whole-cell spectra, which supports strong induction of non-photochemical quenching under low to moderate light, even if xanthophyll cycling is blocked. This non-photochemical quenching, however, relaxes during subsequent darkness with kinetics nearly comparable to the temperate Micromonas NCMA 1646, thereby limiting the opportunity cost of sustained downregulation of PSII function after a decrease in light.

The genus Micromonas comprises distinct genetic clades that commonly dominate eukaryotic phytoplankton community from polar to tropical waters. This phytoplankter is also recurrently infected by abundant and genetically diverse prasinoviruses. Here we report on the interplay between prasinoviruses and Micromonas with regard to the genetic diversity of this host. For 1 year, we monitored the abundance of three clades of Micromonas and their viruses in the Western English Channel, both in the environment using clade-specific probes and flow cytometry, and in the laboratory using clonal strains of Micromonas clades to assay for their viruses by plaque-forming units. We showed that the seasonal fluctuations of Micromonas clades were closely mirrored by the abundance of their corresponding viruses, indicating that the members of Micromonas genus are susceptible to viral infection, regardless of their genetic affiliation. The characterization of 45 viral isolates revealed that Micromonas clades are attacked by specific virus populations, which exhibit distinctive clade specificity, life strategies and genetic diversity. However, some viruses can also cross-infect different host clades, suggesting a mechanism of horizontal gene transfer within the Micromonas genus. This study provides novel insights into the impact of viral infection for the ecology and evolution of the prominent phytoplankter Micromonas.

We confirmed multiple picoeukaryotic algae, Ostreococcus, Micromonas, and Pelagomonas spp., as thiamine (vitamin B1) auxotrophs in laboratory experiments with axenic cultures. Examined strains have half saturation growth constants (Ks) for B1 between 1.26 and 6.22 pmol B1 L−1, which is higher than reported seawater concentrations. Minimum B1 cell quotas for Ostreococcus and Micromonas spp. are high (2.20 × 10−8–4.46 × 10−8 pmol B1 cell−1) relative to other B1 auxotrophic phytoplankton, potentially making them B1 rich prey for zooplankton and significant B1 reservoirs in oligotrophic marine habitats. Ostreococcus and Micromonas genomes are nonuniformly missing portions of the B1 biosynthesis pathway. Given their gene repertoires, Ostreococcus lucimarinus CCE9901 and Ostreococcus tauri OTH95 are expected to salvage B1 from externally provided 4-methyl-5-thiazoleethanol (HET) and 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). However, in culture, neither could use HET plus HMP instead of B1, highlighting current limitations of genome-based prediction of B1 salvaging by picoeukaryotic algae. HMP and phosphorylated B1 use varied amongst tested strains and notably all Prasinophytes tested could not use HMP. B1-limited O. lucimarinus CCE9901 could not grow on added thiamine diphosphate (TDP), a phosophorylated B1 form. However, in co-culture with Pseudoalteromonas sp. TW7, a bacterium known to exhibit phosphatase activity, O. lucimarinus CCE9901 exhibited increased growth following TDP additions. This demonstrates that bacteria influence vitamin B1 availability beyond de novo synthesis and consumption; they can also serve as conduits that chemically alter, but not completely degrade or retain B1 analogs (e.g., TDP), and make them accessible to a broader range of microbes.

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

I here present a survey of generic names with standing in the prokaryotic nomenclature that have homonyms with standing under the International Code of Zoological Nomenclature and/or the International Code of Nomenclature for algae, fungi, and plants. I especially discuss such names added after Principle 2 of the Bacteriological Code/Prokaryotic Code was changed in 1999 to make the prokaryote nomenclature not independent of botanical and zoological nomenclature. Cases include the genera Micromonas , Quadrococcus, Yania , Sinococcus, and Meganema . The generic name Meganema was not previously recognized as a homonym of two genera with standing in the zoological nomenclature. Therefore, I here propose renaming Meganema and Meganema perideroedes as Neomegalonema gen. nov. and Neomegalonema perideroedes comb. nov., respectively.

We have available several plasmids designed for expression of Cas9, guide RNA, chloramphenicol acetyl-transferase, GFP, and beta-lactamase in Micromonas CCMP1545. For protein expression we used the promotor and 3' end elements from the endogenous RPS9 gene, and codon optimized the coding region. For expression of guide RNAs we used the Micromonas U6 snRNA promoter. DNA is available by contacting M. Ares . We have constructed and sequence verified 9 plasmids which we would like to make available to others attempting to detect transformation of DNA into Micromonas. Using the CCMP1545genome as a source for the U6 promoter sequence, and for the promoter and 3' UTR sequences of ribosomal protein RPS9, we built the following plasmids: CRISPR/Cas9 plasmids for Micromonas 1. Mp U6 promoter driving Bae cassette for guide RNA expression inpUC13 2. Mp RPS9-Cas9SV40-RPS9 in pUC13 3. BothMp U6 promoter driving Bae cassette for guide RNA expression andMp RPS9-Cas9SV40-RPS9 in pUC13 These first three plasmids were anticipated to enable stable incorporation of transgenes at specific genomic locations. BaeI is a type IIS restriction enzyme that leaves noncompatible sticky ends. In the context of the gRNA cassette, a pair of 24 nt oligos designed to have the sticky ends compatible with BaeI cleaved plasmid are annealed and cloned into the plasmid, replacing the BaeI cassette while adding the 20 nt target complementary sequence of the desired guide RNA. Selectable/Detectable Marker genes 4. Mp RPS9-codon optimized GFPsv40-RPS9 in pUC13 5. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pUC13 6. Mp RPS9-codon optimized beta-lactamase-RPS9 in pUC13 These three plasmids have the indicated coding regions codon optimized for Micromonas flanked by RPS9 promoter and 3' end sequences. In the case of GFP, a nuclear localization signal from SV40 has been added to the C-terminus. Plasmids for Agrobacterium-mediated gene transfer 7. Mp RPS9-codon optimized GFPsv40-RPS9 in pOSCAR 8. Mp RPS9-codon optimized chloramphenicol acetyltransferase-RPS9 in pOSCAR 9. Mp RPS9-codon optimized beta-lactamase-RPS9 in pOSCAR

Contains fulltext : 174127.pdf (Publisher’s version ) (Open Access) Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter Micromonas. We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 +/- 2 vs. 191 +/- 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term (seasonal cycles) and long term (global warming).