Marine Stramenopiles Research Articles

Taxon-specific contributions of microeukaryotes to biological carbon pump in the Oyashio region

Abstract Microeukaryotes are critical components of sinking particles contributing to carbon export from the surface to deep oceans. However, the knowledge of the sinking microeukaryotic communities and their dynamics is currently limited. In this study, we applied 18S rRNA gene metabarcoding to investigate the microeukaryotic communities in sinking and suspended particles distinguished by marine snow catchers (MSC) during spring in the Oyashio region. Sinking particles displayed distinct communities and lower diversity than suspended particles. The community compositions of the sinking particles varied with depth, suggesting that microeukaryotes were selectively disaggregated or decomposed during settling. Prymnesiophyceae and diatoms were effectively removed, as indicated by their decreased abundance in sinking particles at increasing depths. Conversely, Dinophyceae maintained a higher abundance in sinking particles across depths, indicating resistance to disaggregation and decomposition. Spirotrichea and heterotrophic Dinophyceae were enriched in sinking particles, while marine stramenopiles (MAST) groups were enriched in suspended particles. The heterotrophs in the deeper layers were mainly transported from the surface layers by increasing their relative abundance towards deep layers, indicating that they contributed to the transformation processes of sinking particles. Overall, our results demonstrate the functional differences among microeukaryotes in the biological carbon pump.

How marine are Marine Stramenopiles (MAST)? A cross-system evaluation.

Marine Stramenopiles (MAST) were first described two decades ago through ribosomal RNA gene (rRNA gene) sequences from marine surveys of microbial eukaryotes. MAST comprise several independent lineages at the base of the Stramenopiles. Despite their prevalence in the ocean, the majority of MAST diversity remains uncultured. Previous studies, mainly in marine environments, have explored MAST's cell morphology, distribution, trophic strategies, and genomics using culturing-independent methods. In comparison, less is known about their presence outside marine habitats. Here, we analyse the extensive EukBank dataset to assess the extent to which MAST can be considered marine protists. Additionally, by incorporating newly available rRNA gene sequences, we update Stramenopiles phylogeny, identifying three novel MAST lineages. Our results indicate that MAST are primarily marine with notable exceptions within MAST-2 and MAST-12, where certain subclades are prevalent in freshwater and soil habitats. In the marine water column, only a few MAST species, particularly within clades -1, -3, -4, and -7, dominate and exhibit clear latitudinal distribution patterns. Overall, the massive sequencing dataset analysed in our study confirms and partially expands the previously described diversity of MASTs groups and underscores the predominantly marine nature of most of these uncultured lineages.

A removable and cosmopolitan dsRNA Toti-like virus causes asymptomatic but productive infection in a model diatom strain

Diatoms contribute to 20 % of global primary productivity. Although some diatom viruses have been identified, the molecular mechanisms underlying their interactions with the host remain poorly understood. In this study, we report the discovery of an RNA molecule in the DNA extracts of the Phaeodactylum tricornutum strain Pt1, which possesses a well-annotated genome and has been used as a diatom model system since 1956. We confirmed this molecule to be a double-stranded linear RNA molecule and, through sequencing, demonstrated it to be a virus in the Totiviridae family that is prevalent among marine stramenopiles. We also detected this virus in Phaeodactylum tricornutum strain Pt3, which was collected in 1930s from a similar geographic location to Pt1, suggesting its prevalence within the region. By employing various inhibitors of the viral RNA-dependent RNA polymerase, we successfully generated a virus-free line isogenic to Pt1, establishing a model system to investigate the impact of RNA viruses on diatom physiology. The virus-free lines did not display obvious growth advantages or defects, indicating a tendency of the virus towards asymptomatic infection. Furthermore, we generated a robust antibody against the coat protein of this virus. By performing immunoprecipitation coupled with mass spectrometry, we found that translation-related proteins are enriched as potential interacting partners of the coat protein. Our results suggest that potential viral impacts in molecular research should be considered when Pt1 and Pt3 are used for studying translation-related processes. Additionally, our study unveiled a mode of asymptomatic but productive infection between viruses and marine algal hosts that differs from the commonly-reported virulent, lytic infections.

Spatial distribution and diversity of the heterotrophic flagellates in the Cosmonaut Sea, Antarctic

As predators of bacteria and viruses and as food sources for microzooplankton, heterotrophic flagellates (HFs) play an important role in the marine micro-food web. Based on the global climate change’s impact on marine ecosystems, particularly sea ice melting, we analyzed the community composition and diversity of heterotrophic flagellates, focusing on the Antarctic Cosmonaut Sea. During the 36th China Antarctic research expedition (2019-2020), we collected seawater samples, subsequently analyzing HFs through IlluminaMiSeq2000 sequencing to assess community composition and diversity. Notable variations in HFs abundance were observed between the western and eastern sectors of the Cosmonaut Sea, with a distinct concentration at a 100-meter water depth. Different zones exhibited diverse indicators and dominants taxa influenced by local ocean currents. Both the northern Antarctic Peninsula and the western Cosmonaut Sea, where the Weddell Eddy and Antarctic Land Slope Current intersect, showcased marine stramenopiles as dominant HFs species. Our findings offer insights into dominant taxa, spatial distribution patterns among heterotrophic flagellates, correlations between taxa distribution and environmental factors, and the exploration of potential indicator taxa.

Phylogenomic position of genetically diverse phagotrophic stramenopile flagellates in the sediment-associated MAST-6 lineage and a potentially halotolerant placididean

Unlike morphologically conspicuous ochrophytes, many flagellates belonging to basally branching stramenopiles are small and often overlooked. As a result, many of these lineages are known only through molecular surveys and identified as MArine STramenopiles (MAST), and remain largely uncharacterized at the cellular or genomic level. These likely phagotrophic flagellates are not only phylogenetically diverse, but also extremely abundant in some environments, making their characterization all the more important. MAST-6 is one example of a phylogenetically distinct group that has been known to be associated with sediments, but little else is known about it. Indeed, until the present study, only a single species from this group, Pseudophyllomitus vesiculosus (Pseudophyllomitidae), has been both formally described and associated with genomic information. Here, we describe four new species including two new genera of sediment-dwelling MAST-6, Vomastramonas tehuelche gen. et sp. nov., Mastreximonas tlaamin gen. et sp. nov., one undescribed Pseudophyllomitus sp., BSC2, and a new species belonging to Placididea, the potentially halotolerant Haloplacidia sinai sp. nov. We also provide two additional bikosian transcriptomes from a public culture collection, to allow for better phylogenetic reconstructions of deep-branching stramenopiles. With the SSU rRNA sequences of the new MAST-6 species, we investigate the phylogenetic diversity of the MAST-6 group and show a high relative abundance of MAST-6 related to M. tlaamin in samples across various depths and geographical locations. Using the new MAST-6 species, we also update the phylogenomic tree of stramenopiles, particularly focusing on the paraphyly of Bigyra.

Coupling and decoupling of marine stramenopiles and cyanobacteria in eutrophic coastal waters of Korea

The spatiotemporal distribution of MASTs (MArine STramenopiles), mostly affiliated with heterotrophic protists, and their interactions with Synechococcales were investigated in an anthropogenically polluted bay of the East Sea using 18S rRNA and 16S rRNA gene sequences. The bay was characterized by strong stratification between the surface and bottom layers and cold and nutrient-rich water intrusion in summer, whereas the bay water was well mixed in winter. MAST-3, MAST-6, MAST-7, and MAST-9 were the major MAST clades, whereas the dominance of MAST-9 declined from >80 % in summer to <10 % in winter and the diversity of MAST communities increased in winter. Co-occurrence network analysis via the sparse partial least squares revealed that MAST-3 had a Synechococcales-specific interaction during the study periods but prey-specific interactions with other MAST clades were not detected. Temperature and salinity markedly influenced the relative abundance of major MAST clades. The relative abundance of MAST-3 increased at temperatures above 20 °C and salinities above 33 ‰, however, the abundance of MAST-9 decreased under the same conditions. Analysis of the metabolic functions of cyanobacteria using the FAPROTAX (Functional Annotation of Prokaryotic Taxa) indicated that the response of photosynthetic cyanobacteria to NH4+ and PO43− was significant in summer but these functions were not tightly coupled with the abundance of Synechococcales. Similarly, strong associations of MAST-3 with high temperature/salinity and Synechococcales were indicative of coupled cascading during bottom-up processes. However, other major MAST clades were likely decoupled with Synechococcales and environmental conditions in which cyanobacteria can thrive. Therefore, our results demonstrated that MAST communities can couple or decouple with environmental variables and potential prey depending on MAST clades. Collectively, our findings provide novel insights into the role of MAST communities in microbial food webs in eutrophic coastal waters.

A k-mer-Based Approach for Phylogenetic Classification of Taxa in Environmental Genomic Data.

In the age of genome sequencing, whole-genome data is readily and frequently generated, leading to a wealth of new information that can be used to advance various fields of research. New approaches, such as alignment-free phylogenetic methods that utilize k-mer-based distance scoring, are becoming increasingly popular given their ability to rapidly generate phylogenetic information from whole-genome data. However, these methods have not yet been tested using environmental data, which often tends to be highly fragmented and incomplete. Here, we compare the results of one alignment-free approach (which utilizes the D2 statistic) to traditional multi-gene maximum likelihood trees in 3 algal groups that have high-quality genome data available. In addition, we simulate lower-quality, fragmented genome data using these algae to test method robustness to genome quality and completeness. Finally, we apply the alignment-free approach to environmental metagenome assembled genome data of unclassified Saccharibacteria and Trebouxiophyte algae, and single-cell amplified data from uncultured marine stramenopiles to demonstrate its utility with real datasets. We find that in all instances, the alignment-free method produces phylogenies that are comparable, and often more informative, than those created using the traditional multi-gene approach. The k-mer-based method performs well even when there are significant missing data that include marker genes traditionally used for tree reconstruction. Our results demonstrate the value of alignment-free approaches for classifying novel, often cryptic or rare, species, that may not be culturable or are difficult to access using single-cell methods, but fill important gaps in the tree of life.

Assessing Combinatorial Diversity of Aureochrome Basic Leucine Zippers through Genome-Wide Screening.

Aureochromes are unique blue light-responsive light-oxygen-voltage (LOV) photoreceptors cum basic leucine zipper (bZIP) transcription factors (TFs), present exclusively in photosynthetic marine stramenopiles. Considering the availability of the complete genome sequence, this study focuses on aureochromes from Ectocarpus siliculosus. Aureochromes mediate light-regulated developmental responses in these brown photosynthetic algae. Both the LOV sensor and the bZIP effector show overall sequence-structure conservation. The structurally similar LOV + bZIP modules of aureochrome homologs/paralogs prefer a dimeric state. Besides a heterogeneous linker connecting the sensor-effector and a flexible N-terminal region, the sequence composition of both domains is vital. Aureochromes execute diverse cellular responses in different photosynthetic stramenopiles - though their activities can vary even within a given algal species. Therefore, it is important to understand whether aureochromes select dimerization partners from the same family or interact with other bZIPs as well. To regulate multifarious biological activities, it is possible that aureochromes activate the global TF interaction network. Following homo/heterodimer modeling, we address the compatibility of dimerization partners by screening through heptad repeats. We evaluate the dimer interface area in terms of gain in solvation energy and the number of hydrogen bonds/salt bridge interactions. We further explore the relative stability of these structures from a graph-theoretic perspective through well-studied measures such as the energy of the graph, average participation coefficient, and betweenness centrality. Furthermore, we also conduct an information-theoretic analysis using hitherto understudied measures such as network information centrality and Kullback-Leibler divergence. We find that all our investigations into the relative stability of the dimers using diverse methods from bioinformatics, network science, and information theory are in harmonious agreement. Coupling preferences of monomers in aureochromes can be further translated to design novel optogenetic tools useful for understanding human development and disease.

Planktonic protist diversity across contrasting Subtropical and Subantarctic waters of the southwest Pacific

Planktonic protists are an essential component of marine pelagic ecosystems where they mediate important trophic and biogeochemical functions. Although these functions are largely influenced by their taxonomic affiliation, the composition and spatial variability of planktonic protist communities remain poorly characterized in vast areas of the ocean. Here, we investigated the diversity of these communities in contrasting oceanographic conditions of the southwest Pacific (33–58 °S) using DNA metabarcoding of the 18S rRNA gene. Seawater samples collected during twelve cruises (n = 482, 0–3100 m) conducted east of New Zealand were used to characterize protist communities in Subtropical (STW) and Subantarctic (SAW) surface water masses and the Subtropical Front (STF) that separates them. Diversity decreased with increasing latitude and increasing temperature but tended to be lowest in the STF. Sample ordination resulting from the abundance of amplicon single variants (ASVs) corresponded to the different water masses. Overall, Dinoflagellata (Syndiniales, 27%; Dinophyceae, 24% of standardized number of reads) dominated the euphotic zone followed by Chlorophyta (20%), but their relative abundance and composition at class and lower taxonomic levels varied consistently between water masses. Among Chlorophyta, several picoplanktonic algae species of the Mamiellophyceae class including Ostreococcus lucimarinus dominated in STW, while the Chloropicophyceae species Chloroparvula pacifica was most abundant in SAW. Bacillariophyta (5%), Prymnesiophyceae (5%), and Pelagophyceae (2%) classes were less abundant but showed analogous water mass specificity at class and finer taxonomic levels. Protist community composition in the STF had mixed characteristics and showed regional differences with the southern STF (50 °S) having more resemblance with subantarctic communities than the STF over the Chatham Rise region (42–44 °S). Below the euphotic zone, Syndiniales sequences (40%) dominated the dataset followed by Radiolaria (31%), Dinophyceae (14%) and other heterotrophic groups like Marine Stramenopiles and ciliates (1–1.5%). Among Radiolaria, several unidentified ASVs assigned to Spumellaria were most abundant, but showed significantly different distributions between STW and SAW highlighting the need to further investigate the taxonomy and ecology of this group. The present study represents a significant step forward towards characterizing protistan communities composition in relation to major physical oceanographic features in the southwest Pacific providing new insights about the biogeography and ecological preferences of different planktonic protist taxa from class to species and genotypic level.

Differences in the Composition of Abundant Marine Picoeukaryotes in the Marginal Sea Derived from Flooding

The transient impact of flooding on the community composition of marine picoeukaryotes (PEs, cell size ≤5 μm) in the East China Sea (ECS) was revealed in this study. In a summer without flooding (i.e., July 2009), photosynthetic picoeukaryotes (PPEs) were more abundant in the area covered by the Changjiang River diluted water (CDW, salinity ≤31) than in the non-CDW affected area. According to the 18S ribosomal RNA phylogeny, Alveolata (all from the superclass Dinoflagellata) was the main community component accounting for 72 to 99% of the community at each sampling station during the nonflooded summer. In addition to Dinoflagellata, diatoms or Chlorophyta also contributed a considerable proportion to the PE assemblage at the stations close to the edge of CDW coverage. In July 2010, an extreme flooding event occurred in the Changjiang River basin and led to the CDW covering nearly half of the ECS. In the flooded summer, the abundance of PPEs in the CDW-covered area decreased significantly to less than 1 × 104 cells ml-1. Compared to that during the nonflooded summer, the diversity of the PE composition was increased. While Dinophyceae still dominated the surface waters, Syndiniophyceae, which were represented by the uncultured Marine Alveolata Group (MALV)-I and MALV-II, accounted for a substantial amount in the Dinoflagellata superclass relative to this community composition in the nonflooded summer. Furthermore, a variety of plankton, including Cryptophyta, Haptophyta, Picobiliphyta, the uncultured Marine Stramenopiles (MASTs) and heterotrophic nanoflagellates, were observed. The nutrition modes of these PEs have been reported to be mixotrophic or heterotrophic. Therefore, it was inferred that the potentially mixotrophic and heterotrophic PE compositions might be favored in the marginal sea in the flooded summer.

Diversity and distribution of heterotrophic flagellates in seawater of the Powell Basin, Antarctic Peninsula

Heterotrophic flagellates are essential components of the marine microbial food web. However, how the changes in flagellate populations reflect environmental changes in marine ecosystems is still unclear, especially in polar regions. In this study, we used pyrosequencing to examine the community structure of heterotrophic flagellates (HFs) in the Powell Basin’s surface waters of the northern Antarctic Peninsula. OTUs (operational taxonomic units) of different taxa and the correlations between community structure and environmental factors were analysed. Eight taxa of HFs were selected for the principal analysis: Telonemia, Picozoa, Rhizaria, Amoebozoa, Apusomonas, Centrohelida, Choanomonada and marine stramenopiles (MASTs). The HFs were defined as heterotrophic picoflagellates (HPFs; &lt;3 μm) and heterotrophic nanoflagellates (HNFs; &gt;3 μm, &lt;20 μm), which had similar dominant phyla (MASTs and Telonemia). However, their taxonomic composition differed. Environmental factors exerted similar effects on the community structure of both HPFs and HNPs. Compared with the correlation between HPF and environmental factors, the correlation between HNF and environmental factors was stronger. Salinity, bacterial biomass and the biological interactions amongst dominant taxa were the main variables to influence the diversity and community structure of HFs.

How Communities of Marine Stramenopiles Varied with Environmental and Biological Variables in the Subtropical Northwestern Pacific Ocean

MArine STramenopiles (MASTs) have been recognized as parts of heterotrophic protists and contribute substantially to protist abundances in the ocean. However, little is known about their spatiotemporal variations with respect to environmental and biological factors. The objectives of this study are to use canonical correspondence analysis to investigate how MASTs communities are shaped by environmental variables, and co-occurrence networks to examine their potential interactions with prokaryotic communities. Our dataset came from the southern East China Sea (sECS) in the subtropical northwestern Pacific, and involved 14 cruises along a coastal-oceanic transect, each of which sampled surface water from 4 to 7 stations. MASTs communities were revealed by metabarcoding of 18S rDNA V4 region. Most notably, MAST-9 had a high representation in warm waters in terms of read number and diversity. Subclades of MAST-9C and -9D showed slightly different niches, with MAST-9D dominating in more coastal waters where concentrations of nitrite and Synechococcus were higher. MAST-1C was a common component of colder water during spring. Overall, canonical correspondence analysis showed that MASTs communities were significantly influenced by temperature, nitrite and Synechococcus concentrations. The co-occurrence networks showed that certain other minor prokaryotic taxa can influence MAST communities. This study provides insight into how MASTs communities varied with environmental and biological variables.

Niche adaptation promoted the evolutionary diversification of tiny ocean predators

Unicellular eukaryotic predators play a crucial role in the functioning of the ocean ecosystem by recycling nutrients and energy that are channeled to upper trophic levels. Traditionally, these evolutionarily diverse organisms have been combined into a single functional group (heterotrophic flagellates), overlooking their organismal differences. Here, we investigated four evolutionarily related species belonging to one cosmopolitan group of uncultured marine picoeukaryotic predators: marine stramenopiles (MAST)-4 (species A, B, C, and E). Co-occurrence and distribution analyses in the global surface ocean indicated contrasting patterns in MAST-4A and C, suggesting adaptation to different temperatures. We then investigated whether these spatial distribution patterns were mirrored by MAST-4 genomic content using single-cell genomics. Analyses of 69 single cells recovered 66 to 83% of the MAST-4A/B/C/E genomes, which displayed substantial interspecies divergence. MAST-4 genomes were similar in terms of broad gene functional categories, but they differed in enzymes of ecological relevance, such as glycoside hydrolases (GHs), which are part of the food degradation machinery in MAST-4. Interestingly, MAST-4 species featuring a similar GH composition (A and C) coexcluded each other in the surface global ocean, while species with a different set of GHs (B and C) appeared to be able to coexist, suggesting further niche diversification associated with prey digestion. We propose that differential niche adaptation to temperature and prey type has promoted adaptive evolutionary diversification in MAST-4. We show that minute ocean predators from the same phylogenetic group may have different biogeography and genomic content, which needs to be accounted for to better comprehend marine food webs.

Diversity of picoeukaryotes in the eastern equatorial Indian Ocean revealed by metabarcoding

We used 18S rRNA gene metabarcoding to investigate picoeukaryotic diversity and distribution at the surface and deep chlorophyll maximum (DCM) of 4 stations in the eastern equatorial Indian Ocean (EEIO). The results showed that picoeukaryotic communities were dominated by 5 phyla: Dinoflagellata, Radiolaria, Chlorophyta, Ochrophyta and Ciliophora. The picoeukaryotic communities were classified into 3 groups matching their water mass origins and depth: (1) Group I was in the surface waters of the Bay of Bengal, which had low salinity, and was dominated by Radiolaria Group A, Spirotrichea and marine stramenopiles; (2) Group II was in the DCM within the intrusion of Arabian Sea high salinity water, in which Chloropicophyceae and Pelagophyceae were more abundant; and (3) Group III was located in the 0°-5°S surface water, which was enriched by Dinophyceae. In addition, Caecitellaceae paraparvulus was abundant at 4°S, where weak vertical mixing occurred. This study provides the first baseline of picoeukaryotic diversity in the EEIO.

Investigating the microbial ecology of coastal hotspots of marine nitrogen fixation in the western North Atlantic

Variation in the microbial cycling of nutrients and carbon in the ocean is an emergent property of complex planktonic communities. While recent findings have considerably expanded our understanding of the diversity and distribution of nitrogen (N2) fixing marine diazotrophs, knowledge gaps remain regarding ecological interactions between diazotrophs and other community members. Using quantitative 16S and 18S V4 rDNA amplicon sequencing, we surveyed eukaryotic and prokaryotic microbial communities from samples collected in August 2016 and 2017 across the Western North Atlantic. Leveraging and significantly expanding an earlier published 2015 molecular dataset, we examined microbial community structure and ecological co-occurrence relationships associated with intense hotspots of N2 fixation previously reported at sites off the Southern New England Shelf and Mid-Atlantic Bight. Overall, we observed a negative relationship between eukaryotic diversity and both N2 fixation and net community production (NCP). Maximum N2 fixation rates occurred at sites with high abundances of mixotrophic stramenopiles, notably Chrysophyceae. Network analysis revealed such stramenopiles to be keystone taxa alongside the haptophyte diazotroph host Braarudosphaera bigelowii and chlorophytes. Our findings highlight an intriguing relationship between marine stramenopiles and high N2 fixation coastal sites.

Biogeographical Distribution and Community Assembly of Active Protistan Assemblages along an Estuary to a Basin Transect of the Northern South China Sea

Marine protists are essential for globally critical biological processes, including the biogeochemical cycles of matter and energy. However, compared with their prokaryotic counterpart, it remains largely unclear how environmental factors determine the diversity and distribution of the active protistan communities on the regional scale. In the present study, the biodiversity, community composition, and potential drivers of the total, abundant, and rare protistan groups were studied using high throughput sequencing on the V9 hyper-variable regions of the small subunit ribosomal RNA (SSU rRNA) along an estuary to basin transect in the northern South China Sea. Overall, Bacillariophyta and Cercozoa were abundant in the surface water; heterotrophic protists including Spirotrichea and marine stramenopiles 3 (MAST-3) were more abundant in the subsurface waters near the heavily urbanized Pearl River estuary; Chlorophyta and Pelagophyceae were abundant at the deep chlorophyll maximum depth, while Hacrobia, Radiolaria, and Excavata were the abundant groups in the deep water. Salinity, followed by water depth, temperature, and other biological factors, were the primary factors controlling the distinct vertical and horizontal distribution of the total and abundant protists. Rare taxa were driven by water depth, followed by temperature, salinity, and the concentrations of PO43−. The active protistan communities were mainly driven by dispersal limitation, followed by drift and other ecological processes.

Comparative genomics reveals new functional insights in uncultured MAST species

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.

Rapid succession drives spring community dynamics of small protists at Helgoland Roads, North Sea.

The dynamics of diatoms and dinoflagellates have been monitored for many decades at the Helgoland Roads Long-Term Ecological Research site and are relatively well understood. In contrast, small-sized eukaryotic microbes and their community changes are still much more elusive, mainly due to their small size and uniform morphology, which makes them difficult to identify microscopically. By using next-generation sequencing, we wanted to shed light on the Helgoland planktonic community dynamics, including nano- and picoplankton, during a spring bloom. We took samples from March to May 2016 and sequenced the V4 region of the 18S rDNA. Our results showed that mixotrophic and heterotrophic taxa were more abundant than autotrophic diatoms. Dinoflagellates dominated the sequence assemblage, and several small-sized eukaryotic microbes like Haptophyta, Choanoflagellata, Marine Stramenopiles and Syndiniales were identified. A diverse background community including taxa from all size classes was present during the whole sampling period. Five phases with several communities were distinguished. The fastest changes in community composition took place in phase 3, while the communities from phases 1 to 5 were more similar to each other despite contrasting environmental conditions. Synergy effects of next-generation sequencing and traditional methods may be exploited in future long-term observations.

The zoospores of the thraustochytrid Aurantiochytrium limacinum: Transcriptional reprogramming and lipid metabolism associated to their specific functions.

Aurantiochytrium limacinum (Thraustochytriaceae, class Labyrinthulomycetes) is a marine Stramenopile and a pioneering mangrove decomposer. Its life cycle involves a non-motile stage and zoospore production. We observed that the composition of the medium, the presence of amino acids in particular, affects the release of zoospores. Two opposite conditions were defined, one with a cell population mainly composed of zoospores and another one with almost only non-motile cells. In silico allelic frequency analysis and flow cytometry suggest that zoospores and non-motile cells share the same ploidy level and are diploid. Through an RNA-seq approach, the transcriptional reprogramming accompanying the formation of zoospores was investigated, with a particular focus on their lipid metabolism. Based on a differential expression analysis, zoospores are characterized by high motility, very active signal transduction, an arrest of the cell division, a low amino acid metabolism and low glycolysis. Focusing on lipid metabolism, genes involved in lipase activities and peroxisomal β-oxidation are upregulated. qRT-PCR of selected lipid genes and lipid analyses during the life span of zoospores confirmed these observations. These results highlight the importance of the lipid dynamics in zoospores and show the metabolic processes required to use these energy-dense molecules as fuel for zoospore survival during their quest of new territories.

Expression of genes involved in phagocytosis in uncultured heterotrophic flagellates

AbstractEnvironmental molecular sequencing has revealed an abundance of microorganisms that were previously unknown, mainly because most had not been cultured in the laboratory. Within this novel diversity, there are the uncultured MAST clades (MArine STramenopiles), which are major components of marine heterotrophic flagellates (HFs) thought to be active bacterial grazers. In this study, we investigated the gene expression of natural HFs in a mixed community where bacterivory was promoted. Using fluorescence in situ hybridization and 18S rDNA derived from metatranscriptomics, we followed the taxonomic dynamics during the incubation, and confirmed the increase in relative abundance of different MAST lineages. We then used single cell genomes of several MAST species to gain an insight into their most expressed genes, with a particular focus on genes related to phagocytosis. The genomes of MAST‐4A and MAST‐4B were the most represented in the metatranscriptomes, and we identified highly expressed genes of these two species involved in motility and cytoskeleton remodeling, as well as many lysosomal enzymes. Particularly relevant were the cathepsins, which are characteristic digestive enzymes of the phagolysosome and the rhodopsins, perhaps used for vacuole acidification. The combination of single cell genomics and metatranscriptomics gives insights on the phagocytic capacity of uncultured and ecologically relevant HF species.