Nested PCR Approach for petB Gene Metabarcoding of Marine Synechococcus Populations.

The molecular diversity of marine picocyanobacterial populations, an important component of phytoplankton communities, is better characterized using high-resolution marker genes than the 16S rRNA gene as they have greater sequence divergence to differentiate between closely related picocyanobacteria groups. Although specific ribosomal primers have been developed, another general disadvantage of bacterial ribosome-based diversity analyses is the variable number of rRNA gene copies. To overcome these issues, the single-copy petB gene, encoding the cytochrome b6 subunit of the cytochrome b6f complex, has been used as a high-resolution marker gene to characterize Synechococcus diversity. We have designed new primers targeting the petB gene and proposed a nested PCR method (termed Ong_2022) for metabarcoding of marine Synechococcus populations obtained by flow cytometry cell sorting. We evaluated the specificity and sensitivity of Ong_2022 against the standard amplification protocol (termed Mazard_2012) using filtered seawater samples. The Ong_2022 approach was also tested on flow cytometry-sorted Synechococcus populations. Samples (filtered and sorted) were obtained in the Southwest Pacific Ocean, from subtropical (ST) and subantarctic (SA) water masses. The two PCR approaches using filtered samples recovered the same dominant subclades, Ia, Ib, IVa, and IVb, with small differences in relative abundance across the distinct samples. For example, subclade IVa was dominant in ST samples with the Mazard_2012 approach, while the same samples processed with Ong_2022 showed similar contributions of subclades IVa and Ib to the total community. The Ong_2022 approach generally captured a higher genetic diversity of Synechococcus subcluster 5.1 than the Mazard_2012 approach while having a lower proportion of incorrectly assigned amplicon sequence variants (ASVs). All flow cytometry-sorted Synechococcus samples could be amplified only by our nested approach. The taxonomic diversity obtained with our primers on both sample types was in agreement with the clade distribution observed by previous studies that applied other marker genes or PCR-free metagenomic approaches under similar environmental conditions. IMPORTANCE The petB gene has been proposed as a high-resolution marker gene to access the diversity of marine Synechococcus populations. A systematic metabarcoding approach based on the petB gene would improve the characterization/assessment of the Synechococcus community structure in marine planktonic ecosystems. We have designed and tested specific primers to be applied in a nested PCR protocol (Ong_2022) for metabarcoding the petB gene. The Ong_2022 protocol can be applied to samples with low DNA content, such as those obtained by flow cytometry cell sorting, allowing the simultaneous assessment of the genetic diversity of Synechococcus populations and cellular properties and activities (e.g., nutrient cell ratios or carbon uptake rates). Our approach will allow future studies using flow cytometry to investigate the link between ecological traits and taxonomic diversity of marine Synechococcus.

metaPR2 : A database of eukaryotic 18S rRNA metabarcodes with an emphasis on protists.

In recent years, metabarcoding has become the method of choice for investigating the composition and assembly of microbial eukaryotic communities. The number of environmental data sets published has increased very rapidly. Although unprocessed sequence files are often publicly available, processed data, in particular clustered sequences, are rarely available in a usable format. Clustered sequences are reported as operational taxonomic units (OTUs) with different similarity levels or more recently as amplicon sequence variants (ASVs). This hampers comparative studies between different environments and data sets, for example examining the biogeographical patterns of specific groups/species, as well analysing the genetic microdiversity within these groups. Here, we present a newly-assembled database of processed 18S rRNA metabarcodes that are annotated with the PR2 reference sequence database. This database, called metaPR2 , contains 41 data sets corresponding to more than 4000 samples and 90,000 ASVs. The database, which is accessible through both a web-based interface (https://shiny.metapr2.org) and an R package, should prove very useful to all researchers working on protist diversity in a variety of systems.

Coral-associated nitrogen fixation rates and diazotrophic diversity on a nutrient-replete equatorial reef.

The role of diazotrophs in coral physiology and reef biogeochemistry remains poorly understood, in part because N2 fixation rates and diazotrophic community composition have only been jointly analyzed in the tissue of one tropical coral species. We performed field-based 15N2 tracer incubations during nutrient-replete conditions to measure diazotroph-derived nitrogen (DDN) assimilation into three species of scleractinian coral (Pocillopora acuta, Goniopora columna, Platygyra sinensis). Using multi-marker metabarcoding (16S rRNA, nifH, 18S rRNA), we analyzed DNA- and RNA-based communities in coral tissue and skeleton. Despite low N2 fixation rates, DDN assimilation supplied up to 6% of the holobiont's N demand. Active coral-associated diazotrophs were chiefly Cluster I (aerobes or facultative anaerobes), suggesting that oxygen may control coral-associated diazotrophy. Highest N2 fixation rates were observed in the endolithic community (0.20 µg N cm-2 per day). While the diazotrophic community was similar between the tissue and skeleton, RNA:DNA ratios indicate potential differences in relative diazotrophic activity between these compartments. In Pocillopora, DDN was found in endolithic, host, and symbiont compartments, while diazotrophic nifH sequences were only observed in the endolithic layer, suggesting a possible DDN exchange between the endolithic community and the overlying coral tissue. Our findings demonstrate that coral-associated diazotrophy is significant, even in nutrient-rich waters, and suggest that endolithic microbes are major contributors to coral nitrogen cycling on reefs.

Freshwater protists: unveiling the unexplored in a large floodplain system.

Protists play a fundamental role in all ecosystems, but we are still far from estimating the total diversity of many lineages, in particular in highly diverse environments, such as freshwater. Here, we survey the protist diversity of the Paraná River using metabarcoding, and we applied an approach that includes sequence similarity and phylogeny to evaluate the degree of genetic novelty of the protists' communities against the sequences described in the reference database PR2 . We observed that ~28% of the amplicon sequence variants were classified as novel according to their similarity with sequences from the reference database; most of them were related to heterotrophic groups traditionally overlooked in freshwater systems. This lack of knowledge extended to those groups within the green algae (Archaeplastida) that are well documented such as Mamiellophyceae, and also to the less studied Pedinophyceae, for which we found sequences representing novel deep-branching clusters. Among the groups with potential novel protists, Bicosoecida (Stramenopiles) were the best represented, followed by Codosiga (Opisthokonta), and the Perkinsea (Alveolata). This illustrates the lack of knowledge on freshwater planktonic protists and also the need for isolation and/or cultivation of new organisms to better understand their role in ecosystem functioning.

Annual phytoplankton dynamics in coastal waters from Fildes Bay, Western Antarctic Peninsula.

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.

Author Correction: Chloropicophyceae, a new class of picophytoplanktonic prasinophytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mantoniella beaufortii and Mantoniella baffinensis sp. nov. (Mamiellales, Mamiellophyceae), two new green algal species from the high arctic1.

Members of the class Mamiellophyceae comprise species that can dominate picophytoplankton diversity in polar waters. Yet, polar species are often morphologically indistinguishable from temperate species, although clearly separated by molecular features. Here we examine four Mamiellophyceae strains from the Canadian Arctic. The 18S rRNA and Internal Transcribed Spacer 2 (ITS2) gene phylogeny place these strains within the family Mamiellaceae (Mamiellales, Mamiellophyceae) in two separate clades of the genus Mantoniella. ITS2 synapomorphies support their placement as two new species, Mantoniella beaufortii and Mantoniella baffinensis. Both species have round green cells with diameter between 3 and 5 µm, one long flagellum and a short flagellum (~1 µm) and are covered by spiderweb-like scales, making both species similar to other Mantoniella species. Morphologically, M. beaufortii and M. baffinensis are most similar to the cosmopolitan M. squamata with only minor differences in scale structure distinguishing them. Screening of global marine metabarcoding data sets indicates M. beaufortii has only been recorded in seawater and sea ice samples from the Arctic, while no environmental barcode matches M. baffinensis. Like other Mamiellophyceae genera that have distinct polar and temperate species, the polar distribution of these new species suggests they are cold or ice-adapted Mantoniella species.

Temporal and spatial dynamics of Bacteria, Archaea and protists in equatorial coastal waters.

Singapore, an equatorial island in South East Asia, is influenced by a bi-annual reversal of wind directions which defines two monsoon seasons. We characterized the dynamics of the microbial communities of Singapore coastal waters by collecting monthly samples between February 2017 and July 2018 at four sites located across two straits with different trophic status, and sequencing the V6-V8 region of the small sub-unit ribosomal RNA gene (rRNA gene) of Bacteria, Archaea, and Eukaryota. Johor Strait, which is subjected to wider environmental fluctuations from anthropogenic activities, presented a higher abundance of copiotrophic microbes, including Cellvibrionales and Rhodobacterales. The mesotrophic Singapore Strait, where the seasonal variability is caused by changes in the oceanographic conditions, harboured a higher proportion of typically marine microbe groups such as Synechococcales, Nitrosupumilales, SAR11, SAR86, Marine Group II Archaea and Radiolaria. In addition, we observed seasonal variability of the microbial communities in the Singapore Strait, which was possibly influenced by the alternating monsoon regime, while no seasonal pattern was detected in the Johor Strait.

Tracing the Evolution of the Plastome and Mitogenome in the Chloropicophyceae Uncovered Convergent tRNA Gene Losses and a Variant Plastid Genetic Code.

The tiny green algae belonging to the Chloropicophyceae play a key role in marine phytoplankton communities; this newly erected class of prasinophytes comprises two genera (Chloropicon and Chloroparvula) containing each several species. We sequenced the plastomes and mitogenomes of eight Chloropicon and five Chloroparvula species to better delineate the phylogenetic affinities of these taxa and to infer the suite of changes that their organelle genomes sustained during evolution. The relationships resolved in organelle-based phylogenomic trees were essentially congruent with previously reported rRNA trees, and similar evolutionary trends but distinct dynamics were identified for the plastome and mitogenome. Although the plastome sustained considerable changes in gene content and order at the time the two genera split, subsequently it remained stable and maintained a very small size. The mitogenome, however, was remodeled more gradually and showed more fluctuation in size, mainly as a result of expansions/contractions of intergenic regions. Remarkably, the plastome and mitogenome lost a common set of three tRNA genes, with the trnI(cau) and trnL(uaa) losses being accompanied with important variations in codon usage. Unexpectedly, despite the disappearance of trnI(cau) from the plastome in the Chloroparvula lineage, AUA codons (the codons recognized by this gene product) were detected in certain plastid genes. By comparing the sequences of plastid protein-coding genes from chloropicophycean and phylogenetically diverse chlorophyte algae with those of the corresponding predicted proteins, we discovered that the AUA codon was reassigned from isoleucine to methionine in Chloroparvula. This noncanonical genetic code has not previously been uncovered in plastids.

Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications.

Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

Diversity of photosynthetic picoeukaryotes in eutrophic shallow lakes as assessed by combining flow cytometry cell-sorting and high throughput sequencing.

Photosynthetic picoeukaryotes (PPE) are key components of primary production in marine and freshwater ecosystems. In contrast with those of marine environments, freshwater PPE groups have received little attention. In this work, we used flow cytometry cell sorting, microscopy and metabarcoding to investigate the composition of small photosynthetic eukaryote communities from six eutrophic shallow lakes in South America, Argentina. We compared the total molecular diversity obtained from PPE sorted populations as well as from filtered total plankton samples (FTP). Most reads obtained from sorted populations belonged to the classes: Trebouxiophyceae, Chlorophyceae and Bacillariophyceae. We retrieved sequences from non-photosynthetic groups, such as Chytridiomycetes and Ichthyosporea which contain a number of described parasites, indicating that these organisms were probably in association with the autotrophic cells sorted. Dominant groups among sorted PPEs were poorly represented in FTP and their richness was on average lower than in the sorted samples. A significant number of operational taxonomic units (OTUs) were exclusively found in sorting samples, emphasizing that sequences from FTP underestimate the diversity of PPE. Moreover, 22% of the OTUs found among the dominant groups had a low similarity (<95%) with reported sequences in public databases, demonstrating a high potential for novel diversity in these lakes.

High contribution of Rhizaria (Radiolaria) to vertical export in the California Current Ecosystem revealed by DNA metabarcoding.

Passive sinking of particulate organic matter (POM) is the main mechanism through which the biological pump transports surface primary production to the ocean interior. However, the contribution and variability of different biological sources to vertical export is not fully understood. Here, we use DNA metabarcoding of the 18S rRNA gene and particle interceptor traps (PITs) to characterize the taxonomic composition of particles sinking out of the photic layer in the California Current Ecosystem (CCE), a productive system with high export potential. The PITs included formalin-fixed and 'live' traps to investigate eukaryotic communities involved in the export and remineralization of sinking particles. Sequences affiliated with Radiolaria dominated the eukaryotic assemblage in fixed traps (90%), with Dinophyta and Metazoa making minor contributions. The prominence of Radiolaria decreased drastically in live traps, possibly due to selective consumption by copepods, heterotrophic nanoflagellates, and phaeodarians that were heavily enriched in these traps. These patterns were consistent across the water masses surveyed extending from the coast to offshore, despite major differences in productivity and trophic structure of the epipelagic plankton community. Our findings identify Radiolaria as major actors in export fluxes in the CCE.

Small eukaryotic phytoplankton communities in tropical waters off Brazil are dominated by symbioses between Haptophyta and nitrogen-fixing cyanobacteria.

Symbioses between eukaryotic algae and nitrogen-fixing cyanobacteria have been recognized in recent years as a key source of new nitrogen in the oceans. We investigated the composition of the small photosynthetic eukaryote communities associated with nitrogen-fixing cyanobacteria in the Brazilian South Atlantic Bight using a combination of flow cytometry sorting and high throughput sequencing of two genes: the V4 region of 18S rRNA and nifH. Two distinct eukaryotic communities were often encountered, one dominated by the Mamiellophyceae Bathycoccus and Ostreococcus, and one dominated by a prymnesiophyte known to live in symbiosis with the UCYN-A1 nitrogen-fixing cyanobacterium. Among nifH sequences, those from UCYN-A1 were most abundant but three other UCYN-A clades (A2, A3, A4) were also found. Network analysis confirmed the relation between A1 and A2 clades and their hypothesized hosts and pointed out to the potential association between novel clade A4 with Braarudosphaera bigelowii, previously hypothesized to host A2.

Chloropicophyceae, a new class of picophytoplanktonic prasinophytes.

Prasinophytes are a paraphyletic group of nine lineages of green microalgae that are currently classified either at the class or order level or as clades without formal taxonomic description. Prasinophyte clade VII comprises picoplanktonic algae that are important components of marine phytoplankton communities, particularly in moderately oligotrophic waters. Despite first being cultured in the 1960s, this clade has yet to be formally described. Previous phylogenetic analyses using the 18S rRNA gene divided prasinophyte clade VII into three lineages, termed A, B and C, the latter formed by a single species, Picocystis salinarum, that to date has only been found in saline lakes. Strains from lineages A and B cannot be distinguished by light microscopy and have very similar photosynthetic pigment profiles corresponding to the prasino-2A pigment group. We obtained phenotypic and genetic data on a large set of prasinophyte clade VII culture strains that allowed us to clarify the taxonomy of this important marine group. We describe two novel classes, the Picocystophyceae and the Chloropicophyceae, the latter containing two novel genera, Chloropicon and Chloroparvula, and eight new species of marine picoplanktonic green algae.

Diversity and oceanic distribution of prasinophytes clade VII, the dominant group of green algae in oceanic waters.

Prasinophytes clade VII is a group of pico/nano-planktonic green algae (division Chlorophyta) for which numerous ribosomal RNA (rRNA) sequences have been retrieved from the marine environment in the last 15 years. A large number of strains have also been isolated but have not yet received a formal taxonomic description. A phylogenetic analysis of available strains using both the nuclear 18S and plastidial 16S rRNA genes demonstrates that this group composes at least 10 different clades: A1-A7 and B1-B3. Analysis of sequences from the variable V9 region of the 18S rRNA gene collected during the Tara Oceans expedition and in the frame of the Ocean Sampling Day consortium reveal that clade VII is the dominant Chlorophyta group in oceanic waters, replacing Mamiellophyceae, which have this role in coastal waters. At some location, prasinophytes clade VII can even be the dominant photosynthetic eukaryote representing up to 80% of photosynthetic metabarcodes overall. B1 and A4 are the overall dominant clades and different clades seem to occupy distinct niches, for example, A6 is dominant in surface Mediterranean Sea waters, whereas A4 extend to high temperate latitudes. Our work demonstrates that prasinophytes clade VII constitute a highly diversified group, which is a key component of phytoplankton in open oceanic waters but has been neglected in the conceptualization of marine microbial diversity and carbon cycle.

Pico and nanoplankton abundance and carbon stocks along the Brazilian Bight.

Pico and nanoplankton communities from the Southwest Atlantic Ocean along the Brazilian Bight are poorly described. The hydrography in this region is dominated by a complex system of layered water masses, which includes the warm and oligotrophic Tropical Water (TW), the cold and nutrient rich South Atlantic Central Water (SACW) and the Coastal Water (CW), which have highly variable properties. In order to assess how pico- and nanoplankton communities are distributed in these different water masses, we determined by flow cytometry the abundance of heterotrophic bacteria, Prochlorococcus, Synechococcus and autotrophic pico and nanoeukaryotes along three transects, extending from 23°S to 31°S and 39°W to 49°W. Heterotrophic bacteria (including archaea, maximum of 1.5 × 106 cells mL-1) were most abundant in Coastal and Tropical Water whereas Prochlorococcus was most abundant in open-ocean oligotrophic waters (maximum of 300 × 103 cells mL-1). Synechococcus(up to 81 × 103 cells mL-1), as well as autotrophic pico and nanoeukaryotes seemed to benefit from the influx of nutrient-rich waters near the continental slope. Autotrophic pico and nanoeukaryotes were also abundant in deep chlorophyll maximum (DCM) layers from offshore waters, and their highest abundances were 20 × 103 cells mL-1 and 5 × 103 cells mL-1, respectively. These data are consistent with previous observations in other marine areas where Synechococcus and autotrophic eukaryotes dominate mesotrophic waters, whereas Prochlorococcus dominate in more oligotrophic areas. Regardless of the microbial community structure near the surface, the carbon stock dominance by autotrophic picoeukaryotes near the DCM is possibly linked to vertical mixing of oligotrophic surface waters with the nutrient-rich SACW and their tolerance to lower light levels.

Photosynthetic pigments of oceanic Chlorophyta belonging to prasinophytes clade VII.

The ecological importance and diversity of pico/nanoplanktonic algae remains poorly studied in marine waters, in part because many are tiny and without distinctive morphological features. Amongst green algae, Mamiellophyceae such as Micromonas or Bathycoccus are dominant in coastal waters while prasinophytes clade VII, yet not formerly described, appear to be major players in open oceanic waters. The pigment composition of 14 strains representative of different subclades of clade VII was analyzed using a method that improves the separation of loroxanthin and neoxanthin. All the prasinophytes clade VII analyzed here showed a pigment composition similar to that previously reported for RCC287 corresponding to pigment group prasino-2A. However, we detected in addition astaxanthin for which it is the first report in prasinophytes. Among the strains analyzed, the pigment signature is qualitatively similar within subclades A and B. By contrast, RCC3402 from subclade C (Picocystis) lacks loroxanthin, astaxanthin, and antheraxanthin but contains alloxanthin, diatoxanthin, and monadoxanthin that are usually found in diatoms or cryptophytes. For subclades A and B, loroxanthin was lowest at highest light irradiance suggesting a light-harvesting role of this pigment in clade VII as in Tetraselmis.

Interactions between SIVNef, SIVGagPol and Alix correlate with viral replication and progression to AIDS in rhesus macaques.

Infection with Simian Immunodeficiency Virus (SIV) leads to high viral loads and progression to Simian AIDS (SAIDS) in rhesus macaques. The viral accessory protein Nef is required for this phenotype in monkeys as well as in HIV-infected humans. Previously, we determined that HIVNef binds HIVGagPol and Alix for optimal viral replication in cells. In this study, we demonstrated that these interactions could correlate with high viral loads leading to SAIDS in the infected host. By infecting rhesus macaques with a mutant SIV(mac239), where sequences in the nef gene that are required for these interactions were mutated, we observed robust viral replication and disease in two out of four monkeys, where they reverted to the wild type genotype and phenotype. These two rhesus macaques also died of SAIDS. Two other monkeys did not progress to disease and continued to harbor mutant nef sequences. We conclude that interactions between Nef, GagPol and Alix contribute to optimal viral replication and progression to disease in the infected host.