Diverse patterns of correspondence between protist metabarcodes and protist metagenome-assembled genomes.

Two common approaches to study the composition of environmental protist communities are metabarcoding and metagenomics. Raw metabarcoding data are usually processed into Operational Taxonomic Units (OTUs) or amplicon sequence variants (ASVs) through clustering or denoising approaches, respectively. Analogous approaches are used to assemble metagenomic reads into metagenome-assembled genomes (MAGs). Understanding the correspondence between the data produced by these two approaches can help to integrate information between the datasets and to explain how metabarcoding OTUs and MAGs are related with the underlying biological entities they are hypothesised to represent. MAGs do not contain the commonly used barcoding loci, therefore sequence homology approaches cannot be used to match OTUs and MAGs. We made an attempt to match V9 metabarcoding OTUs from the 18S rRNA gene (V9 OTUs) and MAGs from the Tara Oceans expedition based on the correspondence of their relative abundances across the same set of samples. We evaluated several metrics for detecting correspondence between features in these two datasets and developed controls to filter artefacts of data structure and processing. After selecting the best-performing metrics, ranking the V9 OTU/MAG matches by their proportionality/correlation coefficients and applying a set of selection criteria, we identified candidate matches between V9 OTUs and MAGs. In some cases, V9 OTUs and MAGs could be matched with a one-to-one correspondence, implying that they likely represent the same underlying biological entity. More generally, matches we observed could be classified into 4 scenarios: one V9 OTU matches many MAGs; many V9 OTUs match many MAGs; many V9 OTUs match one MAG; one V9 OTU matches one MAG. Notably, we found some instances in which different OTU-MAG matches from the same taxonomic group were not classified in the same scenario, with all four scenarios possible even within the same taxonomic group, illustrating that factors beyond taxonomic lineage influence the relationship between OTUs and MAGs. Overall, each scenario produces a different interpretation of V9 OTUs, MAGs and how they compare in terms of the genomic and ecological diversity they represent.

How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation.

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.

The global genetic diversity of planktonic foraminifera reveals the structure of cryptic speciation in plankton.

The nature and extent of diversity in the plankton has fascinated scientists for over a century. Initially, the discovery of many new species in the remarkably uniform and unstructured pelagic environment appeared to challenge the concept of ecological niches. Later, it became obvious that only a fraction of plankton diversity had been formally described, because plankton assemblages are dominated by understudied eukaryotic lineages with small size that lack clearly distinguishable morphological features. The high diversity of the plankton has been confirmed by comprehensive metabarcoding surveys, but interpretation of the underlying molecular taxonomies is hindered by insufficient integration of genetic diversity with morphological taxonomy and ecological observations. Here we use planktonic foraminifera as a study model and reveal the full extent of their genetic diversity and investigate geographical and ecological patterns in their distribution. To this end, we assembled a global data set of ~7600 ribosomal DNA sequences obtained from morphologically characterised individual foraminifera, established a robust molecular taxonomic framework for the observed diversity, and used it to query a global metabarcoding data set covering ~1700 samples with ~2.48 billion reads. This allowed us to extract and assign 1 million reads, enabling characterisation of the structure of the genetic diversity of the group across ~1100 oceanic stations worldwide. Our sampling revealed the existence of, at most, 94 distinct molecular operational taxonomic units (MOTUs) at a level of divergence indicative of biological species. The genetic diversity only doubles the number of formally described species identified by morphological features. Furthermore, we observed that the allocation of genetic diversity to morphospecies is uneven. Only 16 morphospecies disguise evolutionarily significant genetic diversity, and the proportion of morphospecies that show genetic diversity increases poleward. Finally, we observe that MOTUs have a narrower geographic distribution than morphospecies and that in some cases the MOTUs belonging to the same morphospecies (cryptic species) have different environmental preferences. Overall, our analysis reveals that even in the light of global genetic sampling, planktonic foraminifera diversity is modest and finite. However, the extent and structure of the cryptic diversity reveals that genetic diversification is decoupled from morphological diversification, hinting at different mechanisms acting at different levels of divergence.

A predictive mortality score in ANCA-associated renal vasculitis.

BACKGROUND: Several scores have been developed to predict mortality at anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) diagnosis. Their prognostic value in Caucasian patients with kidney involvement (AAV-GN) remains uncertain as none has been developed in this specific population. We aimed to propose a novel and more accurate score specific for them. METHODS: This multicentric study included patients diagnosed with AAV-GN since January 2000 in four nephrology centers (recorded in the Maine-Anjou AAV-GN Registry). Existing scores and baseline characteristics were assessed at diagnosis before any therapeutic intervention. A multivariable analysis was performed to build a new predictive score for death. Its prognosis performance (area under receiving operating curve and C-index) and accuracy (Brier score) was compared with existing scores. One hundred and eighty-five patients with AAV-GN from the RENVAS registry were used as a validation cohort. RESULTS: A total of 228 patients with AAV-GN from the Maine-Anjou registry were included to build the new score. It included the four components most associated with death: age, history of hypertension or cardiac disease, creatinine and hemoglobin levels at diagnosis. Overall, 194 patients had all the data available to determine the performance of the new score and existing scores. The new score performed better than the previous ones in the development and in the validation cohort. Among the scores tested, only Five-Factor Score and Japanese Vasculitis Activity Score had good performance in predicting death in AAV-GN. CONCLUSIONS: This original score, named DANGER (Death in ANCA Glomerulonephritis-Estimating the Risk), may be useful to predict the risk of death in AAV-GN patients. Validation in different populations is needed to clarify its role in assisting clinical decisions.

Adding 6-month parameters for the prediction of kidney prognosis in ANCA-associated glomerulonephritis.

Background: Antineutrophil-cytoplasmic antibody (ANCA)-associated vasculitis (AAV) with kidney involvement (AAV-GN) frequently evolves to end-stage kidney disease (ESKD) despite aggressive immunosuppressive treatment. Several risk scores have been used to assess renal prognosis. We aimed to determine whether kidney function and markers of AAV-GN activity after 6 months could improve the prediction of ESKD. Methods: This retrospective and observational study included adult patients with AAV-GN recruited from six French nephrology centers (including from the Maine-Anjou AAV registry). The primary outcome was kidney survival. Analyses were conducted in the whole population and in a sub-population that did not develop ESKD early in the course of the disease. Results: When considering the 102 patients with all data available at diagnosis, Berden classification and Renal Risk Score (RRS) were not found to be better than kidney function [estimated glomerular filtration rate (eGFR)] alone at predicting ESKD (C-index = 0.70, 0.79, 0.82, respectively). Multivariables models did not indicate an improved prognostic value when compared with eGFR alone.When considering the 93 patients with all data available at 6 months, eGFR outperformed Berden classification and RRS (C-index = 0.88, 0.62, 0.69, respectively) to predict ESKD. RRS performed better when it was updated with the eGFR at 6 months instead of the baseline eGFR. While 6-month proteinuria was associated with ESKD and improved ESKD prediction, hematuria and serological remission did not. Conclusion: This work suggests the benefit of the reassessment of the kidney prognosis 6 months after AAV-GN diagnosis. Kidney function at this time remains the most reliable for predicting kidney outcome. Of the markers tested, persistent proteinuria at 6 months was the only one to slightly improve the prediction of ESKD.

Kidney injury molecule 1 (KIM-1): a potential biomarker of acute kidney injury and tubulointerstitial injury in patients with ANCA-glomerulonephritis.

Background: Kidney injury molecule 1 (KIM-1) is a transmembrane glycoprotein expressed by proximal tubular cells, recognized as an early, sensitive and specific urinary biomarker for kidney injury. Blood KIM-1 was recently associated with the severity of acute and chronic kidney damage but its value in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis with glomerulonephritis (ANCA-GN) has not been studied. Thus, we analyzed its expression at ANCA-GN diagnosis and its relationship with clinical presentation, kidney histopathology and early outcomes. Methods: We assessed KIM-1 levels and other pro-inflammatory molecules (C-reactive protein, interleukin-6, tumor necrosis factor α, monocyte chemoattractant protein-1 and pentraxin 3) at ANCA-GN diagnosis and after 6 months in patients included in the Maine-Anjou registry, which gathers data patients from four French Nephrology Centers diagnosed since January 2000. Results: Blood KIM-1 levels were assessed in 54 patients. Levels were elevated at diagnosis and decreased after induction remission therapy. KIM-1 was associated with the severity of renal injury at diagnosis and the need for kidney replacement therapy. In opposition to other pro-inflammatory molecules, KIM-1 correlated with the amount of acute tubular necrosis and interstitial fibrosis/tubular atrophy (IF/TA) on kidney biopsy, but not with interstitial infiltrate or with glomerular involvement. In multivariable analysis, elevated KIM-1 predicted initial estimated glomerular filtration rate (ß = -19, 95% CI -31, -7.6, P = .002). Conclusion: KIM-1 appears as a potential biomarker for acute kidney injury and for tubulointerstitial injury in ANCA-GN. Whether KIM-1 is only a surrogate marker or is a key immune player in ANCA-GN pathogenesis remain to be determined.

Predicting global distributions of eukaryotic plankton communities from satellite data.

Satellite remote sensing is a powerful tool to monitor the global dynamics of marine plankton. Previous research has focused on developing models to predict the size or taxonomic groups of phytoplankton. Here, we present an approach to identify community types from a global plankton network that includes phytoplankton and heterotrophic protists and to predict their biogeography using global satellite observations. Six plankton community types were identified from a co-occurrence network inferred using a novel rDNA 18 S V4 planetary-scale eukaryotic metabarcoding dataset. Machine learning techniques were then applied to construct a model that predicted these community types from satellite data. The model showed an overall 67% accuracy in the prediction of the community types. The prediction using 17 satellite-derived parameters showed better performance than that using only temperature and/or the concentration of chlorophyll a. The constructed model predicted the global spatiotemporal distribution of community types over 19 years. The predicted distributions exhibited strong seasonal changes in community types in the subarctic-subtropical boundary regions, which were consistent with previous field observations. The model also identified the long-term trends in the distribution of community types, which suggested responses to ocean warming.

Inter-comparison of marine microbiome sampling protocols.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22-3 µm, 3-20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and "whole water" (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

Ocean-wide comparisons of mesopelagic planktonic community structures.

For decades, marine plankton have been investigated for their capacity to modulate biogeochemical cycles and provide fishery resources. Between the sunlit (epipelagic) layer and the deep dark waters, lies a vast and heterogeneous part of the ocean: the mesopelagic zone. How plankton composition is shaped by environment has been well-explored in the epipelagic but much less in the mesopelagic ocean. Here, we conducted comparative analyses of trans-kingdom community assemblages thriving in the mesopelagic oxygen minimum zone (OMZ), mesopelagic oxic, and their epipelagic counterparts. We identified nine distinct types of intermediate water masses that correlate with variation in mesopelagic community composition. Furthermore, oxygen, NO3- and particle flux together appeared as the main drivers governing these communities. Novel taxonomic signatures emerged from OMZ while a global co-occurrence network analysis showed that about 70% of the abundance of mesopelagic plankton groups is organized into three community modules. One module gathers prokaryotes, pico-eukaryotes and Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) from oxic regions, and the two other modules are enriched in OMZ prokaryotes and OMZ pico-eukaryotes, respectively. We hypothesize that OMZ conditions led to a diversification of ecological niches, and thus communities, due to selective pressure from limited resources. Our study further clarifies the interplay between environmental factors in the mesopelagic oxic and OMZ, and the compositional features of communities.

Diversity of the Pacific Ocean coral reef microbiome.

Coral reefs are among the most diverse ecosystems on Earth. They support high biodiversity of multicellular organisms that strongly rely on associated microorganisms for health and nutrition. However, the extent of the coral reef microbiome diversity and its distribution at the oceanic basin-scale remains to be explored. Here, we systematically sampled 3 coral morphotypes, 2 fish species, and planktonic communities in 99 reefs from 32 islands across the Pacific Ocean, to assess reef microbiome composition and biogeography. We show a very large richness of reef microorganisms compared to other environments, which extrapolated to all fishes and corals of the Pacific, approximates the current estimated total prokaryotic diversity for the entire Earth. Microbial communities vary among and within the 3 animal biomes (coral, fish, plankton), and geographically. For corals, the cross-ocean patterns of diversity are different from those known for other multicellular organisms. Within each coral morphotype, community composition is always determined by geographic distance first, both at the island and across ocean scale, and then by environment. Our unprecedented sampling effort of coral reef microbiomes, as part of the Tara Pacific expedition, provides new insight into the global microbial diversity, the factors driving their distribution, and the biocomplexity of reef ecosystems.