New sensitive tools to characterize meta-metabolome response to short- and long-term cobalt exposure in dynamic river biofilm communities.

Untargeted metabolomics is a non-a priori analysis of biomolecules that characterizes the metabolome variations induced by short- and long-term exposures to stressors. Even if the metabolite annotation remains lacunar due to database gaps, the global metabolomic fingerprint allows for trend analyses of dose-response curves for hundreds of cellular metabolites. Analysis of dose/time-response curve trends (biphasic or monotonic) of untargeted metabolomic features would thus allow the use of all the chemical signals obtained in order to determine stress levels (defense or damage) in organisms. To develop this approach in a context of time-dependent microbial community changes, mature river biofilms were exposed for 1 month to four cobalt (Co) concentrations (from background concentration to 1 × 10-6 M) in an open system of artificial streams. The meta-metabolomic response of biofilms was compared against a multitude of biological parameters (including bioaccumulation, biomass, chlorophyll a content, composition and structure of prokaryotic and eukaryotic communities) monitored at set exposure times (from 1 h to 28 d). Cobalt exposure induced extremely rapid responses of the meta-metabolome, with time range inducing defense responses (TRIDeR) of around 10 s, and time range inducing damage responses (TRIDaR) of several hours. Even in biofilms whose structure had been altered by Co bioaccumulation (reduced biomass, chlorophyll a contents and changes in the composition and diversity of prokaryotic and eukaryotic communities), concentration range inducing defense responses (CRIDeR) with similar initiation thresholds (1.41 ± 0.77 × 10-10 M Co2+ added in the exposure medium) were set up at the meta-metabolome level at every time point. In contrast, the concentration range inducing damage responses (CRIDaR) initiation thresholds increased by 10 times in long-term Co exposed biofilms. The present study demonstrates that defense and damage responses of biofilm meta-metabolome exposed to Co are rapidly and sustainably impacted, even within tolerant and resistant microbial communities.

Seasonal dynamics of eukaryotic microbial diversity in hypersaline Tuz Lake characterized by 18S rDNA sequencing.

Microbial diversity found in hypersaline ecosystems is structurally unique and essential in many microbiological and ecological processes. Tuz Lake, the second biggest lake in Türkiye, is a talassohaline (over 32% [w/v]) lake with near-neutral pH. The aim of study was to investigate the composition of the eukaryotic microbial community in Tuz Lake by 18S rDNA amplicon sequencing, as well as its relationship and change with environmental factors during 1-year period. Next-generation sequencing and bioinformatic analysis were applied to describe the eukaryotic microbial community in Tuz Lake. As a result of bioinformatics analysis, Archaeplastida (39%) and Stramenopiles, Alveolata, Rhizaria (SAR) (51%) were the most abundant taxa represented in the dataset. The Archaeplastida phylum showed a significant difference between winter and summer and higher abundance in summer in contrast to the SAR group, which represented higher abundance in winter. Genus level assessment showed that the most abundant genera were Navicula, Chlorophyta;unclassified_taxa, Dunaliella, Cladosporium, Paraphelidium, Scuticociliates;unclassified_taxa, and Chlamydomonadales;unclassified_taxa. Navicula abundance was significantly different and overwhelmingly dominant in winter. On the other hand, Cladosporium and Chlorophyta; unclassified_taxa represented a significant difference between seasons and high abundance in summer. Furthermore, Dunaliella populations were not detected in midsummer and early fall when the temperature increased and water volume in the lake decreased.

Protist taxonomic and functional diversity in aquatic ecosystems of the Brazilian Atlantic Forest.

The Brazilian Atlantic Forest and its associated ecosystems are highly biodiverse but still understudied, especially with respect to eukaryotic microbes. Protists represent the largest proportion of eukaryotic diversity and play important roles in nutrient cycling and maintenance of the ecosystems in which they occur. However, much of protist diversity remains unknown, particularly in the Neotropics. Understanding the taxonomic and functional diversity of these organisms is urgently needed, not only to fill this gap in our knowledge, but also to enable the development of public policies for biological conservation. This is the first study to investigate the taxonomic and trophic diversity of the major protist groups in freshwater systems and brackish coastal lagoons located in fragments of the Brazilian Atlantic Forest by DNA metabarcoding, using high-throughput sequencing of the gene coding for the V4 region of the 18S rRNA gene. We compared α and ß diversity for all protist communities and assessed the relative abundance of phototrophic, consumer, and parasitic taxa. We found that the protist communities of coastal lagoons are as diverse as the freshwater systems studied in terms of α diversity, although differed significantly in terms of taxonomic composition. Our results still showed a notable functional homogeneity between the trophic groups in freshwater environments. Beta diversity was higher among freshwater samples, suggesting a greater level of heterogeneity within this group of samples concerning the composition and abundance of OTUs.Ciliophora was the most represented group in freshwater, while Diatomea dominated diversity in coastal lagoons.

Vertical changes in water depth and environmental variables drove the antibiotics and antibiotic resistomes distribution, and microbial food web structures in the estuary and marine ecosystems.

The influence of vertical changes in water depth on emerging pollutants distribution and microbial food web remains elusive. We investigated the influence of vertical transition in water depth on the environmental variables, antibiotics and antibiotic resistomes, and microbial community structures in estuary and marine ecosystems (0-50 m). Stepwise multiple linear regression model showed that among investigated environmental variables, change in water salinity was the most influential factor dictating the fluoroquinolone and macrolides concentrations, while dissolved oxygen and turbidity were the key influencers of sulfonamides and beta-lactam concentrations, respectively. Bacterial and eukaryotic diversity and niche breadth significantly increased with the increasing water depth. Ecosystem food web structure at the bottom depths was more stable than at the middle and surface depths. At the surface depth, the top 5 keystone genera were Cryothecomonas, Syndiniales, Achromobacter, Pseudopirsonia, and Karlodinium. Whereas Eugregarinorida, Neptuniibacter, Mychonastes, Novel_Apicomplexa_Class_1, Aplanochytrium and Dietzia, Halodaphnea, Luminiphilus, Aplanochytrium, Maullinia dominated the top 5 genera at the middle and the bottom depth, respectively. Absolute abundance of antibiotic resistance genes (ARGs) was drastically increased at the surface depth compared with the middle and bottom depths. Abundance of the top 10 ARGs and mobile genetic elements (MGEs) detected including tnpA-05, aadA2-03, mexF, aadA1, intI-1(clinic), qacEdelta1-02, aadA-02, qacEdelta1-01, cmlA1-01, and aadA-01 were amplified at the surface depth. This study demonstrated that ARGs abundance was disproportionate to bacterial diversity, and anthropogenic disturbances, confinement, MGEs, and ecosystem stability play primary roles in the fate of ARGs. The findings of this study also implicate that vertical changes in the water depth on environmental conditions can influence antibiotic concentrations and microbial community dramatically.

The Diversity Patterns of Rare to Abundant Microbial Eukaryotes Across a Broad Range of Salinities in a Solar Saltern.

Solar salterns are excellent artificial systems for examining species diversity and succession along salinity gradients. Here, the eukaryotic community in surface water of a Korean solar saltern (30 to 380 practical salinity units) was investigated from April 2019 to October 2020 using Illumina sequencing targeting the V4 and V9 regions of 18S rDNA. A total of 926 operational taxonomic units (OTUs) and 1,999 OTUs were obtained with the V4 and V9 regions, respectively. Notably, most of the OTUs were microbial eukaryotes, and the high-abundance groups (> 5% relative abundance (RA), Alveolata, Stramenopila, Archaeplastida, and Opisthokonta) usually accounted for > 90% of the total cumulative read counts and > 80% of all OTUs. Moreover, the high-abundance Alveolata (larger forms) and Stramenopila (smaller forms) groups displayed a significant inverse relationship, probably due to predator-prey interactions. Most of the low-abundance (0.1-5% RA) and rare (< 0.1% RA) groups remained small portion during the field surveys. Taxonomic novelty (at < 90% sequence identity) was high in the Amoebozoa, Cryptista, Haptista, Rhizaria, and Stramenopila groups (69.8% of all novel OTUs), suggesting the presence of a large number of hidden species in hypersaline environments. Remarkably, the high-abundance groups had little overlap with the other groups, implying the weakness of rare-to-prevalent community dynamics. The low-abundance Discoba group alone temporarily became the high-abundance group, suggesting that it is an opportunistic group. Overall, the composition and diversity of the eukaryotic community in hypersaline environments may be persistently stabilized, despite diverse disturbance events.

Salinity is the major driver of the global eukaryotic community structure in fish-canning wastewater treatment plants.

Fish-canning wastewater is characterized frequently by a high content of salt (NaCl), making its treatment particularly difficult; however, the knowledge of the effect of NaCl on eukaryotic communities is very limited. In the present study, the global diversity of eukaryotes in activated sludges (AS) from 4 different wastewater treatment plants (WWTPs) treating fish-canning effluents varying in salinity (0.47, 1.36, 1.72 and 12.76 g NaCl/L) was determined by sequencing partial 18S rRNA genes using Illumina MiSeq. A greater diversity than previously reported was observed in the AS community, which comprised 37 and 330 phylum-like and genera-like groups, respectively. In this sense, the more abundant genus-like groups (average relative abundance (RA) > 5%) were Adineta (6.80%), Lecane (16.80%), Dictyostelium (7.36%), Unclassified_Fungi7 (6.94%), Procryptobia (5.13) and Oocystis (5.07%). The eukaryotic communities shared a common core of 25 phylum-like clades (95% of total sequences); therefore, a narrow selection of the eukaryotic populations was found, despite the differences in the abiotic characteristics of fish-canning effluents and reactor operational conditions inflicted. The differences in NaCl concentration were the main factor that influenced the structure of the eukaryotic community, modulating the RAs of the different phylum-like clades of the common core. Higher levels of salt increased the RAs of Ascomycota, Chlorophyta, Choanoflagellata, Cryptophyta, Mollusca, Nematoda, Other Protists and Unclassified Fungi. Among the different eukaryotic genera here found, the RA of Oocystis (Chlorophyta) was intimately correlated to increasing NaCl concentrations and it is proposed as a bioindicator of the global eukaryotic community of fish-canning WWTPs.

Opinion: Genetic Conflict With Mobile Elements Drives Eukaryotic Genome Evolution, and Perhaps Also Eukaryogenesis.

Through analyses of diverse microeukaryotes, we have previously argued that eukaryotic genomes are dynamic systems that rely on epigenetic mechanisms to distinguish germline (i.e., DNA to be inherited) from soma (i.e., DNA that undergoes polyploidization, genome rearrangement, etc.), even in the context of a single nucleus. Here, we extend these arguments by including two well-documented observations: (1) eukaryotic genomes interact frequently with mobile genetic elements (MGEs) like viruses and transposable elements (TEs), creating genetic conflict, and (2) epigenetic mechanisms regulate MGEs. Synthesis of these ideas leads to the hypothesis that genetic conflict with MGEs contributed to the evolution of a dynamic eukaryotic genome in the last eukaryotic common ancestor (LECA), and may have contributed to eukaryogenesis (i.e., may have been a driver in the evolution of FECA, the first eukaryotic common ancestor). Sex (i.e., meiosis) may have evolved within the context of the development of germline-soma distinctions in LECA, as this process resets the germline genome by regulating/eliminating somatic (i.e., polyploid, rearranged) genetic material. Our synthesis of these ideas expands on hypotheses of the origin of eukaryotes by integrating the roles of MGEs and epigenetics.

Benthic monitoring of oil and gas offshore platforms in the North Sea using environmental DNA metabarcoding.

Since 2010, considerable efforts have been undertaken to monitor the environmental status of European marine waters and ensuring the development of methodological standards for the evaluation of this status. However, the current routine biomonitoring implicates time-consuming and costly manual sorting and morphological identification of benthic macrofauna. Environmental DNA (eDNA) metabarcoding represents an alternative to the traditional monitoring method with very promising results. Here, we tested it further by performing eDNA metabarcoding of benthic eukaryotic communities in the vicinity of two offshore oil and gas platforms in the North Sea. Three different genetic markers (18S V1V2, 18S V9 and COI) were used to assess the environmental pressures induced by the platforms. All markers showed patterns of alpha and beta diversity consistent with morphology-based macrofauna analyses. In particular, the communities' structure inferred from metabarcoding and morphological data significantly changed along distance gradients from the platforms. The impact of the operational discharges was also detected by the variation of biotic index values, AMBI index showing the best correlation between morphological and eDNA data sets. Finally, the sediment physicochemical parameters were used to build a local de novo pressure index that served as benchmark to test the potential of a taxonomy-free approach. Our study demonstrates that metabarcoding approach outperforms morphology-based approach and can be used as a cost and time-saving alternative solution to the traditional morphology-based monitoring in order to monitor more efficiently the impact of industrial activities on marine biodiversity.

Characterization of Chlorella vulgaris (Trebouxiophyceae) associated microbial communities1.

Microalgae exhibit extensive potential for counteracting imminent challenges in the nutraceutical, pharmaceutical, and biomaterial sectors, but lack economic viability. Biotechnological systems for contamination control could advance the economic viability of microalgal feedstock, but the selection of suitable strains that sustainably promote microalgal productivity remains challenging. In this study, total diversity in phototrophic Chlorella vulgaris cultures was assessed by amplicon sequencing comparing cultures subjected to five different cultivation conditions. Overall, 12 eukaryotic and 53 prokaryotic taxa were identified; Alphaproteobacteria (36.7%) dominated the prokaryotic and C. vulgaris (97.2%) the eukaryotic community. Despite altering cultivation conditions, 2 eukaryotic and 40 prokaryotic taxa remained stably associated with C. vulgaris; diversity between systems did not significantly differ (P > 0.05). Among those, 20 cultivable taxa were isolated and identified by 16S rDNA sequencing. Subsequently, controlled co-cultures were investigated showing stable associations of C. vulgaris with Sphingopyxis sp. and Pseudomonas sp.. Out-competition of C. vulgaris due to ammonium or phosphate limitation was not observed, despite significantly elevated growth of Sphingopyxis sp. and Tistrella sp.. (P < 0.05). Nevertheless, C. vulgaris growth was impaired by Tistrella sp.. Hence, the study provides a selection of stable indigenous prokaryotes and eukaryotes for artificially tailoring microbial biocenoses. Following a bottom-up approach, it provides a base for controlled co-cultures and thus the establishment of even more complex biocenoses using interkingdom assemblages. Such assemblages can benefit from functional richness for improved nutrient utilization, as well as bacterial load control, which can enhance microalgal feedstock production through improved culture stability and productivity.

Exploring the eukaryotic diversity in rumen of Indian camel (Camelus dromedarius) using 18S rRNA amplicon sequencing.

In addition to a wide variety of anaerobic and facultative anaerobic bacteria, camel rumen also harbors a diverse of eukaryotic organisms. In the present study, the eukaryotic communities of camel rumen were characterized using 18S rRNA amplicon sequencing. Metagenomic DNA was isolated from rumen samples of fourteen adult Bikaneri and Kachchhi breeds of camel fed different diets containing Jowar, Bajra, Maize, and Guar. Illumina sequencing generated 27,161,904 number of reads corresponding to 1543 total operational taxonomic units (OTUs). Taxonomic classification of community metagenome sequences from all the samples revealed the presence of 92 genera belonging to 16 different divisions, out of which Ciliophora (73%), Fungi (13%) and Streptophyta (9%) were found to be the most dominant. Notably, the abundance of Ciliophora was significantly higher in the case of Guar feed, while Fungi was significantly higher in the case of Maize feed, indicating the influence of cellulose and hemicellulose content of feedstuff on the composition of eukaryotes. The results suggest that the camel rumen eukaryotes are highly dynamic and depend on the type of diet given to the animal. Pearson's correlation analysis suggested the ciliate protozoa and fungi were negatively correlated with each other. To the best of our knowledge, this is first systematic study to characterize camel rumen eukaryotes, which has provided newer information regarding eukaryotic diversity patterns amongst camel fed on different diets.

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence-capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence-capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence-capture recovered 80%-90% of the control sample species. DNA sequence-capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low-cost, whereas DNA-sequence capture for biodiversity assessment is still in its infancy, is more time-consuming but provides more taxonomic assignments.

Microbial Diversity in the Eukaryotic SAR Clade: Illuminating the Darkness Between Morphology and Molecular Data.

Despite their diversity and ecological importance, many areas of the SAR-Stramenopila, Alveolata, and Rhizaria-clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well-sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the "dark area" of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and "non-vertebrate" hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.

Analysis of phytoplankton assemblage structure in the Mediterranean Sea based on high-throughput sequencing of partial 18S rRNA sequences.

Studying taxonomic and ecological diversity of phytoplankton assemblages is often difficult because morphological analysis cannot provide a complete description of their composition. Therefore, more robust and feasible approaches have to be chosen to elucidate the interactions between environmental and human pressures and phytoplankton assemblages. The Ocean Sampling Day (OSD) allowed collecting seawater samples from a wide range of oceanic regions including the Mediterranean Sea. In this study, a total of 754,167 V4-18S ribosomal DNA (rDNA) metabarcodes derived from 20 plankton samples collected at 19 sampling sites across the coastal areas of the Mediterranean Sea were analyzed to explore the relationships between phytoplankton assemblages' composition, sub-regional environmental features and human pressures. We reduced the whole set of autotroph plankton (1398 OTUs) to a smaller number of ecologically relevant entities (205 taxa) and used the latter for analysing the structure of phytoplankton assemblages. Chaetoceros was the only genus occurring in all the samples, while the number of taxa was maximum in the W Mediterranean. Based on the assigned OTUs, the structure of E Mediterranean phytoplankton was the most homogeneous. Further, phytoplankton assemblages from the three Mediterranean sub-regions (Western, Adriatic and Eastern) were significantly different (R=0.25, p=0.0136) based on Jaccard similarity. We also observed that phytoplankton diversity and human impact on marine ecosystems were not significantly related to each other based on Mantel's test.

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics.

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule-mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current-day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co-evolved with one another, albeit in different manners. These co-evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

Spatial Distribution of Eukaryotic Communities Using High-Throughput Sequencing Along a Pollution Gradient in the Arsenic-Rich Creek Sediments of Carnoulès Mine, France.

Microscopic eukaryotes play a key role in ecosystem functioning, but their diversity remains largely unexplored in most environments. To advance our knowledge of eukaryotic microorganisms and the factors that structure their communities, high-throughput sequencing was used to characterize their diversity and spatial distribution along the pollution gradient of the acid mine drainage at Carnoulès (France). A total of 16,510 reads were retrieved leading to the identification of 323 OTUs after normalization. Phylogenetic analysis revealed a quite diverse eukaryotic community characterized by a total of eight high-level lineages including 37 classes. The majority of sequences were clustered in four main groups: Fungi, Stramenopiles, Alveolata and Viridiplantae. The Reigous sediments formed a succession of distinct ecosystems hosting contrasted eukaryotic communities whose structure appeared to be at least partially correlated with sediment mineralogy. The concentration of arsenic in the sediment was shown to be a significant factor driving the eukaryotic community structure along this continuum.

Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions.

High throughput sequencing technology has opened a window into the vast communities of bacteria that live on and in humans, demonstrating tremendous variability, and that they play a large role in health and disease. The eukaryotic component of the human gut microbiome remains relatively unexplored with these methods, but turning these tools toward microbial eukaryotes in the gut will likely yield myriad insights into disease as well as the ecological and evolutionary principles that govern the gut microbiota. Microbial eukaryotes are common inhabitants of the human gut worldwide and parasitic taxa are a major source of morbidity and mortality, especially in developing countries, though there are also taxa that cause no harm or are beneficial. While the role microbial eukaryotes play in healthy individuals is much less clear, there are likely many complex interactions between the bacterial, archaeal, and eukaryotic microbiota that influence human health. Integrating eukaryotic microbes into a broad view of microbiome function requires an integrated ecological approach rather than one focused on specific, disease-causing taxa. Moving forward, we expect broad surveys of the eukaryotic microbiota and associated bacteria from geographically and socioeconomically diverse populations to paint a more complete picture of the human gut microbiome in health and disease.