Scientists' call to action: Microbes, planetary health, and the Sustainable Development Goals.

Microorganisms, including bacteria, archaea, viruses, fungi, and protists, are essential to life on Earth and the functioning of the biosphere. Here, we discuss the key roles of microorganisms in achieving the United Nations Sustainable Development Goals (SDGs), highlighting recent and emerging advances in microbial research and technology that can facilitate our transition toward a sustainable future. Given the central role of microorganisms in the biochemical processing of elements, synthesizing new materials, supporting human health, and facilitating life in managed and natural landscapes, microbial research and technologies are directly or indirectly relevant for achieving each of the SDGs. More importantly, the ubiquitous and global role of microbes means that they present new opportunities for synergistically accelerating progress toward multiple sustainability goals. By effectively managing microbial health, we can achieve solutions that address multiple sustainability targets ranging from climate and human health to food and energy production. Emerging international policy frameworks should reflect the vital importance of microorganisms in achieving a sustainable future.

SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

The prokaryotic and eukaryotic microbiome of Pacific oyster spat is shaped by ocean warming but not acidification.

Pacific oysters (Magallana gigas, a.k.a. Crassostrea gigas), the most widely farmed oysters, are under threat from climate change and emerging pathogens. In part, their resilience may be affected by their microbiome, which, in turn, may be influenced by ocean warming and acidification. To understand these impacts, we exposed early-development Pacific oyster spat to different temperatures (18°C and 24°C) and pCO2 levels (800, 1,600, and 2,800 µatm) in a fully crossed design for 3 weeks. Under all conditions, the microbiome changed over time, with a large decrease in the relative abundance of potentially pathogenic ciliates (Uronema marinum) in all treatments with time. The microbiome composition differed significantly with temperature, but not acidification, indicating that Pacific oyster spat microbiomes can be altered by ocean warming but is resilient to ocean acidification in our experiments. Microbial taxa differed in relative abundance with temperature, implying different adaptive strategies and ecological specializations among microorganisms. Additionally, a small proportion (~0.2% of the total taxa) of the relatively abundant microbial taxa were core constituents (>50% occurrence among samples) across different temperatures, pCO2 levels, or time. Some taxa, including A4b bacteria and members of the family Saprospiraceae in the phyla Chloroflexi (syn. Chloroflexota) and Bacteroidetes (syn. Bacteroidota), respectively, as well as protists in the genera Labyrinthula and Aplanochytrium in the class Labyrinthulomycetes, and Pseudoperkinsus tapetis in the class Ichthyosporea were core constituents across temperatures, pCO2 levels, and time, suggesting that they play an important, albeit unknown, role in maintaining the structural and functional stability of the Pacific oyster spat microbiome in response to ocean warming and acidification. These findings highlight the flexibility of the spat microbiome to environmental changes.IMPORTANCEPacific oysters are the most economically important and widely farmed species of oyster, and their production depends on healthy oyster spat. In turn, spat health and productivity are affected by the associated microbiota; yet, studies have not scrutinized the effects of temperature and pCO2 on the prokaryotic and eukaryotic microbiomes of spat. Here, we show that both the prokaryotic and, for the first time, eukaryotic microbiome of Pacific oyster spat are surprisingly resilient to changes in acidification, but sensitive to ocean warming. The findings have potential implications for oyster survival amid climate change and underscore the need to understand temperature and pCO2 effects on the microbiome and the cascading effects on oyster health and productivity.

Biological Interactions and Environmental Influences Shift Microeukaryotes in Permafrost Active Layer Soil Across the Qinghai-Tibet Plateau.

Permafrost active layer soils are harsh environments with thaw/freeze cycles and sub-zero temperatures, harboring diverse microorganisms. However, the distribution patterns, assembly mechanism, and driving forces of soil microeukaryotes in permafrost remain largely unknown. In this study, we investigated microeukaryotes in permafrost active layer across the Qinghai-Tibet Plateau (QTP) using 18S rRNA gene sequencing. The results showed that the microbial eukaryotic communities were dominated by Nematozoa, Ciliophora, Ascomycota, Cercozoa, Arthropoda, and Basidiomycota in terms of relative abundance and operational taxonomic unit (OTU) richness. Nematozoa had the highest relative abundance, while Ciliophora had the highest OTU richness. These phyla had strong interactions between each other. Their alpha diversity and community structure were differently influenced by the factors associated to location, climate, and soil properties, particularly the soil properties. Significant but weak distance-decay relationships with different slopes were established for the communities of these dominant phyla, except for Basidiomycota. According to the null model, community assemblies of Nematozoa and Cercozoa were dominated by heterogeneous selection, Ciliophora and Ascomycota were dominated by dispersal limitation, while Arthropoda and Basidiomycota were highly dominated by non-dominant processes. The assembly mechanisms can be jointly explained by biotic interactions, organism treats, and environmental influences. Modules in the co-occurrence network of the microeukaryotes were composed by members from different taxonomic groups. These modules also had interactions and responded to different environmental factors, within which, soil properties had strong influences on these modules. The results suggested the importance of biological interactions and soil properties in structuring microbial eukaryotic communities in permafrost active layer soil across the QTP.

Contrasting Mechanisms Determine the Microeukaryotic and Syndiniales Community Assembly in a Eutrophic bay.

Syndiniales is a diverse parasitic group, increasingly gaining attention owing to its high taxonomic diversity in marine ecosystems and inhibitory effects on the dinoflagellate blooms. However, their seasonal dynamics, host interactions, and mechanisms of community assembly are largely unknown, particularly in eutrophic waters. Here, using 18S rRNA gene amplicon sequencing, we intended to elucidate the interactions between Syndiniales and microeukaryotes, as well as community assembly processes in a eutrophic bay. The results showed that Syndiniales group II was dominating throughout the year, with substantially higher abundance in the winter and spring, whereas Syndiniales group I was more abundant in the summer and autumn. Temperature and Dinoflagellata were the most important abiotic and biotic factors driving variations of the Syndiniales community, respectively. The assembly processes of microeukaryotes and Syndiniales were completely different, with the former being controlled by a balance between homogeneous selection and drift and the latter being solely governed by drift. Network analysis revealed that Syndiniales group II had the largest number of interactions with microeukaryotes, and they primarily associated with Dinoflagellata in the winter, while interactions with Chlorophyta and Bacillariophyta increased dramatically in summer and autumn. These findings provide significant insights in understanding the interactions and assembly processes of Syndiniales throughout the year, which is critical in revealing the roles of single-celled parasites in driving protist dynamics in eutrophic waters.

Lemur Gut Microeukaryotic Community Variation Is Not Associated with Host Phylogeny, Diet, or Habitat.

Identifying the major forces driving variation in gut microbiomes enhances our understanding of how and why symbioses between hosts and microbes evolved. Gut prokaryotic community variation is often closely associated with host evolutionary and ecological variables. Whether these same factors drive variation in other microbial taxa occupying the animal gut remains largely untested. Here, we present a one-to-one comparison of gut prokaryotic (16S rRNA metabarcoding) and microeukaryotic (18S rRNA metabarcoding) community patterning among 12 species of wild lemurs. Lemurs were sampled from dry forests and rainforests of southeastern Madagascar and display a range of phylogenetic and ecological niche diversity. We found that while lemur gut prokaryotic community diversity and composition vary with host taxonomy, diet, and habitat, gut microeukaryotic communities have no detectable association with any of these factors. We conclude that gut microeukaryotic community composition is largely random, while gut prokaryotic communities are conserved among host species. It is likely that a greater proportion of gut microeukaryotic communities comprise taxa with commensal, transient, and/or parasitic symbioses compared with gut prokaryotes, many of which form long-term relationships with the host and perform important biological functions. Our study highlights the importance of greater specificity in microbiome research; the gut microbiome contains many "omes" (e.g., prokaryome, eukaryome), each comprising different microbial taxa shaped by unique selective pressures.

Integrated Biogeography and Assembly Mechanisms of Microeukaryotic Communities in Coastal Waters Near Shellfish Cultivation.

The Lianjiang coast in the East China Sea is a typical subtropical marine ecosystem, and shellfish cultivation occupies almost all of the available tidal flats. Many studies have investigated the effects of shellfish cultivation on benthic organisms and sediments, while the impact of shellfish cultivation on plankton ecosystems is still poorly understood. This study investigated the biogeographical patterns of microeukaryotic communities from Lianjiang coastal waters in four seasons using 18S ribosomal RNA gene amplicon sequencing. Microeukaryotes were mainly comprised of Dinoflagellata, Diatomea, Arthropoda, Ciliophora, Chlorophyta, Protalveolata, Cryptophyceae, and Ochrophyta, and presented significant differences in three habitats (the aquaculture area, confluent area, and offshore area) and four seasons. Similarity percentage analysis revealed that Paracalanus parvus, Heterocapsa rotundata, Bestiolina similis, and five additional key taxa contributed to spatio-temporal differences. Seasonal environmental and spatial factors explained 27.47% of microeukaryotic community variation on average, with 11.11% of the variation shared. Environmental variables, particularly depth, pH, and nitrite concentration, were strongly associated with the microeukaryotic community compositions. The neutral community model further demonstrated that stochastic processes were sufficient in shaping substantial variation in microeukaryotic communities across four seasons, which may reveal the remaining unexplained microeukaryotic community variation. We further divided four seasons into the aquaculture stages and non-aquaculture stages, and speculated that aquaculture activities may increase the dispersal limitation of microeukaryotes in coastal waters, especially for the big bodied-microbes like Arthropoda. The results provide a better understanding of the biogeographical patterns, processes, and mechanisms of microeukaryotic communities near shellfish cultivation.

The effect of metabarcoding 18S rRNA region choice on diversity of microeukaryotes including phytoplankton.

Metabarcoding using high throughput sequencing of amplicons of the 18S rRNA gene is one of the widely used methods for assessing the diversity of microeukaryotes in various ecosystems. We investigated the effectiveness of the V4 and V8-V9 regions of the 18S rRNA gene by comparing the results of metabarcoding microeukaryotic communities using the DADA2 (ASV), USEARCH-UNOISE3 (ZOTU), and USEARCH-UPARSE (OTU with 97% similarity) algorithms. Both regions showed similar levels of genetic variability and taxa identification accuracy. Richness for DADA2 datasets of both regions was lower than for UNOISE3 and UPARSE datasets, which is due to more accurate error correction in amplicons. Microeukaryotic communities (autotrophs and heterotrophs) structure identified using both regions showed a significant relationship with phytoplankton (autotrophs) communities structure based on microscopy in a seasonal freshwater sample series. The strongest relationship was found between the phytoplankton species and V8-V9 ASVs produced by DADA2.

Seasonal Succession and Coherence Among Bacteria and Microeukaryotes in Lake Baikal.

Microorganisms exhibit seasonal succession governed by physicochemical factors and interspecies interactions, yet drivers of this process in different environments remain to be determined. We used high-throughput sequencing of 16S rRNA and 18S rRNA genes to study seasonal dynamics of bacterial and microeukaryotic communities at pelagic site of Lake Baikal from spring (under-ice, mixing) to autumn (direct stratification). The microbial community was subdivided into distinctive coherent clusters of operational taxonomic units (OTUs). Individual OTUs were consistently replaced during different seasonal events. The coherent clusters change their contribution to the microbial community depending on season. Changes of temperature, concentrations of silicon, and nitrates are the key factors affected the structure of microbial communities. Functional prediction revealed that some bacterial or eukaryotic taxa that switched with seasons had similar functional properties, which demonstrate their functional redundancy. We have also detected specific functional properties in different coherent clusters of bacteria or microeukaryotes, which can indicate their ability to adapt to seasonal changes of environment. Our results revealed a relationship between seasonal succession, coherency, and functional features of freshwater bacteria and microeukaryotes.

Biogeographic patterns of abundant and rare bacterial and microeukaryotic subcommunities in connected freshwater lake zones subjected to different levels of nutrient loading.

AIMS: To reveal whether the patterns of abundant and rare subcommunity composition of both bacteria and microeukaryotes vary between connected regions with different levels of nutrient loading in freshwater lakes. METHODS AND RESULTS: We investigated the abundant and rare subcommunity composition of both bacteria and microeukaryotes in two connected zones (Meiliang Bay (MLB) and Xukou Bay (XKB)) of a large shallow freshwater Lake Taihu via the high-throughput sequencing of bacterial 16S rRNA and microeukaryotic 18S rRNA genes. Even though these two lake zones are connected and share a species bank, they diverge in community composition. Significantly higher alpha diversity was observed for the abundant bacterial subcommunity in the MLB. However, no significant difference in alpha diversity between the rare bacterial subcommunities, as well as both rare and abundant microeukaryotic subcommunities were observed between MLB and XKB. It is demonstrated that both environmental factors and geographic distance play central roles in controlling the rare and abundant microbial subcommunities in the two connected lake zones. CONCLUSIONS: The abundant subcommunity composition of bacteria and microeukaryotes vary between connected regions with different levels of nutrient loading. Dispersal limitation plays a vital role in shaping microbial communities even in connected zones of freshwater lakes. SIGNIFICANCE AND IMPACT OF THE STUDY: Leading to a comprehensive understanding of the characteristics of microbial community in connected lake regions with different levels of nutrient loading.

BIOCOM-PIPE: a new user-friendly metabarcoding pipeline for the characterization of microbial diversity from 16S, 18S and 23S rRNA gene amplicons.

BACKGROUND: The ability to compare samples or studies easily using metabarcoding so as to better interpret microbial ecology results is an upcoming challenge. A growing number of metabarcoding pipelines are available, each with its own benefits and limitations. However, very few have been developed to offer the opportunity to characterize various microbial communities (e.g., archaea, bacteria, fungi, photosynthetic microeukaryotes) with the same tool. RESULTS: BIOCOM-PIPE is a flexible and independent suite of tools for processing data from high-throughput sequencing technologies, Roche 454 and Illumina platforms, and focused on the diversity of archaeal, bacterial, fungal, and photosynthetic microeukaryote amplicons. Various original methods were implemented in BIOCOM-PIPE to (1) remove chimeras based on read abundance, (2) align sequences with structure-based alignments of RNA homologs using covariance models, and (3) a post-clustering tool (ReClustOR) to improve OTUs consistency based on a reference OTU database. The comparison with two other pipelines (FROGS and mothur) and Amplicon Sequence Variant definition highlighted that BIOCOM-PIPE was better at discriminating land use groups. CONCLUSIONS: The BIOCOM-PIPE pipeline makes it possible to analyze 16S, 18S and 23S rRNA genes in the same packaged tool. The new post-clustering approach defines a biological database from previously analyzed samples and performs post-clustering of reads with this reference database by using open-reference clustering. This makes it easier to compare projects from various sequencing runs, and increased the congruence among results. For all users, the pipeline was developed to allow for adding or modifying the components, the databases and the bioinformatics tools easily, giving high modularity for each analysis.

Distribution Patterns of Microbial Community Structure Along a 7000-Mile Latitudinal Transect from the Mediterranean Sea Across the Atlantic Ocean to the Brazilian Coastal Sea.

A central goal in marine microecology is to understand the ecological factors shaping spatiotemporal microbial patterns and the underlying processes. We hypothesized that abiotic and/or biotic interactions are probably more important for explaining the distribution patterns of marine bacterioplankton than environmental filtering. In this study, surface seawater samples were collected about 7000 miles from the Mediterranean Sea, transecting the North Atlantic Ocean, to the Brazilian marginal sea. In bacterial biosphere, SAR11, SAR86, Rhodobacteraceae, and Rhodospiriaceae were predominant in the Mediterranean Sea; Prochlorococcus was more frequent in Atlantic Ocean; whereas in the Brazilian coastal sea, the main bacterial members were Synechococcus and SAR11. With respect to archaea, Euryarchaeota were predominant in the Atlantic Ocean and Thaumarchaeota in the Mediterranean Sea. With respect to the eukaryotes, Syndiniales, Spumellaria, Cryomonadida, and Chlorodendrales were predominant in the open ocean, while diatoms and microzooplankton were dominant in the coastal sea. Distinct clusters of prokaryotes and eukaryotes displayed clear spatial heterogeneity. Among the environmental parameters measured, temperature and salinity were key factors controlling bacterial and archaeal community structure, respectively, whereas N/P/Si contributed to eukaryotic variation. The relative contribution of environmental parameters to the microbial distribution pattern was 45.2%. Interaction analysis showed that Gammaproteobacteria, Alphaproteobacteria, and Flavobacteriia were the keystone taxa within the positive-correlation network, while Thermoplasmata was the main contributor in the negative-correlation network. Our study demonstrated that microbial communities are co-governed by environmental filtering and biotic interactions, which are the main deterministic driving factors modulating the spatiotemporal patterns of marine plankton synergistically at the regional or global levels.

The heterogeneity of composition and assembly processes of the microbial community between different nutrient loading lake zones in Taihu Lake.

To investigate the differences in the microbial community composition and assembly process in two lake zones (Meiliang Bay (MLB) and Xukou Bay (XKB) in Taihu Lake, China) with different nutrient loadings, water samples were collected. Both the 16S ribosomal RNA (rRNA) gene for the bacterial community and the 18S rRNA gene for the microeukaryote community were investigated using the Illumina second-generation sequencing platform (2 × 250 paired-end). The results indicated that both the bacterioplankton and microeukaryote community composition derived from the two lake zones were significantly different. Significantly higher operational taxonomic unit (OTU) richness (P < 0.01) and phylogenetic diversity (P < 0.05) were found for the bacterioplankton community of MLB. However, a comparable alpha diversity was found between the microeukaryote communities of MLB and XKB (P > 0.05). Environmental factors significantly affected the community compositions in XKB for both the bacterioplankton and microeukaryotes. However, they did not significantly influence the microbial community composition in MLB, except for a weak correlation between dissolved organic carbon (DOC) and the microeukaryote community. The microbial communities tended to be more phylogenetically clustered than expected by chance in the two lake zones. Moreover, the results of the phylogenetic structure suggest that deterministic processes played overwhelming roles in driving the assembly of both the bacterioplankton and microeukaryote community in XKB.

Testate Amoebae Like It Hot: Species Richness Decreases Along a Subalpine-Alpine Altitudinal Gradient in Both Natural Calluna vulgaris Litter and Transplanted Minuartia sedoides Cushions.

Most groups of higher organisms show a decrease in species richness toward high altitude, but the existence of such a pattern is debated for micro-eukaryotes. Existing data are scarce and mostly confounded with the diversity of habitats that also decreases with elevation. In order to disentangle these two factors, one approach is to consider only similar types of habitats occurring across an elevational gradient. We assessed the diversity and community structure of testate amoebae in two specific habitats: (1) natural Calluna vulgaris litter and (2) Minuartia sedoides cushions 7 years after their transplantation along a vertical transect from 1770 to 2430 m in the subalpine and alpine zones of the Swiss Alps. Analyses of co-variance and variance showed that testate amoeba species richness, equitability, and diversity declined with elevation and were significantly correlated to habitat type. In a redundancy analysis, the variation in the relative abundance of the testate amoeba taxa in Calluna vulgaris litter was equally explained by elevation and litter pH. This is the first study documenting a monotonic decrease of protist diversity in similar habitats across an elevational gradient.

Nanoscale sulfur alters the bacterial and eukaryotic communities of the tomato rhizosphere and their interactions with a fungal pathogen.

Nanoformulations of sulfur have demonstrated the potential to enhance plant growth and reduce disease incidence when plants are confronted with pathogens. However, the impact of nanoscale sulfur on microbial communities in close contact with the plant root, known as the rhizosphere, remain poorly characterized. In this study, we investigate the impact of three formulations of sulfur; bulk sulfur, uncoated (pristine) sulfur nanoparticles, and stearic acid coated sulfur nanoparticles, on the rhizosphere of tomato plants. Tomato plants were additionally challenged by the pathogenic fungus Fusarium oxysporum f. sp. Lycopersici. Employing bacterial 16S rRNA gene sequencing, along with recently in-house designed peptide nucleic acid clamps to facilitate the recovery of microeukaryote sequences, we performed a comprehensive survey of rhizosphere microbial populations. We found the largest influence on the composition of the rhizosphere microbiome was the presence of the fungal pathogen. However, sulfur amendments also drove state changes in the rhizosphere populations; for example, enriching the relative abundance of the plant-beneficial sulfur-oxidizing bacterium Thiobacillus. Notably, when investigating the response of the rhizosphere community to the different sulfur amendments, there was a strong interaction between the fungal pathogen and sulfur treatments. This resulted in different bacterial and eukaryotic taxa being enriched in association with the different forms of sulfur, which was dependent on the presence of the pathogen. These data point to nano formulations of sulfur exerting unique shifts in the rhizosphere community compared to bulk sulfur, particularly in association with a plant pathogen, and have implications for the sustainable use of nanoscale strategies in sustainable agriculture.

Soil microeukaryotic communities and phosphorus-cycling microorganisms respond to chloropicrin fumigation and azoxystrobin application.

Fumigants and fungicides are effective at controlling soil-borne pathogens but might also adversely affect soil beneficial microbes, such as soil phosphorus (P) solubilizing microbes, further altering nutrient cycling processes. Therefore, this study investigated the effects of the fumigant chloropicrin (CP) and the fungicide azoxystrobin (AZO) on soil microeukaryotes and P-cycling related soil bacteria through a greenhouse experiment. Soil microeukaryotic communities and bacterial communities containing two phosphomonoesterase encoding genes (phoC and phoD) were analysed using high-throughput sequencing methods. Results showed that, when applied at the field recommended application dosage, the fungicide AZO had no significant influence on the community structure of soil microeukaryotes and phoD-containing bacteria. However, in CP-fumigated soils, the soil microeukaryotic community composition changed from fungi-dominated to protist-dominated. CP fumigation significantly decreased the total phoC/phoD gene copy number but increased the relative abundance of some phoC/phoD-containing bacteria (such as Sinorhizobium and Streptomyces), which are significantly positively correlated to available P compositions in soil. The structural equation model (SEM) confirmed that CP fumigation could affect soil available P content directly by altering phoC-/phoD-containing bacteria, or indirectly by affecting phoC/phoD gene abundance and acid/alkaline phosphatases activity in soil. The inconsistent changes in phoC/phoD-containing bacteria, phoC/phoD gene number, and the phosphomonoesterase activities indicated that enzyme secretion may not be the only way for P solubilizing soil microorganisms to regulate P availability after soil fumigation. The outcome of this study can provide theoretical support for the design of soil beneficial microorganism recovery strategies and the regulation of phosphate fertilizer after soil fumigation.

Microeukaryotes Associated with Freshwater Mussels in Rivers of the Southeastern United States.

Microeukaryotes are a diverse and often overlooked group of microbes that are important in food webs and other ecological linkages. Little is known about microeukaryotes associated with aquatic invertebrates, although filter feeders such as mussels are likely to take in and potentially retain microeukaryotes in their gut while feeding. Microeukaryotes such as apicomplexans have been reported in marine mussel species, but no studies have examined the presence of these microorganisms in freshwater mussels or how they relate to mussel host species or environmental conditions. In this study, microbial community DNA was extracted from the gut tissue of over 300 freshwater mussels, representing 22 species collected from rivers in the southeastern USA. Microeukaryote DNA was detected using PCR amplification, followed by the sequencing of positive amplicons. Microeukaryotes were found in 167 individual mussels (53%) of those tested. Amplicons included dinoflagellates/algae that differed between mussel species and are likely food sources that were distinct from those found in water and sediment samples analyzed concurrently. A total of 5% of the positive amplicons were non-photosynthetic alveolates that could represent parasitic microeukaryotes. Understanding the distribution of microeukaryotes in the freshwater mussel gut microbiome could further our understanding of the ongoing decline of mussel populations.

Ciliate diversity and growth rates in experimental recirculating aquaculture and aquaponics systems using microscopy.

The function of recirculating aquaculture systems (RAS) relies on microbial communities, which convert toxic, fish-excreted ammonia into substances that can provide nutrients to plants as in the case of aquaponics systems. In the present study, heterotrophic protist communities of experimental sea water RAS and freshwater aquaponics systems were investigated using microscopy to characterize their diversity, natural abundance, and potential growth rates. Heterotrophic protist abundance was low (732 ± 21 to 5451 ± 118 ciliates L-1 and 58 ± 8 to 147 ± 18 nanoflagellates mL-1 in the aquaponics system and 78 ± 28 to 203 ± 48 ciliates L-1 in the RAS), which is in line with values typically reported for rivers. In the aquaponics system, ciliates grew faster in the fish rearing tanks (1.9 ± 0.01 to 1.21 ± 0.03 d-1 compared to 0.54 ± 0.03 to 0.79 ± 0.05 d-1 in the other compartments), while heterotrophic nanoflagellates grew slower in drain tanks downstream of the hydroponics compartment (0.5 ± 0.3 to 1.37 ± 0.05 d-1 and 4.09 ± 0.11 d-1 to 6.03 ± 0.34 d-1in the other compartments). Results indicated distinct niches and reduced microeukaryotic diversity at the end of the system's operation cycle.

Different Microeukaryotic Trophic Groups Show Different Latitudinal Spatial Scale Dependences in Assembly Processes across the Continental Shelves of China.

The relative role of stochasticity versus determinism is critically dependent on the spatial scale over which communities are studied. However, only a few studies have attempted to reveal how spatial scales influence the balance of different assembly processes. In this study, we investigated the latitudinal spatial scale dependences in assembly processes of microeukaryotic communities in surface water and sediment along the continental shelves of China. It was hypothesized that different microeukaryotic trophic groups (i.e., autotroph, heterotroph, mixotroph, and parasite) showed different latitudinal scale dependences in their assembly processes. Our results disclosed that the relative importance of different assembly processes depended on a latitudinal space scale for planktonic microeukaryotes. In surface water, as latitudinal difference increased, the relative contributions of homogenous selection and homogenizing dispersal decreased for the entire community, while those of heterogeneous selection and drift increased. The planktonic autotrophic and heterotrophic groups shifted from stochasticity-dominated processes to heterogeneous selection as latitudinal differences surpassed thresholds of 8° and 16°, respectively. For mixotrophic and parasitic groups, however, the assembly processes were always dominated by drift across different spatial scales. The balance of different assembly processes for the autotrophic group was mainly driven by temperature, whereas that of the heterotrophic group was driven by salinity and geographical distance. In sediment, neither the entire microeukaryotic community nor the four trophic groups showed remarkable spatial scale dependences in assembly processes; they were always overwhelmingly dominated by the drift. This work provides a deeper understanding of the distribution mechanisms of microeukaryotes along the continental shelves of China from the perspective of trophic groups.

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes.

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operon clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing strategies (PacBio circular consensus sequencing and two Nanopore approaches using unique molecular identifiers) and three tools for resolving amplicons sequence variants (ASVs) (USEARCH, VSEARCH, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essentially error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples, which enables us to identify previously undescribed microeukaryotic lineages.