Metabarcoding Approach Research Articles

Eukaryotic diversity of marine biofouling from coastal to offshore areas

Marine biofouling communities, including biofilms, are composed of many eukaryotes with high taxonomic and functional diversities. However, molecular characterization of eukaryotic diversity of marine biofouling has been barely developed due to the only recent interest in research areas such as marine renewable energies, antifouling technologies, or plastic pollution. The aim of this study was to compare the diversity and taxonomic composition of biofouling through different metabarcoding approaches used to detect the widest range of taxa from samples collected in several contrasted marine environments (French Atlantic and Mediterranean coasts). Thus, we assessed four DNA extraction methods and six primers pairs targeting the 18S rDNA gene (including the V1-V2, V4TAR, V4UNI, V7 and V9 regions) and the COI gene, the latter with two databases (BOLD and MIDORI). In addition the influence of primers selection was analyzed at three sites to compare geographic variations in eukaryotic diversity. Although none of the extraction methods greatly altered the community diversity or composition. we have observed that eukaryotic biofouling community diversity and structure varied depending on primers pairs, reference databases and sites. 18S rDNA regions allowed the detection of more taxa at the species level, including microeukaryotes, while the COI recovered more ASVs, but with a large proportion that remained taxonomically unassigned probably because BOLD and MIDORI specifically targeted metazoans. Interestingly, the spatial pattern obtained with both COI and 18S rDNA markers were similar showing that spatial selection occurred throughout a wide diversity of eukaryotic taxa. These results encouraged the use of these two complementary markers for future metabarcoding investigations but also highlighted the relevance of completing databases to enhance the identification of biofouling eukaryotes.

New Knowledge on Distribution and Abundance of Toxic Microalgal Species and Related Toxins in the Northwestern Black Sea.

Numerous potentially toxic plankton species commonly occur in the Black Sea, and phycotoxins have been reported. However, the taxonomy, phycotoxin profiles, and distribution of harmful microalgae in the basin are still understudied. An integrated microscopic (light microscopy) and molecular (18S rRNA gene metabarcoding and qPCR) approach complemented with toxin analysis was applied at 41 stations in the northwestern part of the Black Sea for better taxonomic coverage and toxin profiling in natural populations. The combined dataset included 20 potentially toxic species, some of which (Dinophysis acuminata, Dinophysis acuta, Gonyaulax spinifera, and Karlodinium veneficum) were detected in over 95% of the stations. In parallel, pectenotoxins (PTX-2 as a major toxin) were registered in all samples, and yessotoxins were present at most of the sampling points. PTX-1 and PTX-13, as well as some YTX variants, were recorded for the first time in the basin. A positive correlation was found between the cell abundance of Dinophysis acuta and pectenotoxins, and between Lingulodinium polyedra and Protoceratium reticulatum and yessotoxins. Toxic microalgae and toxin variant abundance and spatial distribution was associated with environmental parameters. Despite the low levels of the identified phycotoxins and their low oral toxicity, chronic toxic exposure could represent an ecosystem and human health hazard.

Co-occurrences enhance our understanding of aquatic fungal metacommunity assembly and reveal potential host-parasite interactions.

Our knowledge of aquatic fungal communities, their assembly, distributions and ecological roles in marine ecosystems is scarce. Hence, we aimed to investigate fungal metacommunities of coastal habitats in a subarctic zone (northern Baltic Sea, Sweden). Using a novel joint species distribution model and network approach, we quantified the importance of biotic associations contributing to the assembly of mycoplankton, further, detected potential biotic interactions between fungi-algae pairs, respectively. Our long-read metabarcoding approach identified 493 fungal taxa, of which a dominant fraction (44.4%) was assigned as early-diverging fungi (i.e. Cryptomycota and Chytridiomycota). Alpha diversity of mycoplankton declined and community compositions changed along inlet-bay-offshore transects. The distributions of most fungi were rather influenced by environmental factors than by spatial drivers, and the influence of biotic associations was pronounced when environmental filtering was weak. We found great number of co-occurrences (120) among the dominant fungal groups, and the 25 associations between fungal and algal OTUs suggested potential host-parasite and/or saprotroph links, supporting a Cryptomycota-based mycoloop pathway. We emphasize that the contribution of biotic associations to mycoplankton assembly are important to consider in future studies as it helps to improve predictions of species distributions in aquatic ecosystems.

Finding a Needle in a Haystack - In Silico Search for Environmental Traces of Candida auris.

Candida auris, first described from an ear infection in Japan, is an important emerging multidrug-resistant pathogenic fungal species. Its environmental niche remained a mystery until its isolation from the wetlands of the Andaman Islands, India, in 2020. We screened a subset of the world's largest sequence repository, the Sequence Read Archive at National Center for Biotechnology Information, using a DNA metabarcoding approach based on either the internal transcribed spacer (ITS)1 or ITS2 region of the official primary fungal DNA barcode, to identify potential environmental sources of C. auris. Our search identified 34 matches with partial C. auris ITS sequences from seven metabarcoding studies, providing wider evidence for the presence of C. auris outside human-maintained facilities.

Individual Dietary Specialization in a Generalist Bee Varies across Populations but Has No Effect on the Richness of Associated Microbial Communities.

AbstractDespite the increasingly documented occurrence of individual specialization, the relationship between individual consumer interactions and diet-related microbial communities in wild populations is still unclear. Using data from nests of Ceratina australensis from three different wild bee populations, we combine metabarcoding and network approaches to explore the existence of individual variation in resource use within and across populations and whether dietary specialization affects the richness of pollen-associated microbes. We reveal the existence of marked dietary specialization. In the most specialized population, we also show that individuals' diet breadth was positively related to the richness of fungi but not bacteria. Overall, individual specialization appeared to have a weak or negligible effect on the microbial richness of nests, suggesting that different mechanisms beyond environmental transmission may be at play regarding microbial acquisition in wild bees.

Factors affecting soil invertebrate biodiversity in agroecosystems of the Po Plain area (Italy)

Soil is a fundamental component of the biosphere, whose properties and quality are affected by human activities, such as agriculture. Soil health is fundamental for different ecosystem services and soil biota has a crucial role in maintaining it. Elucidating how different crops and agricultural practices affect soil invertebrates communities is of relevance. In the present study, a DNA metabarcoding approach was adopted to evaluate the effects of different biotic and abiotic factors, including agricultural practices, on the composition and diversity of soil invertebrate communities of different agro-ecosystems (Po Plain-Italy). At this aim, the DNA markers and the more effective primers in retrieving soil metazoan communities were established. Bulk soil samples from different agro-ecosystems (i.e., cornfield, alfalfa, paddy fields, vineyard, stable meadow, woodland) were collected and, processed for obtaining 18S rRNA and coi sequences (raw reads analyzed using QIIME2 and R). Soil physical and chemical parameters were measured for each soil sample (e.g., pH, carbon-nitrogen ratio, texture, porosity) and metadata on farms management were retrieved. The most efficient primer pairs in recovering soil metazoans were M620F/M1260R for 18S rRNA, and mlCOIintF/jgHCO2198R for coi gene. Soil communities resulted dominated by Nematoda, Arthropoda, Annelida, Rotifera and Tardigrada. The most diverse invertebrate communities have been found in the soil of stable meadows and woodlands, while cornfields showed the lowest level of diversity. The diversity of soil invertebrate communities (Hill numbers) was positively correlated with the level of porosity and carbon-nitrogen ratio, while it was negatively correlated with the phosphate abundance. This pattern probably reflects the negative effect of excessive fertilization with phosphates on soil fauna, while the abundance of organic matter and microhabitats were found to enhance the presence of more complex communities. Other soil properties were correlated only with specific taxa (e.g., pH was negatively correlated with the diversity of Annelida and Rotifera).

Metabarcoding data analysis revealed the plant dietary variation of long-tailed macaque Macacafascicularis (Cercopithecidae, Cercopithecinae) living in disturbed habitats in Peninsular Malaysia.

The long-tailed macaque (Macacafascicularis) has a wide range in both Peninsular Malaysia and Borneo. Although the primates are especially vulnerable to habitat alterations, this primate lives in disturbed habitats due to human-induced land-use. Thus, this study presents a faecal metabarcoding approach to clarify the plant diet of long-tailed macaques from five locations in Peninsular Malaysia to represent fragmented forest, forest edge, island and recreational park habitats. We extracted genomic DNA from 53 long-tailed macaque faecal samples. We found 47 orders, 126 families, 609 genera and 818 species across these five localities. A total of 113 plant families were consumed by long-tailed macaques in Universiti Kebangsaan Malaysia, 61 in the Malaysia Genome and Vaccine Institute, 33 in Langkawi Island, 53 in Redang Island and 44 in the Cenderawasih Cave. Moraceae (33.24%) and Fabaceae (13.63%) were the most common families consumed by long-tailed macaques from the study localities. We found that habitat type impacted diet composition, indicating the flexibility of foraging activities. This research findings provide an understanding of plant dietary diversity and the adaptability of this macaque with the current alteration level that applies to long-tailed macaque conservation management interest in the future.

DNA metabarcoding of gut contents reveals key habitat and seasonal drivers of trophic networks involving generalist predators in agricultural landscapes.

Understanding the networks of trophic interactions into which generalist predators are embedded is key to assessing their ecological role of in trophic networks and the biological control services they provide. The advent of affordable DNA metabarcoding approaches greatly facilitates quantitative understanding of trophic networks and their response to environmental drivers. Here, we examine how key environmental gradients interact to shape predation by Lycosidae in highly dynamic vegetable growing systems in China. For the sampled Lycosidae, crop identity, pesticide use and seasons shape the abundance of prey detected in spider guts. For the taxonomic richness of prey, local- and landscape-scale factors gradients were more influential. Multivariate ordinations confirm that these crop-abundant spiders dynamically adjust their diet to reflect environmental constraints and seasonal availability to prey. Plasticity in diet composition is likely to account for the persistence of spiders in relatively ephemeral brassica crops. Our findings provide further insights into the optimization of habitat management for predator-based biological control practices. © 2022 Society of Chemical Industry.

Diversity and distribution of Kinorhyncha in abyssal polymetallic nodule areas of the Clarion-Clipperton Fracture Zone and the Peru Basin, East Pacific Ocean, with the description of three new species and notes on their intraspecific variation

Polymetallic nodule fields represent a large reservoir of undiscovered biodiversity that becomes particularly evident for meiobenthic organisms, the smallest-sized faunal group. Knowledge gaps are especially noticeable for the generally low-density metazoan groups, such as Kinorhyncha, the so-called mud dragons. Using both morphological and genetic (metabarcoding) approaches, we provide a general overview and comparison of the diversity of kinorhynchs collected during nine sampling campaigns (2016–2019) that targeted abyssal environments in several contract areas for exploration in the Clarion-Clipperton Fracture Zone (CCZ) and in the Peru Basin. Our findings from morphological analyses reveal a highly diverse mud dragon community, with 16 species present in the CCZ. Of these, 12 appear in the German contract area, including three new species described in the present contribution: Echinoderes delaordeni sp. nov., Echinoderes sanctorum sp. nov., and Echinoderes zeppilliae sp. nov. Furthermore, metabarcoding data of the kinorhynch community gathered from the area is provided, together with the geographic distribution of the known species stated per contractor area, including new records and still undescribed species. Most of the identified species in the CCZ seem to have a wide distribution, with Echinoderes sp.4 being the most common and abundant species with a distribution spreading across the CCZ and also present in the Peru Basin. Metabarcoding analyses targeting the V1V2 hypervariable region of the 18S gene from the 253 stations of the CCZ revealed 14 amplicon sequence variants (ASVs) belonging to Kinorhyncha with grade values higher than 98% detected at 15 different stations within six different areas along the CCZ. Concurring with morphology, the family Echinoderidae was the most diverse as the genus Cephalorhyncha had five ASVs, followed by Echinoderes with four ASVs. Semnoderes, however, showed the widest spread ASV, being detected at six stations. In the CCZ, the metabarcoding data showed there were no shared ASVs between the CCZ areas as well as the highest number of uniques, which was 11. Our morphological study showed a low number of specimens inhabiting nodules (surface/crevices), suggesting that specific kinorhynch species do not typically inhabit the nodules in addition to the surrounding sediment.

More than just hitchhikers: a survey of bacterial communities associated with diatoms originating from sea turtles.

Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

Global patterns in endemicity and vulnerability of soil fungi.

Fungi are highly diverse organisms, which provide multiple ecosystem services. However, compared with charismatic animals and plants, the distribution patterns and conservation needs of fungi have been little explored. Here, we examined endemicity patterns, global change vulnerability and conservation priority areas for functional groups of soil fungi based on six global surveys using a high-resolution, long-read metabarcoding approach. We found that the endemicity of all fungi and most functional groups peaks in tropical habitats, including Amazonia, Yucatan, West-Central Africa, Sri Lanka, and New Caledonia, with a negligible island effect compared with plants and animals. We also found that fungi are predominantly vulnerable to drought, heat and land-cover change, particularly in dry tropical regions with high human population density. Fungal conservation areas of highest priority include herbaceous wetlands, tropical forests, and woodlands. We stress that more attention should be focused on the conservation of fungi, especially root symbiotic arbuscular mycorrhizal and ectomycorrhizal fungi in tropical regions as well as unicellular early-diverging groups and macrofungi in general. Given the low overlap between the endemicity of fungi and macroorganisms, but high conservation needs in both groups, detailed analyses on distribution and conservation requirements are warranted for other microorganisms and soil organisms.

Association between parasite microbiomes and caste development and colony structure in a social trematode.

Division of labour through the formation of morphologically and functionally distinct castes is a recurring theme in the evolution of animal sociality. The mechanisms driving the differentiation of individuals into distinct castes remain poorly understood, especially for animals forming clonal colonies. We test the association between microbiomes and caste formation within the social trematode Philophthalmus attenuatus, using a metabarcoding approach targeting the bacterial 16S SSU rRNA gene. Clonal colonies of this trematode within snail hosts comprise large reproductive individuals which produce dispersal stages, and small, non-reproducing soldiers which defend the colony against invaders. In colonies extracted directly from field-collected snails, reproductives harboured more diverse bacterial communities than soldiers, and reproductives and soldiers harboured distinct bacterial communities, at all taxonomic levels considered. No single bacterial taxon showed high enough prevalence in either soldiers or reproductives to be singled out as a key driver, indicating that the whole microbial community contributes to these differences. Other colonies were experimentally exposed to antibiotics to alter their bacterial communities, and sampled shortly after treatment and weeks later after allowing for turnover of colony members. At those time points, bacterial communities of the two castes still differed across all antibiotic treatments; however, the caste ratio within colonies changed: after antibiotic disruption and turnover of individuals, new individuals were more likely to become reproductives than in undisturbed control colonies. Our results reveal that each caste has a distinct microbiome; whether the social context affects the microbiota, or whether microbes contribute to modulating the phenotype of individuals, remains to be determined.

Detection of human pathogenic bacteria in rectal DNA samples from Zalophus californianus in the Gulf of California, Mexico

Human intrusions into undisturbed wildlife areas greatly contribute to the emergence of infectious diseases. To minimize the impacts of novel emerging infectious diseases (EIDs) on human health, a comprehensive understanding of the microbial species that reside within wildlife species is required. The Gulf of California (GoC) is an example of an undisturbed ecosystem. However, in recent decades, anthropogenic activities within the GoC have increased. Zalophus californianus has been proposed as the main sentinel species in the GoC; hence, an assessment of sea lion bacterial microbiota may reveal hidden risks for human health. We evaluated the presence of potential human pathogenic bacterial species from the gastrointestinal (GI) tracts of wild sea lions through a metabarcoding approach. To comprehensively evaluate this bacterial consortium, we considered the genetic information of six hypervariable regions of 16S rRNA. Potential human pathogenic bacteria were identified down to the species level by integrating the RDP and Pplacer classifier outputs. The combined genetic information from all analyzed regions suggests the presence of at least 44 human pathogenic bacterial species, including Shigella dysenteriae and Bacillus anthracis. Therefore, the risks of EIDs from this area should be not underestimated.

Simultaneous monitoring of SARS-CoV-2 and bacterial profiles from the air of hospital environments with COVID-19-affected patients

The SARS-CoV-2 presence and the bacterial community profile in air samples collected at the Intensive Care Unit (ICU) of the Operational Unit of Infectious Diseases of Santa Caterina Novella Hospital in Galatina (Lecce, Italy) have been evaluated in this study. Air samplings were performed in different rooms of the ICU ward with and without COVID-19 patients. No sample was found positive to SARS-CoV-2, according to Allplex 2019-nCoV Assay. The airborne bacterial community profiles determined by the 16S rRNA gene metabarcoding approach up to the species level were characterized by richness and biodiversity indices, Spearman correlation coefficients, and Principal Coordinate Analysis. Pathogenic and non-pathogenic bacterial species, also detected in outdoor air samples, were found in all collected indoor samples. Staphylococcus pettenkoferi, Corynebacterium tuberculostearicum, and others coagulase-negative staphylococci, detected at high relative abundances in all the patients’ rooms, were the most abundant pathogenic species. The highest mean relative abundance of S. pettenkoferi and C. tuberculostearicum suggested that they were likely the main pathogens of COVID-19 patients at the ICU ward of this study. The identification of nosocomial pathogens representing potential patients’ risks in ICU COVID-19 rooms and the still controversial airborne transmission of the SARS-CoV-2 are the main contributions of this study.

Diversity of <i>Heterocapsa</i> (Dinophyceae) and the algal bloom event in the mariculture areas of Johor Strait, Malaysia

In November 2020, a high biomass multi-species algal bloom caused heavy water discoloration in the fish and mussel farm areas of the Johor Strait, Malaysia. A total of 19 microalgal taxa were identified from the plankton samples collected during the bloom event. Eleven genera were diatoms, and eight genera were dinophytes. The microalgal composition was dominated by the diatom Guinardia sp., with an average cell density of 1.7×106 cells L−1, making up 65–80% of the phytoplankton composition. Concomitantly, high densities of the dinophytes Heterocapsa minima (3.8–5.3×105 cells L−1) and Karlodinium spp. (3.5–6.6×103 cells L−1) were found. This is the first record of the occurrence of H. minima in Malaysian waters. Detailed morphological observations of H. minima based on scanning electron microscopy are presented in this study. To have a better insight into the Heterocapsa species assemblages in the Johor Strait, the diversity of Heterocapsa species assemblages along the strait was investigated based on a metabarcoding approach. Environmental DNA collected between 2018 and 2019 was used for high throughput amplicon sequencing targeting the small subunit (SSU) ribosomal RNA gene marker. The metabarcoding analysis detected three rare Heterocapsa species in the waters, H. niei, H. rotundata, and H. steinii. The results showed that Heterocapsa species assemblages varied temporally across the strait, with higher species diversity and amplicon sequence variant (ASV) read abundances detected in the Eastern Johor Strait. Although no fish/shellfish kills were sighted during the 2020 bloom event, the presence of harmful microalgal species, such as Heterocapsa minima and Karlodinium spp., urged the need for a comprehensive HAB monitoring program in the Strait to safeguard the aquaculture industry in the areas.

Spatial and temporal variation in New Hampshire bat diets

Insectivorous bats consume a diverse array of arthropod prey, with diets varying by bat species, sampling location, and season. North American bat diets remain incompletely described, which is concerning at a time when many bat and insect populations appear to be declining. Understanding the variability in foraging is thus an essential component for effective bat conservation. To comprehensively evaluate local foraging, we assessed the spatial and temporal variability in prey consumed by the little brown bat, Myotis lucifugus, in New Hampshire, USA. We collected bat guano samples from 20 sites over 2 years and analyzed sequence data for 899 of these samples using a molecular metabarcoding approach targeting the cytochrome oxidase I subunit (COI) gene. Some prey items were broadly shared across locations and sampling dates, with the most frequently detected arthropod orders broadly similar to previous morphological and molecular analyses; at least one representative sequence variant was assigned to Coleoptera in 92% of samples, with other frequently detected orders including Diptera (73%), Lepidoptera (65%), Trichoptera (38%), and Ephemeroptera (32%). More specifically, two turf and forest pests were routinely detected: white grubs in the genus Phyllophaga (50%), and the Asiatic Garden beetle, Maladera castanea (36%). Despite the prevalence of a few taxa shared among many samples and distinct seasonal peaks in consumption of specific arthropods, diet composition varied both temporally and spatially. However, species richness did not strongly vary indicating consumption of a broad diversity of taxa throughout the summer. These data characterize little brown bats as flexible foragers adept at consuming a broad array of locally available prey resources.

A DNA Metabarcoding Workflow to Identify Species in Spices and Herbs.

Spices and herbs are food categories regularly cited as highly susceptible to be adulterated. To detect potential adulteration with undeclared species, DNA-based methods are considered the most suitable tools. In this study, the performance of the ready-to-use Thermo Scientific™ NGS Food Authenticity Workflow (Thermo Fisher Scientific)-a commercial DNA metabarcoding approach-is described. The tool was further applied to analyze 272 commercial samples of spices and herbs. Pure samples of spices and herbs were analyzed with the Thermo Scientific NGS Food Authenticity Workflow to assess its specificity, and spikings down to 1% (w/w) allowed evaluation of its sensitivity. Commercial samples, 62 and 210, were collected in Asian and European markets, respectively. All tested species were correctly identified often down to the species level, while spikings at 1% (w/w) confirmed a limit of detection at this level, including in complex mixtures composed of five different spices and/or herbs. The analysis of 272 commercial samples showed that 78% were compliant with the declared content, whereas the rest were shown to contain undeclared species that were in a few cases allergenic or potentially toxic. The Thermo Scientific NGS Food Authenticity Workflow was found to be suitable to identify food plant species in herbs and spices, not only when tested on pure samples, but also in mixtures down to 1% (w/w). The overall workflow is user-friendly and straightforward, which makes it simple to use and facilitates data interpretation. The Thermo Scientific NGS Food Authenticity Workflow was found to be suitable for species identification in herbs and spices, and it allowed the detection of undeclared species in commercial samples. Its ease of use facilitates its implementation in testing laboratories.

Sequencing introduced false positive rare taxa lead to biased microbial community diversity, assembly, and interaction interpretation in amplicon studies

BackgroundIncreasing studies have demonstrated potential disproportionate functional and ecological contributions of rare taxa in a microbial community. However, the study of the microbial rare biosphere is hampered by their inherent scarcity and the deficiency of currently available techniques. Sample-wise cross contaminations might be introduced by sample index misassignment in the most widely used metabarcoding amplicon sequencing approach. Although downstream bioinformatic quality control and clustering or denoising algorithms could remove sequencing errors and non-biological artifact reads, no algorithm could eliminate high quality reads from sample-wise cross contaminations introduced by index misassignment, making it difficult to distinguish between bona fide rare taxa and potential false positives in metabarcoding studies.ResultsWe thoroughly evaluated the rate of index misassignment of the widely used NovaSeq 6000 and DNBSEQ-G400 sequencing platforms using both commercial and customized mock communities, and observed significant lower (0.08% vs. 5.68%) fraction of potential false positive reads for DNBSEQ-G400 as compared to NovaSeq 6000. Significant batch effects could be caused by stochastically introduced false positive or false negative rare taxa. These false detections could also lead to inflated alpha diversity of relatively simple microbial communities and underestimated that of complex ones. Further test using a set of cow rumen samples reported differential rare taxa by different sequencing platforms. Correlation analysis of the rare taxa detected by each sequencing platform demonstrated that the rare taxa identified by DNBSEQ-G400 platform had a much higher possibility to be correlated with the physiochemical properties of rumen fluid as compared to NovaSeq 6000 platform. Community assembly mechanism and microbial network correlation analysis indicated that false positive or negative rare taxa detection could lead to biased community assembly mechanism and identification of fake keystone species of the community.ConclusionsWe highly suggest proper positive/negative/blank controls, technical replicate settings, and proper sequencing platform selection in future amplicon studies, especially when the microbial rare biosphere would be focused.

Deciphering Eurasian otter (Lutra lutra L.) and seal (Phoca vitulina L.; Halichoerus grypus F.) diet: Metabarcoding tailored for fresh and saltwater fish species.

Long-lived top predators shape biodiversity structure in their ecosystems and predator-prey interactions are critical in decoding how communities function. Studies on the foraging ecology of seals and Eurasian otters in Western Europe are outdated and most studies solely performed traditional hard part analysis. Molecular metabarcoding can be used as an innovative noninvasive diet analysis tool, which has proven efficient and complementary to hard part analysis, however, lacking application in the wider North Sea area. In this study, DNA from digesta, collected between 2014-2020, were used to identify fish species in the diet of 47 Eurasian otters, 54 harbour seals and 21 grey seals by applying a next-generation metabarcoding approach. A newly designed 16S rRNA primer, providing the best coverage of >130 local marine and freshwater fish species, was used to amplify prey DNA from seal scats and otter gut content sampled from the North Sea and regional freshwater bodies. Frequent fish species included tench, ninespine stickleback and white bream in otters; hooknose and common roach in grey seals and Pleuronectidae and sand gobies in harbour seals. Bipartite network analysis showed a strong overlap of harbour and grey seal diets. Otter diet intersected with both seal species in terms of freshwater species. This study provides new knowledge about dietary composition and community assemblage of fish prey in otters and seals in the North Sea and regional freshwaters, and a new molecular tool to elucidate predator-prey interactions and interspecies competition in complex and changing ecosystems under pressure from anthropogenic activities.

Spatio-temporal variability of eDNA signal and its implication for fish monitoring in lakes.

Environmental DNA (eDNA) metabarcoding is revolutionizing the monitoring of aquatic biodiversity. The use of eDNA has the potential to enable non-invasive, cost-effective, time-efficient and high-sensitivity monitoring of fish assemblages. Although the capacity of eDNA metabarcoding to describe fish assemblages is recognised, research efforts are still needed to better assess the spatial and temporal variability of the eDNA signal and to ultimately design an optimal sampling strategy for eDNA monitoring. In this context, we sampled three different lakes (a dam reservoir, a shallow eutrophic lake and a deep oligotrophic lake) every 6 weeks for 1 year. We performed four types of sampling for each lake (integrative sampling of sub-surface water along transects on the left shore, the right shore and above the deepest zone, and point sampling in deeper layers near the lake bottom) to explore the spatial variability of the eDNA signal at the lake scale over a period of 1 year. A metabarcoding approach was applied to analyse the 92 eDNA samples in order to obtain fish species inventories which were compared with traditional fish monitoring methods (standardized gillnet samplings). Several species known to be present in these lakes were only detected by eDNA, confirming the higher sensitivity of this technique in comparison with gillnetting. The eDNA signal varied spatially, with shoreline samples being richer in species than the other samples. Furthermore, deep-water samplings appeared to be non-relevant for regularly mixed lakes, where the eDNA signal was homogeneously distributed. These results also demonstrate a clear temporal variability of the eDNA signal that seems to be related to species phenology, with most of the species detected in spring during the spawning period on shores, but also a peak of detection in winter for salmonid and coregonid species during their reproduction period. These results contribute to our understanding of the spatio-temporal distribution of eDNA in lakes and allow us to provide methodological recommendations regarding where and when to sample eDNA for fish monitoring in lakes.