scholarly journals DNA metabarcoding successfully quantifies relative abundances of arthropod taxa in songbird diets: a validation study using camera-recorded diets

2020 ◽  
Author(s):  
Yvonne I. Verkuil ◽  
Marion Nicolaus ◽  
Richard Ubels ◽  
Maurine M. Dietz ◽  
Jelmer M. Samplonius ◽  
...  
Keyword(s):  
Validation Study ◽  
Large Scale ◽  
Taxonomic Resolution ◽  
List Type ◽  
Nest Boxes ◽  
Relative Contribution ◽  
Quantitative Accuracy ◽  
Dna Metabarcoding ◽  
Relative Abundances ◽  
Diet Analyses

AbstractEcological research is often hampered by the inability to quantify animal diets. Large-scale changes in arthropod diversity, abundance and phenologies urge the need to understand the consequences for trophic interactions. Diet composition of insectivorous predators can be tracked through DNA metabarcoding of faecal samples, but to validate the quantitative accuracy of metabarcoding, validation using free-living animals for which their diet can be approximated, is needed.This validation study assesses the use of DNA metabarcoding in quantifying diets of an insectivorous songbird. Using camera footage, we documented food items delivered to nestling Pied Flycatchers Ficedula hypoleuca, and subsequently sequenced the Cytochrome Oxidase subunit I (COI) in their faeces. Our special interest was to retrieve the relative contribution of arthropod taxa with a PCR-based protocol.Assessment of taxonomic coverage of the invertebrate COI primers LCO1490 and HCO1777, previously applied in insectivorous songbirds, demonstrated that COI barcodes were predominantly assigned to arthropod taxa, however, substantial amounts of reads (2–60%) were assigned to flycatchers. Modified primers reduced vertebrate reads to 0.03%, and yielded more spider DNA without significant changing the recovery of other arthropod taxa.To explore digestive biases, contents of stomachs and lower intestines were compared in eight adult male flycatchers, victims of competitors for nest boxes. Similarity in arthropod community composition between stomach and intestines confirmed limited digestive bias.With nest box cameras in 2013, 2015 and 2016, size-adjusted counts of prey items fed to nestlings were recorded, to approximate provided biomass of arthropod orders and families which allowed comparison with abundance of COI barcode reads in nestling faeces. The relative abundances of size-adjusted prey counts and COI reads correlated at R = 0.85 (CI:0.68-0.94) at order level and at R=0.75 (CI:0.67-0.82) at family level. Each common order and all common taxa within Lepidoptera, Diptera and Coleoptera were retrieved in similar proportions, while the taxonomic resolution was higher in barcodes than in camera records.This DNA metabarcoding validation demonstrates that quantitative arthropod diet analyses is possible in songbirds. We discuss the ecological application of our approach in approximating the arthropod diets in insectivorous birds.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Strengths and weaknesses of DNA-based monitoring: Assessing macroinvertebrates in 18 Finnish streams with metabarcoding and morphology

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for assessment of stream ecosystems with macroinvertebrates. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We tested metabarcoding using 18 macroinvertebrate samples from Finland using four primer sets. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Morphological identification was performed to the taxonomic level at which identification was reliable following standardized protocols. 3) We identified over twice the number of taxa, with greater species-level resolution, using DNA metabarcoding than morphology-based identification. For each sample, we detected more taxa by metabarcoding than by previous morphological methods, and all four primer sets showed similarly good performance. There was a significant linear correlation between sequence abundance and the number of taxa in each sample, but the scatter was up to two orders of magnitude. Ecological status assessment indices calculated from morphological and DNA metabarcoding datasets were mostly similar, with a few exceptions. With the recent drop in sequencing costs per sample, both methods identification are currently equally expensive. 4) We used actual samples for monitoring to demonstrate that DNA metabarcoding can achieve similar results and better taxonomic resolution than current morphological identification methods. Metabarcoding has thus already become a viable and reliable invertebrate identification method for stream assessment. However, to unlock the full potential of DNA metabarcoding for ecosystem assessment key problems in current laboratory protocols and reference databases, specified in this work, will require further attention.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals metabaR : an R package for the evaluation and improvement of DNA metabarcoding data quality

2020 ◽  
Author(s):  
Lucie Zinger ◽  
Clément Lionnet ◽  
Anne-Sophie Benoiston ◽  
Julian Donald ◽  
Céline Mercier ◽  
...  
Keyword(s):  
Data Quality ◽  
Large Scale ◽  
Environmental Gradients ◽  
R Package ◽  
Data Curation ◽  
Environmental Research ◽  
List Type ◽  
Sequencing Platform ◽  
Dna Metabarcoding ◽  
Ecological Patterns

AbstractDNA metabarcoding is becoming the tool of choice for biodiversity studies across taxa and large-scale environmental gradients. Yet, the artefacts present in metabarcoding datasets often preclude a proper interpretation of ecological patterns. Bioinformatic pipelines removing experimental noise have been designed to address this issue. However, these often only partially target produced artefacts, or are marker specific. In addition, assessments of data curation quality and the appropriateness of filtering thresholds are seldom available in existing pipelines, partly due to the lack of appropriate visualisation tools.Here, we present metabaR, an R package that provides a comprehensive suite of tools to effectively curate DNA metabarcoding data after basic bioinformatic analyses. In particular, metabaR uses experimental negative or positive controls to identify different types of artefactual sequences, i.e. reagent contaminants and tag-jumps. It also flags potentially dysfunctional PCRs based on PCR replicate similarities when those are available. Finally, metabaR provides tools to visualise DNA metabarcoding data characteristics in their experimental context as well as their distribution, and facilitate assessment of the appropriateness of data curation filtering thresholds.metabaR is applicable to any DNA metabarcoding experimental design but is most powerful when the design includes experimental controls and replicates. More generally, the simplicity and flexibility of the package makes it applicable any DNA marker, and data generated with any sequencing platform, and pre-analysed with any bioinformatic pipeline. Its outputs are easily usable for downstream analyses with any ecological R package.metabaR complements existing bioinformatics pipelines by providing scientists with a variety of functions with customisable methods that will allow the user to effectively clean DNA metabarcoding data and avoid serious misinterpretations. It thus offers a promising platform for automatised data quality assessments of DNA metabarcoding data for environmental research and biomonitoring.

scholarly journals Multi-marker DNA metabarcoding reflects tardigrade diversity in different habitats

Genome ◽  
2020 ◽  
Author(s):  
Lasse Topstad ◽  
Roberto Guidetti ◽  
Markus Majaneva ◽  
Torbjørn EKREM
Keyword(s):  
Large Scale ◽  
Environmental Dna ◽  
Distribution Patterns ◽  
Taxonomic Resolution ◽  
Dna Metabarcoding ◽  
The World ◽  
Microhabitat Distribution ◽  
Environmental Surveys ◽  
Reference Sequences ◽  
Alpine Environments

Like meiofauna in general, tardigrades are often neglected in ecological and environmental surveys. Tardigrades occur in all parts of the world, from deep marine sediments to alpine environments, and are present in most ecosystems. They are therefore potentially good candidates for biomonitoring programs. However, sampling of these minute animals is both tedious and time-consuming, impeding their inclusion in large-scale ecological surveys. In this study we argue that using a multi-marker metabarcoding approach on environmental DNA partly can overcome this barrier. Samples of moss, lichens and leaf litter were investigated both by morphology-based methods and DNA metabarcoding, and the results were compared in terms of tardigrade diversity and community composition of the sampled microhabitats. DNA metabarcoding using three markers detected more species of tardigrades than identification by morphology in most samples. Also, metabarcoding detected the same community differences and microhabitat distribution patterns as morphology-based methods. In general, metabarcoding of litter samples was unreliable, with only one out of three markers consistently amplifying and detecting tardigrades. The low availability of tardigrade reference sequences in public databases restricts the taxonomic resolution in eDNA surveys, but this impediment is partly circumvented by utilizing multiple markers

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardized protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (proxy for biomass) were significantly correlated in each sample, although adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately equal priced per sample to morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve similar assessment results as those of current protocols for morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Strategic resources and smallholder performance at the bottom of the pyramid

2019 ◽  
Vol 22 (3) ◽  
pp. 365-380 ◽  
Author(s):  
Matthias Olthaar ◽  
Wilfred Dolfsma ◽  
Clemens Lutz ◽  
Florian Noseleit
Keyword(s):  
Large Scale ◽  
Business Environment ◽  
Agricultural Economics ◽  
Local Market ◽  
Bottom Of The Pyramid ◽  
Fine Grained ◽  
Strategic Resources ◽  
Relative Contribution ◽  
The Relationship ◽  
Resource Based Theory

In a competitive business environment at the Bottom of the Pyramid smallholders supplying global value chains may be thought to be at the whims of downstream large-scale players and local market forces, leaving no room for strategic entrepreneurial behavior. In such a context we test the relationship between the use of strategic resources and firm performance. We adopt the Resource Based Theory and show that seemingly homogenous smallholders deploy resources differently and, consequently, some do outperform others. We argue that the ‘resource-based theory’ results in a more fine-grained understanding of smallholder performance than approaches generally applied in agricultural economics. We develop a mixed-method approach that allows one to pinpoint relevant, industry-specific resources, and allows for empirical identification of the relative contribution of each resource to competitive advantage. The results show that proper use of quality labor, storage facilities, time of selling, and availability of animals are key capabilities.

scholarly journals MEDITERATRI project - assessing the trophic ecology of predatory arthropods in Mediterranean agriculture via DNA metabarcoding diet analyses

2019 ◽  
Vol 2 ◽  
Author(s):  
Lucija Šerić Jelaska ◽  
Barbara Anđelić ◽  
Mišel Jelić ◽  
Tomislav Kos
Keyword(s):  
Pest Management ◽  
Trophic Ecology ◽  
Arable Land ◽  
Carabid Beetles ◽  
Diet Analysis ◽  
Gut Contents ◽  
Dna Metabarcoding ◽  
Potential Benefits ◽  
Diet Analyses ◽  
Predatory Arthropods

A type of management and the use of pesticides in arable land may negatively affect a range of soil biota and thus their food webs important for ecosystem functioning. By analysing trophic interactions we could reveal the extent of potential benefits that certain organisms can provide in biocontrol and maintaining healthy ecosystems. To evaluate the role of predatory arthropods within olive orchards and vineyards under Integrated Pest Management (IPM) and Ecological Pest Management (EPM) we collected carabid beetles together with other dominant predatory arthropods in the field (e.g. ladybugs, antlions, spiders, centipedes) and subdued the individuals to molecular gut content analyses using NGS. DNA metabarcoding diet analysis approach allowed detecting a wide variety of taxa from gut contents of the predators. In addition, using ICP-MS and LC-MS/MS we quantified Cu, pesticides and its residues in soil and animals representing different trophic guilds. Since concentrations of some toxic compounds detected in carabids body were negatively correlated with those in the soil, we aim to identify a potential vector for possible transfer of toxicants to general predators via predation. The results contribute to the risk assessment of proliferation of detected chemical compounds including copper in the ecosystem and to the knowledge on the overall field sustainability of predatory invertebrates to maximize their role in pest control. The study was conducted under the project activity of HRZZ – Mediteratri.

scholarly journals In Vivo CRISPR Screens Identify E3 Ligase Cop1 as a Modulator of Macrophage Infiltration and Cancer Immunotherapy Target

2020 ◽  
Author(s):  
Xiaoqing Wang ◽  
Collin Tokheim ◽  
Binbin Wang ◽  
Shengqing Stan Gu ◽  
Qin Tang ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Large Scale ◽  
Integrated Approach ◽  
Immune Checkpoint Blockade ◽  
Macrophage Infiltration ◽  
List Type

SUMMARYDespite remarkable clinical efficacies of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits in triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that inhibition of the E3 ubiquitin ligase Cop1 in cancer cells decreases the secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, and shows synergy in anti-tumor immunity with ICB. Transcriptomics, epigenomics, and proteomics analyses revealed Cop1 functions through proteasomal degradation of the C/ebpδ protein. Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. Cop1 inhibition stabilizes C/ebpδ to suppress the expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy by regulating chemokine secretion and macrophage levels in the TNBC tumor microenvironment.HighlightsLarge-scale in vivo CRISPR screens identify new immune targets regulating the tumor microenvironmentCop1 knockout in cancer cells enhances anti-tumor immunityCop1 modulates chemokine secretion and macrophage infiltration into tumorsCop1 targets C/ebpδ degradation via Trib2 and influences ICB response

scholarly journals The use of eDNA and DNA metabarcoding in monitoring the ecological condition of Norwegian lakes

2021 ◽  
Vol 4 ◽  
Author(s):  
Sara Atienza Casas ◽  
Markus Majaneva ◽  
Thomas Jensen ◽  
Marie Davey ◽  
Frode Fossøy ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Ecological Status ◽  
Ecological Condition ◽  
Molecular Techniques ◽  
Taxonomic Resolution ◽  
Potential Cost ◽  
Running Water ◽  
National Monitoring ◽  
Monitoring Programs ◽  
Dna Metabarcoding

Biodiversity assessments using molecular identification of organisms through high-throughput sequencing techniques have been a game changer in ecosystem monitoring, providing increased taxonomic resolution, more objective identifications, potential cost reductions, and reduced processing times. The use of DNA metabarcoding of bulk samples and environmental DNA (eDNA) is now widespread but is not yet universally implemented in national monitoring programs. While bulk sample metabarcoding involves extraction of DNA from organisms in a sample, eDNA analysis involves obtaining DNA directly from environmental samples, which can include microorganisms, meiofauna-size taxa and macrofauna traces such as larval stages, skin and hair cells, gametes, faeces and free DNA bound to particles. In Norway, freshwater biomonitoring in compliance with the EU Water Framework Directive (WFD) is conducted on several administrative levels, including national monitoring programs for running water, small and large lakes. These programs typically focus on a fraction of the actual biodiversity present in the monitored habitats (Weigand 2019). DNA metabarcoding of both bulk samples and eDNA samples are relevant tools for future freshwater biomonitoring in Norway. The aim of this PhD project is to develop assessment protocols based on DNA-metabarcoding and eDNA of benthic invertebrates, microcrustaceans and fish that can be used as standard biomonitoring tools to assess the ecological condition of lakes. The main topics addressed will be: - Development of protocols throughout the eDNA-metabarcoding workflow (i.e. sampling, filtration, preservation, extraction, amplification and sequencing) suitable to execute biodiversity assessments and determine the ecological status of lakes. - Comparison of the results obtained using molecular tools and traditional morphology-based approaches in order to assess the feasibility of such techniques to be incorporated as standard biomonitoring tools, such as the ones implemented under the provisions of the WFD. - Evaluate the effect of improved taxonomic resolution from molecular techniques on determining the ecological status of lakes, both by broadening the number of taxa analyzed and by identifying more taxa to species level. - Assess the feasibility of using eDNA extracted from water samples, taken at different depths and fish densities, to measure fish abundance/biomass as a proxy to calculate the ecological quality indices regulated in the WFD. - Analyze the coverage and resolution provided by reference libraries for certain taxa, such as crustacea, in order to assess the reliability and precision of taxonomic assignments.

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