Standards for Methods Utilizing Environmental DNA for Detection of Fish Species

Environmental DNA (eDNA) techniques are gaining attention as cost-effective, non-invasive strategies for acquiring information on fish and other aquatic organisms from water samples. Currently, eDNA approaches are used to detect specific fish species and determine fish community diversity. Various protocols used with eDNA methods for aquatic organism detection have been reported in different eDNA studies, but there are no general recommendations for fish detection. Herein, we reviewed 168 papers to supplement and highlight the key criteria for each step of eDNA technology in fish detection and provide general suggestions for eliminating detection errors. Although there is no unified recommendation for the application of diverse eDNA in detecting fish species, in most cases, 1 or 2 L surface water collection and eDNA capture on 0.7-μm glass fiber filters followed by extraction with a DNeasy Blood and Tissue Kit or PowerWater DNA Isolation Kit are useful for obtaining high-quality eDNA. Subsequently, species-specific quantitative polymerase chain reaction (qPCR) assays based on mitochondrial cytochrome b gene markers or eDNA metabarcoding based on both 12S and 16S rRNA markers via high-throughput sequencing can effectively detect target DNA or estimate species richness. Furthermore, detection errors can be minimized by mitigating contamination, negative control, PCR replication, and using multiple genetic markers. Our aim is to provide a useful strategy for fish eDNA technology that can be applied by researchers, advisors, and managers.

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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Environmental DNA bioassays corroborate field data for detection of overwintering species at risk Blanding’s turtles (Emydoidea blandingii)

Genome ◽

10.1139/gen-2020-0043 ◽

2021 ◽

pp. 1-12

Author(s):

Scott A. Tarof ◽

Steven Crookes ◽

Kelsey Moxley ◽

Jeff Hathaway ◽

Graham Cameron ◽

...

Keyword(s):

At Risk ◽

Field Data ◽

Quantitative Polymerase Chain Reaction ◽

A Priori ◽

Environmental Dna ◽

Positive Control ◽

Negative Control ◽

Species At Risk ◽

Emydoidea Blandingii ◽

Order Of Magnitude

Environmental DNA (eDNA) is gaining traction in conservation ecology as a powerful tool for detecting species at risk. We developed a quantitative polymerase chain reaction assay to detect a DNA amplicon fragment of the mitochondrial nicotinamide adenine dinucleotide locus of the Blanding’s turtle (Emydoidea blandingii) for detecting overwintering individuals. Seventy-eight water samples were collected from 17 wetland sites in Ontario, Canada. We used traditional field data to identify a priori positive and negative control sites. Fifty percent of positive control sites amplified. Detection was related to the number of individuals estimated from field observations in at least one region surveyed. Positive control sites had lower total dissolved solids and electrical conductivity in relation to negative control sites. Shedding rates were within the same order of magnitude for brumating and active turtles. We recommend collecting additional samples at a larger number of locations to maximize detection. Recommended sampling design changes may overshadow the additional effects of water chemistry and low eDNA shedding rates. eDNA offers tremendous potential to practitioners conducting species at risk assessments in environmental consulting by providing a faster, more efficient method of detection compared with traditional surveys.

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Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

10.1101/113472 ◽

2017 ◽

Cited By ~ 7

Author(s):

Masayuki Ushio ◽

Hiroaki Murakami ◽

Reiji Masuda ◽

Tetsuya Sado ◽

Masaki Miya ◽

...

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Marine Ecosystem ◽

Internal Standard ◽

Environmental Dna ◽

Illumina Miseq ◽

Pcr Inhibition ◽

Copy Numbers ◽

Quantitative Monitoring ◽

Monitoring Of Biodiversity

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

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From Rare Species Detection to Whole-Community Diversity Using High-Throughput Sequencing of Freshwater eDNA

Biodiversity Information Science and Standards ◽

10.3897/biss.3.37975 ◽

2019 ◽

Vol 3 ◽

Author(s):

Maria Judite Alves ◽

Hugo Gante ◽

Ana Veríssimo ◽

Filipe Ribeiro ◽

Carlos David Santos ◽

...

Keyword(s):

High Throughput ◽

Rare Species ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Community Diversity ◽

Census Methods ◽

Recent Approach ◽

Fish Samples ◽

Technological Developments

Understanding natural communities and ecosystems and the services they provide to humanity is highly dependent on knowledge about species composition and diversity through space and time. This is especially difficult in aquatic systems where traditional census methods provide species compositions that are usually truncated since rare species tend to go undetected. Detection of the rare species is important because they are either threatened or invasives at the earliest stage of invasion. One recent approach allowing detection of rare species uses environmental DNA (eDNA), present in water or soil, as traces of their existence. Here we propose to make use of recent technological developments in the area of high throughput sequencing to characterize freshwater fish communities and detect rare species, using a combination of eDNA metabarcoding and bulk eDNA metagenomics. A case-study will be conducted on the River Tagus (Portugal), which is inhabited by several rare fish species including both native and introduced taxa. In addition, the applicability of eDNA metagenomics for estimating the genetic diversity of populations will be assessed by comparing the results against those produced by traditional genetic screening of individual fish samples.

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Anacapa Toolkit: an environmental DNA toolkit for processing multilocus metabarcode datasets

10.1101/488627 ◽

2018 ◽

Cited By ~ 1

Author(s):

Emily E. Curd ◽

Zack Gold ◽

Gaurav S Kandlikar ◽

Jesse Gomer ◽

Max Ogden ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sequence Data ◽

Environmental Dna ◽

Taxonomic Diversity ◽

R Package ◽

Community Diversity ◽

Taxonomic Assignment ◽

Lowest Common Ancestor ◽

Reference Databases ◽

Comprehensive Reference

Abstract1. Environmental DNA (eDNA) metabarcoding is a promising method to monitor species and community diversity that is rapid, affordable, and non-invasive. Longstanding needs of the eDNA community are modular informatics tools, comprehensive and customizable reference databases, flexibility across high-throughput sequencing platforms, fast multilocus metabarcode processing, and accurate taxonomic assignment. As bioinformatics tools continue to improve, addressing each of these demands within a single bioinformatics toolkit is becoming a reality.2. We present the modular metabarcode sequence toolkit Anacapa (https://github.com/limey-bean/Anacapa/), which addresses the above needs, allowing users to build comprehensive reference databases and assign taxonomy to raw multilocus metabarcode sequence data A novel aspect of Anacapa is our database building module, Creating Reference libraries Using eXisting tools (CRUX), which generates comprehensive reference databases for specific user-defined metabarcode loci. The Quality Control and Dereplication module sorts and processes multiple metabarcode loci and processes merged, unmerged and unpaired reads maximizing recovered diversity. Followed by amplicon sequence variants (ASVs) detection using DADA2. The Anacapa Classifier module aligns these ASVs to CRUX-generated reference databases using Bowtie2. Taxonomy is assigned to ASVs with confidence scores using a Bayesian Lowest Common Ancestor (BLCA) method. The Anacapa Toolkit also includes an R package, ranacapa, for automated results exploration through standard biodiversity statistical analysis.3. We performed a series of benchmarking tests to verify that the Anacapa Toolkit generates comprehensive reference databases that capture wide taxonomic diversity and that it can assign high-quality taxonomy to both MiSeq-length and Hi-Seq length sequence data. We demonstrate the value of the Anacapa Toolkit to assigning taxonomy to eDNA sequences from seawater samples from southern California including capability of this tool kit to process multilocus metabarcoding data.4. The Anacapa Toolkit broadens the exploration of eDNA and assists in biodiversity assessment and management by generating metabarcode specific databases, processing multilocus data, retaining all read types, and expanding non-traditional eDNA targets. Anacapa software and source code are open and available in a virtual container to ease installation.

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Exploratory field study on the effects of porcine circovirus 2 (PCV-2) sow vaccination at different physiological stages mimicking blanket vaccination

Porcine Health Management ◽

10.1186/s40813-021-00213-2 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Patricia Pleguezuelos ◽

Marina Sibila ◽

Raúl Cuadrado ◽

Rosa López-Jiménez ◽

Diego Pérez ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Negative Control Group ◽

Negative Control ◽

Late Gestation ◽

Antibody Detection ◽

Porcine Circovirus ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Physiological Status ◽

Porcine Circovirus 2

Abstract Background The objective of the present study was to explore the benefits of Porcine circovirus 2 (PCV-2) blanket vaccination in a sow herd on productive parameters, PCV-2 infection and immune status in sows and their progeny. For this purpose, 288 sows were distributed among four balanced experimental groups. One group remained as negative control group and the other three received 1 mL of PCV-2 Ingelvac Circoflex® intramuscularly at different productive cycle moments: before mating, mid gestation (42–49 days post-insemination) or late gestation (86–93 days post-insemination); phosphate buffered saline (PBS) was used as negative control item. Reproductive parameters from sows during gestation and body weight of their progeny from birth to weaning were recorded. Additionally, blood was collected from sows at each vaccination time and piglets at 3 weeks of age. Moreover, up to 4 placental umbilical cords (PUC) per sow were taken at peri-partum. Sera from sows and piglets were analysed for PCV-2 antibody detection using an enzyme-linked immunosorbent assay (ELISA). Sera from sows and PUC were tested to quantify viraemia using a real time quantitative polymerase chain reaction (qPCR) assay. Results Globally, results indicated that vaccinated sows showed heavier piglets at birth and at weaning, less cross-fostered piglets, lower viral load at farrowing as well as in PUC, and higher antibody levels at farrowing, compared to non-vaccinated ones. When all groups were compared among them, sows vaccinated at mid or late gestation had heavier piglets at birth than non-vaccinated sows, and lower proportion of PCV-2 positive PUC. Also, cross-fostering was less frequently practiced in sows vaccinated at pre-mating or mid gestation compared to non-vaccinated ones. Conclusions In conclusion, the present study points out that PCV-2 sow vaccination at different time points of their physiological status (mimicking blanket vaccination) offers benefits at production and serological and virological levels.

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Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

Antarctic Science ◽

10.1017/s0954102021000274 ◽

2021 ◽

pp. 1-10

Author(s):

Micheline Carvalho-Silva ◽

Luiz Henrique Rosa ◽

Otávio H.B. Pinto ◽

Thamar Holanda Da Silva ◽

Diego Knop Henriques ◽

...

Keyword(s):

High Throughput Sequencing ◽

Crater Lake ◽

Environmental Dna ◽

Food Sources ◽

South Shetland Islands ◽

Deception Island ◽

Shetland Islands ◽

Operational Taxonomic Units ◽

First Time ◽

Impacted Site

Abstract The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

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Tree Diversity Reduces Fungal Endophyte Richness and Diversity in a Large-Scale Temperate Forest Experiment

Diversity ◽

10.3390/d11120234 ◽

2019 ◽

Vol 11 (12) ◽

pp. 234 ◽

Cited By ~ 2

Author(s):

Eric A. Griffin ◽

Joshua G. Harrison ◽

Melissa K. McCormick ◽

Karin T. Burghardt ◽

John D. Parker

Keyword(s):

Phylogenetic Diversity ◽

Large Scale ◽

High Throughput Sequencing ◽

Spatial Scales ◽

Fungal Endophytes ◽

Fungal Endophyte ◽

Tree Diversity ◽

Trophic Levels ◽

Community Diversity ◽

And Function

Although decades of research have typically demonstrated a positive correlation between biodiversity of primary producers and associated trophic levels, the ecological drivers of this association are poorly understood. Recent evidence suggests that the plant microbiome, or the fungi and bacteria found on and inside plant hosts, may be cryptic yet important drivers of important processes, including primary production and trophic interactions. Here, using high-throughput sequencing, we characterized foliar fungal community diversity, composition, and function from 15 broadleaved tree species (N = 545) in a recently established, large-scale temperate tree diversity experiment using over 17,000 seedlings. Specifically, we tested whether increases in tree richness and phylogenetic diversity would increase fungal endophyte diversity (the “Diversity Begets Diversity” hypothesis), as well as alter community composition (the “Tree Diversity–Endophyte Community” hypothesis) and function (the “Tree Diversity–Endophyte Function” hypothesis) at different spatial scales. We demonstrated that increasing tree richness and phylogenetic diversity decreased fungal species and functional guild richness and diversity, including pathogens, saprotrophs, and parasites, within the first three years of a forest diversity experiment. These patterns were consistent at the neighborhood and tree plot scale. Our results suggest that fungal endophytes, unlike other trophic levels (e.g., herbivores as well as epiphytic bacteria), respond negatively to increasing plant diversity.

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