A qPCR MGB probe based eDNA assay for European freshwater pearl mussel (<scp><i>Margaritifera margaritifera</i></scp> L.)

Carlsson, Jeanette E. L.; Egan, Damian; Collins, Patrick C.; Farrell, Edward D.; Igoe, Fran

doi:10.1002/aqc.2788

Cited by 26 publications

(26 citation statements)

References 15 publications

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“…Species-specific primers for FPM (Carlsson et al, 2017) and brown trout (Gustavson et al, 2015) were multiplexed in a droplet-digital-PCR (ddPCR) (Bio-rad Laboratories, Inc), using a 6-FAM labelled and a VIC-labelled TaqMan MGB-probe. Brown trout was well suited as a control because it occurs at all parts of the river, including sections were FPM is absent or occurs at low density.…”

Section: Pcr-setup Was Carried Out In Uv-benches and All Work Relatedmentioning

confidence: 99%

“…Brown trout was well suited as a control because it occurs at all parts of the river, including sections were FPM is absent or occurs at low density. Species-specific primers for FPM (Carlsson et al, 2017) and brown trout (Gustavson et al, 2015) were multiplexed in a droplet-digital-PCR (ddPCR) (Bio-rad Laboratories, Inc), using a 6-FAM labelled and a VIC-labelled TaqMan MGB-probe. Both primer pairs target regions within the mitochondrial cytochrome oxidase I (COI) gene and amplify fragments of 83 bp and 61 bp, respectively.…”

Section: Dna Extraction and Genetic Analysismentioning

confidence: 99%

“…Visual searches for mussels can be hampered when adult individuals are partly or fully buried in the substratum or when the visibility is poor. Environmental DNA is a highly promising tool for monitoring freshwater mussels and previous studies revealed that freshwater mussels shed DNA that can be detected in water samples (Carlsson et al, 2017;Currier, Morris, Wilson, & Freeland, 2018;Deiner & Altermatt, 2014;Dysthe et al, 2018;Sansom & Sassoubre, 2017;Stoeckle, Kuehn, & Geist, 2016). However, empirical work needs to reveal how eDNA concentrations are affected by stream characteristics such as downstream transport and by seasonal variation.…”

Section: Introductionmentioning

confidence: 99%

“…) Carlsson et al (2017). report reliable detection of an FPM aggregation of 62 individuals, but it is unknown to what extent downstream transport of eDNA may have explained those results.…”

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confidence: 99%

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Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

et al. 2019

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Environmental DNA (eDNA) can be used to detect the presence and abundance of aquatic organisms from water samples. Before implementing this methodology as a tool for monitoring, more knowledge is needed on variation in eDNA concentrations in relation to species abundance and potential confounding factors. Shedding and decay of eDNA may vary extensively over the season and are dependent on environmental factors such as water temperature and on biological processes such as activity level and reproduction. In lotic systems, eDNA concentrations are also affected by downstream transport of eDNA. Sessile freshwater mussels provide an ideal study system for investigating the relationship between species spatial distribution and eDNA concentrations in lotic systems. We quantified freshwater pearl mussel (Margaritifera margaritifera) eDNA concentrations at four localities in a natural river with detailed knowledge of mussel spatial distribution: (a) upstream of the known species distribution, just downstream (b) a small and (c) a large aggregation and (d) 1,700 m downstream of the large aggregation. To study seasonal variation, we quantified eDNA concentrations during three periods: (a) in late spring, with cold water and relatively inactive mussels; (b) in mid-summer, with higher water temperature and active mussel filtration; and (c) in late summer, during the release of larvae.Species detection was highly reliable, with no detection of eDNA upstream of the species distribution and complete detection downstream of the large aggregation.Detection success of the small aggregation was low, with 13% of the samples testing positive. Downstream transport was efficient, with no significant decrease in eDNA concentrations over 1,700 m river distance. Seasonal variation was strong, with a 20-fold increase in eDNA concentrations from late spring to late summer, during reproduction. Our results highlight both the potential and challenges of eDNA monitoring in lotic systems. K E Y W O R D Sconservation genetics, environmental DNA, freshwater mussels, species detection, unionidae | 65 WACKER Et Al.

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Section: Pcr-setup Was Carried Out In Uv-benches and All Work Relatedmentioning

confidence: 99%

Section: Dna Extraction and Genetic Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…) Carlsson et al (2017). report reliable detection of an FPM aggregation of 62 individuals, but it is unknown to what extent downstream transport of eDNA may have explained those results.…”

mentioning

confidence: 99%

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Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

et al. 2019

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“…Quantitative PCR (qPCR) and digital droplet (dd) PCR are currently the main methods used to detect eDNA of focal species (Baker, Steel, Nieukirk, & Klinck, 2018;Boothroyd, Mandrak, Fox, & Wilson, 2016;Carlsson et al, 2017;Dejean et al, 2011;Gargan et al, 2017;Rusch et al, 2018;Thomsen, Kielgast, Iversen, Møller, et al, 2012;Uthicke, Lamare, & Doyle, 2018). However, the need to cycle from 95°C to lower temperatures during the PCR process makes the adaptation of PCR-based techniques to a portable biosensor device challenging (Zhang & Xing, 2007).…”

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confidence: 99%

The application of CRISPR‐Cas for single species identification from environmental DNA

Williams

O’Grady

Ball

et al. 2019

Molecular Ecology Resources

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We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.

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Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae)

Currier

Morris

Wilson

et al. 2018

Aquatic Conservation

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1. Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms.2. Previous eDNA studies have yet to develop species-specific markers that target taxa with many potentially sympatric confamilials. Forty-one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity.3. Species-specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. 4. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. 5. The paired sampling design showed that species-specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. 6. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.

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A qPCR MGB probe based eDNA assay for European freshwater pearl mussel (Margaritifera margaritifera L.)

Cited by 26 publications

References 15 publications

Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration

The application of CRISPR‐Cas for single species identification from environmental DNA

Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae)

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