Development of environmental DNA (eDNA) methods for detecting high-risk freshwater fishes in live trade in Canada

Roy, Mélanie; Belliveau, Valérie; Mandrak, Nicholas E.; Gagné, Nellie

doi:10.1007/s10530-017-1532-z

Cited by 26 publications

(22 citation statements)

References 40 publications

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“…We performed TaqMan qPCRs for S. glanis and A. anguilla using previously published protocols (Jensen, Knudsen, Munk, Thomsen, & Møller, ; Roy, Belliveau, Mandrak, & Gagné, ). Sampling sites which either represent suitable habitat for target species or for which recent sightings were reported were identified (Figure ).…”

Section: Re‐evaluation Of a Metabarcoding Study In The Volga Headwatersmentioning

confidence: 99%

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

Schenekar

Schletterer

Lecaudey

et al. 2020

River Research & Apps

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Biodiversity monitoring via environmental DNA, particularly metabarcoding, is evolving into a powerful assessment tool for riverine systems. However, for metabarcoding to be fully integrated into standardized monitoring programmes, some current challenges concerning sampling design, laboratory workflow, and data analysis need to be overcome. Here, we review some of these major challenges and potential solutions. We further illustrate three potential pitfalls, namely the choice of suitable metabarcoding primers, the necessity of complete reference databases, and varying assay sensitivities, by a reappraisal of our‐own recently carried out metabarcoding study in the Volga headwaters. TaqMan qPCRs had detected catfish (Silurus glanis) and European eel (Anguilla anguilla), whereas metabarcoding had not, in the same samples. Furthermore, after extending the genetic reference database by 12 additional species and re‐analysing the metabarcoding data, we additionally detected the Siberian spiny loach (Cobitis sibirica) and Ukrainian brook lamprey (Eudontomyzon mariae) and reassigned the operational taxonomic units previously assigned to Misgurnus fossilis to Cobitis sibirica. In silico analysis of metabarcoding primer efficiencies revealed considerable variability among primer pairs and among target species, which could lead to strong primer bias and potential false‐negatives in metabarcoding studies if not properly compensated for. These results highlight some of the pitfalls of eDNA‐metabarcoding as a means of monitoring fish biodiversity in large rivers, which need to be considered in order to fully unleash the full potential of these approaches for freshwater biodiversity monitoring.

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Section: Re‐evaluation Of a Metabarcoding Study In The Volga Headwatersmentioning

confidence: 99%

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

Schenekar

Schletterer

Lecaudey

et al. 2020

River Research & Apps

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“…Environmental DNA methods development often utilizes gDNA for testing 14,38–40 but low concentrations of eDNA could be lost due to insufficient pelleting during isolations 4,25,41 . DNA is also thought to adhere to many plastics, further reducing eDNA yields.…”

Section: Introductionmentioning

confidence: 99%

Improving eDNA yield and inhibitor reduction through increased water volumes and multi-filter isolation techniques

Hunter

Ferrante

Meigs‐Friend

et al. 2019

Sci Rep

131

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To inform management and conservation decisions, environmental DNA (eDNA) methods are used to detect genetic material shed into the water by imperiled and invasive species. Methodological enhancements are needed to reduce filter clogging, PCR inhibition, and false-negative detections when eDNA is at low concentrations. In the first of three simple experiments, we sought to ameliorate filter clogging from particulates and organic material through a scaled-up, multi-filter protocol. We combined four filters in a 5 mL Phenol-Chloroform-Isoamyl (PCI) procedure to allow for larger volumes of water (~1 L) to be filtered rapidly. Increasing the filtered water volume by four times resulted in 4.4X the yield of target DNA. Next, inhibition from organic material can reduce or block eDNA detections in PCR-based assays. To remove inhibitory compounds retained during eDNA isolation, we tested three methods to chemically strip inhibitors from eDNA molecules. The use of CTAB as a short-term (5–8 day) storage buffer, followed by a PCI isolation, resulted in the highest eDNA yields. Finally, as opposed to a linear relationship among increasing concentrations of filtered genomic eDNA, we observed a sharp change between the lower (70–280 ng) and higher (420–560 ng) amounts. This may be important for effectively precipitating eDNA during protocol testing.

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“…invasion in the Great Lakes, USA (Farrington et al, 2015). A quantitative polymerase chain reaction (qPCR) assay targeting crucian carp was also published in the context of early warning invasion monitoring for fish species that may arrive in Canada (Roy, Belliveau, Mandrak, & Gagné, 2017), but was only tested on tissue-derived DNA. Of equal importance to invasion monitoring, eDNA analysis has enhanced surveys for threatened and endangered freshwater fish (Bylemans et al, 2017;Piggott, 2016;Schmelzle & Kinziger, 2016;Sigsgaard et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

et al. 2018

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The crucian carp (Carassius carassius) is one of few fish species associated with small ponds in the UK. These populations contain genetic diversity not found in Europe and are important to conservation efforts for the species which has declined across its range in Europe. Detection and monitoring of extant crucian carp populations are crucial for conservation success. Environmental DNA (eDNA) analysis could be very useful in this respect as a rapid, cost‐efficient monitoring tool. We developed a species‐specific quantitative polymerase chain reaction (qPCR) assay for eDNA surveillance of crucian carp to enable non‐invasive, large‐scale distribution monitoring. We compared fyke netting and eDNA analysis at ponds with (n = 10) and without (n = 10) crucian carp for presence–absence detection. We examined biotic (crucian carp density represented by catch‐per‐unit‐effort [CPUE] estimate) and abiotic influences on eDNA detection probability using a hierarchical occupancy model, and eDNA quantification using a mixed‐effects model. eDNA analysis achieved 90% detection for crucian carp (n = 10), failing in only one pond where presence was known. CPUE estimate and conductivity had positive and negative influences on eDNA detection probability in qPCR replicates respectively. Similarly, conductivity had a negative effect on DNA copy number, whereas copy number increased with CPUE estimate. Our results demonstrate that eDNA analysis could enable detection of crucian carp populations in ponds and benefit ongoing conservation efforts, but imperfect species detection in relation to biotic and abiotic factors and eDNA workflow requires further investigation. Nonetheless, we have established an eDNA framework for the crucian carp as well as sources of imperfect detection which future investigations can build upon.

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Development of environmental DNA (eDNA) methods for detecting high-risk freshwater fishes in live trade in Canada

Cited by 26 publications

References 40 publications

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

Improving eDNA yield and inhibitor reduction through increased water volumes and multi-filter isolation techniques

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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