Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples

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Abstract

The zebra mussel ( Dreissena polymorpha Pallas, 1771) and the quagga mussel ( D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations.

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Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds

Teruhiko Takahara, Toshifumi Minamoto, Hideyuki Doi (2013)

Knowledge of the presence of an invasive species is critical to monitoring the sustainability of communities and ecosystems. Environmental DNA (eDNA), DNA fragments that are likely to be bound to organic matters in the water or in shed cells, has been used to monitor the presence of aquatic animals. Using an eDNA-based method, we estimated the presence of the invasive bluegill sunfish, Lepomis macrochirus, in 70 ponds located in seven locales on the Japanese mainland and on surrounding islands. We quantified the concentration of DNA copies in a 1 L water sample using quantitative real-time polymerase chain reaction (qPCR) with a primer/probe set. In addition, we visually observed the bluegill presence in the ponds from the shoreline. We detected bluegill eDNA in all the ponds where bluegills were observed visually and some where bluegills were not observed. Bluegills were also less prevalent on the islands than the mainland, likely owing to limited dispersal and introduction by humans. Our eDNA method simply and rapidly detects the presence of this invasive fish species with less disturbance to the environment during field surveys than traditional methods.

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The Relationship between the Distribution of Common Carp and Their Environmental DNA in a Small Lake

Jessica J. Eichmiller, Przemyslaw Bajer, Peter W. Sorensen (2014)

Although environmental DNA (eDNA) has been used to infer the presence of rare aquatic species, many facets of this technique remain unresolved. In particular, the relationship between eDNA and fish distribution is not known. We examined the relationship between the distribution of fish and their eDNA (detection rate and concentration) in a lake. A quantitative PCR (qPCR) assay for a region within the cytochrome b gene of the common carp (Cyprinus carpio or ‘carp’), an ubiquitous invasive fish, was developed and used to measure eDNA in Lake Staring (MN, USA), in which both the density of carp and their distribution have been closely monitored for several years. Surface water, sub-surface water, and sediment were sampled from 22 locations in the lake, including areas frequently used by carp. In water, areas of high carp use had a higher rate of detection and concentration of eDNA, but there was no effect of fish use on sediment eDNA. The detection rate and concentration of eDNA in surface and sub-surface water were not significantly different (p≥0.5), indicating that eDNA did not accumulate in surface water. The detection rate followed the trend: high-use water > low-use water > sediment. The concentration of eDNA in sediment samples that were above the limit of detection were several orders of magnitude greater than water on a per mass basis, but a poor limit of detection led to low detection rates. The patchy distribution of eDNA in the water of our study lake suggests that the mechanisms that remove eDNA from the water column, such as decay and sedimentation, are rapid. Taken together, these results indicate that effective eDNA sampling methods should be informed by fish distribution, as eDNA concentration was shown to vary dramatically between samples taken less than 100 m apart.

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Routes to improving the reliability of low level DNA analysis using real-time PCR

Stephen Ellison, Claire A English, Malcolm J Burns … (2006)

Background Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated. Results At very low target concentrations of 0.5–10 genome equivalents (g.e.) eliminating any replicates within each DNA standard concentration with no measurable signal (non-detects) compromised calibration. Improved calibration could be achieved by eliminating all calibration replicates for any calibration standard concentration with non-detects ('elimination by sample'). Test samples also showed positive bias if non-detects were removed prior to averaging; less biased results were obtained by converting to concentration, including non-detects as zero concentration, and averaging all values. Tube plastic proved to have a strongly significant effect on DNA quantitation at low levels (p = 1.8 × 10-4). At low concentrations (under 10 g.e.), results for assays prepared in standard plastic were reduced by about 50% compared to the low-retention plastic. Preparation solution (carrier DNA or stabiliser) was not found to have a significant effect in this study. Detection probabilities were calculated using logistic regression. Logistic regression over large concentration ranges proved sensitive to non-detected replicate reactions due to amplification failure at high concentrations; the effect could be reduced by regression against log (concentration) or, better, by eliminating invalid responses. Conclusion Use of low-retention plastic tubes is advised for quantification of DNA solutions at levels below 100 g.e. For low-level calibration using linear least squares, it is better to eliminate the entire replicate group for any standard that shows non-detects reasonably attributable to sampling effects than to either eliminate non-detects or to assign arbitrary high Ct values. In calculating concentrations for low-level test samples with non-detects, concentrations should be calculated for each replicate, zero concentration assigned to non-detects, and all resulting concentration values averaged. Logistic regression is a useful method of estimating detection probability at low DNA concentrations.

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Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 14 December 2016

Publication date Collection: 2016

Volume: 6

Electronic Location Identifier: 39067

Affiliations

[1 ]Laboratori d’Ictiologia Genètica, Department of Biology, Universitat de Girona, LEAR Building , Girona, 17003, Spain

[2 ]IRTA Aquatic Ecosystems, Sant Carles de la Ràpita , 43540, Spain

[3 ]Waterfauna Hydrobiologisch Adviesbureau , Oostrandpark 30, 8212 AP Lelystad, The Netherlands

Author notes

[a ] oriol.vidal@ 123456udg.edu

[b ] jordi.vinas@ 123456udg.edu

Article

Publisher Item ID: srep39067

DOI: 10.1038/srep39067

PMC ID: 5155230

PubMed ID: 27966602

SO-VID: 5943cf7c-bd01-4b9d-9a04-8b0f56fb4056

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Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples

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Environmental, Social, and Governance

Most cited references 11

Using Environmental DNA to Estimate the Distribution of an Invasive Fish Species in Ponds

The Relationship between the Distribution of Common Carp and Their Environmental DNA in a Small Lake

Routes to improving the reliability of low level DNA analysis using real-time PCR

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