Laboratory experiments for the detection of environmental DNA of crayfish: Examining the potential

2015 ◽  
Vol 21 (1) ◽  
pp. 159-163 ◽  
Author(s):  
Chester R. Figiel ◽  
Sandra Bohn
Keyword(s):  
Water Samples ◽  
Dna Detection ◽  
Environmental Dna ◽  
Sampling Period ◽  
Sediment Samples ◽  
Target Dna ◽  
Invasive Crayfish ◽  
In The Wild ◽  
Species Specific ◽  
Crayfish Species

Abstract We examined methods for detecting environmental DNA of the invasive white river crayfish Procambarus zonangulus. In a laboratory experiment, we investigated detection capability in benthic sediment samples and in water samples in six flow-through tanks. Additionally we determined whether crayfish density (low = 0.67 or high = 2.69 crayfish·m-2) or crayfish time in tanks influenced DNA detectability (collection of samples on Days 2, 5, 8 and 15). Species-specific primers and probes were designed for P. zonangulus and their specificity was tested against other crayfish species. Limits of detection and quantification were specified for the target DNA sequence by means of quantitative PCR amplifications on dilution series of known amounts of P. zonangulus DNA. We detected crayfish DNA in 14 of the 24 benthic sediment samples and in two of the 24 water samples. DNA detection was found in benthic sediment samples in at least two tanks at every sampling period, while DNA detection was found in water samples only on Day 8. Crayfish DNA was detected in benthic sediment and water samples independently of crayfish density. Crayfish at both densities were observed to ‘explore’ all areas of the tank and move irrespective of diurnal time or conspecific presence. These behavior patterns were observed throughout the 15 day experiment and likely resulted in the positive detections, especially in benthic sediment samples. We believe that these methods could benefit monitoring of invasive crayfish species, although there is no doubt that further optimization and more research is needed to evaluate these techniques in the wild.

scholarly journals Seasonal variation in environmental DNA detection in sediment and water samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (1) ◽  
pp. e0191737 ◽  
Author(s):  
Andrew S. Buxton ◽  
Jim J. Groombridge ◽  
Richard A. Griffiths
Keyword(s):  
Seasonal Variation ◽  
Water Samples ◽  
Dna Detection ◽  
Environmental Dna

Improving reliability in environmental DNA detection surveys through enhanced quality control

2017 ◽  
Vol 68 (2) ◽  
pp. 388 ◽  
Author(s):  
Elise M. Furlan ◽  
Dianne Gleeson
Keyword(s):  
Quality Control ◽  
Native Species ◽  
Conservation Management ◽  
Environmental Dna ◽  
Positive Control ◽  
False Negatives ◽  
Method Error ◽  
Target Dna ◽  
Species Specific ◽  
Australian Freshwater

Species-specific environmental DNA (eDNA) surveys are increasingly being used to infer species presence in an environment. Current inadequacies in quality control increase concern for false negatives, which can have serious ramifications for both the management of invasive species and the conservation of native species. eDNA surveys involve a multi-step process to sample, capture, extract and amplify target DNA from the environment. We outline various positive control options and show that many of the commonly used controls are capable of detecting false negatives arising during the amplification stage only. We suggest a secondary, generic primer, designed to co-amplify endogenous DNA sampled during species-specific eDNA surveys, constitutes a superior positive control to monitor method success throughout all stages of eDNA analysis. We develop a species-specific European carp (Cyprinus carpio) assay and a generic fish assay for use as an endogenous control for eDNA surveys in Australian freshwater systems where fish are known to be abundant. We use these assays in a multiplex on eDNA samples that are simultaneously sampled, captured, extracted and amplified. This positive control allows us to distinguish method error from informative non-amplification results, improving reliability in eDNA surveys, which will ultimately lead to better informed conservation management decisions.

scholarly journals Utilizing environmental DNA for wide-range distributions of reproductive area of an invasive terrestrial toad in Ishikari river basin in Japan

2021 ◽  
Author(s):  
Hiroki Mizumoto ◽  
Osamu Kishida ◽  
Kotaro Takai ◽  
Naru Matsuura ◽  
Hitoshi Araki
Keyword(s):  
Invasive Species ◽  
River Basin ◽  
Water Samples ◽  
Environmental Dna ◽  
Watershed Scale ◽  
Efficient Tool ◽  
Occupancy Model ◽  
Wide Range ◽  
Powerful Means ◽  
In The Wild

AbstractUnderstanding the distribution of invasive species and their reproductive area is crucial for their managements after invasion. While catch and observation surveys are still embraced, environmental DNA (eDNA) has been increasingly utilized as an efficient tool for identifying these species in the wild. In this study, we developed a Bufo-specific eDNA assay for detecting an invasive, toxic, and terrestrial toad species Bufo japonicus formosus in Hokkaido, Japan, and applied it to their reproductive area at watershed scale. The eDNA assay was field-validated in ponds where B. japonicus were observed, as well as in rivers downstream of the reproductive ponds. Thus, the assay provided us an opportunity to screen watersheds that include their reproductive area by collecting downstream water samples. Applying it to the Ishikari river basin, the largest river basin in Hokkaido (c.a., 14,330 km2), we detected toad eDNA at 32 out of 73 sampling sites. They are composed of eleven sites with species observation records nearby (all the sites with observation records within a 500 m radius) and 21 sites without such records. And those eDNA detections were from twelve out of 31 river systems in the entire river basin. A Bayesian, multiscale occupancy model supported high eDNA detectability among those sites. These results suggest that the eDNA assay can efficiently estimate the presence of reproductive area of the terrestrial toad even from a distant downstream of the watershed, and that it provides a powerful means of detecting new reproductive area and monitoring further spread of invasive species.

scholarly journals Environmental DNA detection of Arctic char ( Salvelinus alpinus ) in Irish lakes: Development and application of a species‐specific molecular assay

2020 ◽  
Vol 2 (2) ◽  
pp. 221-233
Author(s):  
Luca Mirimin ◽  
Aaron Hickey ◽  
Dylan Barrett ◽  
Fergus DeFaoite ◽  
Simona Boschetti ◽  
...  
Keyword(s):  
Salvelinus Alpinus ◽  
Dna Detection ◽  
Environmental Dna ◽  
Arctic Char ◽  
Molecular Assay ◽  
Species Specific

An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water

2012 ◽  
Vol 39 (7) ◽  
pp. 629 ◽  
Author(s):  
Zachary H. Olson ◽  
Jeffrey T. Briggler ◽  
Rod N. Williams
Keyword(s):  
Water Samples ◽  
Effective Means ◽  
Environmental Dna ◽  
Cost Effective ◽  
Negative Control ◽  
Specificity And Sensitivity ◽  
Genetic Sampling ◽  
Conservation Concern ◽  
Species Specific ◽  
Eastern Hellbender

Context Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa. Aims We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments. Methods We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis. Key results Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control. Conclusion eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders. Implications eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.

scholarly journals Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels

Genome ◽  
2016 ◽  
Vol 59 (12) ◽  
pp. 1141-1149 ◽  
Author(s):  
Anna Cho ◽  
Todd Morris ◽  
Chris Wilson ◽  
Joanna Freeland
Keyword(s):  
Water Samples ◽  
Freshwater Mussel ◽  
Environmental Dna ◽  
Target Species ◽  
Habitat Occupancy ◽  
Unionid Mussels ◽  
Sufficient Variation ◽  
Species Specific ◽  
Taxonomic Groups ◽  
Tank Water

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species’ presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

scholarly journals Utilizing environmental DNA for wide-range distributions of reproductive area of an invasive terrestrial toad in Ishikari river basin in Japan

2021 ◽  
Author(s):  
Hiroki Mizumoto ◽  
Osamu Kishida ◽  
Kotaro Takai ◽  
Hitoshi Araki
Keyword(s):  
Invasive Species ◽  
River Basin ◽  
Water Samples ◽  
Detection System ◽  
Environmental Dna ◽  
Watershed Scale ◽  
Efficient Tool ◽  
Wide Range ◽  
Powerful Means ◽  
In The Wild

Abstract Understanding the distribution of invasive species and their reproductive area is crucial for their managements after invasion. While catch and observation surveys are still embraced, environmental DNA (eDNA) has been increasingly utilized as an efficient tool for identifying these species in the wild. In this study, we developed an eDNA detection system for an invasive, toxic, and terrestrial toad species Bufo japonicus in Hokkaido, Japan, and applied it to their reproductive area at watershed scale. We found that our system successfully detected their eDNA not only in ponds where their larvae were observed, but also in rivers downstream of the reproductive ponds. Thus, the system provided us an opportunity to estimate watersheds that include their reproductive area by collecting downstream water samples. Applying it to the Ishikari river basin, the largest river basin in Hokkaido (c.a., 14,330 km2), we detected their eDNA at 32 out of 73 river sampling sites. They are composed of eleven sites with species observation records nearby (all the sites with observation records within a 500 m radius) and21 sites without such records. And those eDNA detections were from 14 out of 31 river systems, and they were widespread across the river basin. These results suggest that the eDNA detection system can efficiently estimate the presence of reproductive area of the terrestrial toad even from a distant downstream of the watershed, and that it provides a powerful means of detecting new reproductive area and monitoring further spread of invasive species.

scholarly journals Influence of Preservation Methods, Sample Medium and Sampling Time on eDNA Recovery in a Neotropical River

2018 ◽  
Author(s):  
Naiara Guimarães Sales ◽  
Owen Simon Wangensteen ◽  
Daniel Cardoso Carvalho ◽  
Stefano Mariani
Keyword(s):  
Water Samples ◽  
Fish Assemblages ◽  
Environmental Dna ◽  
Cost Effective ◽  
Southeastern Brazil ◽  
Similar Amount ◽  
Biodiversity Monitoring ◽  
Sediment Samples ◽  
Preservation Methods ◽  
Sampling Event

ABSTRACTEnvironmental DNA (eDNA) has rapidly emerged as a promising biodiversity monitoring technique, proving to be a sensitive and cost-effective method for species detection. Despite the increasing popularity of eDNA, several questions regarding its limitations remain to be addressed. We investigated the effect of sampling medium and time, and preservation methods, on fish detection performance based on eDNA metabarcoding of neotropical freshwater samples. Water and sediment samples were collected from 11 sites along the Jequitinhonha River, Southeastern Brazil; sediment samples were stored in ethanol, while the same amounts of water per sample (3L) were stored in a cool box with ice, as well as by adding the cationic surfactant Benzalkonium chloride (BAC). Sediment and water samples yielded a similar amount of fish MOTUs (237 vs 239 in the first sampling event, and 153 vs 142 in the second sampling event). Water stored in ice provided better results than those preserved in BAC (239 and 142 vs 194 and 71 MOTUs). While documenting the effectiveness of eDNA surveys as practical tools for fish biodiversity monitoring in poorly accessible areas, we showed that keeping water samples cooled results in greater eDNA recovery and taxon detection than by adding cationic surfactants as sample preservatives. Furthermore, by comparing two sets of samples collected from the same locations at a three-week interval, we highlight the importance of conducting multiple sampling events when attempting to recover a realistic picture of fish assemblages in lotic systems.

scholarly journals Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254356
Author(s):  
Bettina Thalinger ◽  
Yannick Pütz ◽  
Michael Traugott
Keyword(s):  
Capillary Electrophoresis ◽  
Water Samples ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Dilution Series ◽  
Target Dna ◽  
Cost Efficient ◽  
Detection And Quantification

The use of sensitive methods is key for the detection of target taxa from trace amounts of environmental DNA (eDNA) in a sample. In this context, digital PCR (dPCR) enables direct quantification and is commonly perceived as more sensitive than endpoint PCR. However, endpoint PCR coupled with capillary electrophoresis (celPCR) potentially embodies a viable alternative as it quantitatively measures signal strength after PCR in Relative Fluorescence Units (RFU). Provided comparable levels of sensitivity are reached, celPCR permits the development of cost-efficient multiplex reactions, enabling the simultaneous detection of several target taxa. Here, we compared the sensitivity of singleplex and multiplex celPCR to dPCR for species-specific primer pairs amplifying mitochondrial DNA (COI) of fish species occurring in European freshwaters by analyzing dilution series of tissue extracts as well as field-collected water samples. Both singleplex and multiplex celPCR and dPCR displayed comparable sensitivity with reliable positive amplifications starting at two to 10 target DNA copies per μl extract. celPCR was suitable for quantifying target DNA and direct inference of copy numbers from RFU was possible after accounting for primer effects in linear mixed-effects models and calibration via dPCR. Furthermore, multiplex celPCR and dPCR were successfully used for the detection and quantification of fish-eDNA in field-collected water samples, confirming the results of the dilution series experiment and exemplifying the high sensitivity of the two approaches. The possibility of detection and quantification via multiplex celPCR is appealing for the cost-efficient screening of high sample numbers. The present results confirm the sensitivity of this approach thus enabling its application for future eDNA-based monitoring efforts.

scholarly journals Simultaneous detection of native and invasive crayfish and Aphanomyces astaci from environmental DNA samples in a wide range of habitats in Central Europe

NeoBiota ◽  
2020 ◽  
Vol 58 ◽  
pp. 1-32 ◽  
Author(s):  
Johannes C. Rusch ◽  
Michaela Mojžišová ◽  
David A. Strand ◽  
Jitka Svobodová ◽  
Trude Vrålstad ◽  
...  
Keyword(s):  
Central Europe ◽  
Western Europe ◽  
Low Cost ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Aphanomyces Astaci ◽  
Wide Range ◽  
Species Specific ◽  
Native Crayfish ◽  
Crayfish Species

Crayfish of North American origin are amongst the most prominent high-impact invasive invertebrates in European freshwaters. They contribute to the decline of European native crayfish species by spreading the pathogen causing crayfish plague, the oomycete Aphanomyces astaci. In this study we validated the specificity of four quantitative PCR (qPCR) assays, either published or newly developed, usable for environmental DNA (eDNA) screening for widely distributed native and non-native crayfish present in Central Europe: Astacus astacus, Pacifastacus leniusculus, Faxonius limosus and Procambarus virginalis. We then conducted an eDNA monitoring survey of these crayfish as well as the crayfish plague pathogen in a wide variety of habitat types representative for Central and Western Europe. The specificity of qPCR assays was validated against an extensive collection of crayfish DNA isolates, containing most crayfish species documented from European waters. The three assays developed in this study were sufficiently species-specific, but the published assay for F. limosus displayed a weak cross-reaction with multiple other crayfish species of the family Cambaridae. In the field study, we infrequently detected eDNA of A. astaci together with the three non-native crayfish species under examination. We never detected eDNA from A. astaci together with native crayfish, but in a few locations eDNA from both native and non-native crayfish was captured, due either to passive transport of eDNA from upstream populations or co-existence in the absence of infected crayfish carriers of A. astaci. In the study, we evaluated a robust, easy-to-use and low-cost version of the eDNA sampling equipment, based mostly on items readily available in garden stores and hobby markets, for filtering relatively large (~5 l) water samples. It performed just as well as the far more expensive equipment industrially designed for eDNA water sampling, thus opening the possibility of collecting suitable eDNA samples to a wide range of stakeholders. Overall, our study confirms that eDNA-based screening for crayfish and their associated pathogen is a feasible alternative to traditional monitoring.

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