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 a quantitative PCR assay for detection of redside shiner (Richardsonius balteatus) from environmental DNA

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Marshal S. Hoy ◽  
Carl O. Ostberg
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Effective Means ◽  
Environmental Dna ◽  
Sympatric Species ◽  
Negative Control ◽  
Control Site ◽  
Qpcr Assay ◽  
Redside Shiner ◽  
Richardsonius Balteatus

Abstract Objective A quantitative PCR (qPCR) assay for the detection of redside shiner (Richardsonius balteatus) environmental DNA (eDNA) was designed as a side product of a larger project aimed at using eDNA to determine the presence and geographic extent of native and non-native fishes in the reservoirs and associated tributaries above the three mainstem dams (Ross, Diablo, Gorge) on the Skagit River, Washington, USA. The eDNA survey results can be used to help guide additional sampling efforts that include traditional sampling methods, such as electrofishing and netting. Results The redside shiner qPCR assay (RSSCOI_540-601) was validated by testing for sensitivity using redside shiner genomic DNA from three different populations and by testing for specificity against 30 potentially sympatric species. No non-target amplification was observed in our validation tests. We then evaluated the assay on field-collected water samples where there are known populations of redside shiner and a negative control site where the target species is known to be absent. The field-collected water samples tested positive at the redside shiner sites and tested negative at the negative control site. The assay could provide resource managers with an effective means for surveying and monitoring redside shiner populations.

scholarly journals Development of an eDNA metabarcoding tool for surveying the world’s largest amphibian

2021 ◽  
Author(s):  
Jie Wang ◽  
Ping Liu ◽  
Jiang Chang ◽  
Cheng Li ◽  
Feng Xie ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Survey Methods ◽  
Environmental Dna ◽  
Cost Effective ◽  
Isolated Populations ◽  
Specificity And Sensitivity ◽  
Chinese Giant Salamander ◽  
Elusive Species ◽  
Primer Sets ◽  
Species Specific

Abstract Due to the overexploitation of farming, as well as habitat loss or degradation, the wild population of Chinese giant salamander Andrias davidianus (CGS), a species with seven genetically distinct lineages, has decreased by over 80% in the past 70 years. Traditional survey methods have proven to be unsuitable for finding this rare and elusive species. We evaluated the efficacy of environmental DNA (eDNA) sampling to detect CGS indirectly from its aquatic environment. We developed several species-specific primer sets; validated their specificity and sensitivity; and assessed their utility in silico, in the laboratory, and in two field sites having released farm-bred CGSs. We detected the presence of CGS DNA by using polymerase chain reaction (PCR) and Sanger sequencing. We also sequenced an amplicon mixture of seven haplotype-represented samples using high-throughput sequencing. Our eDNA methods could detect the presence of CGS at moderate densities reported across its range, proving them as a cost-effective way to establish broad-scale patterns of occupancy for CGS. In addition, our primers enabled the detection of a mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.

scholarly journals Detection of four imperiled western North American freshwater mussel species from environmental DNA with multiplex qPCR assays

2020 ◽  
Author(s):  
Torrey W. Rodgers ◽  
Joseph C. Dysthe ◽  
Cynthia Tait ◽  
Thomas W. Franklin ◽  
Michael K. Schwartz ◽  
...  
Keyword(s):  
Freshwater Mussel ◽  
Environmental Dna ◽  
Vulnerable Species ◽  
Western North America ◽  
Mussel Species ◽  
Multiplex Assays ◽  
Specificity And Sensitivity ◽  
Pcr Assays ◽  
Species Specific ◽  
Management And Conservation

AbstractWe developed multiplexed, species-specific, quantitative PCR assays for the detection of four freshwater mussel species native to western North America, Gonidea angulata, Margaritifera falcata, Anodonta nuttalliana and Anodonta oregonensis, from environmental DNA (eDNA). These species have experienced dramatic declines over the last century and are currently threatened in many portions of their ranges. Therefore, improved tools for detecting and monitoring these species are needed. Species-specificity and sensitivity of assays were empirically tested in the lab, and multiplex assays were also validated with field collected eDNA samples. All assays were species-specific, sensitive, and effective for detection from eDNA samples collected from streams and rivers. These assays will aid in the detection, monitoring, management, and conservation of these vulnerable species.

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 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 Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

2017 ◽  
Author(s):  
Lynsey R. Harper ◽  
Lori Lawson Handley ◽  
Christoph Hahn ◽  
Neil Boonham ◽  
Helen C. Rees ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Detection Threshold ◽  
Environmental Dna ◽  
Cost Effective ◽  
Triturus Cristatus ◽  
Species Detection ◽  
Survey Tool ◽  
Crested Newt ◽  
Species Specific ◽  
Freshwater Monitoring

SummaryEnvironmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

scholarly journals Development of a 16S metabarcoding assay for the environmental DNA (eDNA) detection of aquatic reptiles across northern Australia

2020 ◽  
Author(s):  
Katrina West ◽  
Matthew Heydenrych ◽  
Rose Lines ◽  
Tony Tucker ◽  
Sabrina Fossette ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Environmental Dna ◽  
Cost Effective ◽  
Distribution Data ◽  
Freshwater Turtles ◽  
Northern Australia ◽  
Non Invasive ◽  
Sea Snakes ◽  
Reptile Species ◽  
Species Specific

AbstractA severe lack of distribution data for aquatic reptiles in northern Australia leaves many taxa vulnerable to extirpation and extinction. Environmental DNA (eDNA) technologies offer sensitive and non-invasive genetic alternatives to trapping and visual surveys and are increasingly employed for the detection of aquatic and semi-aquatic reptiles. However, at present, these studies have largely applied species-specific primers which do not provide a cost-effective avenue for the simultaneous detection of multiple reptilian taxa. Here, we present a 16S rRNA metabarcoding assay for the broad detection of aquatic and semi-aquatic reptile species. This assay is tested on water samples collected at multiple sampling sites at two tropical locations: 12 marine/estuarine sites in Roebuck Bay, Western Australia, and 4 estuarine sites in Cooktown, Queensland, Australia. A total of nine reptile taxa were detected from 10 of the 16 sampled sites, including marine and freshwater turtles, aquatic and semi-aquatic/terrestrial snakes, and terrestrial skinks. However, inconsistencies in the detection of previously observed aquatic reptiles at our sampled sites, such as saltwater crocodile and sea snakes, indicates that further research is required to assess the reliability, strengths and limitations of eDNA methods for aquatic reptile detection before it can be integrated as a broad-scale bioassessment tool.

scholarly journals Applications and integration of chromogenic culture media in clinical microbiology

2010 ◽  
Vol 31 (3) ◽  
pp. 127
Author(s):  
John Merlino
Keyword(s):  
Culture Media ◽  
Effective Means ◽  
Traditional Culture ◽  
Cost Effective ◽  
Conventional Culture ◽  
Chromogenic Agar ◽  
Isolation Media ◽  
Species Specific ◽  
Micro Organisms ◽  
New Generation

The ability to rapidly detect and identify micro-organisms is of paramount importance for many microbiology laboratories. The application of new classes of enzymatic-based chromogenic compounds has revolutionised traditional culture media, leading to the developments of a new generation of chromogenic media. Many studies have shown that the chromogenic agar has become more than an isolation media when compared to conventional culture media. A newer, faster, more cost-effective means in the presumptive identification or screening of microorganisms which are either genus, or in some cases species-specific, and allows superior differentiation of mixed micro-organisms in cultures. When supplemented with antibiotics or other agents, chromogenic media allows the screening for multidrug resistance and virulence in many micro-organisms and can be easily integrated with other automated and/or molecular-based methods.

scholarly journals Numerous new mitogenomic sequences and multiple new environmental DNA markers for invasive bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) populations in North America

2014 ◽  
Author(s):  
Richard F. Lance ◽  
Heather L. Farrington ◽  
Christine E. Edwards ◽  
Xin Guan ◽  
Matthew R. Carr ◽  
...  
Keyword(s):  
Genetic Markers ◽  
North American ◽  
Complete Mitochondrial Genome ◽  
Silver Carp ◽  
Environmental Dna ◽  
Invasion Front ◽  
Specificity And Sensitivity ◽  
Introduced Range ◽  
Hypophthalmichthys Nobilis ◽  
Species Specific

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American waterways. Recently, environmental DNA (eDNA), the use of species-specific genetic assays to detect the DNA of a particular species in a water sample, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American waterways. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with other common and closely related species to identify potential eDNA markers. We then field-tested these genetic markers for species-specificity and sensitivity in environmental samples. Newly developed markers performed well in field trials, had low false positive rates and had comparable sensitivity compared to current markers. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp, and can be used to improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.

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.

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