scholarly journals Long distance (>20 km) downstream detection of endangered stream frogs suggests an important role for eDNA in surveying for remnant amphibian populations

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12013
Author(s):  
Cecilia Villacorta-Rath ◽  
Conrad J. Hoskin ◽  
Jan M. Strugnell ◽  
Damien Burrows
Keyword(s):  
Water Samples ◽  
Survey Methods ◽  
Environmental Dna ◽  
Small Population ◽  
Population Declines ◽  
Long Distance ◽  
Amphibian Species ◽  
Filtration Method ◽  
North East ◽  
Water Volumes

Background Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for ‘missing’ and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. However, the utility of this survey technique is dependent on quantifying downstream detection probability and distances. Methods Here we tested downstream detection distances in two endangered stream frogs (Litoria lorica and L. nannotis) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the two last populations of L. lorica persist in this catchment: one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at a series of sites kilometers downstream from the populations using precipitation from two fixed water volumes (15 and 100 mL) and via water filtering (mean 1,480 L). Results We detected L. nannotis and the small L. lorica population (~1,000 frogs) at most sampling sites, including 22.8 km downstream. The filtration method was highly effective for far-downstream detection, as was precipitation from 100 mL water samples, which also resulted in consistent detections at the far-downstream sites (including to 22.8 km). In contrast, we had limited downstream detection success for the very small L. lorica population (~100 frogs). Discussion The ecological aspects of our study system, coupled with thorough traditional surveys, enabled us to measure downstream eDNA detection distances with accuracy. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the population. Water filtration is considered best for eDNA detection of rare aquatic species—indeed it was effective in this study—but we also achieved far-downstream detections when precipitating eDNA from 100 mL water samples. Collecting small water volumes for subsequent precipitation in the lab is more practical than filtration when surveying remote areas. Our downstream detection distances (>20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey methods.

scholarly journals Assessment of Environmental DNA for Detecting Presence of Imperiled Aquatic Amphibian Species in Isolated Wetlands

2015 ◽  
Vol 6 (2) ◽  
pp. 498-510 ◽  
Author(s):  
Anna M. McKee ◽  
Daniel L. Calhoun ◽  
William J. Barichivich ◽  
Stephen F. Spear ◽  
Caren S. Goldberg ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Water Samples ◽  
Survey Methods ◽  
Pinus Palustris ◽  
Environmental Dna ◽  
Breeding Habitat ◽  
Target Species ◽  
Amphibian Species ◽  
Ambystoma Bishopi ◽  
Ambystoma Cingulatum

Abstract Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.

scholarly journals Environmental DNA sampling in a terrestrial environment: methods to detect a critically endangered frog and a global pathogen

2020 ◽  
Author(s):  
Thomas J. Burns ◽  
Nick Clemann ◽  
Anthony R. van Rooyen ◽  
Ben C. Scheele ◽  
Andrew R. Weeks ◽  
...  
Keyword(s):  
Field Experiments ◽  
Survey Methods ◽  
Environmental Dna ◽  
Wash Water ◽  
Critically Endangered ◽  
Chytrid Fungus ◽  
Amphibian Skin ◽  
Natural Habitats ◽  
Amphibian Species ◽  
Terrestrial Environment

AbstractEnvironmental DNA techniques have become established as a useful tool for biological monitoring and are used extensively to determine species presence in aquatic systems. However, their application in terrestrial systems has been more limited, likely in part due to difficulties in choosing where to sample and ensuring that collected DNA reflects current species presence. We developed methods to sample eDNA in the terrestrial environment and trialled them under controlled and field conditions. We targeted three species, an elusive critically endangered frog, an abundant non-threatened frog, and the globally distributed amphibian skin pathogen chytrid fungus, which has been implicated in the decline of over 500 amphibian species. We used a sandpaper-sampling surface to ‘trap’ DNA. After sampling, we washed the surface and filtered the wash water to gather material for DNA extraction and subsequent qPCR. Our controlled condition experiments demonstrated that frog and chytrid fungus DNA was detectable after as few as five contacts between a frog and the sampling surface. Furthermore, this DNA remained detectable after two weeks in cool, shaded, outdoor conditions. Our field experiments demonstrated that these techniques were transferable to natural habitats, where we detected both the common and rare amphibian target species, as well as chytrid fungus. Field sampling eDNA results were broadly consistent with those derived from conventional survey methods. Our methods have potential application in non-invasive sampling of amphibians and other species in terrestrial systems, broadening the applicability of eDNA techniques for species detection and monitoring.

Monitoring post-release survival of the northern corroboree frog, Pseudophryne pengilleyi, using environmental DNA

2018 ◽  
Vol 45 (7) ◽  
pp. 620 ◽  
Author(s):  
Jack Rojahn ◽  
Dianne Gleeson ◽  
Elise M. Furlan
Keyword(s):  
Water Samples ◽  
Survey Methods ◽  
Quantitative Polymerase Chain Reaction ◽  
Environmental Dna ◽  
Life Stages ◽  
Breeding Programs ◽  
Non Invasive ◽  
Naturally Occurring ◽  
Cause Of Failure

Context Translocations are becoming an increasingly important conservation tool to combat rising levels of species extinction. Unfortunately, many translocation efforts fail; yet, the timing and cause of failure often remain unknown. Monitoring individuals in the days and weeks following release can provide valuable information on their capacity to survive this initial hurdle. In Australia, breeding programs have been established for the endangered northern corroboree frog, Pseudophryne pengilleyi, to enable reintroduction to the wild via captive-reared individuals, typically, early life stages such as eggs or juvenile frogs that cannot be monitored via traditional survey methods that target adult frogs (e.g. shout–response). Environmental DNA (eDNA) detects trace amounts of DNA that organisms release into their environment and could provide a means to infer population persistence for wildlife releases and translocations. Aims In the present study, we aim to develop an eDNA assay capable of detecting both sexes of P. pengilleyi across multiple life stages, and use it to monitor their survival. Methods An eDNA assay was developed to target the two corroboree frog species (P. pengilleyi and P. corroboree, the southern corroboree frog) and was tested for its sensitivity and specificity in silico and in vitro. Pseudophryne pengilleyi eggs were released into three naturally occurring ponds and water samples were, subsequently, collected from each pond on several occasions over a period of 78 days. Quantitative polymerase chain reaction was used to detect P. pengilleyi eDNA from water samples. Key Results The developed assay was shown to be sensitive and specific to corroboree frogs. eDNA monitoring of reintroduced P. pengilleyi detected the species’ DNA at three of three release ponds and DNA remained detectable until at least 78 days post-release at two of three ponds. Conclusions We show how the development of a corroboree frog-specific assay allowed us to monitor the post-release survival of P. pengilleyi in naturally occurring pools. Implications eDNA surveys may provide a useful tool to monitor post-release survival of translocated populations in a non-invasive manner, with the potential to identify the timing and causes of failure. Such knowledge can be used to inform the management of translocated populations and future release strategies.

scholarly journals eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 526 ◽  
Author(s):  
Claude Miaud ◽  
Véronique Arnal ◽  
Marie Poulain ◽  
Alice Valentini ◽  
Tony Dejean
Keyword(s):  
Water Samples ◽  
Rana Temporaria ◽  
Clinical Signs ◽  
Environmental Dna ◽  
Diagnostic Techniques ◽  
Mitigation Measures ◽  
Activity Period ◽  
Amphibian Species ◽  
Tissue Samples ◽  
Species Activity

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

scholarly journals DNA-based biomonitoring in the tropics: Detection and control of Batrachochytrium dendrobatidis in Ecuadorian ecosystem

2021 ◽  
Vol 4 ◽  
Author(s):  
Lenin Riascos-Flores ◽  
Andrea Carrera ◽  
Leopoldo Naranjo ◽  
Jomira Yanez ◽  
Peter Goethals ◽  
...  
Keyword(s):  
Invasive Species ◽  
Water Samples ◽  
Large Scale ◽  
Batrachochytrium Dendrobatidis ◽  
Action Plan ◽  
Detection Methods ◽  
Population Declines ◽  
Amphibian Species ◽  
Tissue Samples ◽  
And Control

Batrachochytrium dendrobatidis (Bd) is a fungus that parasites vertebrates, and is associated with population declines worldwide in endemic amphibian species. As such, it is one of several invasive species which pose a serious threat to a variety of vertebrate hosts, in casu: amphibians. Detection of such invasive species is generally based on DNA-based methods where, for instance, swabs or tissue samples of candidate hosts are analysed for their presence. Any management strategy of these invasive species would greatly benefit from sensitive and rapid detection methods which can be applied at a large scale. The analysis of eDNA from the habitat of candidate host organisms may hold significant potential for this purpose. In this study, we compare the ability of eDNA from habitat samples with that of swab and/or tissue samples of candidate hosts to detect the presence of Bd in Ecuador. We collected individuals from the amphibians: Pristimantis (Anura: Craugastoridae), Rhinella (Anura: Bufonidae), Gastroteca (Anura: Hemiphractidae), from the endangered toad species of the genus Atelopus (Anura: Bufonidae) as well as water samples from different water bodies in Andean and coastal Ecuadorian areas. Samples were processed using a portable field molecular laboratory. Commercial primers for the internal transcribed spacer (ITS), in combination with a new set of primers designed from Bd sequences from tropical countries, were used. Positive PCR results from both types of samples were obtained within eight hours after sampling. Prevalence of BD was detected in eDNA, swab and tissue samples in four of the six ecosystems monitored -14 out of 26 water samples and 27 out of 43 amphibian of in total 12 species- including three endangered toad species (Atelopus balios, A. nanay, and the rediscovered A. bomolochos). Our results highlight the potential of eDNA-based monitoring to assess the presence and prevalence of Bd in Ecuadorian aquatic ecosystems, in accordance with the National Action Plan for the Conservation of Ecuadorian Amphibians. Furthermore, our field lab approach leads to reliable and fast results for the monitoring of invasive species in a tropical context of a pandemic.

scholarly journals Evaluating eDNA-based monitoring of fire salamander larvae under field conditions

2021 ◽  
Vol 4 ◽  
Author(s):  
Bianca Spitzl ◽  
Daniela Sint ◽  
Florian Glaser ◽  
Gerda Ludwig ◽  
Michael Traugott
Keyword(s):  
Water Samples ◽  
Field Conditions ◽  
Population Declines ◽  
Amphibian Species ◽  
Batrachochytrium Salamandrivorans ◽  
Small Streams ◽  
Key Species ◽  
Fire Salamander ◽  
Regional Population ◽  
Salamander Larvae

Amphibians globally belong to the most threatened animal groups and monitoring their populations is of critical importance for their conservation. The fire salamander (Salamandra salamandra) is one of the European amphibian species which has been experiencing drastic regional population declines due to the spread of the invasive chytrid fungus Batrachochytrium salamandrivorans, making it a key species for monitoring efforts. Here, we evaluated how the sampling and analysis of eDNA can aid the monitoring of larval salamander populations in small streams under field conditions. Nine small streams with known adult and larval fire salamander populations were investigated in Tirol (Austria). Per steam a stretch of 30 m downstream from its source was divided into 10 m sections where salamander larvae were counted. Water samples were taken at the end of each section and filtered on site. The DNA extracted from these filters was tested by a new PCR assay developed for the detection of mitochondrial DNA of S. salamandra. This assay combines endpoint PCR with capillary electrophoresis, allowing to relatively quantify the amount of fire salamander eDNA present in the water samples. In two of the nine streams no eDNA of S. Salamandra could be detected. The outcomes of an analysis of how larval densities, discharge and volume of filtered water affected the detection of salamander eDNA will be presented. Finally, we will conclude on the practical implications of the current findings for eDNA-based monitoring of fire salamander populations.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals A Simple Technique for the Identification of Environmental DNA (eDNA ) in the Water Samples

2021 ◽  
pp. 77-80
Author(s):  
Chitra K. Y.
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Dna Isolation ◽  
Simple Technique ◽  
Environmental Dna ◽  
Water Bodies ◽  
Easy Method ◽  
Selective Staining ◽  
Generation Sequencing ◽  
Dark Pink

The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

scholarly journals Towards environmental DNA-based bioassessment of freshwater reservoirs with small volumes of water: robust molecular protocols

2021 ◽  
Author(s):  
Rebecca Ker Loh ◽  
Sujatha Narayanan Kutty ◽  
Darren Chong Jinn Yeo ◽  
Rudolf Meier
Keyword(s):  
Water Sample ◽  
Environmental Dna ◽  
Cost Effective ◽  
Sampling Time ◽  
Small Water ◽  
Tropical Reservoirs ◽  
Frequent Monitoring ◽  
Planktonic Crustaceans ◽  
Water Volumes ◽  
Dna Template

Bioassessment of freshwater quality via eDNA is rapidly developing into a powerful alternative to traditional methods involving collecting, sorting, and identifying macroinvertebrates based on morphology. Particularly attractive would be methods that can use remote-controlled boats for sampling because it would allow for cost-effective, and frequent monitoring at multiple sites. The latter will be particularly important for tropical reservoirs that require year-around surveillance. We here optimize molecular protocols for capturing reservoir-specific differences in metazoan communities based on small water volumes (15 mL). The optimization is based on samples from two freshwater reservoirs with very different water qualities ("reservoir signal"). Each reservoir was sampled at three sites ("biological replicates"). For each water sample, the DNA was extracted twice ("technical replicates"). We then tested how much DNA template (0.1 ng to 15 ng) and how many PCR cycles (25 or 35) minimized variance between technical replicates. We find that 15 mL is sufficient for capturing the reservoir signal regardless of sampling time, template amounts, or PCR cycle numbers. Indeed, extrapolation from our results suggests that <1 mL would be sufficient because only 17 of 59 metazoan mOTUs (mainly planktonic crustaceans and rotifers) detected with a 313bp COI minibarcode were shared. We find that the use of 35 PCR cycles significantly lowered the number of detected species and that template amounts <0.5 ng yielded somewhat higher variance between technical replicates. Despite extensive trials, the variance between technical replicates remained high (Bray-Curtis: 5-20%; Jaccard: 10-40%) and we predict that it will be difficult to reduce this variance further. However, the overall reservoir differences are so strong that all biological and technical replicates can be correctly assigned.

scholarly journals Environmental DNA (eDNA) metabarcoding surveys extend the range of invasion for non-indigenous freshwater species in Eastern Europe.

2021 ◽  
Author(s):  
Gert-Jan Jeunen ◽  
Tatsiana Lipinskaya ◽  
Helen Gajduchenko ◽  
Viktoriya Golovenchik ◽  
Michail Moroz ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
False Negative ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Reference Database ◽  
Indigenous Species ◽  
Freshwater Species ◽  
Traditional Approaches ◽  
Surface Water Samples

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e., an ichthyological, hydrobiological, and eDNA survey, whereby DNA was extracted from 500 mL surface water samples and amplified with two 16S rRNA primer assays targeting the fish and macro-invertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous sediment-living macro-invertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two sediment-living macro-invertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey could be attributed to (i) preferential amplification of aquatic over sediment-living macro-invertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximize the probability of early detection of non-native organisms.

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