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 A non-lethal method for detection of Bonamia ostreae in flat oyster (Ostrea edulis) using environmental DNA

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Louise von Gersdorff Jørgensen ◽  
Johan Wedel Nielsen ◽  
Mikkel Kehler Villadsen ◽  
Bent Vismann ◽  
Sussie Dalvin ◽  
...  
Keyword(s):  
Water Samples ◽  
Sea Water ◽  
Diagnostic Technique ◽  
Environmental Dna ◽  
Detection Methods ◽  
Ostrea Edulis ◽  
Breeding Programs ◽  
Bonamia Ostreae ◽  
Flat Oyster ◽  
Selection Of

Abstract Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.

scholarly journals In vitro activity of naturally occurring peptides (defensins) against Listeria monocytogenes

1994 ◽  
Vol 10 (4) ◽  
pp. 440-445
Author(s):  
Maria da Graça F. Nascimento ◽  
James S. Cullor ◽  
Michael E. Selsted
Keyword(s):  
Antibacterial Activity ◽  
Listeria Monocytogenes ◽  
Water Samples ◽  
Water System ◽  
Growth Curves ◽  
Distilled Water ◽  
In Vitro Activity ◽  
Cationic Peptide ◽  
Naturally Occurring

Autoclaved distilled water samples were inoculated with L. monocytogenes strain V7 and strain VPH-1, and incubated aerobically, at 30 C for 48 hours. Each strain was tested individually, and growth curves were determined at 1, 2, 3, 4, 5, 21, 24, and 48 hours. The growth or survival of L. monocytogenes was similar for both strains, with survivors at 24 hour-incubation. The microbicidal activity of one synthetic cationic peptide (NP-2) was examined against L. monocytogenes strain V7, in a water system. Antibacterial activity of NP-2 (1, 5, and 10 g/ml) was best expressed at 60 minute-incubation, with 10 g/ml of peptide, at 30 C.

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 Quantitative PCR assays to detect whales, rockfish, and common murre environmental DNA in marine water samples of the Northeastern Pacific

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242689
Author(s):  
Elizabeth A. Andruszkiewicz ◽  
Kevan M. Yamahara ◽  
Collin J. Closek ◽  
Alexandria B. Boehm
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Environmental Dna ◽  
Humpback Whale ◽  
Environmental Water ◽  
Individual Species ◽  
Common Murre ◽  
Assay Design

Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

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 Development of an environmental DNA method for monitoring fish communities: ground truthing in diverse lakes with characterised fish faunas

2018 ◽  
Author(s):  
Jianlong Li ◽  
Tristan W. Hatton-Ellis ◽  
Lori-Jayne Lawson Handley ◽  
Helen S. Kimbell ◽  
Marco Benucci ◽  
...  
Keyword(s):  
Water Samples ◽  
Fish Communities ◽  
Environmental Dna ◽  
Quantitative Information ◽  
12S Rrna ◽  
Similar Species ◽  
List Type ◽  
Large Lakes ◽  
Non Invasive ◽  
Wide Range

AbstractAccurate, cost-effective monitoring of fish is required to assess the quality of lakes under the European Water Framework Directive (WFD). Recent studies have shown that environmental DNA (eDNA) metabarcoding is an effective and non-invasive method, which can provide semi-quantitative information on fish communities in large lakes.This study further investigated the potential of eDNA metabarcoding as a tool for WFD status assessment by collecting and analysing water samples from eight Welsh lakes and six meres in Cheshire, England, with well described fish faunas. Water samples (N = 252) were assayed using two mitochondrial DNA regions (Cytb and 12S rRNA).eDNA sampling indicated the presence of very similar species in the lakes compared to those expected on the basis of existing and historical information. In total, 24 species were detected with a total of 111 species occurrences in the lakes studied using eDNA. Secondly, there was a significant positive correlation between expected faunas and eDNA data in terms of confidence of species occurrence (Spearman’s r = 0.74, df = 109, p <; 0.001). Thirdly, eDNA data can estimate relative abundance with the standard five-level classification scale (“DAFOR”). Lastly, four ecological fish communities were characterised using eDNA data which agrees with the pre-defined lake types according to environmental characteristics.Synthesis and applications. This study provides further evidence that eDNA metabarcoding could be a powerful and non-invasive monitoring tool for WFD purpose in a wide range of lake types, considerably outperforming other methods for community level analysis.

scholarly journals Detection and persistence of environmental DNA (eDNA) of a vector mosquito, Culex pipiens pallens

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Megumi Sato ◽  
Marcello Sato ◽  
Tomoe Watanabe ◽  
Honami Mitsuishi ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Water Samples ◽  
In Silico ◽  
Culex Pipiens ◽  
Developmental Stages ◽  
Environmental Dna ◽  
In Vitro Tests ◽  
Culex Pipiens Pallens ◽  
Pipiens Pallens

Abstract Introduction: Preventing mosquito-borne infectious diseases requires that vector mosquitoes are monitored and controlled. Targeting immature mosquitoes (eggs, larvae, and pupae), which have less mobility than adults, is an effective management approach. However, conducting these surveys is often difficult due to the limitations of morphological classification and survey costs. The application of environmental DNA (eDNA) analysis can solve these issues because it allows easy estimation of species distribution and morphology-independent species identification. Although a few previous studies have reported mosquito eDNA detection, there is a gap in knowledge regarding the dynamics of mosquito eDNA during the developmental stages of immature mosquitoes. Methods: We used Culex pipiens pallens, a vector of West Nile fever, as a model species. First, we developed a species-specific detection assay and confirmed its specificity using in silico and in vitro tests. Next, we conducted laboratory experiments using breeding tanks. Water samples were collected at each developmental stage. In addition, water samples were collected daily until the seventh day after emergence from the pupae. We quantified eDNA using real-time PCR with the developed assay to investigate the dynamics of mosquito eDNA. Results: The specificity of the developed assay was confirmed by in silico and in vitro tests. Mosquito eDNA was detected at all developmental stages and detected up to seven days after emergence of pupae. In particular, high concentrations of eDNA were detected immediately after hatching from eggs and after emergence from pupae. Highly frequent positive eDNA signals were continuously detected between egg hatching and pupa hatching. Conclusions: Mosquito eDNA was detected immediately after the eggs were introduced, and eDNA-positive detections continued until pupae emergence, suggesting that eDNA analysis is useful for monitoring mosquito larvae. The results show that eDNA analysis provides valuable information about the water sources inhabited by immature mosquitoes in mosquito control. In the future, monitoring immature mosquitoes using eDNA analysis will aid in preventing mosquito-borne infectious diseases.

Follicular fluid and assisted reproductive technology programs outcomes (literature review)

GYNECOLOGY ◽  
2020 ◽  
Vol 21 (6) ◽  
pp. 36-40
Author(s):  
Anna G. Burduli ◽  
Natalia A. Kitsilovskaya ◽  
Yuliya V. Sukhova ◽  
Irina A. Vedikhina ◽  
Tatiana Y. Ivanets ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Assisted Reproductive Technology ◽  
Follicular Fluid ◽  
Economic Cost ◽  
Reproductive Technology ◽  
Art Programs ◽  
Vitro Fertilization ◽  
Non Invasive ◽  
Established Fact

The review presents data on metabolites in the follicular fluid (FF) from the perspective of reproductive medicine and their use in order to predict outcomes of assisted reproductive technology (ART) programs. It considers various components of this biological medium (hormones, lipids, melatonin, etc.) with an assessment of their predictive value in prognosis of the effectiveness of in vitro fertilization (IVF) programs. The data on experimental directions in this field and the prospects for their use in clinical practice are presented. The article emphasizes that the growing clinical need and the unsolved problem of increasing the effectiveness of ART programs determine the need for further studies of the FF composition. Materials and methods. The review includes data related to this topic from foreign and Russian articles found in PubMed which were published in recent years. Results. Given the established fact of a direct effect of FF composition on growth and maturation of oocytes, and further, on the fertilization process, various FF metabolites are actively investigated as non-invasive markers of quality of oocytes/embryos. The article provides data on the experimental directions in this field and the prospects for their use in clinical practice. However, clinical studies of a relation between various FF metabolites levels and outcomes of IVF programs are contradictory. Conclusion. Owing large economic cost for treatment of infertility with IVF, there is need for expansion and intensification of studies to identify and use reliable predictors in prognosis of ART programs outcomes.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

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