scholarly journals Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague pathogen using environmental DNA

2018 ◽  
Vol 222 ◽  
pp. 241-252 ◽  
Author(s):  
Chloe Victoria Robinson ◽  
Tamsyn M. Uren Webster ◽  
Joanne Cable ◽  
Joanna James ◽  
Sofia Consuegra
Keyword(s):  
Simultaneous Detection ◽  
Environmental Dna ◽  
Signal Crayfish

scholarly journals Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague pathogen using environmental DNA

2018 ◽  
Author(s):  
Chloe Victoria Robinson ◽  
Tamsyn M. Uren Webster ◽  
Joanne Cable ◽  
Joanna James ◽  
Sofia Consuegra
Keyword(s):  
Native Species ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Conservation Strategies ◽  
Accurate Information ◽  
Essential Information ◽  
Signal Crayfish ◽  
Aphanomyces Astaci ◽  
Field Samples ◽  
Native Crayfish

ABSTRACTAquatic Invasive Species (AIS) are important vectors for the introduction of novel pathogens which can, in turn, become drivers of rapid ecological and evolutionary change, compromising the persistence of native species. Conservation strategies rely on accurate information regarding presence and distribution of AIS and their associated pathogens to prevent or mitigate negative impacts, such as predation, displacement or competition with native species for food, space or breeding sites. Environmental DNA is increasingly used as a conservation tool for early detection and monitoring of AIS. We used a novel eDNA high-resolution melt curve (HRM) approach to simultaneously detect the UK endangered native crayfish (Austropotamobius pallipes), the highly invasive signal crayfish (Pacifastacus leniusculus) and their dominant pathogen, Aphanomyces astaci, (causative agent of crayfish plague). We validated the approach with laboratory and field samples in areas with known presence or absence of both crayfish species as well as the pathogen, prior to the monitoring of areas where their presence was unknown. We identified the presence of infected signal crayfish further upstream than previously detected in an area where previous intensive eradication attempts had taken place, and the coexistence of both species in plague free catchments. We also detected the endangered native crayfish in an area where trapping had failed. With this method, we could estimate the distribution of native and invasive crayfish and their infection status in a rapid, cost effective and highly sensitive way, providing essential information for the development of conservation strategies in catchments with populations of endangered native crayfish.

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.

A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan

Nematology ◽  
2011 ◽  
Vol 13 (6) ◽  
pp. 713-720 ◽  
Author(s):  
Yu Yu Min ◽  
Keita Goto ◽  
Koki Toyota ◽  
Erika Sato
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Single Species ◽  
The Other ◽  
Parasitic Nematodes ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Assays

AbstractMultiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

scholarly journals Searching for a signal: Environmental DNA (eDNA) for the detection of invasive signal crayfish, Pacifastacus leniusculus (Dana, 1852)

2018 ◽  
Vol 9 (2) ◽  
pp. 137-148 ◽  
Author(s):  
Kirsten Harper ◽  
Patrick Anucha ◽  
James Turnbull ◽  
Colin Bean ◽  
Michael Leaver
Keyword(s):  
Environmental Dna ◽  
Pacifastacus Leniusculus ◽  
Signal Crayfish

scholarly journals Scanning ferry routes: looking for eDNA traces of marine mammals and their preys

2021 ◽  
Vol 4 ◽  
Author(s):  
Elena Valsecchi
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Trophic Levels ◽  
Systematic Sampling ◽  
Marine Biodiversity ◽  
Tyrrhenian Sea ◽  
Night Time ◽  
Species Specific

Marine environmental DNA (eDNA) surveys are becoming a promising approach to monitor biodiversity status and its variation over time. However, monitoring offshore areas could be extremely costly when using dedicated vessels, beside the impossibility to sample simultaneously geographically distant (even if adjacent) areas. The unexplored possibility of availing on operating ferries as an opportunistic platform for eDNA sampling offers several advantages besides opening limitless opportunities for systematic surveys on marine biodiversity.We present the results of both metabarcoding and barcoding approaches obtained from the analysis of water samples collected on board of a ferry boat along a pilot Mediterranean route crossing the Pelagos Sanctuary for Mediterranean Marine Mammals. The recently described MarVer primer sets (12SrDNA and 16SrDNA regions), specifically designed for the simultaneous detection of marine mammals and other marine vertebrates, were employed. The High Throughput Sequencing (HTS) outcome showed that the markers successfully detected most trophic levels of vertebrate marine communities, and classes, including bony fish, rays, cetaceans and birds. Ferry-based sampling allow to collect sample at any time of the day, and we indeed found diel differences in both quantitative and qualitative distribution of read counts. For instances, we observed an increased abundance of lantern fish amplicons in night-time collect samples (50%), reflecting nocturnal migration through the water column. In general, the number of read counts was significantly higher in nocturnal samples. Such diel differences within our sample indirectly provides evidence of the efficiency of the eDNA approach to detect contemporary signals in the sampled environment. Similarly, cetaceans were detected in correspondence of visual sightings (when these occurred, supplementary samples were collected). Rare species, such as the monk seal, are difficult to be detected in metabarcoding surveys, thus we opted to side the screening of the ferry-samples with a panel of species-specific qPCR assays, which were able to detect DNA traces of the endangered pinniped in the Tuscany archipelago (Tyrrhenian Sea) long before visual observations witnessed its presence in the same area. The study demonstrates the feasibility of using commercial shipping as a platform for eDNA marine sampling without dedicated survey cruises. Commercial shipping routes have potential to act as regular systematic sampling transects which can contribute to evaluating and monitoring marine biodiversity.

scholarly journals MiFish metabarcoding: a high-throughput approach for simultaneous detection of multiple fish species from environmental DNA and other samples

2020 ◽  
Vol 86 (6) ◽  
pp. 939-970
Author(s):  
Masaki Miya ◽  
Ryo O. Gotoh ◽  
Tetsuya Sado
Keyword(s):  
Fishery Management ◽  
Massively Parallel Sequencing ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Single Species ◽  
Conservation Strategies ◽  
Estuarine Fish ◽  
12S Rrna ◽  
Rrna Gene ◽  
Biodiversity Monitoring

Abstract We reviewed the current methodology and practices of the DNA metabarcoding approach using a universal PCR primer pair MiFish, which co-amplifies a short fragment of fish DNA (approx. 170 bp from the mitochondrial 12S rRNA gene) across a wide variety of taxa. This method has mostly been applied to biodiversity monitoring using environmental DNA (eDNA) shed from fish and, coupled with next-generation sequencing technologies, has enabled massively parallel sequencing of several hundred eDNA samples simultaneously. Since the publication of its technical outline in 2015, this method has been widely used in various aquatic environments in and around the six continents, and MiFish primers have demonstrably outperformed other competing primers. Here, we outline the technical progress in this method over the last 5 years and highlight some case studies on marine, freshwater, and estuarine fish communities. Additionally, we discuss various applications of MiFish metabarcoding to non-fish organisms, single-species detection systems, quantitative biodiversity monitoring, and bulk DNA samples other than eDNA. By recognizing the MiFish eDNA metabarcoding strengths and limitations, we argue that this method is useful for ecosystem conservation strategies and the sustainable use of fishery resources in “ecosystem-based fishery management” through continuous biodiversity monitoring at multiple sites.

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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