Using environmental DNA methods to survey for rare groundwater fauna: Detection of an endangered endemic cave crayfish in northern Alabama

The conservation and management of subterranean biodiversity is hindered by a lack of knowledge on the true distributions for many species, e.g., the Wallacean shortfall. In recent years, several studies have demonstrated the potential of environmental DNA (eDNA) as an effective approach to detect and monitor biodiversity, including rare, threatened, and endangered taxa. However, there are few eDNA studies of groundwater fauna. Here we report the results of the development and implementation of an eDNA assay targeting a short fragment of the mitochondrial CO1 locus of a critically imperiled cave crayfish, the Sweet Home Alabama Cave Crayfish (Cambarus speleocoopi), known from just four cave systems in the Interior Plateau karst region of northern Alabama. We detected C. speleocoopi DNA from water samples collected at 5 of 16 sites sampled (caves and springs), including two historical sites as well as three additional and potentially new sites in Marshall County, Alabama. All three of these sites were within 2 km of historical sites. Our study is the first to detect a groundwater crustacean in the Interior Plateau karst region. Additionally, our study contributes to the growing literature that eDNA is a viable complementary tool for detection and monitoring of a fauna that is difficult to survey and study using traditional approaches.

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

10.1101/2021.05.16.444374 ◽

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

International Journal of Scientific Research in Science Engineering and Technology ◽

10.32628/ijsrset218421 ◽

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.

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Detection of fish pathogen Saprolegnia parasitica in environmental DNA samples by droplet digital PCR

ARPHA Conference Abstracts ◽

10.3897/aca.4.e64669 ◽

2021 ◽

Vol 4 ◽

Author(s):

Dora Pavić ◽

Anđela Miljanović ◽

Uršula Prosenc-Zmrzljak ◽

Rok Košir ◽

Dorotea Grbin ◽

...

Keyword(s):

Water Samples ◽

Genomic Dna ◽

Environmental Dna ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Economic Losses ◽

Internal Transcribed Spacer Region ◽

Saprolegnia Parasitica ◽

Oomycete Pathogen ◽

Total Dna

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples. Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture. Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively). In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations.

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

Scientific Reports ◽

10.1038/s41598-020-72715-y ◽

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.

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

BMC Research Notes ◽

10.1186/s13104-019-4819-6 ◽

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.

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