scholarly journals Comparison of Two Citizen Scientist Methods for Collecting Pond Water Samples for Environmental DNA Studies

2018 ◽  
Vol 3 (2) ◽  
pp. 2 ◽  
Author(s):  
Andrew Buxton ◽  
Jim Groombridge ◽  
Richard Griffiths
Keyword(s):  
Water Samples ◽  
Environmental Dna ◽  
Pond Water ◽  
Citizen Scientist

scholarly journals Environmental DNA enables detection of terrestrial mammals from forest pond water

2016 ◽  
Author(s):  
Masayuki Ushio ◽  
Hisato Fukuda ◽  
Toshiki Inoue ◽  
Kobayashi Makoto ◽  
Osamu Kishida ◽  
...  
Keyword(s):  
Water Samples ◽  
Cervus Nippon ◽  
Environmental Dna ◽  
Terrestrial Ecosystems ◽  
Illumina Miseq ◽  
Pcr Primers ◽  
Pond Water ◽  
Universal Primers ◽  
Taxonomic Assignment ◽  
Terrestrial Mammals

Terrestrial animals must have frequent contact with water to maintain their lives, implying that environmental DNA (eDNA) originating from terrestrial animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. After verifying the primers’ usefulness in silico and using water samples from zoo cages of animals with known species compositions, we collected five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. Using eDNA extracted from the water samples, we constructed amplicon libraries using MiMammal primers for Illumina MiSeq sequencing. MiMammal metabarcoding yielded a total of 75,214 reads, which we then subjected to data pre-processing and taxonomic assignment. We thereby detected species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Previous applications of the eDNA metabarcoding approach have mostly been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even in forest mammal biodiversity surveys.

scholarly journals Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

2021 ◽  
Author(s):  
Suwimon Taengphu ◽  
Pattanapon Kayansamruaj ◽  
Yasuhiko Kawato ◽  
Jerome Delamare-Deboutteville ◽  
Chadag Vishnumurthy Mohan ◽  
...  
Keyword(s):  
Water Samples ◽  
Detection System ◽  
Environmental Dna ◽  
Pond Water ◽  
Disease Forecasting ◽  
Early Disease ◽  
River Water Samples ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Detection And Quantification

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per uL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 +/- 3.3% of virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 x 102 to 2.79 x 104 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 x 102 to 3.53 x 103 copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.

Investigation of Some Water Quality Parameters of Pond Water under Mymensingh Municipality

2015 ◽  
Vol 8 (1) ◽  
pp. 85-89
Author(s):  
F Zannat ◽  
MA Ali ◽  
MA Sattar
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Chemical Properties ◽  
Pond Water ◽  
Quality Parameters ◽  
Water Quality Parameters ◽  
Urban Region ◽  
Mn Toxicity ◽  
Ph Values ◽  
Drinking Water Standard

A study was conducted to evaluate the water quality parameters of pond water at Mymensingh Urban region. The water samples were collected from 30 ponds located at Mymensingh Urban Region during August to October 2010. The chemical analyses of water samples included pH, EC, Na, K, Ca, S, Mn and As were done by standard methods. The chemical properties in pond water were found pH 6.68 to 7.14, EC 227 to 700 ?Scm-1, Na 15.57 to 36.00 ppm, K 3.83 to 16.16 ppm, Ca 2.01 to 7.29 ppm, S 1.61 to 4.67 ppm, Mn 0.33 to 0.684 ppm and As 0.0011 to 0.0059 ppm. The pH values of water samples revealed that water samples were acidic to slightly alkaline in nature. The EC value revealed that water samples were medium salinity except one sample and also good for irrigation. According to drinking water standard Mn toxicity was detected in pond water. Considering Na, Ca and S ions pond water was safe for irrigation and aquaculture. In case of K ion, all the samples were suitable for irrigation but unsuitable for aquaculture.J. Environ. Sci. & Natural Resources, 8(1): 85-89 2015

scholarly journals Quantitative assessment of ionic status of pond water for irrigation and aquaculture usage in the selected sites of Mymensingh areas, Bangladesh

2019 ◽  
Vol 6 (2) ◽  
pp. 301-313
Author(s):  
Taslima Akter ◽  
Shampa Rani Ghosh ◽  
Sitesh Chandra Sarker ◽  
Md Mokhlesur Rahman ◽  
KM Eadun Nabi
Keyword(s):  
Water Samples ◽  
Quantitative Assessment ◽  
Ph Value ◽  
Pond Water ◽  
The Other ◽  
Acceptable Limit ◽  
Legal Limit ◽  
Other Hand ◽  
Agricultural Chemistry ◽  
Inorganic Components

Ponds are considered to be self-contained, land lock ecosystem which is often teeming with rich vegetation and diverse organismal life. The pond water contains different organic and inorganic components. The experiment was carried out in laboratory, Department of Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh through collection of pond water from Gouripur and Muktagacha under Mymensingh division for assessment of major ionic status and suitability parameters for irrigation and aquaculture usage in quantitative way. Around 30 samples were collected from different location. On the basis of HCO3 ion, all water samples except 3 samples were not suitable for irrigation because this anion exceeded the acceptable limit (1.5 meL-1). On the other hand, HCO3 ion was not treated as problematic in all samples except 2 samples for aquaculture usages. The concentrations of Ca, Mg, Na, K, PO4 and SO4 were far below the recommended limit. Considering aquaculture usage, Cl ion was considered as hazardous in all the pond water samples because this anion was above the legal limit (<0.003mgL-1). pH value of pond ranged from 7.02 to 7.87 indicating alkaline in nature and were not problematic for irrigation and aquaculture usage. Among the major ionic constituents, the remarkable significant correlations existed between Ca vs Mg, Ca vs K, Mg vs SO4, K vs Na, Na vs SO4. Res. Agric., Livest. Fish.6(2): 301-313, August 2019

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 Phytochemical Screening and Antibacterial Evaluation of Conventional Antibiotics, Garlic and Ginger on Isolates from Fish Pond Water Samples in Awka, Anambra State, Nigeria

2021 ◽  
pp. 118-132
Author(s):  
O. R. Umeh ◽  
E. I. Chukwura ◽  
E. L. Okoye ◽  
E. M. Ibo ◽  
P. I. Egwuatu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Klebsiella Pneumoniae ◽  
Water Samples ◽  
Vibrio Parahaemolyticus ◽  
Pond Water ◽  
Inhibitory Effect ◽  
Hot Water ◽  
Aqueous Extracts ◽  
Phytochemical Screening ◽  
Conventional Antibiotics

Medicinal plants are used by almost 80% of the world’s population for their basic health care because of their low cost and ease in availability. In the last few decades, many bacteria have continued to show increasing resistance against current antibiotics. Aim: In this study, phytochemical screening and antibacterial effects of conventional antibiotics, garlic and ginger on test isolates from fish pond water samples were evaluated between May-November, 2019. Methods: Standard methods for phytochemical screening and antibacterial analysis were employed. Results: The results showed that amongst the antibiotics used for susceptibility test, Amoxicilin (30 µg) was mostly resisted by all the bacterial isolates except Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus and Salmonella typhi while erythromycin (10 µg) was unable to inhibit Bacillus subtilis. Ciprofloxacin (10 µg) and pefloxacin (10 µg) inhibited the growth of all the isolates except Pseudomonas aeruginosa. The test isolates showed variable susceptibility to the garlic and ginger extracts (ethanol, methanol and hot water). The extracts inhibited the isolates in descending order; ginger ethanol > ginger methanol > garlic methanol > ginger hot water > garlic ethanol > garlic hot water. Vibrio parahaemolyticus, Vibrio cholerae and Staphylococcus aureus showed little resistant to the extracts while these extracts showed better activity on Klebsiella pneumoniae and Proteus mirabilis. Synergistic effect of garlic and ginger (500mg/ml) inhibited the growth of all the isolates with ethanol extracts having the highest zone diameter (29 mm) on Klebsiella pneumoniae and Proteus mirabilis while hot water extracts had the least zone of inhibition (18 mm) on Acinetobacter calcoaceticus and Vibrio parahaemolyticus. The minimum inhibitory and bactericidal concentration for ethanol, methanol and hot water extracts ranged from 31.25mg/ml to 62.5mg/ml and 62.5mg/ml to 125mg/ml respectively. Conclusion: The outcomes of susceptibility experiment depicted that ethanol and methanol extracts of garlic and ginger (each alone and in combination) showed more inhibitory effect than aqueous extracts and also the combination of ethanol, methanol and aqueous extracts resulted in inhibitory effect greater than each of the extracts when used singly. The use of ginger and garlic for control of fish pathogens appears to be justified.

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.

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

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.

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 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.

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