The experiment's objective is to illustrate a methodology of detecting the presence or absence of the Great Crested Newt (T.cristatus) in water sources. The said amphibian is safeguarded in the United Kingdom under the Wildlife and Countryside Act 1981 because it has undergone a significant decrease in range and number over the past century. It is against the law to harm or disturb both the individual and its habitat at all life stages, including eggs. Great Crested Newts can be spotted in ponds spread across lowland Great Britain in urban, rural, and post-industrial settings. Habitat loss due to destruction and the degradation of water quality, the introduction of fish, and the loss/fragmentation of their terrestrial habitat is the primary cause of the population decline.

To better appreciate change in abundance and make well-informed decisions regarding effective conservation programs, monitoring biodiversity is crucial. This project places its focus on utilizing survey technology, a newly available environmental DNA monitoring technique called the Smith-root eDNA sampler. In aquatic environments, eDNA can find vertebrates without the need for invasive physical contact. Shedding cells containing DNA, such as epithelial cells, mucous, and faeces into the environment, is common in aquatic species. These cells contain mitochondrial genes that are advantageous for identification. The portable sampler backpack system controls filtration pressure and flow rate and captures eDNA-containing cells in a single-use inline filter by drawing water through the sampler. Filter paper is then soaked in 70% ethanol to preserve the sample and prevent degradation.

Although propylene glycol is a viable alternative, Liu et al., 2019, suggests it as the preferred choice for its non-toxicity and non-flammability. Next-generation sequences have dramatically increased the number of reads obtainable from samples containing unknown species and therefore can be applied to samples taken from pitfall traps' arthropods, leaf tissue endophytes, or water samples' organisms. PCR is used to analyze DNA and to detect the species' presence or absence, introducing a fluorescent marker into the reaction that will highlight DNA segments on a gel electrophoresis.

The mitochondrial cytochrome c oxidase gene is useful due to its high mutation rate, ensuring its ability to distinguish animal taxa, as Liu et al., 2019, mentioned. Specific regions of the genome are then amplified using the species-specific primers (or universal which can be used across a wide range of taxa in target organisms in some instances) employing PCR. These areas are then sequenced, and individual operational taxonomic units are compared to existing DNA datasets for identification, known as meta-barcoding.

In one study located in 3 ponds near an area of land in Uttoexeter, Staffordshire, eDNA analysis for Great Crested Newts was conducted. The site, a cattle-grazed pasture, was proposing a planning application for a new solar farm, which would occupy the majority of the fields. There were no records of amphibians or ponds within a kilometer of the site, but 7 water bodies were discovered in the surrounding areas and were subject to the Great Crested Newt Habitat Suitability Index Assessment, determining the potential for presence. Ponds receiving scores below average and poor were deemed potentially suitable for the Great Crested Newt and provided an opportunity for the species to cross the site. The local planning authority then recommended carrying out presence/absence surveys, should the newt be present to implement necessary mitigation strategies.

The study aimed to compare the proficiency of two qPCR methods, Biomeme qPCR analysis and traditional bench PCR, and assess whether the use of the Smith-root eDNA sampler was effective in mapping the mudsnail eDNA distribution and temporal fluctuations. The research concluded that both PCR methods were capable of detecting mudsnail eDNA based on the dilution level, and both Biomeme and eDNA sampler can be utilized to detect, map, and quantify changes in target eDNA in the field. The purpose of this study was to determine whether collecting samples and performing PCR to generate eDNA species detections within an hour in the field would yield identical outcomes as compared to PCR in the lab. The study found that it produced consistent results and was an efficient method that could save a considerable amount of time when rapid and reliable techniques were required to identify invasive species.

According to Kojabad et al. (2021), droplet digital PCR (ddPCR) is a new technology available since 2011, which utilizes Taq Polymerase in a typical PCR reaction to amplify the target DNA fragment. It is a method based on water-oil emulsion droplet technology in which a single sample is fractioned into 20,000 droplets, with nucleic acids randomly encapsulated inside as reaction chambers. Each droplet contains one or more target sequences, and, rather than performing just one PCR analysis on a single sample, each droplet becomes a PCR sample of its own. Despite its risks, such as lack of practicality and pipetting errors (while diluting), the method is considered to be far more precise and reliable than other PCR techniques that employ analog measurements, making it particularly useful when working with eDNA where low levels of DNA may be present (Baker et al., 2018).

The ANDe sampling method under development has a few potential limitations. Some protocols involve transporting large volumes of water back to the lab for analysis, limiting sample size and increasing the possibility of DNA degradation if not stored carefully. Nonetheless, eDNA has already demonstrated significant potential for use in biological monitoring. It can determine relative abundance since some studies have found a correlation between this and biomass (Bohmann et al., 2014). Given the high variance of eDNA concentrations, along with the potential heterogeneity through the water body, it is recommended that the process is optimized, ideally with a pilot study for each application, to ensure the sampling design is appropriate to detect the target species or organism (Ruppert et al., 2019).

References

Bohmann, K., Evans, A., Gilbert, M., Carvalho, G.R., Creer, S. and Knapp, M. (2014) “Environmental DNA for wildlife biology and biodiversity monitoring,” Trends in Ecology & Evolution, 29(6), pp. 358-367. Available at: https://doi.org/10.1016/j.tree.2014.04.003.

Baker, C.S., Steel, D.J., Nieukirk, S.L., Klinck, H., Matsumoto, H., Burtenshaw, J., McLellan, W.M., Thompson, P.O. and Yamamoto, Y. (2018) “Marine mammal biodiversity monitoring using eDNA metabarcoding in the Western North Pacific,” Ecology and Evolution, 8(1), pp. 596-606. Available at: https://doi.org/10.1002/ece3.3600.

Ruppert, K.M., Kline, R.J., Rahman, M.S., Waller, D.L., Holdcraft, R., O’Brien, D.M., Goldberg, C.S., Hunter, M.E. and Kelly, R.P. (2019) “Environmental DNA (eDNA) detection probability is influenced by seasonal activity of organisms,” PLOS ONE, 14(1), p. e0210655. Available at: https://doi.org/10.1371/journal.pone.0210655.

The study conducted by Bohmann et al. in 2014 delves into the use of environmental DNA (eDNA) for wildlife biology and biodiversity monitoring. In their research, they found that eDNA is a highly effective tool for detecting and analyzing the presence and diversity of various animal species in various environments. Their findings highlight the importance of eDNA in conservation and habitat management efforts.

Another study conducted by Ruppert et al. in 2019 provides a comprehensive review of the methods, monitoring, and applications of global eDNA. Their research details the past, present, and future perspectives of eDNA metabarcoding and emphasizes the role of eDNA as a powerful tool for monitoring and conserving diverse ecosystems.

In a more specific application, Liu et al. in 2019 published a practical guide for DNA metabarcoding for entomological ecologists. Their guide provides step-by-step instructions for conducting DNA metabarcoding experiments and collecting and analyzing data on insect populations.

The Forest Research website provides a wealth of information on metabarcoding, detailing the applications, techniques, and benefits of using eDNA for biological monitoring. The site also highlights ongoing research efforts in this area and the potential for eDNA to revolutionize the field of ecology.

Kojabad et al. in 2021 investigated the progress, challenges, and future perspectives of droplet digital PCR for detection and identification of viral DNA/RNA. Their study shows that droplet digital PCR is a highly sensitive and accurate method for detecting and quantifying viral DNA/RNA in various samples, and can be used for a wide range of applications, including disease diagnosis and monitoring.

Finally, Baker et al. in 2018 applied eDNA technology to detect and identify whale species from their wake. Their study demonstrates the power of eDNA as a non-invasive and highly effective tool for species identification and monitoring in marine environments.