By Isoline Donohue, Biological Sciences, ’23

Author’s Note: I wrote this literature review for UWP 102B during the spring quarter of 2022, and learned about the Aggie Transcript from that course. I chose to write about this topic because I am very interested in conservation biology and work as an undergraduate researcher in this field. I had heard of environmental DNA before at my lab, but wanted to learn more by doing my own research. From this piece, I hope readers learn about the new and exciting ways species monitoring is being done to preserve ecosystems.

Introduction

Marine species are experiencing higher population declines than many terrestrial species due to anthropogenic causes [1], such as increased water exports or runoff impacting habitats and behavioral patterns. Aquatic systems require a greater focus on species preservation, but keeping track of different species can be difficult. A first step in conservation involves genetic monitoring to track population decline. Genetic monitoring uses DNA to study variation within a species, as well as to discriminate between different species types in an environment. Monitoring a species may include the use of environmental DNA (eDNA) in lieu of DNA collected off of the organism itself. eDNA is a sample collected from a habitat, such as water from a stream, that contains the DNA of the species present. The skin, mucus, or hair of an organism when shed contains DNA that can be eluted and amplified to detect species [2]. Samples are then used for metabarcoding, or non-species specific detection, as well as for targeting a certain species of interest through assay development of different genetic markers [2]. Overall, this method of sample collection is both non-invasive and applicable for smaller populations when capture methods tend to lose reliability [1]. Traditional capture methods are not always successful in areas where a species may currently or recently inhabit, which is where eDNA can be used to discover new territories and monitor current ones. 

Studies have been conducted with environmental samples to monitor endangered species, as well as non-native species that endanger other populations [1, 3]. eDNA detections often need validation through replicates or prior knowledge of inhabitants, as using eDNA is still a relatively new form of sample collection. Schmelzle et al. (2016) highlights how current eDNA research centers on standardizing DNA capture methods to ensure repeatability [4]. Therefore, studies are being conducted to compare eDNA to traditional capture methods to determine if positive detections can be made the same as if a tissue sample was taken from an organism. 

Currently, there is concern over whether eDNA can be reliably used for instances where seasonality impacts species presence in an area [5], as well as instances where DNA may degrade before proper evaluation [6]. In order to understand how eDNA can be utilized to find the limitations of aquatic species detection, various studies have been compiled. In this review, we evaluate the current methodology, validity, effectiveness, and concerns of eDNA through studies centered on endangered or invasive aquatic species. 

Methodology 

While collection, extraction and analysis methods and materials are not always the same, the general process of going from an environmental sample to an identifiable DNA sequence of a species is. First, the water collected for any aquatic system sampling must be filtered. There are several methods to do this that involve different materials and containers to collect and filter water. Ratcliffe et al. (2020), for example, took water samples from the Irish and Celtic seas to detect key taxa in the area. The researchers strained the water using a syringe and filters, where the water was stored in the filter holder containers at -20°C until DNA extraction [7]. Alternatively, Boothroyd et al. (2016) filtered their water samples with a funnel and vacuum right before DNA extraction to prevent degradation. These samples are generally stored with ethanol at -20°C before DNA extraction and -80°C afterwards [1, 3, 4, 5, 6, 7, 8]. Franklin et al. (2018) and Dubreuil et al. (2021) used the Qiagen DNeasy Blood and Tissue Kit in a two day extraction process of lysis for DNA release, and subsequent washing/elution to isolate the DNA. Afterwards, qPCRs in each study were run against an assay containing the genetic marker for identification of a specific species of interest, or in some cases a range of taxa. Species and their relative abundances have the potential to be identified via DNA sequencing starting with an environmental sample.

Researchers take different approaches in validating positive detections, such as through the use of controls and assessment of assays. For example, Mauvisseau et al. (2019) detected pearl mussels in Lake Windermere. The researchers validated their assays targeting the COI and 16S genes, regions optimal for species identification, with statistical analysis on their level of detection and quantification before using them against the samples collected in the study. The eDNA detections were also tested against tissue samples and positive controls to ensure accuracy. This was a double-blind experiment during the water sample collection and filtering process, where sample site information was not revealed to the researchers until after analysis was done [2]. Dubreuil et al. (2021) cited Mauvisseau et al. (2019) as the researchers followed the same statistical guidelines, and their assays proved to be specific to only their species of interest via the positive detections observed. Lastly, Franklin et al. (2018) also took steps to evaluate an appropriate assay for their species. They found that the COI genetic marker also distinguished their species of interest, smallmouth bass, from other non-target species. They examined COI sequences of bass from different regions to obtain sequence specific primers. The developed assay was tested against database sequences, tissue samples, and finally eDNA itself [9]. These preliminary tests aid in ensuring accurate detections are being made when the assay is used against eDNA samples.

Additionally, repeatability in sample analysis can be used to rule out contamination and strengthen confidence in positive detections. Schmelzle et al. (2016) demonstrated this by testing each water sample with 6 replicate qPCRs alongside positive controls to detect tidewater gobies on the California coast. qPCRs are done to identify and quantify the DNA in the samples collected, while positive controls confirm the accuracy of target detections. Overall, research tends to center on validating eDNA detections, as variability can be high even within the same sampling region.

Genetic information from numerous species may be present in one water sample. Researchers may choose to target a specific species or to identify all of the species in the sample using DNA sequencing methods. National Park Service

Comparison to traditional methods 

A non-invasive method 

A benefit of eDNA sample collection is that it is noninvasive to an ecosystem. This is most beneficial for detecting low density species when capture methods tend to fail. Boothroyd et al. (2016) evaluated the effectiveness of eDNA in relation to traditional capture methods, specifically regarding spotted gar fish that were collected via netting. The downsides of netting include sample size limitations and disruption of habitats. Boothroyd et al. (2016) placed fyke nets (cylindrical fish traps) at the eDNA sample sites for 24 hours during the spotted gar spawning season to gather a representative sample. Fin clips from 12 reference samples of spotted gar captured were used for DNA extraction and downstream analysis to compare to the eDNA samples [1]. eDNA detections, made from 1 liter water samples, were consistent with areas spotted gar were previously known to inhabit, and were even made at sites where capture methods failed to pick up the species of interest when placed [1]. The researchers in this study also captured dozens of other fish species at the different bodies of water sampled while attempting to collect spotted gar. The variability of netting shows that it can cause disruption for more than just the species of interest, as well as limit the sample size. 

Similarly, Schmelzle et al. (2016) used occupancy modeling to compare traditional capture methods against eDNA detection. This study noted the limitations of traditional capture methods in their low capture yield versus high cost and time requirement. A seine haul (vertical net placed in the water) of tidewater goby fish was taken at each sample location to compare to the water samples collected. It was found that eDNA detections from the water samples, validated against known goby presence and replicate qPCRs, were more effective than seining detections in accurately representing goby occupancy [4]. Schmelzle et al. (2016) concluded that eDNA has the potential to be the dominant method for tidewater goby tracking [4]. In another study, Dubreuil et al. (2021) set up baited fish traps for armored catfish at sample sites three times each for 16 hours. eDNA of the species of interest was detected in the water sampled at 18 sites. Alternatively, the fish traps detected catfish at 14 of the 18 sites where positive eDNA detections were made [8]. There were no captures made at sites eDNA did not detect. This study highlights that trapping is variable, as it can depend upon predation, breeding, and food sources [8]. eDNA may be subject to similar variability, however, due to its sensitivity even a species low in numbers can be detected. While trapping non-native species is not as large of a concern in terms of invasiveness, the possibility of capturing at risk species in the area can be avoided with eDNA. 

Reaching new limits with low density species detection 

eDNA has proven to detect species in a wide range of sampling locations, even where inhabitantance has not previously been verified with traditional capture methods [1, 4]. This is in part because traditional capture methods become all the more difficult as a species population size declines. de Souza et al. (2016) monitored the black warrior waterdog salamander and flattened musk turtle, two at risk species from the upper Black Warrior River basin of Alabama that are impacted by habitat degradation, using eDNA sampling. The low payoff of laborious methods such as dip netting, trapping, and electrofishing for these species makes eDNA a better alternative. The researchers highlighted how sampling with eDNA is most effective when used alongside knowledge of a species’s territorial range and migration patterns, which is also true for trapping/fishing except that the latter takes more time to gather the necessary sample size [5]. eDNA also has the sensitivity to detect low density species, colonization events, and target species against similar ones [1, 3, 4, 9]. For instance, Mauvisseau et al. (2019) found that eDNA was able to differentiate between freshwater pearl mussels and non-target species with two different assays used against other mussel species. Meanwhile, Franklin et al. (2018) had success in identifying smallmouth bass, even when simulations predicted the genetic marker of choice would amplify additional species from the eDNA sample. eDNA allows researchers to determine the presence of a species with low population numbers in order to increase the regions being targeted for conservation. 

Detecting non-native species 

Non-native species are found either at a low density due to recent colonization, or a high density due to successful adaptation. Both instances need to be monitored to assess plans of action for restoring ecosystem balance [8]. In a notable study, Franklin et al. (2018) detected smallmouth bass to maintain the population of pacific salmon and other native species of the Pacific Northwest. While the spread of smallmouth bass in the US has had economic benefits, close population management is required for habitat stability [9]. Smallmouth bass have been documented to consume 35% of a salmon run, which has negative consequences on salmon migration and local predator/prey interactions. eDNA, with its high sensitivity, was able to detect smallmouth bass successfully against non-target species to find sample locations that require conservation targeting [9]. Similarly, Dubreuil et al. (2021) also tested eDNA by tracking armored catfish who recently started to inhabit rivers in Martinique and compete with gobies for food sources. 22% of sample sites detected the aquatic invasive species (AIS), armored catfish, using water collections. This AIS did not appear to have habitat preferences, such as pH, oxygen level, or temperature. Therefore, this species is more likely to successfully adapt to new territories [8], hence the need for species monitoring.

Current limitations 

Despite the promise that eDNA shows, there are potential concerns over its consistency and longevity. For example, de Souza et al. (2016) evaluated the effect of species’ seasonality on eDNA detection probabilities. Seasonality differences may limit the time frame eDNA can effectively be utilized for and introduce a sampling bias. The study discovered that warm and cool seasons played a significant role in the detection of their species of interest. Investigating a species’s migratory and spawning behavior can improve the reliability of eDNA in the same way that traditional sampling methods can be improved [5]. 

Detection results can also be impacted by eDNA degradation, as validating a species’s presence depends on whether there is recent DNA in the surrounding area [6]. Barnes et al. (2014) found that freshwater amphibian eDNA lasted for over two weeks, while marine fish eDNA only lasted for seven days. There are many possible influences for eDNA degradation, such as sunlight or pH [6], so it is important to consider how genetic degradation may impact detections, or lack of. Brys et al. (2020) also reported eDNA degradation rates of close to one week for the 7 fish and 2 amphibian species the researchers were sampling as their control, and cited Barnes et al. (2014) in how high temperatures, UV radiation etc. may be to blame. 

eDNA degradation is also accompanied with dispersion, where detections can only be deemed as reliable when they are repeatable in the area. Boothroyd et al. (2016) observed replicate water samples and determined there was variation among the number of positive detections, despite eDNA being highly sensitive to amplification. The study mentions that a downside of eDNA is that actual organisms are not being detected, so DNA could possibly be due to run-off or the remains of a fish [1]. This implies that DNA detections can vary, which may make eDNA collections a better supplement rather than replacement to traditional capture methods. 

The type of aquatic ecosystem and species present can influence detection probabilities in many cases as well. Brys et al. (2020) observed a lentic (standing water) system with high eDNA decay rates and distance limitations for DNA retrieval. Metabarcoding was used to detect various species in a pond, where natural water samples were compared to those taken in proximity to a known variety of locally caged species. The 12S genetic marker detected the known fish and amphibians in the area via DNA sequencing in metabarcoding. Positive detections depend on if a lentic or lotic (rushing water) system is being observed, where the flow of water in a lotic system increases the range of eDNA [3]. Species type and density were shown to impact eDNA dispersal rates as well in this case [3], meaning samples taken in proximity to one another could potentially show different detection results due to spatial limitations. 

Conclusion 

The goal of this review was to demonstrate how eDNA collection and analysis works, and how it can be useful for aquatic species conservation. Various factors, such as the behavior of a species, population density, and sample regions determine whether eDNA can outperform traditional sampling methods. Methodology is often altered to correspond with these factors, as the different species being monitored and types of aquatic systems being observed impact everything from sampling to analyzing. This implies that whether eDNA can replace traditional capture methods will depend on the study itself and what information researchers are seeking. Dubreuil et al. (2021) had greater success in their study using eDNA compared to trapping, noting that eDNA has the potential to detect species at a greater distance than a trap does. Future research should be aimed towards identifying the susceptibility of eDNA degradation and false detections in different bodies of water. The more eDNA is understood, the simpler it becomes to validate findings and lessen reliance on traditional capture methods.

References:

1. Boothroyd M, Mandrak NE, Fox M, Wilson CC. 2016. Environmental DNA (eDNA) detection and habitat occupancy of threatened spotted gar (Lepisosteus oculatus). Aquatic Conserv: Mar. Freshw. Ecosyst. 26: 1107–1119.
2. Mauvisseau Q, Burian A, Gibson C, Byrs R, Ramsey A, Sweet M. 2019. Influence of accuracy, repeatability, and detection probability in the reliability of species-specific eDNA based approaches. Scientific Reports. 9:580.
3. Brys R, Haegeman A, Halfmaerten D, Neyrinck S, Staelens A, Auwerx J, Ruttink T. 2020. Monitoring of spatiotemporal occupancy patterns of fish and amphibian species in a lentic aquatic system using environmental DNA. Molecular Ecology. 30:3097–3110.
4. Schmelzle MC, Kinziger AP. 2016. Using occupancy modeling to compare environmental DNA to traditional field methods for regional-scale monitoring of an endangered aquatic species. Molecular Ecology Resources. 16, 895-908.
5. de Souza LS, Godwin JC, Renshaw MA, Larson E. 2016. Environmental DNA (eDNA) detection probability is influenced by seasonal activity of organisms. PLOS ONE.11(10): e0165273.
6. Barnes MA, Turner CR, Jerde CL, Renshaw MA, Chadderton WL, Lodge DM. 2014. Environmental Conditions Influence eDNA Persistence in Aquatic Systems. Environmental Science & Technology. 48, 1819−1827.
7. Ratcliffe FC, Uren Webster TM, de Leaniz CG. 2020. A drop in the ocean: Monitoring fish communities in spawning areas using environmental DNA. Environmental DNA. 3:43-54.
8. Dubreuil T, Baudry T, Mauvisseau Q, Arqué A, Courty C, Delaunay C, Sweet M, Grandjean F. 2021. The development of early monitoring tools to detect aquatic invasive species: eDNA assay development and the case of the armored catfish Hypostomus robinii. Environmental DNA. 4:349–362.
9. Franklin TW, Dysthe JC, Rubenson ES, Carim KJ, Olden JD. 2018. A non-invasive sampling method for detecting nonnative smallmouth bass (micropterus dolomieu). Northwest Science. 92(2):149-157.