Summary of the research
@Zerina Rahic
eDNA as a possible alternative method
Environmental DNA (eDNA) is nuclear or mitochondrial DNA that is released from an organism into the environment. Currently scientists prefer research methods involving analysis of eDNA as it serves as monitoring molecular tool with an enormous potential to give an insight to aquatic conservation and management. eDNA offers solutions through rapid, sensitive, cost effective, non - invasive monitoring which promises the enhancement of understanding of global biodiversity.
Unlike current methods which are performed by using skin swabs in order to collect samples from a large number of individual hosts - resulting in higher expenses, eDNA detection offers an alternative approach that has been proven to have a higher detection capability and cost‐effectiveness compared with traditional methods.
List of advantages
- Cost effective
- Less time consuming
- More efficient in cases of lower prevalence of the fungus
- Increased biosecurity
- Easier to collect
Because eDNA is sampled from non‐living ecosystem components, it provides an entirely non‐invasive means of conducting large‐scale ecological surveys without physically capturing, handling, or harming organisms . eDNA methods are also proving to be a safer sampling method, with lower sampling effort and cost. By analyzing eDNA from a water sample, it is possible to determine whether a species of interest is present, regardless of life stage or whether specimens are complete or fragmented. Therefore, eDNA provides a useful tool for evaluating biodiversity in remote or challenging regions to access and sample, such as macrotidal and open ocean environments.
Firstly, the most obvious potential of eDNA analysis is that eDNA metabarcoding may be the next logical step to screen for multiple freshwater diseases that threaten biodiversity, or to monitor host, threatened species, and pathogens simultaneously. Specifically talking about Bd research, eDNA sampling techniques accurately detected the presence of Bd at sites that experienced Bd related die-offs, and unlike swabs, did not require the capture or handling of target animals. Using non-invasive techniques such as eDNA is ideal when researching federally or state listed species because it may shorten or avoid lengthy research permitting processes. Environmental DNA techniques are also useful when working with species or habitats sensitive to human impact. In addition to being non-invasive, eDNA sampling techniques detected Bd at a critical time for management.
Secondly, research done on Bsal using eDNA had a noticeable outcome as the detection of Bsal via eDNA was possible unlike via swabs which can be explained by a lower prevalence of the fungus. To detect a pathogen that occurs at low prevalence, sampling of a large number of individuals is required. Using standard methods allows only around 20 swabs to be collected (as no more individuals can be caught). To detect Bsal with this limited sample size, the Bsal prevalence should range, depending on the level of confidence, between 15% and 20%. These researches highlight the high detection capability and advances of eDNA technique. It also shows it is a useful detection method complementary to the collection of skin swab samples. Also, eDNA‐based methods will help to delineate the outbreak and allow for the evaluation of the effectiveness of management measures. Collecting swabs remains necessary in active pathogen surveillance for detecting prevalence and infection intensity.
List of disadvantages
- Properties of eDNA samples are subject to changes
- eDNA is easily transferred causing possible false detection
- Amount of eDNA in water body is highly affected by temp, UV, pH
- PCR inhibition can reduce eDNA recovery and cause false negatives
- Uneven distribution of organisms can cause false positives
- Life stage, history, behavior, etc of a host have to be considered
However, there are challenges that have to be considered. Firstly, environments such as ponds are influenced by the activity of domestic and wild animals which can increase suspended solids within the water column and change the properties of an eDNA sample. These external influences may also transfer eDNA between water bodies and potentially cause false positive detection. Secondly, due to pond nature larger fluctuations in temperature range and potentially greater exposure to ultraviolet (UV) light are common in ponds. Temperature, UV light, and pH all influence eDNA shedding and degradation rates, and can affect the amount of eDNA present within a waterbody. Moreover, as water volume decreases over time, ponds become increasingly ephemeral or seasonal. Accessing these waters via wet, vegetated margins may make cross-contamination between sites. Lastly, anoxic conditions in ponds were shown to slow marine eDNA decay but impacts of anoxia on pond eDNA have not been investigated. Slow decay may affect inferences made from eDNA regarding contemporary species presence.
Moreover, one of the disadvantages of eDNA method is related to possible PCR inhibition. PCR inhibition can affect eDNA samples from any environment, especially if water body containsg hight organic input. Turbid water with high suspended particulate matter not only clogs filters, but blocks extraction spin columns reducing DNA recovery. DNA extracts produced from turbid water often contain humic acid and tannin compounds, created through non-enzymatic decay of the organic material. These compounds can inactivate DNA polymerase and inhibit the PCR amplification process, reducing its efficiency or causing complete failure. PCR inhibition can cause false negatives, and thus it is imperative that eDNA practitioners and researchers test for it by spiking reactions with control DNA that will not be found in the sample.
Use of droplet digital PCR (ddPCR) may overcome the aforementioned limitations for detection and quantification, particularly in turbid waters containing high concentrations of PCR inhibitors. In ponds, ddPCR outperformed qPCR, especially at very low eDNA concentrations, and may be more accurate for abundance or biomass estimation due to lower variability.
Methods
Two broad methods are used in the capture of eDNA: filtration or ethanol precipitation. Comparative studies have generally shown that filtration approaches have higher sample throughput and can process greater water volumes, thereby increasing potential to recover greater amounts of DNA.
- Filtration: Since ponds can contain high levels of suspended solids, filters tend to become blocked when sampling comparatively small water volumes. Where water is turbid, centrifugation, increased pore size, or pre-filtering will be necessary . However, prefilters increase cost and larger pore sizes trade capture of smaller particle sizes for greater proportions of target DNA, reducing total eDNA yield.
- Ethanol precipitation: In contrast to filtration, water volumes are consistent with ethanol precipitation and species recovery may be the same or higher. However, water volume is usually limited to * 90 ml per sample due to logistical and financial constraints on the number of tubes of ethanol that can be taken into the field . Moreover, ethanol is not always easy to obtain and is subject to dangerous goods regulations for transportation.
Metabarcoding
Metabarcoding utilizes universal primers that target taxonomically informative genes such as, the nuclear small subunit ribosomal RNA (18S rRNA) or the mitochondrial Cytochrome c Oxidase subunit I (COI) genes (Tanabe et al., 2016; Stat et al., 2017; Bista et al., 2018; Wangensteen et al., 2018). In the context of surveillance for marine NIS, this approach holds great potential but has limitations, including challenges in identifying NIS at species level due to the lack of sufficiently resolved phylogenetic markers, incomplete reference databases, primer biases and sequencing artifacts, which all may lead to false positive or negative results (Brown et al., 2016; Ammon et al., 2018; Cristescu and Hebert, 2018)
Target method
Targeted methods, e.g., species-specific qPCR, may offer a more sensitive approach for effective detection of specific marine NIS (Wood et al., 2017). However, species-specific assays need to be designed based on a priori knowledge of target organisms. Droplet digital PCR (ddPCR) is a real time PCR technology that divides eDNA/eRNA template into thousands of nanoliter droplets, each containing a single target molecule. Within each droplet, a PCR is conducted, and the outcome visualized via the presence or absence of a fluorescence signal. The number of target copies can be calculated on the positive-negative droplet relation, allowing direct quantification without the need for standard curves (Baker et al., 2018). When using ddPCR, the parallel processing of thousands of reactions enables the detection of very low target concentrations while minimizing PCR inhibition and removing the need for technical replicates, thereby reducing analysis costs and time (Nathan et al., 2014; Doi et al., 2015).
Limitations of both method
There is still limited knowledge on the factors affecting detection probabilities. (Wood et al., 2019b). For example, there is a need for more research to determine if the complexity of sampling matrices affects the detection efficiency and whether eDNA binds to certain environmental matrices for longer periods of time. Furthermore, additional information on the relationship between eDNA and eRNA signals will assist in determining whether the use of eDNA in isolation can accurately predict if living organisms are present near the collection source, thus making these tools more cost-effective for routine biomonitoring programs.
eRNA vs eDNA
Dead biomaterial or extracellular DNA can be transported into a sampling region from a significant distance, therefore the detection of eDNA does not necessarily confirm the presence of living organisms, nor automatically indicate that live organisms occur in close proximity. In contrast, environmental RNA (eRNA) is believed to deteriorate more rapidly due to the chemical composition (hydroxyl groups) which makes this molecule more prone to hydrolysis or degradation. Environmental RNA may therefore provide a better proxy for inferring the presence of living organisms. However, working with eRNA requires specialized storage of samples, and expensive and time-consuming workflow protocols; potentially limiting its applicability to routine monitoring programs.
Since RNA is directly linked with active gene expression of metabolic pathways and deteriorates rapidly after cell death, it may be a better proxy for detecting “live” signals in environmental samples. However, cellular RNA production can vary enormously (over 3 orders of magnitude), largely due to varying transcription rates of ribosomal RNA . Additionally, working with RNA requires the conversion of RNA into cDNA which introduces additional costs and processing time. While ddPCR technology is particularly sensitive, it is not immune to inhibition. The influence of community diversity and different sample matrices is still relatively unexplored.
There is not much empirical information available on eRNA degradation in the marine environment, but it is assumed to degrade significantly faster than eDNA due to its more fragile chemical structure . A recent in situ study specifically on S. spallanzanii could trace DNA signals for up to 42 h, while RNA could not be recovered after 13 h of organism removal from the tank.
References