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1. What is metabarcoding?
Metabarcoding is a method which generates a list of DNA sequences found in a sample, which can be either a sample of multiple animals (‘bulk metabarcoding’, e.g. organisms picked out of a benthic sample or scraped from a hard surface) or a sample of water or sediment (‘eDNA metabarcoding’). These sequences can be assigned to taxonomic group or species, providing information at the community level.
2. What is eDNA?
The definition of environmental DNA (eDNA) is genetic material that can be extracted from environmental samples. According to recent views by e.g. Rodriguez-Ezpeleta et al.(2021), eDNA can be brought under two categories: organismal DNA and extra-organismal DNA, including extracellular DNA. Organismal DNA stems from whole individuals and typically, this type of eDNA is of high quality and significant quantity. Extra-organismal DNA originates from a variety of sources, like shed from an organism, cell lysis or biologically active propagules, and is of highly variable quality and quantity. Therefore, eDNA is composed of a mixture of these two types of DNA, originating from different sources and in varying proportions.
3. What forms of eDNA can be found?
The DNA material could be dissolved, freely floating DNA that has broken out of the tissue cells from where it was originated. But it is thought that the majority of eDNA is often contained within cells, or in clusters of cells, and isn’t evenly distributed in a water body. These clumps can move away from the organism producing them, or even settle out of the water column, and eDNA researchers are trying to learn more about how eDNA moves in the environment and where individuals might have been when their DNA was shed.
4. What are the advantages of using molecular methods for surveying?
Molecular approaches to ecological surveying are becoming increasingly rapid and cost efficient. This is a very useful tool to support and complement ongoing monitoring of marine and terrestrial populations, including invasive and conservation-interest species. Depending on the environment, molecular analysis could be more sensitive to species presence and could be a powerful complementary tool to the traditional field studies. The analysis of sediment or water samples to complement field surveys could result in shortenedtime in the field, reduce costs of sample collection, and reduce disturbance of sensitive habitats. One of the biggest advantages of molecular methods is that they can help to screen and identify multiple groups of species from a single water sample. This includes species groups which are difficult for even experienced taxonomists to identify, but which might provide useful information on benthic ecosystem health. Therefore, molecular sampling could become a much more efficient survey tool to sample multiple species, helping to augment monitoring studies.
5. Can you detect abundance?
The results of metabarcoding analysis are considered reliable, replicable, and meaningful data on presence or absence of aquatic species, and often give a good indication of the relative abundance of aquatic species. However, these values are not considered to be absolute abundance. Some dedicated work has been extensively calibrated (see eDNA work conducted by Levi et al. 2018 on salmonids), which has covered some of these aspects. There are many factors which can affect the amount of DNA detected from a particular species in metabarcoding studies, some related to the methodology (e.g. primer bias towards certain species) and some related to the behavioural characteristics of the animals themselves (e.g. small active species may tend to shed more material thanless active larger species). However, as an increasing number of studies are looking into this issue, it is likely that molecular methods will be more useful for detecting abundance in the near future.
6. Can eDNA be used in deeper areas to track the distribution of deep-water species?
Metabarcoding can be used to estimate the presence of a variety of organisms, helping to generate a high-resolution data setfrom water samples could be collected to inform on macrofauna, meiofauna, fish, marine mammals and microorganisms. This type of information could help to document species biodiversity and community composition (e.g. to provide baselines against which impacts of drilling or other disturbances can be compared).
7. Which molecular methods are best for assessing benthic communities?
One of the aims of the GEANS project has been to trial different methods, to see which are applicable to different situations. Different approaches can be applied depending on the monitoring question of the specific study and the resources available. If trying to detect non-native species in ports, analysis of water samples or of samples scraped from hard surfaces are both valid methods which can detect different sets of species. Molecular sampling of benthic species may be best achieved by picking out organisms for ‘bulk metabarcoding’ if the focus is on macrofauna, but analysis of the sediment itself will be necessary if smaller organisms such as nematodes are of interest. More information about specific methods can be found in the GEANS pilot studies.
8. Are molecular methods cost-effective compared to traditional methods for benthic surveys?
Although the laboratory work involved in molecular methods can be expensive, the costs involved have decreased over time, and will likely continue to decrease. Any assessment of the cost-effectiveness of molecular methods will depend on the questions being asked and the circumstances of that project, but when all factors are taken into account (e.g. amount of time needed for field sampling, amount of laboratory time, requirements for trained staff) it is likely that molecular methods will be equally as cost-effective, or more cost-effective, than traditional morphological identification methods alone. This is particularly the case when presence/absence data is the aim, but in cases where abundance data is necessary, traditional methods (or a combination of the two approaches) may still be necessary.
9. What are the risks of false positives or false negatives?
Any method of ecological sampling, especially at sea, is imperfect in that it will not give you a complete picture of the species present. Molecular analysis of a sediment or water sample will give a snapshot of the DNA present in one location at one time of sampling (in much the same way as a grab or trawl sample also provides a snapshot of the community). Less abundant organisms might therefore not be detected, representing a ‘false negative’. Conversely, the issue of ‘false positives’ needs to be considered with eDNA methods, as there may be cases where DNA is present at a given locality when the organism itself isn’t (e.g., DNA from pelagic species which has been transported some distance in the water before sinking into the sediment). But the risk of such ‘false positives’ is generally considered minimal.
10. How long can DNA remain in sea water?
Although DNA is broken down at varying rates depending on factors such as water temperature, salinity and UV light, recent research has shown that eDNA is detectable for only a limited time after release by an organism. Collins et al. (2018) estimated that 48 hours was the average length of time that eDNA would be reliably detectable in inshore marine waters. This relatively short ‘lifespan’ gives greater confidence that any DNA detected in water or sediment samples does indeed correspond to an organism present in that vicinity, although consideration of whether the DNA could have been transported on water currents in that timeframe may be necessary in some cases.
11. What methods are recommended to avoid risk of contamination?
There are several methods which can be adopted to reduce risk of contamination. Some of these include filters and/or membranes that could be attached within a plastic housing device. There are several steps (e.g. for water sample collection) that you will have to try to minimise further contamination via the vessel, which could enable you to collect water from a dedicated water body or to set up a filter to pass the sample over. The GEANS programme has been testing several protocols that will be easy to guide and adapt whilst you are conducting your sampling. As always, equipment used (e.g. bucket, bags, etc.) will have to be cleaned and the use of gloves during the sampling is also recommended to avoid introducing your own DNA to the samples.
12. How can molecular methods be used to monitor invasive species?
Molecular methods are important for detecting new invaders. Metabarcoding studies, producing inventories of the species present in water or sediment samples, can provide an important early warning system to detect newly invading species. Collection of samples from sites at high risk of invasion, like ports and marinas, is likely to be the most efficient method of detecting non-native species, potentially enabling management action to prevent their spread. In addition to the metabarcoding approach, species of particular interest can also be targeted using specific primers, to map their distribution with a higher degree of precision.
13. Are the sequence reference libraries completed enough to allow us to identify most of the species whose DNA is in the samples?
Currently, a high percentage of marine invertebrates in European waters (including the North Sea) have been barcoded. This proportion is rapidly increasing, and indeed the GEANS project has been sequencing many samples to ultimately provide a more complete list of North Sea species which can be detected by metabarcoding. Over the next decade, reference libraries will be nearing completion for the majority of taxonomic groups in European waters. In fact, an important consideration when using metabarcoding methods is that metabarcoding data collected now can be re-analysed in the future, when most gaps in the sequence databases will have been filled.
14. What are the potential challenges associated with molecular survey methods?
DNA-based methods can describe the presence of species, including target species, pathogens, potentially hybridizing individuals, or even whole communities. These methods can’t, however, tell us about the species’ population size, reproductive status, age structure, disease status, or the presence of hybrids. Advances in technology may someday soon allow us to measure this type of information, but for now it needs to be learned using conventional field methods.
15. What is the current regulatory acceptance of eDNA methods for monitoring and across sampling programmes?
At present there are several R&D programmes and monitoring in pilot studies are looking across these current techniques. For example, the Environment Agency, UK is using eDNA for monitoring fish communities, and is working on a tool that would generate a Water Framework Directive (WFD)-compliant index score for lake fish communities, based on the work carried out in collaboration with the University of Hull (e.g. Lawson-Handley et al., 2019). These techniques are improving and evolving rapidly, so it is best to check directly with the agencies regarding specific projects and dedicated R&D questions.