To ‘e’ or Not to ‘e’: Differences and Applications of Bulk-DNA and eDNA-based Biomonitoring

DNA-based Revolution

Freshwater systems, including rivers, lakes and wetlands, are complex and dynamic habitats, home to a wide variety of species.  This complexity coupled with the often-large surface area covered by freshwater, makes aquatic species difficult to both detect and monitor over time. Traditional monitoring methods, including trapping, electrofishing and seine netting, often miss species which are rare (naturally exist in low abundance) and can be bias towards size of the species targeted, meaning some aquatic species remain undetected despite their presence in the system.

Luckily, as technology advances, so do our methods of monitoring freshwater ecosystems. Through developments in DNA extraction techniques and sequencing, it is now possible to detect and monitor aquatic species using the DNA they leave behind in the water. Environmental DNA is defined as DNA found in an environmental sample (water, soil, permafrost, sediment), which has been shed from an animal in the form of urine, faeces, dead skin cells or through death and decomposition. We are able to collect these environmental samples and accurately identify all of the species which have been present in a freshwater ecosystem across a variety of sample types.

DNA Monitoring Strategies

The variety of environmental sample types targeted for DNA-based biomonitoring of freshwater, i.e. benthos/sediment and water, give rise to different variations of DNA-based terminology. Typically, DNA collected from water is referred to as ‘eDNA’, where as the environmental sample collected when analysing benthos/sediment is called ‘bulk-tissue DNA’ (Deiner et al., 2017; Dickie et al., 2018). This difference primarily refers to the fact actual organisms (bugs) are being collected from benthos/sediment, forming the source of the DNA analysed, whereas in water, often the DNA found in water samples had been previously shed by organisms into the water column.

Water eDNA sample being collected from a river. ©STREAM 2019.

Rivers and the habitats surrounding them are complex systems, which are home to a wide range of species. In addition to DNA from aquatic species (bugs, fish, amphibians etc) being present in the water and river sediment, DNA from land-based species including mammals, birds and plants can be deposited into the river. Rivers can be considered as ‘conveyor belts of information’, as they carry DNA shed by a wide variety of species from the headwaters downstream. Some of this DNA is deposited into the river sediment, in the same way DNA from bugs can leave the sediment and be leached into the river water and carried away by the water flow. This highly dynamic nature of rivers means that to detect and monitor particular species using DNA-based biomonitoring, we need to consider what type of sample we are collecting and also what methods we are using to analyse the DNA.

Diagram of how DNA is deposited and exchanged within a river system. ©STREAM 2019.

DNA-based Methods for Freshwater

In freshwater systems, DNA biomonitoring is conducted using one of two methods; single-species detection or metabarcoding. For water samples, both methods involve collection (and often filtration) of water, which is then processed in the laboratory to separate the DNA from the water (DNA extraction). When sampling benthos/sediment, samples are collected using a kick-net and preserved in ethanol prior to DNA extraction. After the DNA extraction process, both water and benthos/sediment samples are processed in the same way. For single-species detection, DNA is then multiplied in a PCR machine using species-specific primers, using a technique which is designed to produce a ‘flag’ on the screen when DNA from the target species is present in a sample. This enables us to essentially map out where the target species is across samples and sites.

Water sampling in an stream which is also used as a source of water for cattle ©STREAM 2019.

DNA metabarcoding also involves the multiplication of DNA, however the primers here are designed to amplify numerous species as opposed to just one. After DNA replication, every single DNA strand is sorted in a sequencer and matched to a reference database to create a list of species present in the water sample. Check out our DNA metabarcoding PDF for more information on DNA metabarcoding.

DNA-based Applications in Freshwater

DNA-based sampling of freshwater is typically used to detect aquatic species such as fish, amphibians and bugs and answer a variety of ecological questions. Single-species detection methods are often used when trying to detect either rare or invasive aquatic species. Similar to rare species, invasive species are normally in low numbers at the start of their invasion of a new river or lake, meaning they are often missed with traditional monitoring methods such as trapping. Some previous studies have used the single-species detection eDNA method to detect and monitor some aquatic invasive species individually including topmouth gudgeon (Pseudorasbora parva) and Chinese mitten crab (Eriocheir sinensis) (Robinson et al., 2019a; Robinson et al., 2019b).

DNA metabarcoding is an efficient method for ‘cataloging’ the communities of species found within a particular habitat, however, with the DNA exchange which can occur in rivers between sediment and water, it can be difficult to know which sample type is best for assessing bug communities in rivers. A recent study found that bug communities were better represented in river sediment than in the water (Hajibabaei et al., 2019), in particular the species of bug which are good indicators of river health, including Caddisflies and Mayflies.

Overall, there are numerous types of environmental sources of DNA in freshwater systems, from which we can extract DNA and look at the species/s’ living in our rivers. The different methods allow us to answer different questions we have regarding the health status of rivers.

©STREAM 2019.

References

Deiner, K., Bik, H.M., Mächler, E., Seymour, M., Lacoursière‐Roussel, A., Altermatt, F., Creer, S., Bista, I., Lodge, D.M., Vere, N. de, Pfrender, M.E., Bernatchez, L., 2017. Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology 26, 5872–5895. https://doi.org/10.1111/mec.14350

Dickie, I.A., Boyer, S., Buckley, H.L., Duncan, R.P., Gardner, P.P., Hogg, I.D., Holdaway, R.J., Lear, G., Makiola, A., Morales, S.E., Powell, J.R., Weaver, L., 2018. Towards robust and repeatable sampling methods in eDNA-based studies. Molecular Ecology Resources 18, 940–952. https://doi.org/10.1111/1755-0998.12907

Hajibabaei, M., Porter, T.M., Robinson, C.V., Baird, D.J., Shokralla, S., Wright, M., 2019. Watered-down biodiversity? A comparison of metabarcoding results from DNA extracted from matched water and bulk tissue biomonitoring samples. bioRxiv 575928. https://doi.org/10.1101/575928

Robinson, C.V., Leaniz, C.G. de, Consuegra, S., 2019a. Effect of artificial barriers on the distribution of the invasive signal crayfish and Chinese mitten crab. Scientific Reports 9, 7230. https://doi.org/10.1038/s41598-019-43570-3

Robinson, C.V., Leaniz, C.G. de, Rolla, M., Consuegra, S., 2019b. Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay. Environmental DNA, early view. https://doi.org/10.1002/edn3.12

Definitions:

Primer: short DNA sequence which is used as a starting point to multiply copies of DNA.

DNA replication: the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules.

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