A10. Fish eDNA

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1. Objectives

Environmental DNA (eDNA) can have several objectives, e.g.:

  • Assessing species occupancy in water basins, remoted areas, and at precise locations inside a catchment
  • Assessing relative abundance and species distribution over longitudinal patterns (Nakagawa et al., 2018; Pont et al., 2018)
  • Detecting and monitoring invasive species (Darling and Mahon, 2011; Keskin, 2014; Geerts et al., 2018) in aquatic environments, and confirm public sightings (Parrondo et al., 2018)
  • Determine habitat connectivity for fish migration, between the sea and spawning grounds, and track seasonal migration (Yamanaka and Minamoto, 2016)
  • Provides site occupancy inferences of rare species and fish that are hardly detectable by other methods (Turner, Uy and Everhart, 2015), and highlight positive or negative co-occurrence (Balasingham et al., 2018)

2. Method summary

Barcoding and metabarcoding environmental DNA is a valuable tool due to its usefulness in conservation biology and decision making. It consists of a water sample collected by a filtering machine or a sediment sample directly collected from the river bed. Analyzing the filter content in a laboratory will:

  1. Extract the total DNA
  2. Select primers to target mitochondrial or nuclear DNA, cut it, and obtain amplicons (fragment of DNA)
  3. Apply Polymerase Chain Reaction (PCR) to amplify amplicons
  4. Sequence all amplicons through High Throughput Sequencing
  5. Compare the sequences to a species gene database -GENBANK or BOLD- with bioinformatics tools to deduce the probability of species presence in the sample

Determining survey effort is important to get consistent detection probability after laboratory process of replicates PCR. If the species is rare, a larger volume should be filtered. Conversely, if the objective is to study fish communities, the amount of filtered water can be reduced. Shaw et al., (2016) have shown that two 1L water samples per site were insufficient to detect less abundant taxa but a 100% detection was available with five 1L samples per site. The survey effort and sampling strategy also depends on the catchment. If the goal is to identify invasive species – previously detected by eDNA in the lower part of the catchment – the next step would be to reduce the sample volume but increase distribution along tributaries, to precisely locate the target. Deep or shallow samples can be chosen according to species ecology, as well as the time of year. An example of this is accessing the use of tributaries by anadromous species during the mating period.

Many sampling and extracting methods exist and a choice has to be made to maximize eDNA yield and detection probability of the targeted species. Piggott (2016) demonstrated that PCR replication and detection probability success rates are mainly influenced by sampling strategy (timing, filtered volume, sampling depth), the extraction method, and PCR strategy but less by amplicon size. The PCR strategy is crucial and can be related to the potential abundance of the targeted species’ in the sample: the rarer the species, the more PCR amplification is required. A presence probability is deduced by the ratio between the number of detections of the targeted species (x) over the total PCR replicates (y).

Some postulate the advantages of sediment samples, where eDNA appears more concentrated and exists 5 times longer than in flowing water (more than 3 months) (Turner, Uy and Everhart, 2015). However, it has been demonstrated that Silurus glanis eDNA levels are higher in water samples than in sediments (Vautier, 2020 com. pers.), but may be multiple fish (Shaw et al., 2016). Yet, it could provide retrospective data on fish occupancy and aquatic macro-fauna, especially for benthic species. Aqueous and sedimentary analysis of fish eDNA have their own strengths and weaknesses and can be complementary (Shaw et al., 2016), but must be carefully interpreted with the presence of seasonal fish, hydrology, and others factors in mind.

3. Advantages

  • Cost and time effective
  • Non-invasive with no fish handling
  • Can confirm species presence and give an exhaustive overview of the fish community
  • Estimates of relative abundance might be possible (Pont et al., 2018)
  • More effective than traditional methods of biodiversity inventories (Valentini et al., 2016)

4. Disadvantages

  • Difficult to conclude on the possibility of missing species (Balasingham et al., 2018) due to sample deficiency, bad storage, and laboratory problems
  • Impossible (yet) to assess population viability or developmental stages
  • False positive data about fish occupancy can occur due to resuspension of sediments in flowing water reaching surface samples (Turner, Uy and Everhart, 2015)
  • Detection probability is influenced by laboratory methods, DNA extraction, and PCR strategies (Piggott, 2016)
  • Careful data interpretation is necessary at each step of the process
  • Database incomplete for some species
  • eDNA presence does not always mean fish presence (eDNA may originate from or human food or other sources)

5. Recommendations for method application

  • Sample conservation is crucial for reliable data. The filters are frozen at –20 or -80°C, or stored in a buffer solution
  • Primer choice is vital to allow for robust taxonomic assignment (Bylemans et al., 2018)
  • Avoid sampling sedimentary eDNA to assess fish occupancy (bias related to DNA conservation, and resuspension in water during flood)
  • Hydrologic and thermal conditions may influence the levels of eDNA in your sample. Low flow period maximizes the amount of eDNA and planning is needed to consider hydrologic conditions.
  • Careful interpretation of results must be made as mistakes are common in the bioinformatic identification of sequences, analysis, or detection. Logical inference and local knowledge are crucial to avoid erroneous conclusions (Shaw et al., 2016). Taxa presence can come from human consumption and not from its real presence in the catchment.

6. Cost

There are several steps, each with different costs:

  • Fields costs
  • Sampling:
    • £1 - £140 for the material for DNA filtering method (simple filter or sterile cartridge)
  • DNA extraction:
    • £1 - £10 depending on method
  • Sequencing:
    • Approximately £30 – £150 per sample multiplied by the number of PCR replicates (usually between 3 and 12)

Overall cost between £90 and £2000 per sample.

7. Protocol and data analysis

See references and:

8. Acknowledgement and associated authors

Marine Vautier – INRAE CAARTEL – Thonon-Les-Bains – France.

9. Further reading

  • Balasingham, K. D. et al. (2018) ‘Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries’, Molecular Ecology, 27(1), pp. 112–127. doi: 10.1111/mec.14395.
  • Bylemans, J. et al. (2018) ‘Toward an ecoregion scale evaluation of eDNA metabarcoding primers: A case study for the freshwater fish biodiversity of the Murray–Darling Basin (Australia)’, Ecology and Evolution, 8(17), pp. 8697–8712. doi: 10.1002/ece3.4387.
  • Darling, J. A. and Mahon, A. R. (2011) ‘From molecules to management: Adopting DNA-based methods for monitoring biological invasions in aquatic environments’, Environmental Research. Elsevier, 111(7), pp. 978–988. doi: 10.1016/j.envres.2011.02.001.
  • Geerts, A. N. et al. (2018) ‘A search for standardized protocols to detect alien invasive crayfish based on environmental DNA (eDNA): A lab and field evaluation’, Ecological Indicators. Elsevier, 84(September 2017), pp. 564–572. doi: 10.1016/j.ecolind.2017.08.068.
  • Keskin, E. (2014) ‘Detection of invasive freshwater fish species using environmental DNA survey’, Biochemical Systematics and Ecology. Elsevier Ltd, 56, pp. 68–74. doi: 10.1016/j.bse.2014.05.003.
  • Nakagawa, H. et al. (2018) ‘Comparing local- and regional-scale estimations of the diversity of stream fish using eDNA metabarcoding and conventional observation methods’, Freshwater Biology, 63(6), pp. 569–580. doi: 10.1111/fwb.13094.
  • Parrondo, M. et al. (2018) ‘Citizen warnings and post checkout molecular confirmations using eDNA as a combined strategy for updating invasive species distributions’, Journal for Nature Conservation. Elsevier, 43(April 2017), pp. 95–103. doi: 10.1016/j.jnc.2018.02.006.
  • Piggott, M. P. (2016) ‘Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish’, Ecology and Evolution, 6(9), pp. 2739–2750. doi: 10.1002/ece3.2083.
  • Pont, D. et al. (2018) ‘Environmental DNA reveals quantitative patterns of fish biodiversity in large rivers despite its downstream transportation’, Scientific Reports, 8(1), pp. 1–13. doi: 10.1038/s41598-018-28424-8.
  • Shaw, J. L. A. et al. (2016) ‘Comparison of environmental DNA metabarcoding and conventional fish survey methods in a river system’, Biological Conservation. Elsevier Ltd, 197, pp. 131–138. doi: 10.1016/j.biocon.2016.03.010.
  • Turner, C. R., Uy, K. L. and Everhart, R. C. (2015) ‘Fish environmental DNA is more concentrated in aquatic sediments than surface water’, Biological Conservation. Elsevier Ltd, 183, pp. 93–102. doi: 10.1016/j.biocon.2014.11.017.
  • Valentini, A. et al. (2016) ‘Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding’, Molecular Ecology, 25(4), pp. 929–942. doi: 10.1111/mec.13428.
  • Yamanaka, H. and Minamoto, T. (2016) ‘The use of environmental DNA of fishes as an efficient method of determining habitat connectivity’, Ecological Indicators. Elsevier Ltd, 62, pp. 147–153. doi: 10.1016/j.ecolind.2015.11.022.