eDNA for biomonitoring PART 1: general introduction Dr. C. Van der heyden

Steps to be taken… eDNA: what’s that? eDNA sampling of aquatic environment eDNA extraction (q)PCR Observation of results agarose gel electrophoresis eDNA quantitation Next Gen Sequencing Questions

1. eDNA: what’s that? eDNA = environmental DNA All free DNA present in the environment Aquatic, soil,… Originating from bacteria, plants, animals,… http://fishbio.com/field-notes/conservation/traces-left-behind

eDNA-based monitoring: why? Selecting Class Colour code High Blue Good Green Moderate Yellow Low Orange Bad Red Intrusive sampling Sieving Classic biotic index Determining

Disadvantages of classic biotic index Many specific sampling equipment needed (e.g. wading suits, nets, sieves,…) Moving from one sampling site to the other needs good organisation Sampling and sample processing are time consuming Sampling is intrusive and disturbing the environment Animals are caught and killed, ecosystem becomes poorer Not all animals are caught easily Buried/fast swimming animals aren’t caught frequently, though they might be abundantly present Technique is expensive, mostly due to its labor intensity After sampling, samples have to be washed, sieved, picked, determined Specific taxonomic knowledge of the macro-invertebrates indispensable

eDNA-based monitoring: why? DNA extraction Filtration Simple water sampling Klasse Kleurcode Zeer goed Blauw Goed Groen Matig Geel Ontoereikend Oranje Slecht Rood Molecular based evaluation of ecological water quality Agarose gel electroforesis Amplification via PCR using specific primers

Advantages of eDNA based monitoring Much less specific sampling equipment needed Only some clean sampling bottles, clean gloves and boots are needed Sampling and sample processing are done quickly Sampling is not intrusive and does not disturb the environment Only water is sampled, no animals are caught All animals, also buried ones, release DNA which can be found in the water Technique is less expensive Water samples are filtered, DNA extracted and PCR-processed Specific taxonomic knowledge of the macro-invertebrates not needed as primers are species specific

eDNA in the environment Biotic influences Abiotic influences  pH  Oxygen consumption eDNA  UV  Chlorophyll a concentration  Temperature Strickler et al 2015; Barnes & Turner 2015

Pitfalls and chalenges

Contamination Controls are needed: In the field During DNA extraction During PCR Positive controls are added DNA from tissues from the animals tested for Pre- en post-PCR processing performed in different locations indispensable …

PCR inhibition Humic acids Sediment samples vs. Water samples Filtering is performed Serum / PCR Buffers are used …

Wrong DNA sequences Occuring during: PCR Sequencing Trade off Preventing mistakes Maintaining information

DNA reference database The databases should be Reliable & complete Depending on the species Commonly used databases, e.g.: fishbol.org mammaliabol.org barcodingbirds.org Focus on complete sequences Preferably mitochondrial DNA

Be critical! Molecular expertise needed for a correct interpretation of the results Criticism concerning the results and their interpretation is needed! No contamination? Correct amplicons? All controls OK? Positive results:  animal IS present  animal WAS present

Present and Future…

Present and Future… Present: eDNA + PCR: eDNA + Next Gen Sequencing: Presence/absence of specific species can be demonstrated eDNA + Next Gen Sequencing: Aquatic ecosystem can be studied for macro-invertebrates, fish, amphibians,… Future: DNA-based water quality index/DNA-based water quality classification? Standardised protocol?

COST action DNAquaNet http://www.DNAqua.net

COST action DNAquaNet Working Group 1 – DNA Barcode References led by Torbjørn Ekrem, Fedor Čiampor WG2 – Biotic Indices & Metrics Led by Jan Pawlowski, Maria Kahlert WG3 – Field & Lab Protocols Led by Kat Bruce, Emre Keskin WG4 – Data Analysis & Storage Led by Kessy Abarenkov, Diego Fontaneto WG5 – Implementation Strategy & Legal Issues Led by Patricia Mergen, Daniel Hering