ARPHA Conference Abstracts :
Conference Abstract
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Corresponding author: Mandy Sander (mandy.sander1995@gmx.de)
Received: 21 Feb 2021 | Published: 04 Mar 2021
© 2021 Mandy Sander, Arne Beermann, Dominik Buchner, Vasco Elbrecht, Peter Haase, Vera Zizka, Florian Leese
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sander M, Beermann A, Buchner D, Elbrecht V, Haase P, Zizka VMA, Leese F (2021) Improved freshwater macroinvertebrate detection from environmental DNA through minimized nontarget amplification. ARPHA Conference Abstracts 4: e64753. https://doi.org/10.3897/aca.4.e64753
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Environmental DNA (eDNA) metabarcoding is a new, promising, and non-invasive method to detect biodiversity in aquatic environments. So far, it has mainly been used to screen for fish and amphibian diversity and rarely to detect macroinvertebrates. Typically, DNA metabarcoding relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase I (COI) gene with degenerate primers. In comparison to other genes like 16S, COI has a greater taxonomic resolution and availability of an extensive reference database. Benthic stream invertebrates are of critical importance for regulatory biomonitoring, but when using universal primers on eDNA isolated from water, the number of reads and OTUs is “watered down”. This means the target taxa, macroinvertebrates, are underrepresented in comparison to other nontarget taxa, e. g. algae, bacteria, and fungi.
The aim of the project was to design an insect-specific primer, which minimizes nontarget amplification. Therefore, data from a time series of 15 months at the Kinzig (Hesse), a silica-rich low-mountain-range stream, which is part of the Rhine‑Main‑Observatory (LTER site) was generated using the universal primers BF2/BR2. With this data we identified the most abundant nontarget taxa and designed a new reverse primer (EPTDr2n) with 3’ ‐ specificity toward benthic invertebrate taxa. Primer specificity was validated in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. 20 eDNA samples from the Kinzig River and its tributaries were then used to test the new primer in situ together with primer fwhF2.
The new primer combination showed a much higher amplification of benthic invertebrates, insects in particular, than two other universal primer pairs for both, number of target reads (fwhF2/EPTDr2n: 99.6% versus BF2/BR2: 25.89% and fwhF2/fwhR2n: 39.04%; Fig.
bioassessment, bioindication, biomonitoring, COI, eDNA, insects, LTER, metabarcoding, primer bias
Mandy Sander
1st DNAQUA International Conference (March 9-11, 2021)