A simplified DNA extraction protocol for unsorted bulk arthropod samples that maintains exoskeletal integrity

Nielsen, Martin; Gilbert, M. Thomas P.; Pape, Thomas; Bohmann, Kristine

doi:10.1002/edn3.16

Cited by 42 publications

(76 citation statements)

References 43 publications

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“…This approach can likely be automated using incubation shakers, reducing the hands on time and increasing scalability of this size sorting approach by an order of magnitude. Additionally, since only gentle shaking is used with wet sieving, specimens are less damaged and might be used for morphological identification if non destructive metabarcoding is applied (using ethanol (Hajibabaei et al, 2012) or lysis buffer protocols (Nielsen et al, 2019)). However, non destructive methods can introduce additional biases to the metabarcoding process, in some cases detecting substantially less taxa than with morphology or homogenizing bulk samples (Erdozain et al, 2019;Marquina et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

Elbrecht

Bourlat

Hörren³

et al. 2020

Preprint

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1) Small and rare specimens can remain undetected when metabarcoding bulk samples with a high size heterogeneity of specimens. This is especially critical for malaise trap samples, where most of the biodiversity is often contributed by small specimens. How to size sort and in which proportions to pool these samples has not been widely explored. We set out to find a size sorting strategy that maximizes taxonomic recovery but remains highly scalable and time efficient. 2) Three 3 malaise trap samples where size sorted into 4 size classes using dry sieving.Each fraction was homogenized and lysed. The corresponding lysates were pooled to simulate samples never sorted, pooled in equal proportions and in 4 different proportions favoring the small size fractions. DNA from the pooled fractions as well as the individual size classes were extracted and metabarcoded using the FwhF2 and Fol-degen-rev primer set. Additionally wet sieving strategies were explored. 3) The small size fractions harbored the highest diversity, and were best represented when pooling in favor of small specimens. Not size sorting a sample leads to a 45-77% decrease in taxon recovery compared to size sorted samples. A size separation into only 2 fractions (below 4 mm and above) can already double taxon recovery compared to not sorting. However, increasing the sequencing depth 3-4 fold can also increase taxon recovery to comparable levels, but remains biased toward biomass rich taxa in the sample. 4) We demonstrate that size fractionizing bulk malaise samples can increase taxon recovery. The most practical approach is wet sieving into two size fractions, and proportional pooling of the lysates in favor of the small size fraction (80-90% volume).However, in large projects with time constraints, increasing sequencing depth can also be an alternative solution.

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Section: Discussionmentioning

confidence: 99%

Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

Elbrecht

Bourlat

Hörren³

et al. 2020

Preprint

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“…After manual inspection, 192 papers were removed from this list because they clearly were outside a biodiversity context. The full list of all papers considered is available upon request [Colour figure can be viewed at wileyonlinelibrary.com] 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020Microbial Macrobial Both the DNA extracted from bulk samples (see, for example , Lynggaard et al, 2019;Nielsen et al, 2019, both studies working on bulk samples but published in a journal dedicated to eDNA). Hence, the same term is used in slightly different ways for different types of studies, which can lead to misunderstandings or confusion.…”

Section: The E Voluti On Of the Edna Con Cep T: From MI Crob Ial Tomentioning

confidence: 99%

Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring

Pawłowski

Apothéloz-Perret-Gentil

2020

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The last decade brought a spectacular development of so‐called environmental (e)DNA studies. In general, “environmental DNA” is defined as DNA isolated from environmental samples, in contrast to genomic DNA that is extracted directly from specimens. However, the variety of different sources of eDNA and the range of taxonomic groups that are targeted by eDNA studies is large, which has led to some discussion about the breadth of the eDNA concept. In particular, there is a recent trend to restrict the use of the term “eDNA” to the DNA of macro‐organisms, which are not physically present in environmental samples. In this paper, we argue that such a distinction may not be ideal, because the eDNA signal can come from organisms across the whole tree of life. Consequently, we advocate that the term “eDNA” should be used in its generic sense, as originally defined, encompassing the DNA of all organisms present in environmental samples, including microbial, meiofaunal and macrobial taxa. We first suggest specifying the environmental origin of the DNA sample, such as water eDNA, sediment eDNA or soil eDNA. A second specification would then define the taxonomic group targeted through polymerase chain reaction amplification, such as fish eDNA, invertebrate eDNA and bacterial eDNA. This terminology does also not require assumptions about the specific state of the DNA sampled (intracellular or extracellular). We hope that such terminology will help better define the scope of eDNA studies, especially for environmental managers, who use them as reference in routine biomonitoring and bioassessment.

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“…Drying of specimens is included in some non-destructive DNA extraction protocols (e.g. Nielsen, Gilbert, Pape, & Bohmann, 2019, Vesterinen et al, 2016 prior to digestion in the lysis buffer. Samples may also dry up accidentally, especially in difficult field conditions.…”

Section: Experiments 3: Effect Of Storage and Processing Factorsmentioning

confidence: 99%

The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies

Marquina

Ronquist

Łukasik

2020

Preprint

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1.Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well. Highly concentrated ethanol (95-100 %), preferred as a DNA preservative for molecular biology, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies of the correlation between ethanol concentration and specimen preservation are lacking.2. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After the treatments, we counted the number of appendages (legs, wings, antennae or heads) that each specimen had lost. 3.We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90 % made the insects more brittle and resulted in more shriveling, but insects with more robust or sclerotized exoskeletons actually retained more of their appendages at high concentrations. Surprisingly, neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the loss of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. 4.While higher ethanol concentrations might positively affect long-term DNA preservation, there is a clear trade-off between collecting and preserving insects for morphological examination and genetic analysis, since the optimal ethanol concentration for the latter is detrimental for the former and vice versa. The trade-off needs to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of the samples.

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A simplified DNA extraction protocol for unsorted bulk arthropod samples that maintains exoskeletal integrity

Cited by 42 publications

References 43 publications

Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

Pooling size sorted malaise trap fractions to maximise taxon recovery with metabarcoding

Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring

The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies

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