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

Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95–100%), preferred...

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Autores principales: Marquina, Daniel, Buczek, Mateusz, Ronquist, Fredrik, Łukasik, Piotr
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883690/
https://www.ncbi.nlm.nih.gov/pubmed/33614282
http://dx.doi.org/10.7717/peerj.10799
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author Marquina, Daniel
Buczek, Mateusz
Ronquist, Fredrik
Łukasik, Piotr
author_facet Marquina, Daniel
Buczek, Mateusz
Ronquist, Fredrik
Łukasik, Piotr
author_sort Marquina, Daniel
collection PubMed
description Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95–100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. 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 preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, or heads) that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. 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, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.
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spelling pubmed-78836902021-02-19 The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies Marquina, Daniel Buczek, Mateusz Ronquist, Fredrik Łukasik, Piotr PeerJ Biodiversity Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95–100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. 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 preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, or heads) that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. 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, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens. PeerJ Inc. 2021-02-12 /pmc/articles/PMC7883690/ /pubmed/33614282 http://dx.doi.org/10.7717/peerj.10799 Text en ©2021 Marquina et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biodiversity
Marquina, Daniel
Buczek, Mateusz
Ronquist, Fredrik
Łukasik, Piotr
The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title_full The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title_fullStr The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title_full_unstemmed The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title_short The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
title_sort effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
topic Biodiversity
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883690/
https://www.ncbi.nlm.nih.gov/pubmed/33614282
http://dx.doi.org/10.7717/peerj.10799
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