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Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar
Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examinatio...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9931127/ https://www.ncbi.nlm.nih.gov/pubmed/36791141 http://dx.doi.org/10.1371/journal.pone.0281759 |
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author | Fowler, Elizabeth V. Starkie, Melissa L. Zhu, Xiaocheng Piper, Alexander M. Agarwal, Arati Rako, Lea Gardiner, Alexandra Oczkowicz, Sybilla Gopurenko, David Schutze, Mark K. Blacket, Mark J. |
author_facet | Fowler, Elizabeth V. Starkie, Melissa L. Zhu, Xiaocheng Piper, Alexander M. Agarwal, Arati Rako, Lea Gardiner, Alexandra Oczkowicz, Sybilla Gopurenko, David Schutze, Mark K. Blacket, Mark J. |
author_sort | Fowler, Elizabeth V. |
collection | PubMed |
description | Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers–the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives. |
format | Online Article Text |
id | pubmed-9931127 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-99311272023-02-16 Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar Fowler, Elizabeth V. Starkie, Melissa L. Zhu, Xiaocheng Piper, Alexander M. Agarwal, Arati Rako, Lea Gardiner, Alexandra Oczkowicz, Sybilla Gopurenko, David Schutze, Mark K. Blacket, Mark J. PLoS One Research Article Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers–the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives. Public Library of Science 2023-02-15 /pmc/articles/PMC9931127/ /pubmed/36791141 http://dx.doi.org/10.1371/journal.pone.0281759 Text en © 2023 Fowler 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, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Fowler, Elizabeth V. Starkie, Melissa L. Zhu, Xiaocheng Piper, Alexander M. Agarwal, Arati Rako, Lea Gardiner, Alexandra Oczkowicz, Sybilla Gopurenko, David Schutze, Mark K. Blacket, Mark J. Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title | Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title_full | Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title_fullStr | Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title_full_unstemmed | Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title_short | Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar |
title_sort | development of a cost-effective, morphology-preserving method for dna isolation from bulk invertebrate trap catches: tephritid fruit flies as an exemplar |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9931127/ https://www.ncbi.nlm.nih.gov/pubmed/36791141 http://dx.doi.org/10.1371/journal.pone.0281759 |
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