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An improved method for the molecular identification of single dinoflagellate cysts
BACKGROUND: Dinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408718/ https://www.ncbi.nlm.nih.gov/pubmed/28462033 http://dx.doi.org/10.7717/peerj.3224 |
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author | Gao, Yangchun Fang, Hongda Dong, Yanhong Li, Haitao Pu, Chuanliang Zhan, Aibin |
author_facet | Gao, Yangchun Fang, Hongda Dong, Yanhong Li, Haitao Pu, Chuanliang Zhan, Aibin |
author_sort | Gao, Yangchun |
collection | PubMed |
description | BACKGROUND: Dinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species. METHODS: In this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera, Polykrikos kofoidii, Lingulodinium polyedrum, Pyrophacus steinii, Protoperidinium leonis and Protoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification. RESULTS: For the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning. DISCUSSION: The technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts. |
format | Online Article Text |
id | pubmed-5408718 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-54087182017-05-01 An improved method for the molecular identification of single dinoflagellate cysts Gao, Yangchun Fang, Hongda Dong, Yanhong Li, Haitao Pu, Chuanliang Zhan, Aibin PeerJ Biodiversity BACKGROUND: Dinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species. METHODS: In this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera, Polykrikos kofoidii, Lingulodinium polyedrum, Pyrophacus steinii, Protoperidinium leonis and Protoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification. RESULTS: For the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning. DISCUSSION: The technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts. PeerJ Inc. 2017-04-26 /pmc/articles/PMC5408718/ /pubmed/28462033 http://dx.doi.org/10.7717/peerj.3224 Text en ©2017 Gao et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://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 Gao, Yangchun Fang, Hongda Dong, Yanhong Li, Haitao Pu, Chuanliang Zhan, Aibin An improved method for the molecular identification of single dinoflagellate cysts |
title | An improved method for the molecular identification of single dinoflagellate cysts |
title_full | An improved method for the molecular identification of single dinoflagellate cysts |
title_fullStr | An improved method for the molecular identification of single dinoflagellate cysts |
title_full_unstemmed | An improved method for the molecular identification of single dinoflagellate cysts |
title_short | An improved method for the molecular identification of single dinoflagellate cysts |
title_sort | improved method for the molecular identification of single dinoflagellate cysts |
topic | Biodiversity |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408718/ https://www.ncbi.nlm.nih.gov/pubmed/28462033 http://dx.doi.org/10.7717/peerj.3224 |
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