Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry
Abstract Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the ear...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2020-07-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-020-69491-0 |
| _version_ | 1818347086298480640 |
|---|---|
| author | Carly J. Randall Justin E. Speaks Claire Lager Mary Hagedorn Lyndon Llewellyn Rock Pulak Julia Thompson Line K. Bay David Mead Andrew J. Heyward Andrew P. Negri |
| author_facet | Carly J. Randall Justin E. Speaks Claire Lager Mary Hagedorn Lyndon Llewellyn Rock Pulak Julia Thompson Line K. Bay David Mead Andrew J. Heyward Andrew P. Negri |
| author_sort | Carly J. Randall |
| collection | DOAJ |
| description | Abstract Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species. |
| first_indexed | 2024-12-13T17:28:34Z |
| format | Article |
| id | doaj.art-9adccfefcb734ed083ce67846a2488b7 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-13T17:28:34Z |
| publishDate | 2020-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-9adccfefcb734ed083ce67846a2488b72022-12-21T23:37:07ZengNature PortfolioScientific Reports2045-23222020-07-0110111110.1038/s41598-020-69491-0Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometryCarly J. Randall0Justin E. Speaks1Claire Lager2Mary Hagedorn3Lyndon Llewellyn4Rock Pulak5Julia Thompson6Line K. Bay7David Mead8Andrew J. Heyward9Andrew P. Negri10Australian Institute of Marine ScienceAustralian Institute of Marine ScienceHawaiʻi Institute of Marine BiologyHawaiʻi Institute of Marine BiologyAustralian Institute of Marine ScienceUnion Biometrica, Inc.Union Biometrica, Inc.Australian Institute of Marine ScienceAustralian Institute of Marine ScienceAustralian Institute of Marine Science, Indian Ocean Marine Research Centre, University of Western AustraliaAustralian Institute of Marine ScienceAbstract Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.https://doi.org/10.1038/s41598-020-69491-0 |
| spellingShingle | Carly J. Randall Justin E. Speaks Claire Lager Mary Hagedorn Lyndon Llewellyn Rock Pulak Julia Thompson Line K. Bay David Mead Andrew J. Heyward Andrew P. Negri Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry Scientific Reports |
| title | Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry |
| title_full | Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry |
| title_fullStr | Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry |
| title_full_unstemmed | Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry |
| title_short | Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry |
| title_sort | rapid counting and spectral sorting of live coral larvae using large particle flow cytometry |
| url | https://doi.org/10.1038/s41598-020-69491-0 |
| work_keys_str_mv | AT carlyjrandall rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT justinespeaks rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT clairelager rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT maryhagedorn rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT lyndonllewellyn rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT rockpulak rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT juliathompson rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT linekbay rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT davidmead rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT andrewjheyward rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry AT andrewpnegri rapidcountingandspectralsortingoflivecorallarvaeusinglargeparticleflowcytometry |