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 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. Coral populations have suffered widespread losses over the last half century as a result of local, regional, and global pressures driven by human activities 1-4. Research on the early life-history stages of corals is foundational to our understanding of coral bioecology and is an increasingly active area of research as we enter a new era of restoration and adaptation science 5. Yet, research on the early stages of broadcast spawning corals is severely restricted by spawning events that only occur on a few nights of the year for a given species and location 6,7. These arduous periods often are defined by long working hours, demands on equipment and resources shared across research groups, and hence missed research opportunities. Much research relies on the painstaking process of counting and hand-sorting individual sub-millimetre diameter oocytes, embryos and larvae into well plates, test tubes, or petri dishes 8. Furthermore, rapid rates of cleavage 9 leave little time to work with early developmental stages. Investigations that employ CRISPR/Cas9, for example, require fertilized but not yet cleaving eggs 10 , offering as little as a few hours per year for research. A rapid and reliable method for sorting coral eggs, embryos and larvae, capturing population-level data, and sorting the samples based on condition would en...

Coloring coral larvae allows tracking of local dispersal and settlement

“…The peak emission spectra of neutral red (610 to 630 nm; [ 23 ]) and Nile blue (650 to 670 nm; [ 24 ]) are distinct from the spectral signatures of green fluorescent proteins in Acropora millepora larvae (510 to 520 nm; [ 25 ]), highlighting their potential use as fluorochromes in cellular imaging or cytometry applications. For example, combing our method with large-particle flow cytometry of live larvae [ 26 ] would enable sorting of larval cohorts in experiments to assign parentage, or allow rapid separation to recover experimentally colored larvae from mixed wild cultures in large-scale field experiments. Following refinement of concentration and exposure times, our results indicate that for two vital stains (neutral red and Nile blue), coloring coral larvae has minimal direct (reduced larval survival) or indirect (latent effects on settlement and metamorphosis) impacts, with no clear differences observed from controls.…”

A High throughput method for egg size measurement inDrosophila

“…Advent of large particle flow cytometers has made the automated analysis and sorting of large particles up to 1500 μm possible. Analysis and sorting of nematodes (Pillai & Dandurand, n.d.) , D. melanogaster (Furlong et al, 2001) and Anopheles gambiae eggs (Marois et al, 2012), and coral larvae (Randall et al, 2020) have been performed by measuring the fluorescence intensity of the expressed protein reporters. However, the fluorescence -based analysis requires fluorescence labelling or transgenic modification of individuals.…”

“…LPFC has been used for the analysis of fluorescent-labeled or transgenic organisms e.g. egg of nematodes (Pillai & Dandurand, 2021) , D. melanogaster (Furlong et al, 2001) and Anopheles gambiae (Marois et al, 2012), and coral larvae (Randall et al, 2020). Non-fluorescent labeled objects can also be analyzed and sorted on LPFC solely based on their size.…”

A high throughput method for egg size measurement in Drosophila

“…LPFC has been used for the analysis of fluorescent-labeled or transgenic organisms e.g. eggs of nematodes 15 , D. melanogaster 16 and Anopheles gambiae 17 , and coral larvae 18 . Non-fluorescent labeled objects can also be analyzed and sorted on LPFC solely based on their size.…”