Development of a quantitative PCR–high-resolution melting assay for absolute measurement of coral-Symbiodiniaceae associations and its application to investigating variability at three spatial scales
Previous molecular studies have shown that many corals host a dynamic consortium of dominant and background populations of Symbiodiniaceae genotypes with putatively distinct physiological traits. In the present study, we developed a quantitative PCR assay combined with high-resolution melting analysis (qPCR–HRM) to distinguish which Symbiodiniaceae clades are present in a sample. Because the qPCR–HRM used in isolation yielded identical melt profiles for both clades A and C, this analysis was then coupled with further specific qPCR assays to enable the absolute quantification of all Symbiodiniaceae clades and host cells. When the assays were applied to in hospite samples, they had an absolute quantification level corresponding to one coral embryo of two cells and 1000 symbiont cells. The assays were successful on coral fragments from twelve species (eight families). We then used the qPCR–HRM assay in an ecological survey of Acropora pulchra at different spatial scales (within colony, between colonies and between sites). Differences in abundance and composition of Symbiodiniaceae clades in A. pulchra were observed at all spatial scales, suggesting that various environmental factors drove changes in Symbiodiniaceae assemblages among and within coral colonies. The qPCR–HRM assay developed in this study is a relatively simple, cost-effective and reproducible tool that can be used to accurately differentiate and quantify endosymbiont Symbiodiniaceae clades in coral in the field. This will provide new insights into coral-symbiont shuffling mechanisms and the resilience of coral colonies to environmental stressors.
This work was funded by the ANR “R-ECOLOGS” and the ANR JCJC “Live and let die”. Sincere thanks are due to Prof. Mary-Alice Coffroth from State University of New York at Buffalo (USA) and Dr. Hollie Putnam from University of Rhode Island (USA) for providing us with Symbiodiniaceae cultures and DNA samples, as well as Dr. Sarah Nahon from French National Institute for Agricultural Research (France) for providing pictures. The authors would like to thank the staff of the CRIOBE for their help during the experiments, and especially Antoine Puisay for his help in sampling corals.
A-LM and LH designed research; ALM performed research; ALM and XP analyzed data; ALM and XP wrote the paper; and J-FG, SAW and LH revised the paper.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Coral collection was performed according to the French Polynesia regulation.
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