SGER: Proof of concept on using spliced leader to study dinoflagellate gene expression in the ocean

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Intellectual merit: Dinoflagellates are second only to diatoms in abundance and the potential of carbon fixation in the ocean, are essential for growth of reef-building corals, and are the most important contributors to harmful algal blooms. Despite extensive laboratory and field experiments, knowledge of dinoflagellate growth and interaction with the environment and other organisms in natural marine ecosystems is still very limited because research results from manipulated experiments (laboratory and field) often do not explain dinoflagellate dynamics in nature. The ability to measure in situ gene expression for a natural dinoflagellate assemblage amidst other co-existing organisms would increase understanding of dinoflagellate-associated oceanographic processes without experimental manipulation. Recently, the investigator has identified a dinoflagellate-specific trans-spliced leader sequence (DinoSL). These findings strongly suggest that DinoSL can be used to isolate dinoflagellate mRNA from a mixed RNA sample contributed by many organisms, which would facilitate studies of dinoflagellate-associated oceanographic and ecological processes. However, the concept has not been proven with systematic evaluation, and there is skepticism about whether this technique will be 1) specific for dinoflagellates and 2) universal for all mRNAs from the dinoflagellate nuclear genome. Proof of the suitability of the DinoSL-based technique will provide a new tool for dinoflagellate oceanographic research, alleviating technical obstacles for dinoflagellate studies to examine the potential of carbon fixation, grazing, stress, response to climate change, competition with other phytoplankton, growth, and contribution to coral growth in the natural environment. In this project, the investigator will construct and sequence cDNA libraries using DinoSL as the selective primer for dinoflagellate cultures, pure and mixed with other phytoplankton, and verify that only and all dinoflagellate genes will be isolated.Broader impact: If successful, this project will bring about an innovative tool for dinoflagellate research with far-reaching implications for future oceanographic studies. Furthermore, the investigator plans to use Symbiodinium microadriaticum, an endosymbiont essential for reef growth, as a model organism. If the proposed concept is proved, the DinoSL-based technique will be immediately applicable to research on this and other dinoflagellate species. Furthermore, this project will generate a substantial data set for the S. microadriaticum that will be useful for future research.. Furthermore, the proved concept may become an addition to the aquarium exhibit the investigators are working on with Mystic Aquarium. This exhibit will include an introduction to harmful algal blooms, how dinoflagellates contribute to them, how dinoflagellate toxins impact the ecosystem and public health, and how dinoflagellate genomes regulate these activities. In addition, information obtained in this study may be added to the Encyclopedia of Life (EoL) online pages on dinoflagellates.