EAGER: Collaborative Research: Mechanisms of Establishing and Maintaining an Algal Endosymbiont in a Vertebrate Host

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The investigators have recently discovered the first example of a beneficial, symbiotic alga entering cells of a vertebrate host. The host in this association is the common spotted salamander (Ambystoma maculatum) from Eastern North America. The green algal symbiont enters tissues and cells of the salamander embryos soon after they begin development in temporary vernal pools. The project will use several experimental approaches, including cell co-culturing, mRNA sequencing, and laser-based cell sorting, to determine the mechanisms of algal cell entry into the salamander host and the physiological implications of this unique association. The successful completion of this project will also make new tools available for introducing foreign genes (transgenics) in both the algal symbiont and salamander host, which will allow further investigation into the cellular and molecular mechanisms that enable this intimate association. The project will include training of undergraduate students from Gettysburg College in both field sampling of multiple salamander species as well as advanced techniques in cell culture, cell sorting, and next generation sequencing. The project will also support a museum video exhibit through the American Museum of Natural History (AMNH), and fund a post-doctoral researcher at the museum.This project outlines several high-risk high-reward experiments that will significantly advance our understanding of the molecular mechanisms of this intimate association and provide a suite of research tools for future investigations. The methods that will be employed to accomplish these goals include: Collecting salamander embryos from New York, Pennsylvania and Washington State; Culturing embryos and their algae from two salamander species and switching symbionts between hosts; Dissociation and fluorescence-activated cell sorting (FACS) of host cells containing algal endosymbionts; Transcriptomics and accompanying bioinformatics to determine differentially expressed genes in the host+endosymbiont cell population; Establishing host endoderm cell culture for in vitro co-culturing with algal symbionts; Pilot transgene transformation of both host and symbiont cell cultures using protocols and vectors established in closely related model organisms. The results from this work will reveal how a host?s response to a beneficial symbiont can differ from known vertebrate reactions to harmful pathogens that enter host cells.