Photosynthesis plays a fundamental role in understanding plant growth and productivity. The chloroplast, the organelle of photosynthesis, evolved following the endosymbiotic uptake of a cyanobacterium and massive gene transfer. As a result, the chloroplast is highly dependent upon nuclear genes to provide essential chloroplast proteins. A special form of endosymbiosis, kleptoplasty, has evolved in the marine mollusc Elysia chlorotica. This green, leaf-like animal carries out photosynthesis for its entire ten month life-cycle, as if it were a plant, by using chloroplasts it steals and retains from the alga Vaucheria litorea. It is highly likely that horizontal gene transfer (HGT) has occurred between these two unrelated multi-cellular organisms, to support the long-term activity of the chloroplasts in the sea slug. The overall approach of this study is to combine cellular and molecular analyses with studies of whole animal biology to understand how such an endosymbiotic association can form and be sustained, and also influence the evolution of photosynthesis in an animal. These studies will identify specific examples of HGT from the alga to the sea slug, where and how the genes are integrated into the animal DNA, the mechanism of recognition and uptake of the chloroplasts, and the specificity of the association. The broader impact of these studies is seen at many levels. Fascinating organisms can transform the teaching of basic principles in biology; hence, the "solar-powered" sea slugs will be exploited in developing multimedia educational materials for students of all ages. Kleptoplastic sea slugs also potentially have a direct bearing on human health, through their production of anti-cancer compounds. The ability to culture the sea slugs will provide a supply of this unusual organism for classrooms and laboratories, marine hobbyists, and contributes to protection of a rare species and its native habitat.
The importance of symbiotic associations has become increasingly evident in the past few years as advances in technology have aided our understanding of the make-up and resulting new activities/products that result from these associations. This is true whether looking at soil-root microorganisms or inhabitants of the termite or human gut. We examined a unique symbiotic association that has led to the ability of a marine mollusk (the sea slug Elysia chlorotica) to carry out photosynthesis as if it were a plant. This acquired property of the animal results from it forming a long-term, functional symbiotic association with the green, photosynthetic organelles (chloroplasts) of the particular alga (Vaucheria litorea) that it feeds on. Our research focused on characterizing how this rare, yet specific association forms, what sustains the activity of the captured chloroplasts in the foreign host, and what and how other microorganisms contribute to the association (for example, by assimilating nitrogen, producing defense compounds, etc.). Our experimental approaches have ranged from very basic molecular analyses including sequencing the mitochondrial and nuclear genome of the mollusk and the chloroplast genome of the alga, to more applied approaches including establishing an aquatic culture system to raise the mollusks and establish the symbiosis in culture. The results of our sequencing work showed that the algal chloroplasts are typical in terms of gene content and, despite what we observe, they should not be able to survive very long when isolated from the algal cell. Our original hypothesis suggesting gene transfer from the algal nucleus to the mollusk contributes to the long-term functioning of the chloroplasts was not supported after analyzing the sea slug genome and transcriptome and failing to identify any examples of foreign gene insertion. Microscopic and metabolic analyses of the early developmental stages of formation of the symbiosis revealed that the timing of irreversible retention of chloroplasts by the mollusk is associated with massive lipid production by the captured chloroplasts, including lipids known to act as anti-oxidant metabolites. This is particularly important in an organism absorbing the energy of the sun and producing reactive oxygen compounds, and it may be key to the long-term survival of the chloroplasts and the mollusk. The lipids provide a high energy carbon source for the animal while also protecting the chloroplasts and animal from oxidative damage. Understanding this unique association has implications ranging from a) establishing artificial photosynthesis in other organisms or systems, to b) better understanding immune rejection and pathogen infection, to c) the biosynthesis of specific lipid compounds and novel anti-cancer compounds. From a more practical standpoint, the establishment of a culture system for the animals not only allows us to study every step of development, but also provides a system to generate animals for teaching and public outreach activities. Commercial development of this system would likely alleviate stress on the dwindling numbers of these animals in nature and serve to protect their natural habitat. This project contributed to the development of both undergraduate and graduate students, research technicians, and post-doctoral associates. Two different grants were obtained to mentor and train female undergraduates based on this project and we routinely hosted a summer high school student from a disadvantaged background. Collaborations were established with scientists at Rutgers University (NJ), Trinity University (CT), Maine Maritime Academy (ME) and McLennan Community College (TX), whereas international collaborations were established with scientists in Germany, Egypt and Portugal. Several school teachers in ME, MA, and OH used these unusual photosynthetic organisms in teaching, and various school groups were introduced to symbiosis through hands-on experiments in our lab or their classrooms. Most recently, images of the striking "leaves that crawl" formed part of the art exhibit, "Shifting the Paradigm: Microbes as Animal Helpmates," held at Cornell University to promote the beauty, diversity and importance of animal-microbial symbiosis.
