Date of Award

8-2012

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Mary E. Rumpho

Second Committee Member

Keith W. Hutchison

Third Committee Member

Karen N. Pelletreau

Abstract

Microbes, both free-living and symbiotic, are ubiquitous throughout all environments and are essential for eukaryotic life, playing key roles in nutrient recycling and sequestration. Bacteria can also colonize the gut and mucus of organisms where they are crucial for host development and protection. The sacogossan mollusc (sea slug) Elysia chlorotica forms an obligate endosymbiotic relationship with chloroplasts (kleptoplasty) obtained from its algal prey, Vaucheria litorea, allowing the sea slug to sustain itself photoautotrophically for several months. This photosynthetic sea slug also associates with a number of bacteria, which may contribute to the long-term success of the symbiosis through the provision of nutrients as well as developmental and protective benefits.

In this study, 16S rDNA-based metagenomic analyses (clone libraries and amplicon pyrosequencing) were utilized to characterize the microbial diversity associated with two populations of E. chlorotica from Halifax, Nova Scotia, Canada, and from Martha's Vineyard, MA, USA. Whole animals were examined immediately after collection from their native marsh environments, after being starved of their algal prey for several months in the laboratory, and after being bred in the laboratory (second-generation sea slugs) to characterize the effect of varying environmental and culturing conditions on the associated bacteria. Additionally, the microbiome of the algal prey, laboratory-cultured V. litorea, was analyzed to determine whether the lab-bred sea slugs obtained bacteria from their algal food source during development. Alpha-, beta-, and gamma-proteobacteria dominated all of the samples along with Actinobacteria, Bacilli, Flavobacteria, and Sphingobacteria. However, abundances and taxonomic diversity at the order and family levels varied between the two populations and among all samples except for the lab-bred sea slugs, which had very similar bacterial profiles to one another but were dissimilar to the algal microbiome. Bacteria capable of polysaccharide digestion and photosynthesis, as well as putative nitrogen fixation, vitamin B12 production, and natural product biosynthesis were associated with the sea slug and algal samples.

This study also characterized the microbial diversity associated with the digestive gut and secreted mucus of E. chlorotica from Nova Scotia. A comparison of wild-captured vs. lab-starved (1 month) sea slugs showed that a change in habitat and diet affected gut microflora. Likewise, the gut and mucus of wild-captured E. chlorotica revealed different selection pressures for microbes. The most prevalent classes included a-, β-, and y-proteobacteria as well as Actinobacteria, Bacilli, and Mollicutes. All samples had a high number of bacterial families in common, suggesting that these families represent transient environmental microflora acquired from the water of the native salt marsh. Most of the bacteria were chemoorganotrophs, although a number of phototrophs were identified. Putative metabolic functions of identified bacteria included carbon cycling, denitrification, nitrogen fixation, and vitamin B12 production. This study is the first to identify the bacteria living in association with E. chlorotica and V. litorea, but further localization (using fluorescence in situ hybridization) and functional studies (metatranscriptomics and metabolomics) are needed in order to confirm the presence of specific symbionts and define their metabolic function in the Elysia-Vaucheria system.

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