Coral reefs are often considered as the proverbial canary in the mine for the oceans. Their current decline alarmingly correlates with indirect and direct anthropogenic stress resulting in increased frequencies and geographic spread of mass mortality, i.e., �coral bleaching�, and disease events. Thus, a rapid advancement in the understanding of the factors determining coral health and disease is of great importance. The post-genomic era has propelled both the development of high-throughput technologies and a shift in biological research from reductionist to system-level approaches. By viewing an organism as an �integrated and interacting network of genes, proteins, and biochemical reactions�, the nascent field of systems biology seeks to understand biological processes at a holistic level. In this dissertation, corals are considered as superorganisms, or �holobionts�, i.e., biological entities composed of a host organism and all of its associated microorganisms. The microbial component has been shown to play key roles in the functioning of the coral holobiont, but much remains to be studied about its 1) diversity, 2) response to stress, and 3) influence on the physiology, ecology, and evolution of the host. To address these gaps, I have applied a combinatorial approach of 454-sequencing, microbial community profiling (Phylochip), algal and host genotyping, as well as cDNA microarrays. The results include the most comprehensive census of coral-associated bacteria so far and illustrate that corals provide specialized habitats for an extremely diverse consortium of bacteria, including taxa that are often unknown, as well as rarely or not detected in the seawater. In diseased corals, bacterial communities profiles shifted and displayed a higher abundance of bacteria that are commonly found in other diseased marine invertebrates. Finally, correlating algal genotype and coral host transcriptomic data revealed a substantial interaction between microbial symbionts and host gene expression. These results represent initial efforts towards capturing the parts lists, i.e., the microbial diversity in coral holobionts, and integrating them with host transcriptomic data. The relevance of the results are discussed in the context of an envisioned coral (eco)systems biology approach to advance our understanding of coral health and disease.

Due to the advantages of the hard, calcifying shell, the Mollusca are one of the most successful animal phyla. The shell forms during embryonic and larval development; however, many molluscan groups have a highly reduced shell or have lost it completely as development and maturation proceeds. These major developmental transitions in shell morphology frequently correlate with ecological transitions (e.g. diet change/change from planktonic to bethic existence pre- and post-metamorphosis, respectively). While shell loss may leave an organism vulnerable to predation, many have evolved alternative means to deter predators. Here we compare and contrast the post-hatching larval development and shell growth through the use of the life cycle staging of Bursatella leachii and Aplysia californica in laboratory settings. The larval developmental sequence of B. leachii is indistinguishable to other previously described plankotrophic aplysiids. However, the growth rate and size of B. leachii larvae differ from A. californica larvae substantially, growing relatively faster and larger by an average of 10 μm. We also describe the life cycle of B. leachii in context of the development of the larval shell and its subsequent loss in post-metamorphic stages. Comparison of the Stage 6 shells, both whole and cross-sections, of A. californica and B. leachii through the use of SEM showed little difference in morphology. These data indicate that we have established a reliable culturing technique for B. leachii in the laboratory which makes this species can be easily amendable to experimentation at all developmental stages. Metamorphosis and shell loss/reduction in A. californica and B. leachii highlight the differences of the developmental program of both species, which reflects its complexity at a molecular, cellular and organismal level. The comparison of sea hares is an ideal evolutionary comparative model system for the loss of acquired features. Molluscan biomineralization has been of broad scientific interest ranging from paleontological (molluscan shells provide one of the best fossil records for a metazoan phylum), to material science (perl and nacre formation) research. Although the properties (i.e. evolutionary origins, construction, patterning, physical) of the molluscan shell have been studied for decades, the underlying molecular and cellular mechanisms of how shell formation occurs are just recently surfacing with the identification of a handful of shell forming proteins. It is now known that one of the main components involved in the control of shell synthesis are the proteinaceous constituents of the shell matrix with in different kinds of functions (i.e. cell signaling, enzymatic activities), which are contributing to the diversity of different shell types in gastropod, bivalve and scaphopod molluscs. However, the differential gene expression and regulation within the mantle still remains unknown. Here we relate the developmental expression of eleven genes present in the mantle, the organ responsible for the secretion of the shell, in the sea hare Aplysia californica (Opisthobranchia, Anaspidea). Six genes that show very little changes in expression levels (Cluster 1). Three genes shows increased levels of expression during trochophore and veliger stages which then decrease in metamorphic stages (Cluster 3). Two genes had peptide-like profiles, genes that low expression during early development but have high expression levels late in development (Cluster 4). All eleven genes display dynamic spatial and temporal expression profiles within the larval shell field and mantle for the construction of the larval shell. The expression data from these eleven genes reflect the regulatory complexity that underlies the molluscan shell construction during larval stages. While the fabrication of the shell is taking place, the incorporation of both ancient and novel genes during also suggest that there is a core set of mantle-secreting genes for shell construction was provided by a shared metazoan ancestor to produce the range of molluscan shell types we see today.

The robust clade coral Montastraea faveolata engages in a symbiosis with the microeukaryote Symbiodinum. We used this experiment in combination with results from previous microarray experimentation in coral of the same species to provide a time series that begins before metamorphosis and ends after the transition from planula to polyp in Montastraea faveolata. This time series allows us to evaluate the effects of symbiosis upon the host transcriptome at various stages of metamorphosis and days after infection--Abstract.

The reef-building coral species of tropical seas worldwide, together with their algal endosymbionts, drive the productivity of coral reef ecosystems by forming the three-dimensional structure of reefs and by functioning as primary producers. The photosynthetic endosymbionts (of the genus Symbiodinium) are key to the role of primary production. This mutualism is under intense investigation because high temperature anomalies in coastal seas trigger a breakdown in the symbiosis known as coral bleaching. As sea surface temperatures rise due to global climate change, understanding the physiological, cellular, and molecular mechanisms underlying coral bleaching is imperative to predict whether corals will survive this age of human-induced environmental degradation. Operating under this impetus, genomic approaches to studying the coral-algal symbiosis and its breakdown, such as cDNA microarrays, offer great promise. Microarrays allow for the rapid quantification of gene expression for thousands of genes in a single snapshot. In focusing on thermal stress and bleaching microarray experiments in the Caribbean corals Acropora palmata and Montastraea faveolata, I have found that the following cellular processes/components are affected during bleaching: oxidative stress, chaperone activity, the glyoxylate cycle, DNA repair, calcium homeostasis, cell death, the extracellular matrix, cell cycle progression, the actin cytoskeleton, nitric oxide signaling, and metabolite transfer between host and symbiont. Specific differentially expressed genes represent subjects of further investigation that are likely involved in coral-specific processes such as calcification, symbiosis maintenance, and bleaching. Furthermore, my experiments have revealed the striking effects that both stress and different symbiont genotypes can have on the host transcriptome. It is hypothesized that the host transcriptome is shaped by the competing effects of stress and the symbionts, and that this balance depends on both technical parameters (e.g. acclimation time, thermal treatment) and biological factors (e.g. species-specific stress sensitivities).

Sea hare species within the Aplysia clade are distributed worldwide. Their phylogenetic and biogeographic relationships are, however, still poorly known. New molecular evidence is presented from a portion of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) that improves our understanding of the phylogeny of the group. Based on these data a preliminary discussion of the present distribution of sea hares in a biogeographic context is put forward. Our findings are consistent with only some aspects of the current taxonomy and nomenclatural changes are proposed. The first, is the use of a rank free classification for the different Aplysia clades and subclades as opposed to previously used genus and subgenus affiliations. The second, is the suggestion that Aplysia brasiliana (Rang, 1828) is a junior synonym of Aplysia fasciata (Poiret, 1789). The third, is the elimination of Neaplysia since its only member is confirmed to be part of the large Varria clade.

The taxonomic diversity of endosymbiont Symbiodinium species and the physiological contributions they confer to coral host species are challenging to assess. It seems likely the genetic diversity of obligate Symbiodinium species modulates the physiological performance of reef building corals. Here we used deep amplicon sequencing to reveal high genetic diversity of the endosymbiotic dinoflagellate, Symbiodinium, in coral hosts by amplifying ribosomal DNA internal transcribed spacer region 2 (ITS-2). We revealed quantitative proportions of mixed Symbiodinium populations within the Orbicella species complex collected from three Caribbean locations. Symbiodinium diversity at the Flower Garden Banks for Orbicella franksi and Orbicella faveolata demonstrated coral host species contained Symbiodinium clade B type B1 and detected five genetically variable B1 Symbiodinium types, three of which may be endemic to the Flower Garden Banks locations. Our second dataset assessed Symbiodinium diversity in O. faveolata collected from Puerto Morelos, Mexico, previously genotyped using restriction fragment length polymorphism (RFLP) and direct sequencing. We detected an additional background population of clade A, type A13. Additionally we showed one colony hosted a mixed infection of Symbiodinium genotypes B1 and A13 where RFLP and direct sequencing assigned genotypes C7 and B1. Our third dataset assessed Symbiodinium diversity across a depth gradient in O. faveolata collected from Curaçao using the Illumina MiSeq platform. We showed shallow water coral hosted a more complex mixed infection relative to hosts from deeper waters and detected Symbiodinium genotype G3, a genotype not commonly associated with scleractinian hosts. These results highlight the need for consistent molecular genotyping techniques to assess community assemblages of Symbiodinium-host relationships. This deep sequencing approach used to characterize cryptic genetic diversity of Symbiodinium will potentially contribute to the understanding of physiological variations among coral populations.