Coral reefs ecosystems are some of the most productive and diverse in the world. The symbiotic association between cnidarians and their symbiotic microalgae is of great importance in coral reef ecosystems; however, its underlying molecular mechanism remains unclear even after decades of research. Rab small GTP binding proteins are critical regulators of vesicle trafficking. Here we present the experimental evidence supporting a possible association of ApRab37 with the surface of lipid droplets in the endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly known as zooxanthellae). ApRab37, a protein of 215 amino acids, displays strong homology with human Rab37. In transfected COS7 cells, EGFP-ApRab37 localized to lipid droplets and clustered in the peri-nuclear region, which stained positive for the ER (endoplasmic reticulum) marker. Immunostaining analysis found ApRab37 associated with symbiosomes and lipid droplets, which was also confirmed by Western blot analysis of in the enrich lipid droplet fraction. Phagocytosis assay showed that ApRab37 involved in late phase of phagocytosis. DCMU treatment indicates symbiosome association of ApRab37 is mediated by zooxanthellae. I propose that ApRab37 plays a pivotal role in the regulation of lipid trafficking from the symbiosomes to the host cell during the Aiptasia-zooxanthellal endosymbiosis.

Abstract In order to explore the mechanisms causing trade-offs between sexual and asexual reproductions, the sea anemones Aiptasia pulchella were taken as objects since they can reproduce by both modes simultaneously. Specimens were reared under different temperatures, photoperiods, and densities. Total weights of lacerates were used as an index of investment in asexual reproduction, whereas gonad weights as an index of investment in sexual reproduction. Sea anemones reared under low temperature (22¢J) reached larger pedal disks and produced larger, heavier lacerates, they also produced heavier gonads than those under high temperature (29¢J). Those reared under long light hours (15¡G9) reached smaller sizes and produced heavier gonads than those under short light hours (9¡G15). But the effect of photoperiod on lacerate size, number and weight are not significant. Interaction effect was not significant between these two factors. Correlation between sexual and asexual investments was not significant, either. In addition, A. pulchella reared under low density (1 sea anemone/beaker) produced more, heavier lacerates and heavier gonads than those under crowding conditions (4 sea anemones/beaker). There was a significantly positive correlationship between sexual and asexual reproduction investments. It seems there was no trade-offs between sexual and asexual investiments of the sea anemone A. pulchella.

Coral reefs are crucial for the ecological sustainability of the oceans, yet, increasing sea surface temperature is threatening these ecosystems globally. Microbial communities associated with corals have become a recent research focus, as the associated microbiome may contribute to coral resilience to environmental stressors, e.g., heat stress. However, research in this area is hampered by the difficulty of working with corals. This study aims to use Aiptasia, a sea anemone, as a tractable laboratory model system to study the role of the coral microbiome. Analyses of the bacterial compositions associated with different Aiptasia strains across two temperatures (25 °C and 32 °C), based on 16S rRNA gene sequencing. This study aims also to identify a “core” microbiome associated with heat stress acclimation, as well as host-specific differences. In general, results showed that bacterial composition associated with Aiptasia strains differs significantly with temperature. Higher bacterial diversity and richness were observed when all Aiptasia strains were placed under heat stress. Moreover, results showed an increase in beta diversity and dispersion of bacterial communities in response to heat stress. These changes in the bacterial composition are in line with the recently described “Anna Karenina principle” for animal microbiomes, which suggests that the microbiomes of unhealthy individuals vary more than healthy and stable individuals. This study further shows that while temperature had the greatest effect on structuring the bacterial compositions, there were some variations better attributed to batch and host effects. This suggests that technical aspects have to be carefully addressed in the framework of microbiome studies. Members of a putative “core” microbiome associated with 32 °C Aiptasia have been identified as indicator species of heat stress (i.e., Francisella sp.,). Previous reports have shown that these indicator taxa are associated with saline environments and can tolerate high temperatures. Putative functional profiles based on taxonomic inference of associated bacterial taxa (i.e., enrichment and depletion of various metabolic processes) were also identified, implying functional differences of the microbiomes associated with Aiptasia strains in response to heat stress. Future studies should more specifically examine how the microbiome influences the animal ability to respond to environmental changes.

Coral reefs form marine-biodiversity hotspots of enormous ecological, economic, and aesthetic importance that rely energetically on a functional symbiosis between the coral animal and a photosynthetic alga. The ongoing decline of corals worldwide due to anthropogenic influences heightens the need for an experimentally tractable model system to elucidate the molecular and cellular biology underlying the symbiosis and its susceptibility or resilience to stress. The small sea anemone Aiptasia is such a model organism and the main aims of this dissertation were 1) to assemble and analyze its genome as a foundational resource for research in this area and 2) to investigate the role of miRNAs in modulating gene expression during the onset and maintenance of symbiosis. The genome analysis has revealed numerous features of interest in relation to the symbiotic lifestyle, including the evolution of transposable elements and taxonomically restricted genes, linkage of host and symbiont metabolism pathways, a novel family of putative pattern-recognition receptors that might function in host-microbe interactions and evidence for horizontal gene transfer within the animal-alga pair as well as with the associated prokaryotic microbiome. The new genomic resource was used to annotate the Aiptasia miRNA repertoire to illuminate the role of post-transcriptional regulatory mechanisms in regulating endosymbiosis. Aiptasia encodes a majority of species-specific miRNAs and first evidence is presented that even evolutionary conserved miRNAs are undergoing recent differentiations within the Aiptasia genome. The analysis of miRNA expression between different states of Symbiodinium infection further revealed that species-specific and conserved miRNAs are symbiotically regulated. In order to detect functional miRNA-mRNA interactions and to investigate the downstream effects of such miRNA action, a protocol for cross-linking immunoprecipitations of Argonaute, the central protein of the miRNA-induced silencing complex, was developed. This method identified binding sites of miRNAs on a transcriptome-wide scale and revealed target genes of symbiotically regulated miRNAs that were identified previously to be involved in the symbiosis. In summary, this dissertation provides novel insights into miRNA-mediated post-transcriptional modulation of the host transcriptome and by presenting a critically needed genomic resource, lays the foundation for the continued development of Aiptasia as a model for coral symbiosis.

Coral reefs are in global decline due to ocean warming and ocean acidification. While these stressors are commonly studied in climate change predictions, salinity, although being an important environmental factor, is not well understood. The response of the coral holobiont (the association of the coral host, its algal endosymbiont and a suit of other microbes) to changes in salinity and the contribution of each holobiont compartment underlying the necessary osmoadaptation remain especially elusive. Interestingly, we find some of the most thermotolerant corals in some of the most saline seas, e.g. the Red Sea and the Persian Arabian Gulf. This observation sparked the hypothesis of a link between osmoadaptation and coral thermotolerance. Here, we set out to elucidate the putative effects of high salinity on conveying thermotolerance and thereby a possible link to bleaching in the context of the coral holobiont. For this, we conducted a series of heat stress experiments at different salinities in the coral model Aiptasia and subsequently validated our findings in corals from the central Red Sea. We confirm a role of osmoadaptation in increased thermotolerance and reduced bleaching in Aiptasia and Red Sea corals. This salinity-conveyed thermotolerance was characterized by a reduction in algal endosymbiont loss, photosystem damage and leakage of damaging reactive oxygen species (ROS) in high salinity. Further analysis of the osmoadaptation response using targeted GC-MS uncovered high levels of the sugar floridoside at high salinity only in holobionts that show the salinity-conveyed thermotolerance. The increase of floridoside, an osmolyte capable of scavenging ROS, and the concurrent reduction of ROS argues for a mechanistic link of increased thermotolerance and reduced bleaching in high salinities. In addition, the restructuring of the microbiome at high salinity that aligned with the difference in thermotolerance in Aiptasia may be indicative of a microbial contribution towards a more beneficial holobiont composition. Hence, emphasizing the potential cumulative contribution of each holobiont compartment during stress-resilience, as well as highlighting the overall role of osmoadaptation in the thermotolerance of corals.

The symbiotic relationship between cnidarians and their photosynthetic dinoflagellate symbionts underpins the success of coral reef communities in oligotrophic, tropical seas. Despite several decades of study, the cellular and molecular mechanisms that regulate the symbiotic relationship between the dinoflagellate algae and the coral hosts are still not clear. One of the hypotheses on the metabolic interactions between the host and the symbiont suggests that ammonium assimilation by the host can be the underlying mechanism of this endosymbiosis regulation. An essential intermediate of the ammonium assimilation pathway is glutamate, which is also known for its glutamatergic signaling function. Interestingly, recent transcriptomic level and DNA methylation studies on sea anemone Aiptasia showed differences in metabotropic glutamate signaling components when comparing symbiotic and non-symbiotic animals. The changes in this process on transcriptional and epigenetic levels indicate the importance of glutamate signaling in regard to cnidarian symbiosis. In this study, I tested glutamatergic signaling effect on symbiosis in sea anemone Aiptasia using a broad-spectrum glutamate receptor inhibitor 7- CKA and glutamate. Significantly decreased cell density was observed in animals with inhibitor treatment suggesting a possible correlation between glutamate signaling and the establishment or maintenance of symbiosis. Using RNA-Seq, I was able to obtain transcriptional profiles of the animals under inhibitor and glutamate treatment. Differential gene expression and gene ontology analyses indicated changes in amino acid metabolism, lipid metabolism and such signaling pathways as MAPK, NF-kappa B and phospholipase C. Although amino acid and lipid metabolism could be a result of the reduced symbiotic state of inhibitor treated Aiptasia, the signaling pathways which are related to apoptosis and immune response provide an exciting venue for direct regulatory interaction between symbiosis and glutamatergic signaling. However, as these signaling pathways mainly act via signal transduction through protein phosphorylation, further studies looking at changes on a post-translational level might provide further insight into the mechanisms underlying the observed phenotype.

Recent works has highlighted the contribution of microbes to animal function. In this regard, the microbial community associated with corals has become a growing field of research in order to understand how microbes contribute to the host organisms’ response to environmental changes. It has been shown that microbes associated with corals have important functions in the coral holobiont such as immunity and nutrient assimilation. However, corals are notoriously difficult to work with. To this end, the sea anemone Aiptasia is becoming a model organism for coral symbiosis. Given the importance of host-­microbiome interactions, the topic of this thesis is to assess microbial structure of Aiptasia, culture prominent bacterial members, and compare bacterial community structure to corals. Different molecular methods have been applied using 16S rRNA bacterial gene fragments to characterize the microbial composition of Aiptasia. 16S rRNA gene sequence derived from cultured bacteria was compared to 16S rRNA gene sequences retrieved from native Red Sea Aiptasia. Inter-­individual as well as methodological differences were found to account for variance in microbiome composition. However, all approaches showed a highly abundant microbial taxon belonging to the genus Alteromonas in all samples. The Alteromonas species was successfully isolated for further research targeting microbiome selection mechanisms in Aiptasia. Future investigations by using different molecular tools will help to define the functions and relationship between the Aiptasia and its complex microbiome.

As climate change intensifies, the capacity of organisms to adapt to changing environments becomes increasingly relevant. Heat-induced coral bleaching –the breakdown of the symbiotic association between coral hosts and photosynthetic algae of the family Symbiodiniaceae– is rapidly degrading reefs worldwide. Hence, there is a growing interest to study symbioses that can persist in extreme conditions. The Red Sea is such a place, known as one of the hottest seas where healthy coral reef systems thrive. Here (Chapter 1), we tested the potential of symbiont manipulation as means to improve the thermal resilience of the cnidarian holobiont, particularly using heat tolerant symbiont species from the Red Sea. We used clonal lineages of the model system Aiptasia (host and symbiont), originating from different thermal environments to assess how interchanging either partner affected their short- and long-term performance under heat stress. Our findings revealed that symbioses are not only intra-specific but have also adapted to native, local environments, thus potentially limiting the acclimation capacity of symbiotic cnidarians to climate change. As such, infection with more heat resistant species, even if native, might not necessarily improve thermotolerance of the holobiont. We further investigated (Chapter 2) how environment-dependent specificity, in this case elevated temperature, affects the establishment of novel symbioses. That is, if Aiptasia hosts are, despite exhibiting a high degree of partner fidelity, capable of acquiring more thermotolerant symbionts under stress conditions. Thus, we examined the infection dynamics of multi-species symbioses under different thermal environments and assessed their performance to subsequent heat stress. We showed that temperature, more than host identity, plays a critical role in symbiont uptake and overall performance when heatchallenged. Additionally, we found that pre-exposure to high temperature plays a fundamental role in improving the response to thermal stress, yet, this can be heavily influenced by other factors like feeding. Like climate change, ocean acidification is a serious threat to corals. Yet, most research has focused on the host and little is known for the algal partner. Thus, here we studied (Chapter 3) the global transcriptomic response of an endosymbiotic dinoflagellate to long-term seawater acidification stress. Our results revealed that despite observing an enrichment of processes related to photosynthesis and carbon fixation, which might seem beneficial to the symbiont, low pH has a detrimental effect on its photo-physiology. Taken together, this dissertation provides valuable insights into the responses of symbiotic cnidarians to future climate and ocean changes.

Coral reefs are the most diverse marine ecosystems of significant ecological and economic importance, globally. Increasing environmental stress imposed by global warming, ocean acidification and pollution has led to the continuous decline of coral reefs. For reefs to thrive and survive, they rely on the stable endosymbiosis between coral animal and photosynthetic algae. The fragile symbiotic relationship is dependent on a balanced metabolic exchange, which is easily disturbed by stress, consequently leading to the loss of the endosymbiotic algae - a process known as bleaching. Since corals energetically rely on the algae, the breakdown of symbiosis can have fatal consequences. However, the underlying molecular and cellular mechanisms of this symbiosis are thus far poorly understood. The small sea anemone Aiptasia has provided an experimentally tractable model organism, furthering our understanding on the function of symbiosis and hence, coral susceptibility and resilience to stress. Nonetheless, this model organism is comparatively young and therefore, requires innovative approaches as well as establishment and optimization of protocols. In this thesis, we applied transcriptomic, proteomic and epigenomic tools in Aiptasia, with the aim to assess the dynamics of symbiosis and thermotolerance. Heat stress studies, on Aiptasia's originating from geographically distinct regions, provided insight into the cnidarian-algae symbiosis mechanisms and the role of metabolic compatibility in symbiosis. Furthermore, findings elucidated that associating with thermotolerant algae can improve the cnidarian host's tolerance, potentially acting as a form of local adaptation. Finally, the role of epigenetic mechanisms in cnidarian symbiosis was investigated, by optimizing Chromatin Immunoprecipitation (ChIP) and establishing the genomic landscape of histone 3 lysine 9 acetylation (H3K9ac) in Aiptasia. These new results will enable progressing Aiptasia further as a model organism and thus, advance our understanding on the complex mechanisms of coral symbiosis.

Many organisms form mutually beneficial, symbiotic partnerships with other organisms. Corals and sea anemones undergo mutualistic symbioses with photosynthetic algae of the family Symbiodiniaceae, and these partnerships are key for the viability of coral reef ecosystems. Cnidarian-Symbiodiniaceae symbioses are sensitive to climate change-induced ocean warming, which causes the disruption of symbiosis, commonly referred to as bleaching, and can lead to coral mortality. Cellular and molecular aspects of how this symbiosis is established and disrupted by heat stress are not well understood. The research presented herein characterizes immunity transcription factor NF-kappaB in the cnidarian model organism Exaiptasia pallida (Aiptasia). It is shown that the DNA-binding site specificity of Aiptasia NF-kappaB is similar to mammalian NF-kappaB subunit p50 and that this binding specificity is conserved across a broad expanse of metazoans. Moreover, Aiptasia and human IkappaB kinases can phosphorylate serine residues in the C-terminus of NF-kappaB, signaling the protein for proteasomal processing to allow for nuclear localization, DNA binding, and transactivation. In Aiptasia, NF-kappaB expression is downregulated by symbiosis onset in larvae, and NF-kappaB total expression, DNA-binding activity, and tissue-specific expression are increased following laboratory-induced loss of symbiosis in adult Aiptasia. NF-kappaB downregulation during the onset of symbiosis occurs only with the compatible symbiont Breviolum minutum and data suggest that host TGFbeta plays a role in NF-kappaB downregulation. Results demonstrate that aposymbiotic Aiptasia (with high NF-kappaB levels) have increased survival following bacterial infection as compared to symbiotic anemones. A bioinformatic analysis shows that potential NF-kappaB binding sites are enriched in promoter regions of immune-related genes that are upregulated in aposymbiotic Aiptasia. Increased levels of NF-kappaB are also found in a genet of the coral Pocillopora damicornis that exhibits resilience to heat-induced bleaching. Overall, the results in this thesis suggest a role for NF-kappaB-directed immunity in symbiosis onset, bleaching, and resistance to biological stressors in cnidarians. It is proposed that NF-kappaB downregulation in Aiptasia is a mechanism to lower host immunity and promote the establishment of symbiosis, but that this process compromises host immunity to pathogen infection. Nevertheless, constitutively high basal levels of NF-kappaB may be protective against bleaching in cnidarians.