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.

The ability of corals to build reefs can be attributed to their relationship with single-celled algae of the familySymbiodiniaceae.Through the process of photosynthesis, these algae can provide their coral hosts with over 90% of their daily energy requirements. Most coral species acquire multiple species of symbionts from the surrounding water during their larval stage or immediately after settling. However, over time, the coral will select a dominant symbiont speciesthat can depend on the local environment. Until this study, the size or age of the coral at which this transition from multiple Symbiodiniaceaespecies to one dominant species occurs has remained uncertain. Likewise, it was unclear whether the selection of Symbiodiniaceaespeciesis influenced by the environment. The environmental conditions and symbiont composition of one hundred and eighteen juvenile Siderastrea siderea were assessed across four sites in Broward County, Florida. Presuming newly settled corals acquire multiple symbionts and then select just one dominant species, it was determined that the transition from multiple symbiont speciesto one dominant species in Siderastrea sidereaoccurs in the single polyp stage, between the time of settlement and approximately 4 to 6 months of age. The results also suggest that the selection of these dominant symbiont speciesis influenced by the environment, and that juveniles commonly select the same species as adults inhabiting similar environmental conditions. The selection of symbionts homologous to adult corals combined with environmental influences may be an early indicator of acclimatization in Siderastrea siderea.

Microorganisms provide essential services for host organisms and this is especially true for communities of algal symbionts and bacteria hosted by tropical reef-building corals–collectively termed the coral holobiont. Endosymbiotic algae provide essential nutrients to the host, and can impact coral growth and susceptibility to environmental stress. Corals also associate with a diverse microbiome, and specific bacterial taxa have been implicated in important nutritional and immunity roles. However, fundamental questions regarding the environmental factors that govern coral holobiont composition remain unanswered. The overarching goal of my dissertation is to characterize how environmental variation, including disturbance, influences the composition of coral-associated algal and bacterial communities. To achieve this goal, I first compared these communities across reef zones (locales differing in diel thermal variability and other factors) in tandem with host genetic background under baseline conditions. I found that in the more stable reef zone, algal communities were more diverse and that host genetic diversity correlated with bacterial diversity, implying the more variable reef zone constrained diversity of host-microbial partnerships. Next, I characterized how these communities responded to a hurricane disturbance in two stress-tolerant congeneric coral species. Again, I observed stark differences across reef zones in algal symbiont and bacterial communities, but these communities were not exceptionally impacted by disturbance. Finally, I experimentally tested the role of daily thermal variability as a key environmental factor in shaping holobiont community composition and heat tolerance. While thermal variation treatment increased algal photosynthetic efficiency, it did not facilitate coral thermal tolerance to heat stress. Specific bacterial taxa were differentially abundant after 90 days in treatment acclimation, suggesting that thermal variation may contribute to part of the observed community differences across natural reef zones. In sum, this dissertation provides a deeper understanding of the interplay between coral-associated microorganisms and their local reef environments, as well as taxa-specific patterns of interest for monitoring coral holobiont dynamics under rapidly changing oceans.

Functionality of Cnidarian symbiosis with Symbiodiniaceae is fundamental to reef ecosystem success. Symbiodiniaceae cells have a complex life history, which, in hospite, is controlled by the host. In addition to the endosymbiotic lifestyle, they can exist free-living cells which diurnally alternate between a coccoid, vegetative night-time form to a day-time motile, flagellated cell. Their cell division cycle is gated by external light cues, and correlates with transitions in cell morphology. In contrast, endosymbiotic cells have an elongated G1 phase – demonstrating a de-coupling of cell cycle from 24-hour cycle in response to symbiosis. Furthermore, daughters of dividing endosymbiotic Symbiodiniaceae remain as coccoid cells, de-coupling morphological and cell division cycles. How this occurs remains unknown. The answer may lie in crystalline uric acid deposits, which are present only in motile, daytime cells, correlating with G1 and S phase. These store excess nitrogen and are quickly metabolized in low nitrogen availability. They also function as an eyespot. The influence of uric acid on the life cycle of free-living and endosymbiotic Symbiodiniaceae is unknown. In this study, I treated cultures of B. minutum with allopurinol, an inhibitor of uric acid synthesis. Flow cytometry showed that allopurinol the reduced growth rate and ratio of coccoid:motile cell cultures. RNA sequencing and differential gene expression analysis identified biological processes enriched in allopurinol treatment. I hypothesize that an intracellular lack of nitrogen imposed lack of uric acid crystals stimulates the General Amino Acid Control pathway. This represses translation, explaining the downregulation of ribosomal proteins, and upregulates amino acid and purine de novo biosynthesis pathways. Repression of translation may slow cellular growth and the G1 phase of the cell cycle, reducing number of cells meeting the size threshold for G1/S transition. Without uric acid deposits, cells may lack a functioning eyespot and not receive light cues which usually trigger morphological transitioning. This may suppress the motile morphology of free-living Symbiodiniaceae and cells in hospite even though the cell division cycle progresses, albeit more slowly. Genes involved in biosynthesis of flagella, thecal plates and the eyespot are upregulated, suggesting suppression of the motile form may act downstream of transcription.

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.

Symbiodiniaceae is a family of dinoflagellates which often live in a symbiotic relationship with cnidarian hosts such as corals. Symbiodiniaceae are vital for host survival, providing energy from photosynthesis and in return gaining protection from environmental stress and nutrients. However, when these symbiont cells are exposed to environmental stress such as elevated temperatures they can be expelled from their host, leading to the coral bleaching, a global issue. Coral reefs are vital for marine biodiversity and hold a large economic importance due to fishing and tourism.  This thesis aims to develop a computational pipeline to study growth, shape and size of Symbiodiniaceae cells, which takes microscopy images using a mother machine microfluidics device and segments the Symbiodiniaceae cells. This enables extraction ofcellular features such as area, circularity and cell count to study morphology and growth of Symbiodiniaceae based on segmentation labels. To achieve this, pretrained segmentation models from the Cellpose algorithm were evaluated to decide which was the best to use to extract features most accurately. The results showed the pretrained ‘cyto3’ model with default parameters performed the best based on the Dice score. The feature extraction showed indications of division events of Symbiodiniaceae linked to light and dark cycles, suggesting synchronicity among cells. However, segmentation needs further investigation to accurately capture cells and add statistical significance to the feature extraction.

Coral bleaching causes the disappearance of unique ecosystems supporting thousands of different species. The corals health depends on its relationship with its endosymbiont, the zooxanthellate Symbiodiniaceae. This mutualistic relationship can be disrupted by anthropogenic chemical pollution by inducing physiologically stress in Symbiodiniaceae. This thesis investigates the effects of organic ultraviolet filter chemical in Symbiodiniaceae of four genera (Fugacium, Effrenium, Symbiodinium, Breviolum), with regards to their growth and photophysiological performance. Six Symbiodiniaceae strains were exposed to a mixture of the most commonly observed UV-filter at five different concentrations. The cells growth during exposure was followed with spectrophotometry optical density measures and flow cytometry cell counts. The photophysiological performance of Symbiodiniaceae cells in presence of UV-filter chemicals was obtained using chlorophyll fluorometry. This experiment revealed strain-specific sensitivity to UV-filter chemicals, where Symbiodiniaceae strains 421 and 4013 showed highest sensitivity on cell growth. A difference in photophysical performance of Symbiodiniaceae exposed to UV-filter chemicals was observed. The ecotoxicological effects observed in Symbiodiniaceae from UV-filter chemical exposure could indicate a contribution to the disruption of the mutualistic relationship between the coral host and Symbiodiniaceae.