Coral bleaching, i.e. the loss of photosynthetic algal symbionts, caused by ocean warming is now the main factor driving reef decline, but not all corals are affected equally. Corals from the Arabian Seas have unusually high temperature tolerances, and recently studies implicated salinity as one of the contributing factors. In particular, a recent heat stress experiment at different salinities using the model system Aiptasia and Red Sea corals, showed that cnidaria at large bleach less at heat stress under high salinities and that this is associated with an increase of the osmolyte, floridoside Here we were interested to assess microbial community changes under heat stress at different salinity levels and whether this could help to explain the increase in thermal tolerance of the metaorganism at high salinities. We determined microbial community composition via HiSeq 16S rRNA gene amplicon sequencing of two anemone strains that differ in their associated symbionts, namely H2-SSB01 (type B1) and CC7-SSA01 (type A4), after six days under ambient (25 °C) and heat stress (34 °C) temperatures at salinities of 36, 39, and 42. Both anemones harbored distinct microbial communities, irrespective of temperature or salinity, that were also different from the bacteria in surrounding seawater. Within both host-endosymbiont pairings, the bacterial community composition at low (36) and intermediate (39) salinities did not differ between ambient and heat stress, but was significantly different at high (42) salinities. Subsequent elucidation of bacterial indicator species revealed several taxa that could be associated with a response to temperature and salinity. Our results underline that microbial community composition adjusts under different environmental settings. Importantly, microbial community dynamics of H2-SSB01 aligned with observed differences in bleaching susceptibility and thermal tolerance, whereas the pattern remains unclear for CC7-SSA01, which harbors an intrinsically higher thermal tolerance. Such responses could argue for a contribution of the microbiome to the observed increase in temperature tolerance of the Aiptasia metaorganism at increased salinities. An alternative interpretation is that the microbiome changes denotes a parallel response to changing salinities.

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.

Ocean warming is leading to increased occurrence of coral mass bleaching events, threatening the persistence of these ecosystems and the communities that rely on them. While reef recovery is possible, conservation approaches based purely on transplantation/coral-gardening will not suffice to maintain these ecosystems over the projected environmental changes. Assisted evolution approaches aim to boost acclimatization and adaptation processes. A potential approach could be to harness the naturally occurring mechanism of environmental memory that has been observed in corals and other organisms, where an organism remembers a priming stress event to allow a faster/stronger response when the stress re-occurs. In this thesis I aimed to investigate whether this mechanism exists and how it is regulated on a molecular level in the sea anemone Aiptasia. Aiptasia were primed to heat stress by exposing them to 32 °C water for several years, or for one week. After a recovery period of one week at 25 °C, a naïve and the primed treatments were exposed to lethal thermal stress at 34 °C for three days. Primed treatments performed better than the naïve treatment in survival, photosynthetic efficiency and symbiont density for two days, after which the priming advantage was lost. The difference between the primed treatments indicated that the priming dose may affect priming success. There were clear indications of an epigenetic transcriptional memory mechanism on a transcriptional level. I observed a pronounced difference between control and heat-stressed treatments, indicating that transcription returned to near baseline expression after cessation of the priming exposure. The functional categories of differentially expressed genes in heat stress relative to control were similar between naïve and primed treatments, with the main difference observed in a stronger up- and downregulation of stress response genes in the long-term primed treatment. I optimized a chromatin immunoprecipitation protocol for use with Aiptasia by adjusting fixation, sonication and immunoprecipitation conditions. The enrichment of H3K4me2/me3 and poised RNA Pol II in the promoters of stress response genes will be investigated next to elucidate the mechanism of the observed epigenetic transcriptional memory in Aiptasia, and to ultimately inform conservation strategies for coral reefs globally.

Many members of the Phylum Cnidaria are mutualistic with unicellular dinoflagellates belonging to the genus Symbiodinium. Corals are the most widely recognized example of these associations due to their key ecological importance in coral reef ecosystems where they serve as the structural and trophic foundation of these rich ecosystems. Coral reefs are severely threatened by human activities worldwide and are at great risk from global climate change, in particular the increase in seasurface temperatures. Detailed knowledge of how corals respond to stress is scarce. The most serious and immediate response of corals to environmental stress is a process referred to as coral bleaching (a.k.a. cnidarian bleaching). Nevertheless, the cellular and molecular processes by which elevated temperatures elicit the bleaching response are poorly understood. This dissertation deals with this important question by describing two mediators of cnidarian bleaching in the model symbiotic tropical sea anemone Aiptasia pallida (Verril), namely nitric oxide and cyclophilin. After an introduction to the topic of cnidarian-algal symbioses and cnidarian bleaching (Chapter 1), I present results from a study describing the involvement of nitric oxide (NO) in the anemone A. pallida (Chapter 2). Elevated temperature as well as oxidative stress induces production of NO and exposure of A. pallida to NO induces bleaching at non-stressful temperatures. Co-incubation with an NO scavenger suppresses bleaching. I propose that the host up-regulates NO production in response to elevated oxidative stress and that this situation leads to cytotoxicity and bleaching. Chapter 3 examines the role of cyclophilin from A. pallida in the regulation of the symbiosis. Cyclophilins belong to a highly conserved family peptydyl-prolyl cistrans isomerases (PPIases). Incubation of A. pallida with cyclosporin A (CsA), a potent inhibitor of cyclophilin resulted in bleaching and a decrease in tolerance to elevated temperatures. Protein extracts from A. pallida exhibited CsA-sensitive PPIase activity. Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live A. pallida revealed that CsA strongly induced the production reactive oxygen species as well as NO. We tested weather the CsAsensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro. In Chapter 4 I review what is known about the molecular and cellular mechanisms of bleaching and describe a model of bleaching based on the results presented herein as well as studies of non-cnidarian models. / Graduation date: 2007

The effects of energy quantity to the reproductive strategies of the sea anemone Aiptasia pulchella Carlgren 1943 Chien-Hsun Chen (Advisors: Drs. Keryea Soong, Chao-Lun Chen) Institute of Marine Biology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan, R.O.C. Thesis abstract The influences of energy quantity on the sexual and asexual reproduction and possible trade-offs between them in the sea anemone, Aiptasia pulchella were investigated. Different feeding frequencies, light intensities and presence of zooxanthellae were the treatments of energy quantity. Lacerate numbers, lacerate sizes and total weight of lacerates were used to quantify investment in asexual reproduction; oocyte sizes and gonad weights were used as the measures of sexual reproduction. The correlation between total weight of lacerates and that of gonads provided an opportunity for evidence of trade-offs between sexual and asexual reproduction. Under higher feeding frequencies, A. pulchella produced more and larger lacerates as well as larger oocytes and heavier gonads than under lower feeding frequencies. Under lower light intensities, A. pulchella produced more and heavier lacerates with than without zooxanthellae. Lacerate sizes were influenced by light intensities, presence of zooxanthellae and the interaction of the two factors. At intermediate light intensity (2000 lux), Aiptasia pulchella produced heavier gonads than at 0 lux or 10000 lux. A. pulchella produced heavier gonads with than without zooxanthellae. The oocyte sizes of A. pulchella were also influenced by light intensities, zooxanthellae and the interaction of the two. In both sexual or asexual modes of reproduction, A. pulchella performed better under higher than lower feeding frequencies. There were higher level of asexual reproduction for A. pulchella under lower light intensities with zooxanthellae. A. pulchella achieved the highest levels of fecundity, as an indication of sexual reproduction, under a light intensity of 2000 lux in individuals containing zooxanthellae. There is a positive correlation between sexual and asexual investment under different energy regimes. This is not compatible with the prediction of the trade-off hypothesis.