<p>Reef-building corals form critical ecosystems, which provide a diverse range of goods and services. Their success is based on a complex symbiosis between cnidarian host, dinoflagellate algae (genus Symbiodinium) and associated microorganisms (together termed the holobiont). Under functional conditions nutrients are efficiently recycled within the holobiont; however, under conditions of thermal stress, this dynamic relationship can dysfunction, resulting in the loss of symbionts (bleaching). Mass coral bleaching associated with elevated temperatures is a major threat to the long-term persistence of coral reefs. Further study is therefore necessary in order to elucidate the cellular and metabolic networks associated with function in the symbiosis and to determine change elicited by exposure to thermal stress. Metabolomics is the study of small compounds (metabolites) in a cell, tissue or whole organism. The metabolome comprises thousands of components, which will respond rapidly to change, reflecting a combination of genotype, phenotype and the environment. As a result, the study of these metabolic networks serves as a sensitive tool for the detection and elucidation of cellular responses to abiotic stress in complex systems. This thesis presents outputs of gas chromatography-mass spectrometry-based metabolite profiling techniques, which have been applied to the study of thermal stress and bleaching in the cnidarian-dinoflagellate symbiosis. In Chapter 2 these techniques were developed and applied to the model symbiotic cnidarian Aiptasia sp., and its homologous symbiont (Symbiodinium ITS 2 type B1), to characterise both ambient and thermally-induced metabolite profiles (amino and non-amino organic acids) in both partners. Thermal stress, symbiont photodamage and associated bleaching, resulted in characteristic modifications to the free metabolite pools of both partners. These changes differed between partners and were associated with modifications to central metabolism, biosynthesis, catabolism of stores and homeostatic responses to thermal and oxidative stress. In Chapter 3 metabolite profiling techniques (focussing this time on carbohydrate pools) were once again applied to the study of thermally-induced changes to the free pools of the coral Acropora aspera and its symbionts (dominant Symbiodinium ITS 2 type C3) at differing stages of symbiont photodamage and thermal stress. Additionally, targeted analysis was employed to quantify these changes in terms of absolute amounts. Once again exposure to elevated temperatures resulted in symbiont photodamage, bleaching and characteristic modifications to the free metabolite pools of symbiont and host, which differed between partners and with the duration of thermal stress. These changes were associated with increased turnover of a number of networks including: energy-generating pathways, antioxidant networks, ROS-associated damage and damage signalling, and were also indicative of potential alterations to the composition of the associated microbial holobiont. Finally in Chapter 4, metabolite profiling techniques optimized in Chapter 2 and 3 were coupled to 13C labelling in both Aiptasia sp. and A. aspera, in order to further investigate the questions raised in these preceding studies. Once again changes were observed to central metabolism, biosynthesis and alternative energy-generation modes in symbiont and host, in both symbioses. Interestingly however, in all cases there was continued fixation of carbon, production- and translocation of mobile products by the remaining symbionts in hospite. This suggests that even during the later stages of bleaching, symbionts are, at least in part, metabolically functional in terms of photosynthate provision. This study therefore serves as an important first step in developing the application of metabolomics-based techniques to the study of thermal stress in the cnidarian-dinoflagellate symbiosis. The power of these techniques lies in the capacity to simultaneously assess rapid and often post-translational change in a highly repeatable and quantitative manner. With the use of these methods, this study has shown how metabolic, homeostatic and acclimatory networks interact to elicit change in each partner of the symbiosis during thermal stress and how these responses vary between symbiotic partners. Further understanding of these networks, individual sensitivities- and enhanced resistance to thermal stress are essential if we are to better understand the capacity of coral reefs to acclimate and persist in the face of climate change.</p>
Additional Stress on a Buried Pipeline under the Influence of Coal Mining Subsidence