The impact of thermal stress and nutrient availability on the physiology and proteome of symbiotic dinoflagellates

<p>Scleractinian corals, which form the building blocks of tropical reefs, are reliant on a mutualistic symbiosis with phototrophic dinoflagellates of the family Symbiodiniaceae for their metabolic needs and survival. Unfortunately, when subjected to environmental stress this symbiosis can destabilise, culminating in coral bleaching (the loss of symbionts from coral tissue). The most prominent cause of coral bleaching is elevated sea surface temperatures as a result of global warming. However, local stressors such as eutrophication can determine coral reef resilience. Although the physiological responses to temperature and nutrient enrichment are well characterised, the cellular mechanisms underlying these responses are not well understood. This thesis aims to further the understanding of the physiological and cellular responses of Symbiodiniaceae to both thermal stress and nutrient availability. The Symbiodiniaceae species used in this study was Breviolum minutum (ITS2 ‘type’ B1), the homologous symbiont of the model cnidarian Aiptasia. The first objective of this thesis was to compare the physiological response to a rapid versus slow temperature increase, in two strains of the Breviolum minutum (culture IDs: NZ01 and FlAp2), by measuring a range of physiological parameters in cultures exposed to an increase in temperature from 25 to 35°C, either immediately or over one-week. The physiological measurements taken were: population growth, chlorophyll fluorescence and concentration, photosynthetic and respiratory oxygen flux, and alkaline phosphatase activity (APA). Measurements of chlorophyll fluorescence and oxygen flux demonstrated that NZ01 was able to maintain photosynthetic efficiency and metabolic balance at 35˚C, while FlAp2 was experiencing lethal thermal stress. This divergence in physiological plasticity between strains was emphasised by different heating rates. FlAp2 showed more significant thermal stress at a slower heating rate, exemplified by reduced photosynthetic rates relative to cultures exposed to a rapid temperature increase. Alternately, NZ01 cultures exposed to a slow versus rapid heating rate demonstrated greater thermal acclimation, as alkaline phosphatase activity was elevated, and unlike cultures exposed to a rapid temperature increase, respiration and gross photosynthetic rates were equal to cultures at control temperatures. The intraspecies variability in thermal tolerance demonstrated in this thesis adds to the data supporting the intra-species physiological plasticity of the Symbiodiniaceae family. The second objective of this thesis was to determine the influence of nutrient supply on the proteomic response to elevated temperature of B. minutum (using the FlAp2 strain). This was achieved by utilising novel proteomics techniques (Liquid chromatography-electrospray ionisation – tandem mass spectrometry, LC-ESI-MS/MS) and various physiological measurements to corroborate trends of protein expression (population growth, chlorophyll fluorescence and concentration, photosynthetic and respiratory oxygen flux, and alkaline phosphatase activity). Algal cultures were exposed to either ambient (dissolved inorganic nitrogen: DIN ~1.8 µM, dissolved inorganic phosphorus: DIP ~0.2 µM), imbalanced (DIN ~26 µM, DIP ~0.5 µM), or enriched nutrient regimes (DIN ~3 µM, DIP ~0.55 µM), at either 25 or 34˚C. Although it was hypothesised that there would be an interaction between the influence of temperature and nutrient availability on the Symbiodiniaceae proteome, this was not found. However, separately these environmental stressors had a strong influence on protein abundance. Temperature caused a reduction in photosynthesis proteins, ribosomal proteins, metabolic proteins (Calvin cycle/glycolysis) and proteins involved in biosynthesis, and a relative increased abundance of chaperonin proteins and proteins involved in cellular redox homeostasis. Interestingly, the Symbiodiniaceae proteome under the ambient and enriched regimes was very similar, while the proteome under the imbalanced nutrient regime was different to these comparatively balanced regimes. This trend highlights the importance of the nitrogen to phosphorus ratio in determining the cellular response of Symbiodiniaceae to nutrient enrichment. Under an imbalanced nutrient regime, there was a down-regulation in photosynthetic and Calvin cycle proteins and an upregulation of proteins involved in protein translation, energy-generating metabolic pathways and storage-product turn-over. Consistent with previous studies, proteomic and physiological data indicated that B. minutum might have been experiencing phosphorus deficiency under an imbalanced nutrient regime. However, photochemical efficiency and metabolic balance was maintained, indicating metabolic adaption to the skewed nutrient ratio. This thesis provides insight into the physiological and cellular response of Symbiodiniaceae to both temperature and nutrients, highlighting potential avenues of research that could be directed to facilitate the knowledge-based management of coral reefs. The intraspecies plasticity demonstrated in chapter two highlights the need to characterise physiological variability within Symbiodiniaceae species, as this could confer an adaptive advantage to the coral holobiont. In conjunction, the proteomics results of chapter three indicate that the relative availability of nitrogen to phosphorus determines the response of Sybiodiniaceae cellular physiology to nutrient availability. This emphasises the importance of determining the threshold of nitrogen to phosphorus that has a negative influence on the coral holobiont, facilitating the setting of ecologically relevant nutrient input limits by coral reef management.</p>

The impact of thermal stress and nutrient availability on the physiology and proteome of symbiotic dinoflagellates

<p>Scleractinian corals, which form the building blocks of tropical reefs, are reliant on a mutualistic symbiosis with phototrophic dinoflagellates of the family Symbiodiniaceae for their metabolic needs and survival. Unfortunately, when subjected to environmental stress this symbiosis can destabilise, culminating in coral bleaching (the loss of symbionts from coral tissue). The most prominent cause of coral bleaching is elevated sea surface temperatures as a result of global warming. However, local stressors such as eutrophication can determine coral reef resilience. Although the physiological responses to temperature and nutrient enrichment are well characterised, the cellular mechanisms underlying these responses are not well understood. This thesis aims to further the understanding of the physiological and cellular responses of Symbiodiniaceae to both thermal stress and nutrient availability. The Symbiodiniaceae species used in this study was Breviolum minutum (ITS2 ‘type’ B1), the homologous symbiont of the model cnidarian Aiptasia. The first objective of this thesis was to compare the physiological response to a rapid versus slow temperature increase, in two strains of the Breviolum minutum (culture IDs: NZ01 and FlAp2), by measuring a range of physiological parameters in cultures exposed to an increase in temperature from 25 to 35°C, either immediately or over one-week. The physiological measurements taken were: population growth, chlorophyll fluorescence and concentration, photosynthetic and respiratory oxygen flux, and alkaline phosphatase activity (APA). Measurements of chlorophyll fluorescence and oxygen flux demonstrated that NZ01 was able to maintain photosynthetic efficiency and metabolic balance at 35˚C, while FlAp2 was experiencing lethal thermal stress. This divergence in physiological plasticity between strains was emphasised by different heating rates. FlAp2 showed more significant thermal stress at a slower heating rate, exemplified by reduced photosynthetic rates relative to cultures exposed to a rapid temperature increase. Alternately, NZ01 cultures exposed to a slow versus rapid heating rate demonstrated greater thermal acclimation, as alkaline phosphatase activity was elevated, and unlike cultures exposed to a rapid temperature increase, respiration and gross photosynthetic rates were equal to cultures at control temperatures. The intraspecies variability in thermal tolerance demonstrated in this thesis adds to the data supporting the intra-species physiological plasticity of the Symbiodiniaceae family. The second objective of this thesis was to determine the influence of nutrient supply on the proteomic response to elevated temperature of B. minutum (using the FlAp2 strain). This was achieved by utilising novel proteomics techniques (Liquid chromatography-electrospray ionisation – tandem mass spectrometry, LC-ESI-MS/MS) and various physiological measurements to corroborate trends of protein expression (population growth, chlorophyll fluorescence and concentration, photosynthetic and respiratory oxygen flux, and alkaline phosphatase activity). Algal cultures were exposed to either ambient (dissolved inorganic nitrogen: DIN ~1.8 µM, dissolved inorganic phosphorus: DIP ~0.2 µM), imbalanced (DIN ~26 µM, DIP ~0.5 µM), or enriched nutrient regimes (DIN ~3 µM, DIP ~0.55 µM), at either 25 or 34˚C. Although it was hypothesised that there would be an interaction between the influence of temperature and nutrient availability on the Symbiodiniaceae proteome, this was not found. However, separately these environmental stressors had a strong influence on protein abundance. Temperature caused a reduction in photosynthesis proteins, ribosomal proteins, metabolic proteins (Calvin cycle/glycolysis) and proteins involved in biosynthesis, and a relative increased abundance of chaperonin proteins and proteins involved in cellular redox homeostasis. Interestingly, the Symbiodiniaceae proteome under the ambient and enriched regimes was very similar, while the proteome under the imbalanced nutrient regime was different to these comparatively balanced regimes. This trend highlights the importance of the nitrogen to phosphorus ratio in determining the cellular response of Symbiodiniaceae to nutrient enrichment. Under an imbalanced nutrient regime, there was a down-regulation in photosynthetic and Calvin cycle proteins and an upregulation of proteins involved in protein translation, energy-generating metabolic pathways and storage-product turn-over. Consistent with previous studies, proteomic and physiological data indicated that B. minutum might have been experiencing phosphorus deficiency under an imbalanced nutrient regime. However, photochemical efficiency and metabolic balance was maintained, indicating metabolic adaption to the skewed nutrient ratio. This thesis provides insight into the physiological and cellular response of Symbiodiniaceae to both temperature and nutrients, highlighting potential avenues of research that could be directed to facilitate the knowledge-based management of coral reefs. The intraspecies plasticity demonstrated in chapter two highlights the need to characterise physiological variability within Symbiodiniaceae species, as this could confer an adaptive advantage to the coral holobiont. In conjunction, the proteomics results of chapter three indicate that the relative availability of nitrogen to phosphorus determines the response of Sybiodiniaceae cellular physiology to nutrient availability. This emphasises the importance of determining the threshold of nitrogen to phosphorus that has a negative influence on the coral holobiont, facilitating the setting of ecologically relevant nutrient input limits by coral reef management.</p>

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica

Oxygen fluctuations are common in marine waters, and hypoxia/reoxygenation (H/R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h &lt;0.01% O­2) and subsequent reoxygenation (1.5 h 21% O­2) compared to normoxic control animals (21% O­2). We demonstrated that flux capacities of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased oxygen flux capacities of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H/R stress, whereas the control of the proton leak subsystem over LEAK respiration increased during hypoxia and returned to normoxic level during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, while suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

Impact of Upward Oxygen Diffusion From the Oceanic Crust on the Magnetostratigraphy and Iron Biomineralization of East Pacific Ridge-Flank Sediments

Recent measurements of pore-water oxygen profiles in ridge flank sediments of the East Pacific Rise revealed an upward-directed diffusive oxygen flux from the hydrothermally active crust into the overlying sediment. This double-sided oxygenation from above and below results in a dual redox transition from an oxic sedimentary environment near the seabed through suboxic conditions at sediment mid-depth back to oxic conditions in the deeper basal sediment. The potential impact of this redox reversal on the paleo- and rock magnetic record was analyzed for three sediment cores from the Clarion-Clipperton-Zone (low-latitude eastern North Pacific). We found that the upward-directed crustal oxygen flux does not impede high quality reversal-based and relative paleointensity-refined magnetostratigraphic dating. Despite low and variable sedimentation rates of 0.1–0.8 cm/kyr, robust magnetostratigraphic core chronologies comprising the past 3.4 resp. 5.2 million years could be established. These age-models support previous findings of significant local sedimentation rate variations that are probably related to the bottom current interactions with the topographic roughness of the young ridge flanks. However, we observed some obvious paleomagnetic irregularities localized at the lower oxic/suboxic redox boundaries of the investigated sediments. When analyzing these apparently remagnetized sections in detail, we found no evidence of physical disturbance or chemical alteration. A sharp increase in single-domain magnetite concentration just below the present lower oxic/suboxic redox boundary suggests secondary magnetite biomineralization by microaerophilic magnetotactic bacteria living as a separate community in the lower, upward oxygenated part of the sediment column. We therefore postulate a two-phased post-depositional remanent magnetization of ridge flank sediments, first by a shallow and later by a deep-living community of magnetotactic bacteria. These findings are the first evidence of a second, deep population of probably inversely oriented magnetotactic bacteria residing in the inverse oxygen gradient zone of ridge flank sediments.

Simulating the effect of climate change on the performance of a monolayer cover combined with an elevated water table placed on acid generating mine tailings

Several reclamation approaches were developed in the last decades to control acid mine drainage from tailings storage facilities, including the monolayer cover combined with an elevated water table. Its performance is dependent on water table elevation and tailings saturation, and is directly affected by climatic conditions, therefore climate change needs to be taken into account to design resilient reclamation systems. The objective of this research was to evaluate three approaches to simulate climate change and compare the impact on reclamation performance up to year 2100. Numerical simulations were calibrated using experimental field data and future weather conditions were established based on three climate change scenarios adapted for local conditions. Results showed that the projected impact of climate change varied depending on the approach used. Simpler and more conservative approaches indicated that reclamation would eventually fail following an increase of droughts during future summers. However, 80-year simulations showed that reclamation failures (evaluated as oxygen flux) could be limited to a few isolated summers and that a well-designed monolayer cover with elevated water table appeared to remain efficient in the long-term. Overall, the probability to exceed the oxygen flux target of 1 mol/m2/y did not exceed 2% for the simulated conditions.