The following thesis explores the physiological effects on European sea bass (Dicentrarchus labrax) and shore crabs (Carcinus maenas) resulting from the dissolution of anthropogenic carbon dioxide (CO2) into seawater: known as ocean acidification. It assesses how ocean acidification, characterised by elevated seawater pCO2 (1200 µatm) and lowered pH (~7.7), affect the internal chemistry of these animals through the homeostatic process of acid-base regulation. Control conditions used for comparison were close to current ocean average values for CO2 (~400 µatm) and pH (8.2). The proficiency and magnitude of these compensatory mechanisms was explored. Both sea bass and shore crabs were found to be highly effective acid-base regulators and employed the same strategy to compensate the hypercapnia-induced respiratory acidosis: namely an elevation of extracellular bicarbonate (HCO3-). It then considers how these regulatory mechanisms both affect, and are affected by, simultaneous exposure to a ubiquitous coastal metal contaminant, copper. Evidence for a hitherto undocumented protective effect of elevated HCO3- against copper-induced DNA damage was found to be afforded to both sea bass and shore crab cells. DNA damage was used as a sensitive toxicity marker and blood cells were used as proxies for other internal tissues. Erythrocytes exposed in vitro (2 h) to copper (45 µg/L) showed significant DNA damage under control [HCO3-] (6 mM) but were completely protected when exposed under high [HCO3-] (12 mM). A similar protective effect was apparent in crabs under in vivo exposure (14 d) to 10 µg/L waterborne copper. Conversely, during exposure to higher waterborne copper concentrations (sea bass: 80 µg/L, shore crabs: 40 µg/L), animals showed a severe or total inhibition of acid-base regulatory ability in the face of simultaneously elevated seawater CO2 (1200 µatm). The downstream effects of longer-term (28 d) exposure to high CO2 and copper, both individually and in combination was assessed. Food conversion efficiency (FCE), growth and copper accumulation were quantified in juvenile sea bass as economically relevant endpoints. Growth and FCE remained unaffected by either stressor and copper was not accumulated in the muscle tissue: pertinent to human consumption. As a bi-product of this longer term study assessment of gut calcium carbonate production rates in these animals was possible, providing some of the first evidence of excretion rates in fish fed on naturally high calcium diets. A directly proportional influence of feeding rate on gut carbonate excretion rates as a result of increased dietary calcium was observed, and novel evidence provided of the proportional contribution of dietary and seawater calcium to excreted carbonate. Both findings have considerable application to global models of fish contribution to the oceanic carbon cycle.

Increasing atmospheric CO2 concentrations associated with climate change will likely influence a wide variety of ecosystems. Terrestrial research has examined the effects of increasing CO2 concentrations on the functionality of plant systems; with studies ranging in scale from the short-term responses of individual leaves, to long-term ecological responses of complete forests. While terrestrial plants have received much attention, studies on the responses of marine plants (seagrasses) to increased CO2(aq) concentrations remain relatively sparse, with most research limited to small-scale, ex situ experimentation. Furthermore, few studies have attempted to address similarities between terrestrial and seagrass responses to increases in CO2(aq). The goals of this dissertation are to expand the scope of marine climate change research, and examine how the tropical seagrass, Thalassia testudinum responds to increasing CO2(aq) concentrations over multiple spatial and temporal scales. Manipulative laboratory and field experimentation reveal that, similar to terrestrial plants, seagrasses strongly respond to increases in CO2(aq) concentrations. Using a novel field technique, in situ field manipulations show that over short time scales, seagrasses respond to elevated CO2(aq) by increasing leaf photosynthetic rates and the production of soluble carbohydrates. Declines in leaf nutrient (nitrogen and phosphorus) content were additionally detected, paralleling responses from terrestrial systems. Over long time scales, seagrasses increase total above- and belowground biomass with elevated CO2(aq), suggesting that, similar to terrestrial research, pervasive increases in atmospheric and oceanic CO2(aq) concentrations stand to influence the productivity and functionality of these systems. Furthermore, field experiments reveal that seagrass epiphytes, which comprise an important component of seagrass ecosystems, additionally respond to increased CO2(aq) with strong declines in calcified taxa and increases in fleshy taxa. Together, this work demonstrates that increasing CO2(aq) concentrations will alter the functionality of seagrass ecosystems by increasing plant productivity and shifting the composition of the epiphyte community. These results have implications for future rates of carbon storage and sediment production within these widely distributed systems.

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abstract / Biological Sciences / Doctoral / Doctor of Philosophy

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Marine phytoplankton is responsible for ~50% of global primary productivity, it supports the oceanic food web and affects biogeochemical cycles. I participated in a large mesocosm experiment that observed altered community structure and carbon drawdown in response to increased CO2. There was a 27% reduction in community primary production at the peak of an Emiliania huxleyi-dominated bloom in mesocosms initially at 760 ppm CO2 compared to present day pCO2. There were changes in community structure but not dominance; Synechococcus and large pico-eukaryote abundances were reduced by ~60%, E. huxleyi was reduced by ~50%. A number of E. huxleyi strains persisted throughout the experiment in both treatments and no malformation or significant change in lith size occurred at increased CO2. In a second field experiment in the oligotrophic ocean off the Canary Islands, 760 ppm pCO2 did not change community structure or cell division rates of Synechococcus, Prochlorococcus or pico-eukaryotes.In laboratory experiments, I maintained the diatom, Thalassiosira pseudonana CCMP1335 at 760 ppm and present day pCO2 for ~100 generations in gas equilibrated continuous cultures – one of the longest experiments that has been attempted to investigate the effect of increased CO2 on marine phytoplankton. No clear evidence of adaptation or acclimation to increased CO2 was found, neither were there consistent changes in transcription of RuBisCO or carbonic anhydrase genes. Non-calcified E. huxleyi CCMP1516 and calcified CCMP371 grown in gas equilibrated semi-continuous cultures for several weeks showed no change in cell division rate at 760 ppm CO2. An understanding of the underlying changes in communities is required for modelling responses to increasing CO2, molecular tools may prove useful for this task. The strong community response in the mesocosms shows that rising atmospheric CO2 can greatly affect phytoplankton productivity and biogeochemical cycling.

Atmospheric carbon dioxide (CO2) has increased due to human activity from a pre-industrial value of about 280 ppm to the present level of 399 ppm. The ocean acts as an important natural carbon sink that effectively removes 1/3 of this anthropogenic CO2 from the atmosphere, buffering global warming effects. However, the dissolution of CO2 causes a dramatic change in seawater chemistry and ultimately results in the phenomenon commonly known as "ocean acidification" (OA). As a consequence, the pH value and the saturation states for calcium carbonate decline in the surface seawater, posing a threat to calcareous marine organisms that build their shells using exquisite biomineralization mechanisms. Biological minerals produced by marine organisms are compositionally and structurally more complex than geological minerals. Although changes in biomineral formation in response to OA has been intensively investigated, the features of calcified products in terms of their composition, architectures and mechanical properties have been overlooked in climate change research. The tubeworm is a favourite marine model organism in larval biology. Its life cycle is well understood hence provides a good opportunity to study OA impacts on the stochastic early life. In addition, the model enables comprehensive observation of the sophisticated biomineralization events. In this thesis, four studies on the biomineralization of Hydroides elegans, using a multidisciplinary collaborative approach combining larval biology and material science were conducted. (1) The tube mineral composition at different juvenile stages (4, 11, 18, 25 days) were characterized. (2) The impacts of different predicted OA scenarios (pH 8.1, 7.9, 7.6, and 7.4) on the resultant calcification products were compared. (3) A multiple-stressor investigation of OA (pH 8.1 and 7.8), reduced salinity (33 ‰ and 27 ‰) and increased temperature (25 °C and 29 °C) was conducted to further determine the more environmentally realistic OA impacts. (4) Calcification sites were examined by using a microscopy approach The main findings from each study were: (1) H. elegans produced both calcite and aragonite forms of CaCO3, which have distinctive physical and chemical properties. Thus, the tubeworm serves as an interesting model for studying OA impacts on biomineralization. The early juvenile stages are expected to be more sensitive to OA than the later life stages because the juvenile tubes are rich in aragonite and amorphous calcium carbonate. (2) Under experimental OA conditions, the composition and architecture of the tube structures were adversely affected, ultimately producing tubes with weaker mechanical properties. (3) Warming appeared to strengthen the tube structures and mitigated the adverse OA effects. (4) Calcification sites correlated to regions with higher pH values of 8.5 - 9.0. These regions may be sensitive to OA and should be further analyzed to study the mechanisms of OA impacts on calcification. This series of experiments study biomineralization and larval biology using a variety of modern multidisciplinary approaches provided new insights into the impacts of OA and climate change impacts on marine organisms and also helped us to project which species might adapt or succumb to future scenarios. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Ocean acidification has the potential to profoundly affect marine ecosystems before the end of this century, but there are large uncertainties on its effects on temperate benthic communities. Volcanic CO2 seeps provide an opportunity to examine and improve our understanding of community responses to ocean acidification. In this thesis, two Mediterranean CO2 seeps (Methana in Greece and Vulcano in Italy) were used to investigate the responses of macroalgae and their epifaunal communities to increased CO2. Changes in plant-herbivore interactions at elevated CO2, as well as adaptation potential of dominant macroalgae and responses of macroalgae and epifauna to concurrent exposure to elevated CO2 and copper pollution, were also examined. Firstly, I determined that volcanic seeps off Methana (Greece) are suitable for ocean acidification studies as they do not have confounding gradients in temperature, salinity, total alkalinity, nutrients, hydrogen sulphide, heavy metals or wave exposure. Calcifying macroalgae abundance decreased as CO2 increased both at Methana and at Vulcano, while fucoid algae seemed to benefit from elevated pCO2 levels. Seasonality greatly affected macroalgal responses to increasing CO2, according to the annual cycles of dominant species. Epifaunal communities of dominant fucoid algae changed at elevated pCO2 as well, with calcifying invertebrates decreasing and polychaetes increasing near the seeps. Herbivore control of macroalgal biomass did not greatly change at elevated pCO2 levels, as limpets had a minor role in controlling macroalgal biomass off Vulcano (Italy) and sea urchins were replaced by herbivorous fish near seeps off Methana. The two macroalgal species examined for signs of long-term acclimatisation (Cystoseira corniculata (Turner) Zanardini and Jania rubens (Linnaeus) J.V.Lamouroux) to ocean acidification using reciprocal transplants did not appear to have permanently acclimatised to elevated pCO2 levels, but changed their physiology in four to nine months depending on the local environment. Furthermore, when exposed to a 36-hour copper pulse at elevated pCO2 levels both seaweed species accumulated more copper in their tissues compared to those exposed to copper in reference pCO2 conditions, and this resulted in altered epifaunal assemblages on C. corniculata. These observations suggest that benthic communities will significantly change as CO2 levels increase, and that long-term acclimatisation is not likely to play a significant role; this would have profound consequences for benthic ecosystems and the services they provide.

Future changes to the pH and temperature of the oceans are predicted to impact the biodiversity of marine ecosystems, particularly those animals that rely on the process of calcification. The marine intertidal gastropod Littorina littorea can be used as a model of intertidal organism for investigating the effects of ocean acidification and high temperature, alone and in combination because its ability to be quickly adapt against environmental stressor. In the first study a single species population of L. littorea was used to test for physiological and biochemical effects underpinning organismal responses to climate change and ocean acidification. Compared with control conditions, snails decreased metabolic rates by 31% in response to elevated pCO2 while by 15% in response to combined pCO2 and temperature. Decreased metabolic rates were associated with metabolic depression, a strategy to match oxygen demand and availability, and an increase in end-product metabolites in the tissue under acidified treatments, indicating an increased reliance on anaerobic metabolism. This study also showed that anthropogenic alteration of CO2 and temperature may also lead to plastic responses, a fundamental mechanism of many marine gastropods to cope environmental variability. At low pH and elevated temperature in isolation or combined showing lower shell growth than individuals kept under control conditions. Percentage change in shell length and thicknesses was also lower under acidified and temperature in isolation or combined than control condition, making shells were more globular and desiccation rates were higher. Further studies to broader latitudinal ranges for six populations of L. littorea showed that shell growth decreased in all six populations under elevated pCO2 compared to control snails particularly those at range edges. Elevated pCO2 also affected to the reduction of shell length and width that causing shell aspect ratio to increase across latitudinal gradients except individuals from Millport, UK. Percentage changes of aperture width and aperture area were also decrease under elevated pCO2 with greater reduction of aperture area were found at populations in the mid-ranges which is assumed this response might be linked to local adaptation of the individual to microclimatic conditions. This study also showed that metabolic rates were negatively affected by high pCO2 and show non-linear trend across latitudinal gradients in compared to individual kept under normal pCO2 conditions. Metabolomic analysis showed that two northern populations of Trondheim and TromsØ were distinct from other populations when exposed to low temperature (15 °C) with elevated pCO2 due to, in part, high concentrations of thymine, uracil, valine and lysine. A similar separation also occurred under medium (25 °C) and high (35 °C) temperature exposure in which one of northern population (Trondheim) was distinct from other populations and had lower concentrations of alanine, betaine and taurine while higher of valine. These results suggest that populations at northern latitudes may apply different ionic transport mechanisms under elevated pCO2 and elevated temperatures and those populations are likely to vary in terms of their physiological responses to this environmental challenge.