Laurie C. Hofmann

Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis

The concentration of CO2 in global surface ocean waters is increasing due to rising atmospheric CO2 emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO2 concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO2 concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO2 concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO2 and was highest in algae grown at 665 µatm CO2. Nitrate and phosphate uptake rates were inversely related to CO2, while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO2. The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO2 due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO2 are discussed.

Nutrient availability affects the response of the calcifying chlorophyte Halimeda opuntia (L.) J.V. Lamouroux to low pH

Atmospheric carbon dioxide emissions cause a decrease in the pH and aragonite saturation state of surface ocean water. As a result, calcifying organisms are expected to suffer under future ocean conditions, but their physiological responses may depend on their nutrient status. Because many coral reefs experience high inorganic nutrient loads or seasonal changes in nutrient availability, reef organisms in localized areas will have to cope with elevated carbon dioxide and changes in inorganic nutrients. Halimeda opuntia is a dominant calcifying primary producer on coral reefs that contributes to coral reef accretion. Therefore, we investigated the carbon and nutrient balance of H.opuntia exposed to elevated carbon dioxide and inorganic nutrients. We measured tissue nitrogen, phosphorus and carbon content as well as the activity of enzymes involved in inorganic carbon uptake and nitrogen assimilation (external carbonic anhydrase and nitrate reductase, respectively). Inorganic carbon content was lower in algae exposed to high CO2, but calcification rates were not significantly affected by CO2 or inorganic nutrients. Organic carbon was positively correlated to external carbonic anhydrase activity, while inorganic carbon showed the opposite correlation. Carbon dioxide had a significant effect on tissue nitrogen and organic carbon content, while inorganic nutrients affected tissue phosphorus and N:P ratios. Nitrate reductase activity was highest in algae grown under elevated CO2 and inorganic nutrient conditions and lowest when phosphate was limiting. In general, we found that enzymatic responses were strongly influenced by nutrient availability, indicating its important role in dictating the local responses of the calcifying primary producer H.opuntia to ocean acidification.

Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions

The resilience of tropical corals to ocean acidification depends on their ability to regulate the pH within their calcifying fluid (pHcf). Recent work suggests pHcf homeostasis under short-term exposure to pCO2 conditions predicted for 2100, but it is still unclear if pHcf homeostasis can be maintained throughout a corals lifetime. At CO2 seeps in Papua New Guinea, massive Porites corals have grown along a natural seawater pH gradient for decades. This natural gradient, ranging from pH 8.1–7.4, provides an ideal platform to determine corals’ pHcf (using boron isotopes). Porites maintained a similar pHcf (~8.24) at both a control (pH 8.1) and seep-influenced site (pH 7.9). Internal pHcf was slightly reduced (8.12) at seawater pH 7.6, and decreased to 7.94 at a site with a seawater pH of 7.4. A growth response model based on pHcf mirrors the observed distribution patterns of this species in the field. We suggest Porites has the capacity to acclimate after long-time exposure to end-of-century reduced seawater pH conditions and that strong control over pHcf represents a key mechanism to persist in future oceans. Only beyond end-of-century pCO2 conditions do they face their current physiological limit of pH homeostasis and pHcf begins to decrease.

Biotic Control of Surface pH and Evidence of Light-Induced H+ Pumping and Ca2+-H+ Exchange in a Tropical Crustose Coralline Alga

Presently, an incomplete mechanistic understanding of tropical reef macroalgae photosynthesis and calcification restricts predictions of how these important autotrophs will respond to global change. Therefore, we investigated the mechanistic link between inorganic carbon uptake pathways, photosynthesis and calcification in a tropical crustose coralline alga (CCA) using microsensors. We measured pH, oxygen (O2), and calcium (Ca2+) dynamics and fluxes at the thallus surface under ambient (8.1) and low (7.8) seawater pH (pHSW) and across a range of irradiances. Acetazolamide (AZ) was used to inhibit extracellular carbonic anhydrase (CAext), which mediates hydrolysis of HCO3-, and 4,4′ diisothiocyanatostilbene-2,2′-disulphonate (DIDS) that blocks direct HCO3- uptake by anion exchange transport. Both inhibited photosynthesis, suggesting both diffusive uptake of CO2 via HCO3- hydrolysis to CO2 and direct HCO3- ion transport are important in this CCA. Surface pH was raised approximately 0.3 units at saturating irradiance, but less when CAext was inhibited. Surface pH was lower at pHSW 7.8 than pHSW 8.1 in the dark, but not in the light. The Ca2+ fluxes were large, complex and temporally variable, but revealed net Ca2+ uptake under all conditions. The temporal variability in Ca2+ dynamics was potentially related to localized dissolution during epithallial cell sloughing, a strategy of CCA to remove epiphytes. Simultaneous Ca2+ and pH dynamics suggest the presence of Ca2+/H+ exchange. Rapid light-induced H+ surface dynamics that continued after inhibition of photosynthesis revealed the presence of a light-mediated, but photosynthesis-independent, proton pump. Thus, the study indicates metabolic control of surface pH can occur in CCA through photosynthesis and light-inducible H+ pumps. Our results suggest that complex light-induced ion pumps play an important role in biological processes related to inorganic carbon uptake and calcification in CCA.

Ultraviolet radiation modulates the physiological responses of the calcified rhodophyte Corallina officinalis to elevated CO2

Abstract Ocean acidification reduces the concentration of carbonate ions and increases those of bicarbonate ions in seawater compared with the present oceanic conditions. This altered composition of inorganic carbon species may, by interacting with ultraviolet radiation (UVR), affect the physiology of macroalgal species. However, very little is known about how calcareous algae respond to UVR and ocean acidification. Therefore, we conducted an experiment to determine the effects of UVR and ocean acidification on the calcified rhodophyte Corallina officinalis using CO2-enriched cultures with and without UVR exposure. Low pH increased the relative electron transport rates (rETR) but decreased the CaCO3 content and had a miniscule effect on growth. However, UVA (4.25 W m-2) and a moderate level of UVB (0.5 W m-2) increased the rETR and growth rates in C. officinalis, and there was a significant interactive effect of pH and UVR on UVR-absorbing compound concentrations. Thus, at low irradiance, pH and UVR interact in a way that affects the multiple physiological responses of C. officinalis differently. In particular, changes in the skeletal content induced by low pH may affect how C. officinalis absorbs and uses light. Therefore, the light quality used in ocean acidification experiments will affect the predictions of how calcified macroalgae will respond to elevated CO2.

Seagrass biofilm communities at a naturally CO2‐rich vent

Seagrass meadows are a crucial component of tropical marine reef ecosystems. Seagrass plants are colonized by a multitude of epiphytic organisms that contribute to broadening the ecological role of seagrasses. To better understand how environmental changes like ocean acidification might affect epiphytic assemblages, the microbial community composition of the epiphytic biofilm of Enhalus acroides was investigated at a natural CO2 vent in Papua New Guinea using molecular fingerprinting and next-generation sequencing of 16S and 18S rRNA genes. Both bacterial and eukaryotic epiphytes formed distinct communities at the CO2-impacted site compared with the control site. This site-related CO2 effect was also visible in the succession pattern of microbial epiphytes. We further found an increased relative sequence abundance of bacterial types associated with coral diseases at the CO2-impacted site (Fusobacteria, Thalassomonas), whereas eukaryotes such as certain crustose coralline algae commonly related to healthy reefs were less diverse. These trends in the epiphytic community of E. acroides suggest a potential role of seagrasses as vectors of coral pathogens and may support previous predictions of a decrease in reef health and prevalence of diseases under future ocean acidification scenarios.

CO2 and inorganic nutrient enrichment affect the performance of a calcifying green alga and its noncalcifying epiphyte

Ocean acidification studies in the past decade have greatly improved our knowledge of how calcifying organisms respond to increased surface ocean CO2 levels. It has become evident that, for many organisms, nutrient availability is an important factor that influences their physiological responses and competitive interactions with other species. Therefore, we tested how simulated ocean acidification and eutrophication (nitrate and phosphate enrichment) interact to affect the physiology and ecology of a calcifying chlorophyte macroalga (Halimeda opuntia (L.) J.V. Lamouroux) and its common noncalcifying epiphyte (Dictyota sp.) in a 4-week fully crossed multifactorial experiment. Inorganic nutrient enrichment (+NP) had a strong influence on all responses measured with the exception of net calcification. Elevated CO2 alone significantly decreased electron transport rates of the photosynthetic apparatus and resulted in phosphorus limitation in both species, but had no effect on oxygen production or respiration. The combination of CO2 and +NP significantly increased electron transport rates in both species. While +NP alone stimulated H. opuntia growth rates, Dictyota growth was significantly stimulated by nutrient enrichment only at elevated CO2, which led to the highest biomass ratios of Dictyota to Halimeda. Our results suggest that inorganic nutrient enrichment alone stimulates several aspects of H. opuntia physiology, but nutrient enrichment at a CO2 concentration predicted for the end of the century benefits Dictyota sp. and hinders its calcifying basibiont H. opuntia.

Short- and long-term acclimation patterns of the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) along a depth gradient.

The giant kelp, Macrocystis pyrifera, is exposed to highly variable irradiance and temperature regimes across its geographic and vertical depth gradients. The objective of this study was to extend our understanding of algal acclimation strategies on different temporal scales to those varying abiotic conditions at various water depths. Different acclimation strategies to various water depths (0.2 and 4 m) between different sampling times (Jan/Feb and Aug/Sept 2012; long‐term acclimation) and more rapid adjustments to different depths (0.2, 2 and 4 m; short‐term acclimation) during 14 d of transplantation were found. Adjustments of variable Chl a fluorescence, pigment composition (Chl c, fucoxanthin), and the de‐epoxidation state of the xanthophyll cycle pigments were responsible for the development of different physiological states with respect to various solar radiation and temperature climates. Interestingly, the results indicated that phlorotannins are important during long‐term acclimation while antioxidants have a crucial role during short‐term acclimation. Furthermore, the results suggested that modifications in total lipids and fatty acid compositions apparently also might play a role in depth acclimation. In Aug/Sept (austral winter), M. pyrifera responded to the transplantation from 4 m to 0.2 m depth with a rise in the degree of saturation and a switch from shorter‐ to longer‐chain fatty acids. These changes seem to be essential for the readjustment of thylakoid membranes and might, thus, facilitate efficient photosynthesis under changing irradiances and temperatures. Further experiments are needed to disentangle the relative contribution of solar radiation, temperature and also other abiotic parameters in the observed physiological changes.

Arctic Coralline Algae Elevate Surface pH and Carbonate in the Dark

Red coralline algae are projected to be sensitive to ocean acidification, particularly in polar oceans. As important ecosystem engineers, their potential sensitivity has broad implications, and understanding their carbon acquisition mechanisms is necessary for making reliable predictions. Therefore, we investigated the localized carbonate chemistry at the surface of Arctic coralline algae using microsensors. We report for the first time carbonate ion concentration and pH measurements ([CO32-]) at and above the algal surface in the microenvironment. We show that surface pH and [CO32-] are higher than the bulk seawater in the light, and even after hours of darkness. We further show that three species of Arctic coralline algae have efficient carbon concentrating mechanisms including direct bicarbonate uptake and indirect bicarbonate use via a carbonic anhydrase enzyme. Our results suggest that Arctic corallines have strong biological control over their surface chemistry, where active calcification occurs, and that net dissolution in the dark does not occur. We suggest that the elevated pH and [CO32-] in the dark could be explained by a high rate of light independent carbon fixation that reduces respiratory CO2 release. This mechanism could provide a potential adaptation to ocean acidification in Arctic coralline algae, which has important implications for future Arctic marine ecosystems.

Latitudinal trends in stable isotope signatures and carbon concentrating mechanisms of northeast Atlantic rhodoliths

Rhodoliths are free-living calcifying red algae that form extensive beds in shallow marine benthic environments (< 250 m), which provide important habitats and nurseries for marine organisms and contribute to carbonate sediment accumulation. There is growing concern that these organisms are sensitive to global climate change, yet little is known about their physiology. Considering their broad distribution along most continental coastlines, their potential sensitivity to global 10 change could have important consequences for the productivity and diversity of benthic coastal environments. The goal of this study was to determine the plasticity of dissolved inorganic carbon (DIC) uptake mechanisms of rhodoliths along a latitudinal gradient in the Northeast (NE) Atlantic using natural stable isotope signatures. The δC signature of macroalgae can be used to provide an indication of the preferred inorganic carbon source (CO2 vs. HCO3). Here we present the total (δCT) and organic (δCorg) δC signatures of NE Atlantic rhodoliths with respect to changing environmental conditions 15 along a latitudinal gradient from the Canary Islands to Spitsbergen. The δCT signatures (-11.9 to -0.89) of rhodoliths analysed in this study were generally higher than the δCorg signatures, which ranged from -25.7 to -2.8. We observed a decreasing trend in δCT signatures with increasing latitude and temperature, while δCorg signatures were only significantly correlated to DIC. These data suggest that high latitude rhodoliths rely more on CO2 as an inorganic carbon source, while low latitudes rhodoliths likely take up HCO3 directly, but none of our specimens had ∂Corg signatures less than -30, 20 suggesting that none of them relied solely on diffusive CO2 uptake. However, depth also has a significant effect on both skeletal and organic δC signatures, suggesting that both local and latitudinal trends influence the plasticity of rhodolith inorganic carbon acquisition and assimilation. Our results show that many species, particularly those at lower latitudes, have carbon concentrating mechanisms that facilitate HCO3 use for photosynthesis. This is an important adaptation for marine macroalgae, because HCO3 is available at higher concentrations than CO2 in seawater, and this becomes even more extreme 25 with increasing temperature. The flexibility of CCMs in northeast Atlantic rhodoliths observed in our study may provide a key physiological mechanism for potential adaptation of rhodoliths to future global climate change. Copyright statement: The authors have read and agree to the copyright terms outlined by the journal. 30