Tough as a rock-boring urchin: adult Echinometra sp. EE from the Red Sea show high resistance to ocean acidification over long-term exposures

Ocean acidification, a process caused by the continuous rise of atmospheric CO2 levels, is expected to have a profound impact on marine invertebrates. Findings of the numerous studies conducted in this field indicate high variability in species responses to future ocean conditions. This study aimed at understanding the effects of long-term exposure to elevated pCO2 conditions on the performance of adult Echinometra sp. EE from the Gulf of Aqaba (Red Sea). During an 11-month incubation under high pCO2 (1,433 µatm, pHNBS 7.7) and control (435 µatm, pHNBS 8.1) conditions, we examined the urchins' somatic and gonadal growth, gametogenesis and skeletal microstructure. Somatic and gonadal growths were exhibited with no significant differences between the treatments. In addition, all urchins in the experiment completed a full reproductive cycle, typical of natural populations, with no detectable impact of increased pCO2 on the timing, duration or progression of the cycle. Furthermore, scanning electron microscopy imaging of urchin tests and spines revealed no signs of the usual observed effects of acidosis, such as skeletal dissolution, widened stereom pores or non-smoothed structures. Our results, which yielded no significant impact of the high pCO2 treatment on any of the examined processes in the urchins studied, suggest high resistance of adult Echinometra sp. EE to near future ocean acidification conditions. With respect to other findings in this area, the outcome of this study provides an example of the complicated and diverse responses of echinoids to the predicted environmental changes.

Effects of ocean acidification on learning in coral reef fishes

Ocean acidification has the potential to cause dramatic changes in marine ecosystems. Larval damselfish exposed to concentrations of CO2 predicted to occur in the mid- to late-century show maladaptive responses to predator cues. However, there is considerable variation both within and between species in CO2 effects, whereby some individuals are unaffected at particular CO2 concentrations while others show maladaptive responses to predator odour. Our goal was to test whether learning via chemical or visual information would be impaired by ocean acidification and ultimately, whether learning can mitigate the effects of ocean acidification by restoring the appropriate responses of prey to predators. Using two highly efficient and widespread mechanisms for predator learning, we compared the behaviour of pre-settlement damselfish Pomacentrus amboinensis that were exposed to 440 µatm CO2 (current day levels) or 850 µatm CO2, a concentration predicted to occur in the ocean before the end of this century. We found that, regardless of the method of learning, damselfish exposed to elevated CO2 failed to learn to respond appropriately to a common predator, the dottyback, Pseudochromis fuscus. To determine whether the lack of response was due to a failure in learning or rather a short-term shift in trade-offs preventing the fish from displaying overt antipredator responses, we conditioned 440 or 700 µatm-CO2 fish to learn to recognize a dottyback as a predator using injured conspecific cues, as in Experiment 1. When tested one day post-conditioning, CO2 exposed fish failed to respond to predator odour. When tested 5 days post-conditioning, CO2 exposed fish still failed to show an antipredator response to the dottyback odour, despite the fact that both control and CO2-treated fish responded to a general risk cue (injured conspecific cues). These results indicate that exposure to CO2 may alter the cognitive ability of juvenile fish and render learning ineffective.

The influence of ocean acidification on nitrogen regeneration and nitrous oxide production in the northwest European shelf sea

The assimilation and regeneration of dissolved inorganic nitrogen, and the concentration of N2O, was investigated at stations located in the NW European shelf sea during June/July 2011. These observational measurements within the photic zone demonstrated the simultaneous regeneration and assimilation of NH4+, NO2- and NO3-. NH4+ was assimilated at 1.82-49.12 nmol N/L/h and regenerated at 3.46-14.60 nmol N/L/h; NO2- was assimilated at 0-2.08 nmol N/L/h and regenerated at 0.01-1.85 nmol N/L/h; NO3-was assimilated at 0.67-18.75 nmol N/L/h and regenerated at 0.05-28.97 nmol N/L/h. Observations implied that these processes were closely coupled at the regional scale and that nitrogen recycling played an important role in sustaining phytoplankton growth during the summer. The [N2O], measured in water column profiles, was 10.13 ± 1.11 nmol/L and did not strongly diverge from atmospheric equilibrium indicating that sampled marine regions were neither a strong source nor sink of N2O to the atmosphere. Multivariate analysis of data describing water column biogeochemistry and its links to N-cycling activity failed to explain the observed variance in rates of N-regeneration and N-assimilation, possibly due to the limited number of process rate observations. In the surface waters of five further stations, ocean acidification (OA) bioassay experiments were conducted to investigate the response of NH4+ oxidising and regenerating organisms to simulated OA conditions, including the implications for [N2O]. Multivariate analysis was undertaken which considered the complete bioassay data set of measured variables describing changes in N-regeneration rate, [N2O] and the biogeochemical composition of seawater. While anticipating biogeochemical differences between locations, we aimed to test the hypothesis that the underlying mechanism through which pelagic N-regeneration responded to simulated OA conditions was independent of location. Our objective was to develop a mechanistic understanding of how NH4+ regeneration, NH4+ oxidation and N2O production responded to OA. Results indicated that N-regeneration process responses to OA treatments were location specific; no mechanistic understanding of how N-regeneration processes respond to OA in the surface ocean of the NW European shelf sea could be developed.

Short- and long-term conditioning of a temperate marine diatom community to acidification and warming

Ocean acidification and greenhouse warming will interactively influence competitive success of key phytoplankton groups such as diatoms, but how long-term responses to global change will affect community structure is unknown. We incubated a mixed natural diatom community from coastal New Zealand waters in a short-term (two-week) incubation experiment using a factorial matrix of warming and/or elevated pCO2 and measured effects on community structure. We then isolated the dominant diatoms in clonal cultures and conditioned them for 1 year under the same temperature and pCO2 conditions from which they were isolated, in order to allow for extended selection or acclimation by these abiotic environmental change factors in the absence of interspecific interactions. These conditioned isolates were then recombined into 'artificial' communities modelled after the original natural assemblage and allowed to compete under conditions identical to those in the short-term natural community experiment. In general, the resulting structure of both the unconditioned natural community and conditioned 'artificial' community experiments was similar, despite differences such as the loss of two species in the latter. pCO2 and temperature had both individual and interactive effects on community structure, but temperature was more influential, as warming significantly reduced species richness. In this case, our short-term manipulative experiment with a mixed natural assemblage spanning weeks served as a reasonable proxy to predict the effects of global change forcing on diatom community structure after the component species were conditioned in isolation over an extended timescale. Future studies will be required to assess whether or not this is also the case for other types of algal communities from other marine regimes.

Patterns of Starchy Endosperm Acidification and Protease Gene Expression in Wheat Grains following Germination1

Cereal aleurone responses to gibberellic acid (GA3) include activation of synthesis of hydrolytic enzymes and acidification of the external medium. We have studied the effect of the pH of the incubation medium on the response of wheat (Triticum aestivum) aleurone cells to GA3. De-embryonated half grains show the capacity for GA3-activated medium acidification when incubation is carried out at pH 6.0 to 7.0 but not at lower pHs. In addition, the activating effect of GA3 on the expression of carboxypeptidase III and thiol protease genes is more efficient when the hormone treatment is carried out at neutral pH. In situ pH staining showed that starchy endosperm acidification takes place upon imbibition and advances from the embryo to the distal part of the grain. In situ hybridization experiments showed a similar pattern of expression of a carboxypeptidase III gene, which is up-regulated by GA3 in aleurone cells. However, aleurone gene expression precedes starchy endosperm acidification. These findings imply that in vivo GA perception by the aleurone layer takes place at neutral pH and suggest that the acidification of the starchy endosperm is regulated by GA3 in germinated wheat grains.

Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase.

Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program.

Effects of ocean warming and acidification on the early stages of marine fishes

Tese de doutoramento, Ciências do Mar, Universidade de Lisboa, Faculdade de Ciências, 2016

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool-scenario, link to supplementary data

Rising atmospheric CO2-concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO2-concentrations in a rockpool scenario. U. lactuca was cultured under aeraton with air containing either preindustrial pCO2 (280µatm) or for the end of the 21st century predicted (700µatm) pCO2 for 31 days. We addressed the following question: Will elevated CO2-concentrations affect photosynthesis (net photosynthesis, rETR(max), Fv/Fm, pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1-4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO2 condition by continuous aeration with target pCO2 values. In the second phase (day 4-31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO2-concentrations whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO2-concentrations. At low CO2-concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO2-concentrations most thalli remained integer until day 31. Thallus disintegration at low CO2-concentrations was mirrored in a drastic decline in seawater DIC and HCO3-. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO2-concentrations. The accelerated thallus disintegration at high CO2-concentrations under conditions of limited water exchange indicates additional CO2 effects on the life cycle of U. lactuca when living in rockpools.