ALBERT

Description: Southern Ocean organisms are thought to be particularly vulnerable to ocean acidification, as they inhabit cold waters where calcite-aragonite saturation states are naturally low. It is also generally assumed that calcifying animals would be more affected by ocean acidification than non-calcifying ones. In this context, we aimed to study the impacts of reduced pH on the ascidia Cnemidocarpa verrucosa sp. A. Here, we used gene expression profiling and enzymatic activity to study the responses of that Antarctic benthic species to ocean acidification. We sampled Cnemidocarpa verrucosa sp. A. by scuba diving at approximately 15 m depth at Carlini station, Potter Cove, King George Island, Antarctica. Superoxide dismutase (SOD) activity was measured in the ascidia, samples (approximately 70 mg of brachial basket) were homogenized in 20 mM Tris-HCl, 1 mM EDTA, pH 7.6, with a ratio 1:4 w/v. Homogenates were centrifuged at 14,000 x g for 3 min at 4°C and the supernatant was used to measure SOD activity at 20°C following Livingstone et al. (1992) protocol. Supernatant was mixed with the measurement buffer (43 mM K₂HPO₄, 43 mM KH₂PO₄, 0.1 mM EDTA, pH 7.68), 5 mM Xanthina (Sigma X-0626), 100 µM Citocromo-C (Sigma C-2037), 0.3 mU/µl XOD (Xanthin-Oxidasa, Sigma X-4875) in 2 M (NH₄)2SO₄. The measurement was made in a photometer at 20°C, 550 nm wavelength, for 3 minutes, every 10 seconds. For the calculations, the total protein content of the samples was measured using the method of Bradford (1976). Superoxide dismutase activity was expressed in activity in the extract (mU) / amount of protein (mg). All measurements were made in triplicate.

Description: Southern Ocean organisms are thought to be particularly vulnerable to ocean acidification, as they inhabit cold waters where calcite-aragonite saturation states are naturally low. It is also generally assumed that calcifying animals would be more affected by ocean acidification than non-calcifying ones. In this context, we aimed to study the impacts of reduced pH on the ascidia Cnemidocarpa verrucosa sp. A. Here, we used gene expression profiling and enzymatic activity to study the responses of that Antarctic benthic species to ocean acidification. We sampled Cnemidocarpa verrucosa sp. A. by scuba diving at approximately 15 m depth at Carlini station, Potter Cove, King George Island, Antarctica. Caspases 3/7 activity as indicators of apoptosis intensity was measured using the Caspase-Glow 3/7 Assay kit (Promega, USA) following the manufacturer's instructions. Samples were homogenized (16-33 mg) in lysis buffer consisting in 25 mM HEPES, 5 mM MgCl₂·6H₂O, 1 mM EGTA, 1 μg/mL pepstatin, 1 μg/mL leupectin, and 1 μg/mL aprotinin at a ratio 1:100 (Rivera-Ingraham et al., 2013) using a Precellys homogenizer (2 cycles at 5,500 x g at 4°C for 20 s). Homogenates were centrifuged at 13,000 x g at 4°C for 15 min and the supernatant was used to measure luminescence using Tristar LB941 plate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). The total protein content of the samples was measured using the method of Bradford (1976). Caspase/Apoptotic activity was expressed as relative light units (RLU) per μg of protein × 104.

Description: Ocean acidification (OA) could become a serious threat for the Antarctic marine ecosystem over coming years, as the solubility of atmospheric CO2 and CaCO3 minerals increases at lower temperatures. We evaluated the effect of OA on the stress response of the limpet Nacella concinna by measuring gene expression levels. The experiment was performed with the two ecotypes (Littoral and Sublittoral) of the species during 54 days (IPCC, 2019 scenario RCP8.5; control, 375 ppm; low-pH treatment, 923 ppm). Exposure to low-pH treatment during 15 days triggered the down-regulation of two heat-shock protein genes (HSP70A, HSP70B) only in sublittoral individuals. Little variation in the relative expression values of all genes in both ecotypes was observed probably, due to a historical exposure to the substantial daily natural pH fluctuations recorded in the study area during the experiment. This study provides relevant baseline data for future OA experiments on coastal species in Antarctica.

Description: Benthic organisms of the Southern Ocean are particularly vulnerable to ocean acidification (OA), as they inhabit cold waters where calcite-aragonite saturation states are naturally low. OA most strongly affects animals with calcium carbonate skeletons or shells, such as corals and mollusks. We exposed the abundant cold-water coral Malacobelemnon daytoni from an Antarctic fjord to low pH seawater (LpH) (7.68 +/- 0.17) to test its physiological responses to OA, at the level of gene expression (RT-PCR) and enzyme activity. Corals were exposed in short- (3 days) and long-term (54 days) experiments to two pCO2 conditions (ambient and elevated pCO2 equaling RCP 8.5, IPCC 2019, approximately 372.53 and 956.78 μatm, respectively). Of the eleven genes studied through RT-PCR, six were significantly upregulated compared with control in the short-term in the LpH condition, including the antioxidant enzyme superoxide dismutase (SOD), Heat Shock Protein 70 (HSP70), Toll-like receptor (TLR), galaxin and ferritin. After long-term exposure to low pH conditions, RT-PCR analysis showed seven genes were upregulated. These include the mannose-binding C-Lectin and HSP90. Also, the expression of TLR and galaxin, among others, continued to be upregulated after long-term exposure to low pH. Expression of carbonic anhydrase (CA), a key enzyme involved in calcification, was also significantly upregulated after long-term exposure. Our results indicated that, after two months, M. daytoni is not acclimatized to this experimental LpH condition. Gene expression profiles revealed molecular impacts that were not evident at the enzyme activity level. Consequently, understanding the molecular mechanisms behind the physiological processes in the response of a coral to LpH is critical to understanding the ability of polar species to cope with future environmental changes. Approaches integrating molecular tools into Antarctic ecological and/or conservation research make an essential contribution given the current ongoing OA processes.