Molecular responses of calcifying and non-calcifying Antarctic benthic species to Ocean Acidification - Superoxide dismutase activity

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.