Superoxide dismutase measurements as a complement to tackle the responses of Cnemidocarpa verrucosa sp. A to sedimentation due to rapid climate change

Rapid regional warming causes glacial retreat and melting of ice caps along the West Antarctic Peninsula, in consequence, sediment discharge into marine coastal areas reduces food availability and impairs respiratory performance of benthic filter-feeders. Here we analyzed the response of a highly abundant Antarctic ascidian Cnemidocarpa verrucosa sp. A to experimental changes in sedimentation rates through enzymatic measurements: Caspase and Superoxide Dismutase. Experimental work was carried out at Dallmann laboratory, Carlini Station in Potter Cove (58°40'W; 62°14'S), South Shetland Islands, Antarctica; during the summer campaign 2015/2016. Animals were collected by SCUBA diving at 20-30 m water depth (58°39'37,86W; 62°14'6, 153S), placed in containers with seawater from the sampling site, and immediately transferred to the experimental facilities of the laboratory. Specimens were placed in a 100 L container after cleaning their tunics from large epibionts. Both species were kept in an open flow system with unfiltered natural seawater (0 ± 1 °C) directly pumped from the cove for ten days, to allow for recovery from sampling and acclimation to the experimental system. Thereafter, specimens (n=10) with similar body sizes (approximately 10cm high) were selected, and 1-2 individuals were placed randomly in six aquaria (8 L), with individual closed circulation systems (EHEIM universal 300 Water Pump (300l/h)) and a venturi to assure constant aeration inside the aquaria. The six aquaria were placed in a 90 L running seawater bath (open system) for the incubation experiments in order to keep constant temperature (1.76 ± 0.37 °C). Treatment was applied to three aquaria which consisted in applying a single pulse of 200 mg L-1 of sediment per experimental aquarium. The experiment was stopped when turbidity was no longer observed in the water (36 h after exposure). The control treatments were maintained in seawater without sediment addition. Sediment used for the treatment was obtained using a grab sampler at 20 m depth in Potter Cove, from the surface bottom sediments of the inner cove (around 15 m depth, close to the mouth of the major creek), dried at 70 °C and sieved through a 50 μm mesh, following the protocol of Torre et al. (2012). After exposure, animals were dissected on ice, branchial basket, mantle, and gut were separated and frozen at -80ºC. The Superoxide Dimutase data contains the raw data obtained when measuring SOD activity and total protein content, as well as the calculation for the triplicates. Superoxide dismutase (SOD) measurements were carried out according to McCord & Fridovich (1969) in all samples analyzed with transcriptomics (n=3 in each experimental group). Samples were kept on ice throughout the entire extraction process. Branchial basket tissue (>70 mg) was homogenized in extraction buffer (20 mMTris-HCl, 1 mM EDTA, pH 7.6) at a 1:4 (w/v) ratio, manually using an IKA ® stirrer (Staufen, Germany) and a plastic pestle. Homogenates were centrifuged for 3 min at 18,188 g at 4ºC, 10 µl supernatant was extracted, then 870 µl measurement buffer (43 mM K2HPO4, 43 mM KH2PO4, 0,1 mM EDTA, pH 7.68), 100 µl cytochrome c (100 µM, Sigma C-2037), 10 µl xanthine (5 mM, Sigma X-0626), and 10 µl XOD (0,3 mU/µl xanthine oxidase in 2 M (NH4)2SO4, Sigma X-4875), was added to the supernatant. The measurement was done in a spectrophotometer DU800 (Beckman Coulter, Unterschleißheim-Lohhof, Germany) at 550 nm in a thermostatted half-macro cuvettes of optical glass (Hellma, Munich, Germany) at 20°C. All samples were measured in triplicates, and the blank consisted of molgrade water. The principle of the test is based on the fact that SOD is a radical interceptor; O2 radicals reduce the oxidized cytochrome-c, resulting in increased extinction at 550 nm. Thus, by adjusting the amount of XOD (at a slope of 0.02 per minute, 100% of activity), the SOD in the sample traps the O2 radicals and inhibits the increase in extinction, thus the concentration of SOD in the sample can be calculated. The amount of protein in the extract was measured using the protocol described by Bradford (1976), briefly, using bovine serum albumin (BSA, Sigma A-6003) in 0.9% NaCl) as standards (0- 23.3 µg/mL). Biuret reagent (270 μL) was added to the samples (30 μL), mixture was incubated at room temperature for 10 min, directly in the microplate reader. Absorbance was read at 549 nm in a plate photometer TriStar LB941. The results are expressed as activity in the extract (mU) / amount of protein (mg).