DOI: 10.1594/pangaea.945272
title: Caspase measurements as a complement to tackle the responses of Cnemidocarpa verrucosa sp. A to sedimentation due to rapid climate change

description: 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 caspase data contains the raw data obtained, as well as the calculation for the triplicates. Caspase activity was analyzed as an indicator for the intensity of apoptotic cell death under stress exposure in all samples analyzed with transcriptomics (n=3 in each experimental group). The assay was carried out using the Caspase-Glo® 3/7 kit (Promega, USA), according to the standard protocol. The Caspase-Glo® 3/7 assay is based on the cleavage of the DEVDsequence of a luminogenic substrate by the caspases 3 and 7 which results in a luminescent signal. Frozen branchial basket tissue (16-33 mg) was ground in liquid nitrogen and weighed into 2 mL Eppendorf tubes. Homogenization buffer (HEPES 25mM, MgCl2·6H2O 5mM, EGTA 1mM) was added at 1:50 (w/v) ratio before samples were homogenized with a Eurostar digital IKA ® Werke (Staufen, Germany) for 2 minutes at 300 rpm. Homogenates were centrifuged (15 min, 15,682 g at 4 ºC) and 50 µl of supernatant were mixed with 50 µl of Caspase-Glo® 3/7 reagent in 96 well plates (F96 microwell plate, PS from ThermoFisher Scientific, Roskilde, Denmark). Luminescence of this mix was read using a TRISTAR LB941 microplate reader (Berthold, Bad Wildbad, Germany) following 30 seconds of automated sample mixing at 350 rpm and a subsequent reaction time of 30 min at 25 ºC. All samples were measured in triplicates with a blank sample (50 µl supernatant, 50 µl homogenization buffer) and a negative control (50 µl Caspase-Glo® 3/7 reagent, 50 µl homogenization buffer). Given that luminescence is proportional to caspase-3 activity, caspase activity is expressed in luminescence (number of relative light units; RLU).