Molecular responses of calcifying and non-calcifying Antarctic benthic species to Ocean Acidification - Apoptotic 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.
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.

学界普遍认为，南大洋生物对海洋酸化（ocean acidification）尤为敏感，因其栖息于天然方解石-文石饱和度较低的冷水环境中。此外，学界通常假设钙化动物相较于非钙化动物，更易受到海洋酸化的影响。在此背景下，本研究旨在探究pH值降低对海鞘（ascidia）疣茎茎鞘海鞘（Cnemidocarpa verrucosa sp. A）的影响。本研究采用基因表达谱分析与酶活性检测技术，解析该南极底栖物种对海洋酸化的响应。

研究团队于南极乔治王岛波特湾卡尔尼站（Carlini station）约15米水深处，通过水肺潜水采集疣茎茎鞘海鞘（Cnemidocarpa verrucosa sp. A）样本。

超氧化物歧化酶（Superoxide dismutase, SOD）活性检测：取约70 mg的鳃篮（brachial basket）组织样本，以1:4的质量体积比（w/v）置于20 mM Tris-HCl、1 mM EDTA、pH 7.6的缓冲液中进行匀浆。将匀浆样品在4℃、14000×g条件下离心3分钟，取上清液按照Livingstone等（1992）的实验方案，于20℃下检测SOD活性。取上清液与检测缓冲液（含43 mM K₂HPO₄、43 mM KH₂PO₄、0.1 mM EDTA，pH 7.68）、5 mM 黄嘌呤（Xanthine，原文拼写为Xanthina，Sigma X-0626）、100 μM 细胞色素C（Cytochrome C，Sigma C-2037）以及2 M (NH₄)₂SO₄体系中的0.3 mU/μL 黄嘌呤氧化酶（Xanthin-Oxidasa, Sigma X-4875）混合。检测在20℃、550 nm波长下于分光光度计中进行，持续3分钟，每10秒记录一次数据。蛋白定量采用Bradford（1976）法测定样品总蛋白含量，超氧化物歧化酶活性以提取物酶活性（mU）/蛋白质量（mg）表示。所有检测均设置三次平行重复。