Additive effects of ocean acidification (7.8/ 8.1 pH) and reduced light availability (35/ 150 PAR) on growth, photosynthesis, calcification and pigment content of stony coral Acropora millepora (NERP 5.2, AIMS)

This dataset consists of one data file from a 16 day aquarium experiment manipulating pH and light level and measuring growth, photosynthesis, calcification and pigment content of Acropora millepora.

The aim of this study was to test the hypothesis that a combined effect of decreased pH and decreased light on physiological responses of the coral is larger than that of each stressor individually.

Method:

Colonies of the coral Acropora millepora were collected from an inshore fringing reef next to Pelorus Island (central section of the Great Barrier Reef (GBR); 18° 33.001’ S, 146° 29.304’ E) between 2 and 4 m below lowest astronomical tide (LAT). After colonies were fragmented, individual coral nubbins were glued onto stubs and kept at the Australian Institute of Marine Science (AIMS, Townsville) in flow-through (recirculating flow ~1200 l h-1) aquaria facilities under plasma light (150 µmol photons m-2 s-1) for >3 mo. Nubbins were transferred into experimental tanks 2 weeks prior to the start of experiment to acclimate to experimental control conditions. Light levels chosen as the control and low light conditions were well within average ranges found between 3 and 6 m below LAT at midshelf and inshore reefs on the GBR, respectively.

The manipulative aquaria experiment was carried out in flow-through conditions over a period of 16 days between July and August 2012 at AIMS. After two weeks acclimation, four nubbins of A. millepora were allocated to each of the twelve experimental aquaria. Four treatments with three replicate tanks (working volume 17.5 L) were placed in alternating order. Treatments consisted of combinations of ambient pCO2 (427 µatm), high pCO2 (1073 µatm), low light (35 µmol photons m-2 s-1) and control light (150 µmol photons m-2 s-1). Light intensities were chosen from average PAR readings from an inshore and mid-shelf reef at ~ 5 m below LAT, present during the summer months. Water flow was provided with fresh filtered (0.5 µm) seawater at 25 °C, with a salinity of 34.5, at a flow-rate of 150 mL min-1. Irradiance was delivered by white light LED (6000 K, Aqua Illumination), covering the full color spectrum. Light levels were set to 12 h/ 12 h light-dark cycle. Additional aquaria pumps (AquarWorld, Australia, 250 L h-1) were fitted into each tank to assure water movement. Target pH levels were achieved by an automatic CO2 injection system (Aqua Medic, Germany) controlled by potentiometric pH sensors.

Growth of organisms was determined by the buoyant weight technique. Individual specimens were single-weighted (accuracy: 0.1 mg, Mettler Toledo, USA) in a custom build buoyant weight set-up with water jacket and seawater of constant temperature (25 °C) and salinity (34.5 ppt) at the start and at the end of the experiment. Growth of organisms was expressed as daily percentage of change.

After 16 days in experimental conditions, two individuals of each replicate tank were incubated for 1 h in the light and thereafter 1 h in the dark to determine calcification and photosynthesis. Light intensity and seawater pH of incubations corresponded to treatment condition of each organism. One experimental run consisted of 12 parallel incubations in 200 mL incubation chambers, including two blanks per treatment. To assure constant water temperature during incubation, chambers were placed into a flow-through water bath at 25 °C. Additionally, magnetic stirrer bars ensured water movement within the incubation chambers.

Calcification rates in light and dark were determined by the alkalinity anomaly technique (Chisholm & Gattuso 1991). A subsample of 50 mL was pipetted from the incubation seawater and directly titrated for total alkalinity (AT) on a Metrohm 855. A change in one mole of AT corresponds to a change of half a mole of calcium carbonate (Gao and Zheng 2010). Thus, calcification can be expressed by determining the change of carbon in calcium carbonate. Calcium carbonate precipitation or dissolution in µM C h-1 was calculated following Gao and Zheng (2010) and standardized to organisms’ surface area.
Net photosynthesis in the light or dark respiration were monitored consecutively during the incubations by three Firesting 4-channel oxygen meters (Pyroscience, Germany), which were connected to each chamber with fibre optic cables. Gross photosynthesis, net photosynthesis and respiration rates were expressed as µM O2 h-1 and standardized to organism surface area.

Chlorophyll a content of coral A. millepora was determined after coral tissue was stripped from the skeleton with an air gun utilizing fresh, ultra-filtered (0.2 µm) seawater. Zooxanthellae were isolated from the host tissue and re-suspended in 2 mL of ethanol (EtOH 95%), heat shocked and extracted for 24h in the cold. Absorbencies were read on 750 and 664 nm and Chl a content was calculated standardized to nubbin surface area.

References:

Gao K, Zheng Y (2010) Combined effects of ocean acidification and solar UV radiation on photosynthesis, growth, pigmentation and calcification of the coralline alga Corallina sessilis (Rhodophyta). Global Change Biol 16:2388-2398

Chisholm JRM, Gattuso JP (1991) Validation of the alkalinity anomaly technique for investigating calcification and photosynthesis in coral-reef communities. Limnol Oceanogr 36:1232-1239

Format:

A single csv text file, Acid_Light_Millepora_Growth.csv

Data Dictionary:

- Tank: running number of tanks in experiment
- pH: single factor pH
- Light: single factor light
- Treatment: combined factor of pH and light
- Growth: increase in buoyant weight of coral after 16 days of experimental treatment given in % change d-1
- Light calcification: calcification in light, given in µM C h-1 cm-2
- Dark calcification: calcification in darkness, given in µM C h-1 cm-2
- Net calcification: light calcification + dark calcification
- Net photosynthesis: oxygen production during light, given in µM O2 h-1 cm-2
- Respiration: oxygen consumption during darkness, given in µM O2 h-1 cm-2
- Gross photosynthesis: net photosynthesis – respiration
- Chlorophyll: Chlorophyll a content after 16 days in experimental conditions, given in µg cm-2

本数据集包含16天水族箱调控实验的一份数据文件，该实验通过操控pH值与光照水平，测定了微孔鹿角珊瑚(Acropora millepora)的生长、光合作用、钙化作用及色素含量。

本研究旨在验证假说：pH降低与光照降低对珊瑚生理响应的联合效应，强于单一胁迫因子的单独效应之和。

### 实验方法
研究采集的微孔鹿角珊瑚(Acropora millepora)群落采自佩洛鲁斯岛附近的近岸裙礁（大堡礁(Great Barrier Reef, GBR)中部；18° 33.001’ S, 146° 29.304’ E），采样深度为最低天文潮(lowest astronomical tide, LAT)下2~4 m处。将珊瑚群落切割为片段后，将单个珊瑚断枝粘固于基座，随后置于澳大利亚海洋科学研究所(Australian Institute of Marine Science, AIMS，汤斯维尔)的流通式（循环流量约1200 L·h⁻¹）水族箱设施中，采用等离子光源（150 μmol光子·m⁻²·s⁻¹）培育超过3个月。实验开始前2周，将珊瑚断枝转移至实验水箱以适应对照条件。实验设置的对照光照与低光照条件，分别对应大堡礁近岸与陆架中部礁体在最低天文潮下3~6 m处的平均光照范围。

该调控水族箱实验于2012年7月至8月在AIMS开展，采用流通式环境，持续16天。经过2周驯化后，将4株微孔鹿角珊瑚断枝分配至12个实验水族箱中，设置4种处理、3个重复水箱（工作容积17.5 L），并以交替顺序排布。4种处理分别为环境分压二氧化碳(ambient pCO₂, 427 μatm)、高分压二氧化碳(high pCO₂, 1073 μatm)、低光照(35 μmol光子·m⁻²·s⁻¹)与对照光照(150 μmol光子·m⁻²·s⁻¹)的组合。光照强度选取夏季期间大堡礁近岸与陆架中部礁体在最低天文潮下约5 m处的平均光合有效辐射(Photosynthetically Active Radiation, PAR)数值。

实验用水为经0.5 μm过滤的新鲜海水，水温维持25 ℃，盐度34.5，流量为150 mL·min⁻¹。光照由6000 K白光LED（Aqua Illumination）提供，覆盖全光谱。光照周期设置为12 h光照/12 h黑暗。每个水箱额外加装AquarWorld（澳大利亚）品牌的250 L·h⁻¹水族箱水泵，以保证水体流动。目标pH值通过德国Aqua Medic品牌的电位式pH传感器控制的自动二氧化碳注入系统实现。

珊瑚生长通过浮重法(buoyant weight technique)测定：实验开始与结束时，将单个珊瑚样本置于定制的浮重装置中，在水温25 ℃、盐度34.5 ppt的恒温水套与海水中称重（精度0.1 mg，梅特勒托利多(Mettler Toledo)，美国）。珊瑚生长以每日百分比变化量表示。

实验处理16天后，每个重复水箱中的2株珊瑚分别在光照下孵育1小时，随后在黑暗中孵育1小时，以测定钙化作用与光合作用。孵育的光照强度与海水pH值对应样本所在处理组的条件。单次实验包含12个200 mL孵育舱的平行孵育，每个处理设置2个空白对照。为保证孵育期间水温恒定，将孵育舱置于25 ℃的流通式水浴中。此外，使用磁力搅拌棒保证孵育舱内水体流动。

光照与黑暗条件下的钙化速率通过碱度偏差法(alkalinity anomaly technique, Chisholm & Gattuso 1991)测定。从孵育海水中移取50 mL子样本，直接在Metrohm 855型滴定仪上滴定总碱度(total alkalinity, AT)。1摩尔总碱度的变化对应0.5摩尔碳酸钙的变化(Gao and Zheng 2010)，因此可通过测定碳酸钙中碳的变化计算钙化速率。碳酸钙沉淀或溶解速率（以μM C·h⁻¹为单位）参照Gao和Zheng(2010)的方法计算，并标准化至珊瑚样本的表面积。

光照下的净光合作用与黑暗中的呼吸作用，在孵育期间通过3台Firesting 4通道氧气仪（Pyroscience，德国）连续监测，该仪器通过光纤与每个孵育舱连接。总光合作用、净光合作用与呼吸速率以μM O₂·h⁻¹为单位，并标准化至珊瑚样本的表面积。

微孔鹿角珊瑚的叶绿素a含量测定：利用空气喷枪将珊瑚组织从骨骼上剥离，使用新鲜的0.2 μm超滤海水。将虫黄藻从宿主组织中分离出来，重悬于2 mL 95%乙醇中，经热激后在低温下萃取24 h。在750 nm与664 nm波长下读取吸光度，计算得到的叶绿素a含量标准化至珊瑚断枝的表面积。

### 参考文献
1. Gao K, Zheng Y (2010) 海洋酸化与太阳紫外辐射对珊瑚藻*Corallina sessilis*（红藻门）的光合作用、生长、色素沉积与钙化作用的联合效应. 《全球变化生物学(Global Change Biology)》16:2388-2398
2. Chisholm JRM, Gattuso JP (1991) 碱度偏差法在珊瑚礁群落钙化与光合作用研究中的验证. 《湖沼学与海洋学(Limnology and Oceanography)》36:1232-1239

### 数据集格式
单CSV文本文件：*Acid_Light_Millepora_Growth.csv*

### 数据字典
- Tank：实验中水箱的运行编号
- pH：单因子pH值
- Light：单因子光照水平
- Treatment：pH与光照的组合因子
- Growth：经过16天实验处理后，珊瑚浮重的增长，以每日百分比变化量表示
- Light calcification：光照条件下的钙化速率，单位为μM C·h⁻¹·cm⁻²
- Dark calcification：黑暗条件下的钙化速率，单位为μM C·h⁻¹·cm⁻²
- Net calcification：光照钙化速率与黑暗钙化速率之和
- Net photosynthesis：光照下的氧气产生量，单位为μM O₂·h⁻¹·cm⁻²
- Respiration：黑暗中的氧气消耗量，单位为μM O₂·h⁻¹·cm⁻²
- Gross photosynthesis：净光合作用速率减去呼吸速率
- Chlorophyll：经过16天实验处理后的叶绿素a含量，单位为μg·cm⁻²