Biosynthesis of pigments as response to light changes in the up-side down jellyfish Cassiopea andromeda

The published data originate from a laboratory experiment with adult Cassiopea andromeda medusae, which were sourced from an established jellyfish culture bred from polyps (origin: Aquarium Berlin) within the aquaria facilities of the Leibniz Centre for Tropical Marine Research (ZMT), Bremen, Germany. Incubation experiments were conducted in experimental tanks (ETs) in the Marine Experimental Ecology unit (MAREE) of ZMT. The individual ETs function as recirculating aquaculture systems with a water volume of ~120 L, with an upper culture unit and a sump tank equipped with a biofilter system and a protein skimmer below. The temperature and salinity were set at 26°C and 35 SA (Red Sea Salt, Red Sea Fish, Israel), respectively, these conditions were controlled and regulated automatically through submerged sensors. The ETs were each illuminated with an Aquaillumination Hydra FiftyTwo HD (AI Hydra 52 HyperDrive, USA) lamp with seven types of LEDs, emitting the full spectrum (380–680 nm) of photoactive radiation (PAR) with a photon flux density of 100 μmol photons/m²/s. In total, 52 visually healthy (i.e. no injured bells or lost oral arms etc.) Cassiopea andromeda specimens with initial body weights of 111.4 ± 35.7 g and diameters of 10.3 ± 1.3 cm were randomly allocated into 6 ETs. Within these ETs, the animals were individually housed in plastic containers (length 16 cm, width 12 cm, height 12 cm). These containers were fixed just below the water surface, to maintain the same horizontal position and vertical distance under the lamps. This setup allowed for the recognition of individual jellyfish and the precise control of PAR emission on a per-animal basis. Slits on the sides of these plastic containers allowed the exchange of water within the container and the surrounding tank. Over the acclimation and experimental phase, the ETs were cleaned once per week. This included the scratching off of biofilms and the siphoning of feed residues and other particles. During cleaning, approximately one-third of the water volume was exchanged with filtered seawater. In addition, the bacterial film that accumulated at the surface of the water was removed daily with a fine mesh, to prevent the refraction of light through this layer. C. andromeda individuals were target fed daily with 1 mL of dense freshly hatched brine shrimp Artemia nauplii solution using a plastic pipette. One hour after feeding, remaining food residues and any faecal matter were removed from the plastic containers via siphoning with a small plastic pipette. The small plastic boxes were regularly rotated in order to exclude any potential confounding effects associated with different positioning within these tanks. For acclimation purposes, the jellyfish were kept for three weeks in the ETs at a constant PAR intensity of 100 μmol photons/m²/s with 12:12 h light/dark cycle. Light intensities (Li-250A, LI-COR, USA) and spectra (RAMSES ACC-VIS spectroradiometer, TriOS, Germany) were determined at the bottom of the plastic containers. After the acclimation phase, four animals (n = 4) were collected for initial sampling. Subsequently, the PAR intensities in five ETs were changed in steps of no more than 100 μmol photons/m²/s per day, until the desired PAR intensities of 50, 200, 400, and 800 μmol photons/m²/s were reached. For the UVR-treatment, UVB-LEDs (l=285 ± 10 nm) emitting a dose of 1.3 KJ/m²/d were installed above a second ET that reached a final PAR intensity of 200 μmol photons/m²/s. Once the target treatment conditions had been reached, these five different light manipulations remained constant over a four-week period. The sixth ET kept a constant PAR intensity of 100 μmol photons/m²/s throughout the acclimation and experimental phase and served as control treatment. The umbrella pulsation rate, chlorophyll a fluorescence and wet biomass were quantified for each individual C. andromeda medusa initially, after the acclimation phase (day 0) and at the end (day 28) of the different PAR intensity treatments and the exposure to UVB radiation. Umbrella pulsations were counted over 15 s, this number was then extrapolated to determine the number of umbrella pulses per minute and served as proxy for overall organism activity. To exclude the effect of potential handling stress, umbrella pulsations were counted before taking the organisms out of the tanks for further analyses. Chlorophyll a fluorescence was measured using a portable pulse amplitude modulated fluorometer (Diving-PAM, Walz, Effeltrich, Germany). Immediately before the fluorescence measurements, randomly selected C. andromeda specimens (n = 4 per treatment) were collected from the ETs and placed into small glass containers filled with seawater derived from their tanks. In these containers, the animals were kept in darkness for 8 min, to dark-adapt the endosymbiotic dinoflagellates. The subsequent fluorescence measurements included the following steps: 1) a probe with a low-intensity modulated measuring light was placed in close distance to the oral arms and tentacles of each C. andromeda specimen to detect minimal fluorescence (F0) of the dark-adapted endosymbionts. 2) A saturating pulse of light (2700 μmol photons/m²/s) was applied (0.8 s) to obtain the maximum fluorescence (Fm) of the endosymbiotic dinoflagellates. On the basis of F0 and Fm the ratio Fv/Fm was calculated using the formula: Fv = Fm = (Fm − F0) = Fm The ratio Fv/Fm quantifies the maximum quantum efficiency of photosystem II and can serve as a proxy for photosynthetic performance. Right after the fluorescence measurements, the C. andromeda medusa were placed on absorbent tissues for 5 s to remove excess water before determining the wet weight of the jellyfish on a digital scale (Sartorius, Germany). The relative growth rate (RGR) was calculated based on wet biomass weight using the formula: RGR = ln (W2/W1)/DT Where ln is the natural log, W1 is the starting wet weight of one individual jellyfish, W2 is the total jellyfish wet weight after four weeks experimental time and DT is the length of the experiment. For the analyses of pigments and antioxidant activity (AOA), four C. andromeda were sampled after the acclimation phase, after two weeks, and after four weeks (at the end of the experimental period). At each of these time points, whole animals were snap-frozen in liquid N2 and stored at -80°C. Prior to lab-based analyses, the sampled organisms were lyophilized for 72 h at 1 mbar (ALPHA 1-4 LD plus; Christ GmbH, Osterode, Germany). After lyophilization the dry weight of individual organisms were documented before the whole jellyfish were ground to powder for 20 s, using a benchtop homogenizer (FastPrep-24, MP Biomedicals, Germany). For the counting of endosymbiotic algae cells, from each sample, a quantity of ~20 mg lyophilized powder was resuspended in 500 μl of distilled water. For homogenization, the suspension was shaken for 16 h at 60 rpm (Intelli-Mixer RM-2L; ELMI SIA, Riga, Latvia). To prevent cellular clumping, resuspended sample solutions were ultrasonicated twice for 30 s (HD 2070.2; Brandelin electronic GmbH & Co KG, Berlin, Germany) using a Sonotrode (MS 72; 20 KHz, 70 Watt, 285 μm = 100%) with a low amplitude of 20% prior to cell counting. The sample solutions were then pipetted into a Neubauer Hemocytometer (0.1 mm depth), to count the microalgae cells in triplicate under an optical microscope (20x). The total amount of microalgae cells per individual jellyfish, was calculated based on the cell concentration per dry weight of the resuspended jellyfish sub-sample. For pigment analyses, 140 mg of lyophilized sample material was weighed into Eppendorf tubes, after which the pigments therein were extracted in 1 mL of cold 90% acetone for 24 h at 4°C in the dark. After centrifugation (2500g, 4°C, 5 min) and filtration (0.45 μm nylon syringe filters, Nalgene, USA), pigment analyses were performed using reversed-phase high-performance liquid chromatography (HPLC). Pigments (chlorophyll a, peridinin) were separated on a LaChromElite system equipped with a chilled autosampler L-2200 and a DAD detector L-2450 (VWR-Hitachi, Germany) with a LiChropher 100-RP-18 guard cartridge, applying a gradient according to Wright et al. (1991). Peaks were detected at 440 nm, identified, and quantified via co-chromatography with appropriate standards (obtained from DHI Lab Products, Denmark). Pigment concentrations were expressed as μg/g C. andromeda dry weight and as pg/cell of endosymbiotic microalgae. To measure AOA, 200 mg of lyophilized sample was dissolved in 1 mL ethanol (70%) and extracted in a water bath (47°C) for 4 h, vortexing hourly. Prior to this analysis, samples were centrifuged (2500 g, 20°C) for 5 min. The AOA was determined using a modified version of the ABTS+ assay described by Re et al. (1999), also known as the Trolox Equivalent Antioxidant Capacity (TEAC) assay, with Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) serving as a standard. A 2.45 mM ABTS+ stock solution was obtained by oxidizing 7.0 mM of ABTS+ with potassium disulfate (K2S2O8) for 16 h. A working solution with a consistent photometrically measured absorption of 0.7 ± 0.02 at a wavelength of 734 nm (UV/VIS-spectrophotometer, Thermo Scientific Genesys 140/150, Fisher Scientific GmbH, Schwerte, Germany) was obtained via dilution with absolute ethanol. For the AOA analysis, 1 mL of this ABTS+ working solution was added to 10 mL of sample extract, and deradicalization was measured after 6 min. AOA of the samples was expressed as Trolox Equivalents (mmol TE 100 g-1 DW) after adjusting for the appropriate dilution factor. All chemicals were purchased from Sigma (Aldrich/Merck KGaA, Darmstadt, Germany).本公开数据源自一项针对成年倒立水母（Cassiopea andromeda）水母体的实验室实验，实验所用个体取自德国不来梅莱布尼茨热带海洋研究中心（Leibniz Centre for Tropical Marine Research, ZMT）水族养殖设施中，由水螅体（polyp）繁育的成熟水母种群（来源：柏林水族馆）。孵化实验于ZMT海洋实验生态单元（Marine Experimental Ecology unit, MAREE）的实验水箱（ETs）中开展。单个实验水箱为容积约120 L的循环水产养殖系统（recirculating aquaculture systems），包含上层养殖单元与下方配备生物过滤系统（biofilter system）和蛋白质分离器（protein skimmer）的集水箱。实验设置温度为26℃，盐度为35 SA（红海盐（Red Sea Salt），红海鱼类公司，以色列），上述条件通过浸没式传感器自动调控。每个实验水箱均配备Aquaillumination Hydra FiftyTwo HD（"AI Hydra 52 HyperDrive"，美国）灯具，该灯具搭载7组LED，可发射全光谱（380–680 nm）光合有效辐射（photosynthetically active radiation, PAR），光子通量密度为100 μmol photons/m²/s。 共计52只外观健康（即伞部无损伤、口腕未缺失等情况）的倒立水母个体，初始体重为111.4 ± 35.7 g，伞径为10.3 ± 1.3 cm，被随机分配至6个实验水箱中。每个水箱内，实验个体单独饲养于尺寸为长16 cm、宽12 cm、高12 cm的塑料容器内。这些容器固定于水面下方同一深度处，以保证所有个体与灯具的水平位置和垂直距离一致。该设置可实现单只水母的个体识别，并可精准控制每只个体接收的PAR辐射强度。塑料容器侧壁开有缝隙，可实现容器内与周围水箱的水体交换。在驯化与实验阶段，每周对实验水箱清洁一次，包括刮除生物膜、虹吸清除饲料残渣及其他颗粒物。清洁过程中，更换约三分之一容积的过滤海水。此外，每日使用细网去除水面积聚的细菌膜，以避免该层水体对光线产生折射。每日使用塑料滴管向每只倒立水母投喂1 mL高密度新鲜孵化的卤虫无节幼体（Artemia nauplii）溶液。投喂1小时后，使用小型塑料滴管虹吸清除塑料容器内残留的食物残渣与粪便。定期旋转塑料容器，以排除水箱内不同位置带来的潜在混淆效应。驯化阶段，将水母置于PAR强度为100 μmol photons/m²/s、光暗周期为12:12 h的环境中饲养三周。使用照度计（Li-250A，LI-COR，美国）与光谱仪（RAMSES ACC-VIS光谱辐射计，TriOS，德国）在塑料容器底部测定光照强度与光谱。 驯化阶段结束后，收集4只个体（n=4）进行初始采样。随后，将5个实验水箱的PAR强度以每日不超过100 μmol photons/m²/s的幅度逐步调整，直至达到预设的50、200、400和800 μmol photons/m²/s。针对紫外线辐射（ultraviolet radiation, UVR）处理组，在最终PAR强度为200 μmol photons/m²/s的第二个实验水箱上方安装UVB发光二极管（UVB-LEDs，波长285 ± 10 nm），其辐射剂量为1.3 KJ/m²/d。待所有处理组达到预设光照条件后，维持5种不同光照处理4周。第6个实验水箱在驯化与实验全程保持PAR强度为100 μmol photons/m²/s，作为对照组。 在驯化结束时（第0天）、不同PAR强度处理与UVB辐射暴露结束时（第28天），对每只倒立水母的伞脉搏动率、叶绿素a荧光与湿生物量进行定量测定。伞脉搏动计数时长为15 s，将计数结果外推得到每分钟的伞脉搏动次数，以此作为整体生物活动的替代指标。为排除抓取操作带来的应激影响，伞脉搏动计数在将个体从水箱取出进行后续分析前完成。叶绿素a荧光使用便携式脉冲振幅调制荧光计（Diving-PAM，Walz，埃夫尔特里希，德国）测定。荧光测定前，从每个处理组随机选取4只倒立水母，转移至装有对应水箱海水的小型玻璃容器中，置于黑暗环境8 min以暗适应其内共生甲藻。后续荧光测定步骤如下：1）将探头以低强度调制测量光紧贴每只水母的口腕与触手，测定暗适应内共生体的最小荧光（F0）；2）施加0.8 s的饱和光脉冲（2700 μmol photons/m²/s），获取内共生甲藻的最大荧光（Fm）。基于F0与Fm，通过公式计算Fv/Fm比值：Fv = Fm − F0，Fv/Fm = (Fm − F0)/Fm。该比值可量化光系统II的最大量子效率，作为光合性能的替代指标。荧光测定结束后，将水母置于吸水纸上静置5 s以去除多余水分，随后使用电子天平（Sartorius，德国）测定其湿重。基于湿生物量计算相对生长率（RGR），计算公式为：RGR = ln(W2/W1)/DT，其中ln为自然对数，W1为单只水母的初始湿重，W2为4周实验结束时的总湿重，DT为实验时长。 为分析色素与抗氧化活性（AOA），在驯化结束时、实验第2周与实验结束时（第4周）分别采样4只倒立水母。每个时间点均将完整个体快速液氮冷冻后保存于-80℃。实验室分析前，将采样个体在1 mbar压力下冻干72 h（ALPHA 1-4 LD plus；Christ GmbH，奥斯特罗德，德国）。冻干后记录单只个体的干重，随后使用台式均质器（FastPrep-24，MP Biomedicals，德国）将完整水母研磨20 s成粉末。为计数内共生藻类细胞，从每个样品中称取约20 mg冻干粉末，重悬于500 μL蒸馏水中。将悬浮液以60 rpm振荡16 h进行均质化（Intelli-Mixer RM-2L；ELMI SIA，里加，拉脱维亚）。为防止细胞聚集，在细胞计数前，将重悬样品溶液以20%低振幅超声处理两次，每次30 s（HD 2070.2；Brandelin electronic GmbH & Co KG，柏林，德国），使用Sonotrode探头（MS 72；20 KHz，70 W，285 μm = 100%）。随后将样品溶液移至血球计数板（Neubauer Hemocytometer，0.1 mm深度），在光学显微镜（20×）下重复计数三次。基于重悬水母子样品的干重与细胞浓度，计算单只水母的总微藻细胞数。对于色素分析，称取140 mg冻干样品至Eppendorf管中，使用1 mL冷90%丙酮于4℃黑暗环境中萃取24 h。离心（2500g，4℃，5 min）与过滤（0.45 μm尼龙针式过滤器，Nalgene，美国）后，采用反相高效液相色谱（reversed-phase high-performance liquid chromatography, HPLC）进行色素分析。色素（叶绿素a、多甲藻素）在LaChromElite系统上分离，该系统配备冷却自动进样器L-2200与二极管阵列检测器（DAD detector）L-2450（VWR-Hitachi，德国），搭配LiChropher 100-RP-18保护柱，采用Wright等人（1991）的梯度洗脱程序。在440 nm处检测色谱峰，通过与标准品（购自丹麦DHI Lab Products公司）共层析进行定性与定量分析。色素浓度以μg/g倒立水母干重与pg/内共生微藻细胞表示。为测定抗氧化活性（AOA），称取200 mg冻干样品溶于1 mL 70%乙醇，置于47℃水浴中萃取4 h，每小时涡旋一次。萃取前，将样品离心（2500g，20℃，5 min）。采用Re等人（1999）描述的改良ABTS+法（即Trolox等效抗氧化能力（Trolox Equivalent Antioxidant Capacity, TEAC）法）测定AOA，以Trolox（6-羟基-2,5,7,8-四甲基色烷-2-羧酸）作为标准品。将7.0 mM ABTS与过硫酸钾（K2S2O8）氧化16 h，得到2.45 mM ABTS+储备液。使用无水乙醇稀释该储备液，得到在734 nm波长下吸光度稳定为0.7 ± 0.02的工作液（使用UV/VIS分光光度计，Thermo Scientific Genesys 140/150，Fisher Scientific GmbH，施韦尔特，德国）测定吸光度。抗氧化活性分析时，将1 mL ABTS+工作液与10 mL样品提取物混合，6 min后测定自由基清除率。样品的抗氧化活性以Trolox当量（mmol TE 100 g-1 DW）表示，已校正相应稀释因子。所有化学品均购自Sigma（Aldrich/Merck KGaA，达姆施塔特，德国）。