DOI: 10.1594/pangaea.960575
title: Biosynthesis of pigments as response to light changes in the up-side down jellyfish Cassiopea andromeda

description: 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) = FmThe 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)/DTWhere 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).