Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Concentrations of dissolved nitrates, nitrite, silicate and phosphate were analysed from each experimental aquaria after one, six and nine months. Inorganic nutrient concentrations in seawater were determined using a SFA (Segmented Flow Autoanalyzer) (Aminot & Kérouel, 2007) with a SEAL Analytical QuAAtro analyzer using established colorimetric methods (Hydes et al. 2010 & Becker et al. 2020). RMNs (Reference Material for nutrients in seawater) with different concentration ranges were used for quality control and accuracy of our analysis. Low nutrient seawater (surface seawater filtered and aged) is also used to control low nutrient concentrations.

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Dry mass of the coral nubbins (3 per experimental aquarium) was assessed by means of the buoyant weight technique (Jokiel et al. 1978, Davies, 1989), using an analytical balance (OHAUS AX124, precision 0.1 mg). The dry mass was calculated considering the nubbin net weight in water, the water density and the skeletal density of D. cornigera (2.63 g/cm3; Movilla et al. 2014). Measurements were performed just once the acclimation time finished and after 2 , 4, 6 and 9 months under the experimental conditions. Skeletal growth rates were calculated as the slope of the linear regression between the logarithmically transformed dry mass and the experimental time (%/day) (Orejas et al. 2011).

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Prokaryotes were quantified by flow citometry (CytoFLEXflex S, Beckman Coulter) as previously described by Gasol et al. (1999) with our own adjustment of the scatter settings to more accurately resolve these cells with the new CytoFLEX flow cytometry technology. Measurements were performed every month from different parts of the aquaria setup: the input seawater, the reservoir tank (after the filtration of seawater to 5 µm and before the UV lamp) and every experimental aquaria.

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain), from the 6th of May 2022 to the 24th of February 2023. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. This dataset contains the registered values for temperature, pH, DO (% air saturation and mg/L) and sainity from the experimental aquarium over the course of the experiment. Temperature and DO were daily measured with a YSI ProODO dissolved oxygen instrument. Salinity was weekly assessed with a WTW 350i multiparameter device equipped with a ConOx probe. Measurements for pH were performed every 1 – 2 months. Samples for pH were directly collected on cylindrical 10 cm cuvettes and analysed on daily basis. After being thermostated at 25ºC, samples were measured using a manual spectrophotometrical procedure with a Sigma Aldrich impure indicator (Clayton and Byrne, 1993). The uncertainty of pH is about 0.005 pH units.

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Measurements for pH and total alkalinity (TA) were performed on seawater samples from all the experimental aquaria every 1 – 2 months. Samples for pH were directly collected on cylindrical 10 cm cuvettes and analysed on daily basis. After being thermostated at 25ºC, samples were measured using a manual spectrophotometrical procedure with a Sigma Aldrich impure indicator (Clayton and Byrne, 1993). TA was measured following the double end point potentiometric technique by Pérez and Fraga (1987a) and Pérez et al. (2000). Measurements of Certified Reference Material (Dickson's lab, SIO) were performed in order to control the accuracy of the TA measurements. The uncertainty of TA and pH is about 3 μmol/kg and 0.005 pH units, respectively. In situ additional CO2 system variables were calculated using the R package seacarb (Gattuso et al. 2023) using in situ temperature, salinity, measured pH and TA, the dissociation constants of carbonic acid in seawater (K1 and K2) by Lueker et al. (2000), the equilibrium constant of hydrogen fluoride by Perez and Fraga (1987b), the concentration of total boron by Uppström (1974) formulation, and the stability constant of hydrogen sulphate by Dickson (1990). Mean values for the concentration of silicate and phosphate on the aquaria experiments were used in the CO2 calculations, respectively, mean (and standard) deviations as 2.1 (0.4) and 0.31 (0.03) μmol/kg.

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Raspberry-based controllers were set to modify and monitor the water temperature, pH and dissolved oxygen in every treatment. Values for temperature (ºC), pH (NBS scale) and oxygen (% air saturation) were registered every 15 minutes in a database. The parameters were measured using PT100 temperature sensors, and Atlas Scientific Lab Grade pH and Dissolved Oxygen sensors. Every set of sensors was placed on each header tank, corresponding to each treatment (8 in total). Parameter setpoint values for each header tank were finely adjusted on the controller to ensure the treatment target values on each experimental aquaria. Oxygen sensors from treatments with ambient oxygen (~8.63 mg/L) were removed from the system due to calibration issues. Here, an example of the results for temperature, pH and oxygen for every header tank over 24 hours on 31st of December 2022 is presented. Values for pH (total scale) and dissolved oxygen (mg/L) were calculated using AquaEnv (Hofmann et al. 2010) and respR (Harianto et al. 2019) R packages, respectively, using the in situ temperature and salinity (35.1).

Description: A 9-month aquarium experiment with the cold-water Dendrophyllia cornigera was conducted to investigate the single and combined effects of warming, acidification and deoxygenation on its ecophysiological response. The experiment took place at the Aquarium finisterrae (A Coruña, Spain) between 2022-05-06 and 2023-02-24. Treatment values for each parameter (current in situ vs. climate change) were: 12 °C and 15 °C (temperature); ~7.99 and 7.69 (pH); ~8.63 mg/L and 6.45 mg/L (dissolved oxygen concentration). A total of eight treatments (with 3 replicates each, 5 L aquaria) were set up. Respiration rates from each nubbin (3 per aquarium) were assessed after 6 and 9 months under the experimental conditions by means of closed-cell incubations. Oxygen consumption was calculated by measuring the dissolved oxygen concentration of the seawater inside the chamber using an optical oxygen sensor (YSI ProODO) at the beginning and at the end of the incubation time (24 h incubation). Dry mass (g) of the coral nubbins was assessed by means of the buoyant weight technique (Jokiel et al. 1978, Davies, 1989), using an analytical balance (OHAUS AX124, precision 0.1 mg). Tissue surface area (cm2) was assessed on virtual 3D models of each nubbin at the time when the measurements were conducted. Respiration rates were normalised by dry mass and by tissue surface area.

Description: Knowledge on basic biological functions of organisms is essential to understand not only the role they play in the ecosystems but also to manage and protect their populations. The study of biological processes, such as growth, reproduction and physiology, which can be approached in situ or by collecting specimens and rearing them in aquaria, is particularly challenging for deep-sea organisms like cold-water corals. Field experimental work and monitoring of deep-sea populations is still a chimera. Only a handful of research institutes or companies has been able to install in situ marine observatories in the Mediterranean Sea or elsewhere, which facilitate a continuous monitoring of deep-sea ecosystems. Hence, today’s best way to obtain basic biological information on these organisms is (1) working with collected samples and analysing them post-mortem and / or (2) cultivating corals in aquaria in order to monitor biological processes and investigate coral behaviour and physiological responses under different experimental treatments. The first challenging aspect is the collection process, which implies the use of oceanographic research vessels in most occasions since these organisms inhabit areas between ca. 150 m to more than 1000 m depth, and specific sampling gears. The next challenge is the maintenance of the animals on board (in situations where cruises may take weeks) and their transport to home laboratories. Maintenance in the home laboratories is also extremely challenging since special conditions and set-ups are needed to conduct experimental studies to obtain information on the biological processes of these animals. The complexity of the natural environment from which the corals were collected cannot be exactly replicated within the laboratory setting; a fact which has led some researchers to question the validity of work and conclusions drawn from such undertakings. It is evident that aquaria experiments cannot perfectly reflect the real environmental and trophic conditions where these organisms occur, but: (1) in most cases we do not have the possibility to obtain equivalent in situ information and (2) even with limitations, they produce relevant information about the biological limits of the species, which is especially valuable when considering potential future climate change scenarios. This chapter includes many contributions from different authors and is envisioned as both to be a practical “handbook” for conducting cold-water coral aquaria work, whilst at the same time offering an overview on the cold-water coral research conducted in Mediterranean laboratories equipped with aquaria infrastructure. Experiences from Atlantic and Pacific laboratories with extensive experience with cold-water coral work have also contributed to this chapter, as their procedures are valuable to any researcher interested in conducting experimental work with cold-water corals in aquaria. It was impossible to include contributions from all laboratories in the world currently working experimentally with cold-water corals in the laboratory, but at the conclusion of the chapter we attempt, to our best of our knowledge, to supply a list of several laboratories with operational cold-water coral aquaria facilities.