All methods are fully described in:
Lunden et al. 2014 Frontiers in Marine Science “Acute survivorship of the deep-sea coral Lophelia pertusa from the Gulf of Mexico under acidification, warming, and deoxygenation”
From the Paper:
Forty-one nubbins of L. pertusa used in the experiments were collected in November 2010 on the NOAA Ship Ronald H. Brown with ROV Jason II as part of the “Lophelia II” project jointly sponsored by the Bureau of Ocean Energy Management and the NOAA Office of Ocean Exploration and Research in the Gulf of Mexico (GoM). Permits for the collection of corals were obtained from the U.S. Department of the Interior prior to any collection activities. Spatially discrete coral branches were collected with the ROV and placed in temperature-insulated bioboxes (volume = 20 l) at depth. Upon return to the surface, corals were kept alive in 20 l aquaria in the ship’s constant-temperature room. Partial water changes were made regularly while at sea. Upon return to port, corals were immediately transported overnight to the laboratory on wet ice.
In the laboratory, corals were maintained in one of two 570 liter recirculating aquaria systems at temperature 8 degrees celsius and salinity 35 ppt (Lunden et al., 2014). Regular partial water changes (15–20%) were performed with seawater made using Instant Ocean sea salt. Submersible power heads were placed in each holding tank to ensure water movement and turbulence sufficient to cause swaying of coral polyps. Corals were fed three times weekly using a combination of MarineSnow PlanktonDiet (Two Little Fishies, Miami Gardens, FL) and freshly hatched Artemia nauplii.
Survivorship was assessed by daily observations of polyp tissue presence and behavior. Final survivorship counts were taken 3 to 4 days following the end of each treatment after transfer to the maintenance tank. Survivorship is reported as percent cumulative mortality.
Net calcification was measured using the buoyant weight technique (Davies, 1989). Coral nubbins were buoyantly weighed at the start and end of each experimental period (days eight and fifteen) using a Denver Instruments SI-64 analytical balance (d = 0.1mg, Fisher Scientific, Waltham, MA). A weighing chamber was constructed using 1/2” plexiglass to prevent disturbances from air movement during weighing. Each coral nubbin was transported individually from its respective aquarium to the weighing chamber in a four-liter Pyrex beaker and suspended from the balance. The buoyant weight was recorded after the coral nubbin stabilized, typically 2 min. Each coral nubbin was weighed three times to determine measurement precision (2–3 mg). Seawater density was determined in each aquarium by buoyantly weighing a 2.5 cm^2 aluminum block with known density (2.7 g/cm^−3). Coral weight in air (i.e., dry weight) was calculated by the following equation:
Wa = Ww / (1− (Dw/SD))
Where
Wa = coral weight in air (dry weight)
Ww = coral weight in water (buoyant weight)
Dw = density of seawater
SD = coral skeletal density (= 2.82 g/cm^−3, Lunden et al., 2013).
Coral growth rate is reported as percent growth per day (%/d−1), which was calculated by the equation:
Gt = 100 × (Mt2 − Mt1)/(Mt1(T2 −T1))
Where
Gt = growth rate as %/d^−1
Mt2 = mass (mg, dry weight) at time 2 (end of experimental period, day 15)
Mt1= mass (mg, dry weight) at time 1 (start of experimental period, day 8)
T2 = time 2 (end of experimental period, day 15)
T1= time 1 (start of experimental period, day 8)
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