Sampling Details
Between 10–12 November 2014, three weeks following the peak of the 2014 thermal stress event, six visibly non-bleached and six visibly bleached parent colonies of Montipora capitata and Porites compressa were collected from 1–3m depth from the fringing reefs surrounding Moku o Loʻe in Kāneʻohe Bay (centered at 21°26’6” N, 157°47’12” W) and in Waimānalo Bay (centered at 21°19’36” N, 157°40’54” W) using a hammer and chisel.
One ramet from each colony was immediately placed in a -20 °C freezer to evaluate the initial physiology of the parent colonies prior to experimental manipulation. An additional twelve ramets were collected from each parent colony of each species from each location (totaling 576 fragments) and mounted on pre-labeled plastic tiles for use in the manipulative experiment.
Experimental Tank Conditions
All experimental ramets were returned to the Hawai‘i Institute of Marine Biology and allowed to recover under ambient seawater conditions in outdoor, unfiltered, flow-through seawater tanks for three days. Natural light levels were reduced using 40% neutral density shade cloth such that photosynthetically active radiation levels mimicked that at collection depth.
On 15 November 2014, ramets were equally divided among eight outdoor, 38 L experimental tanks supplied with unfiltered, flow-through seawater. Though the incoming seawater was unfiltered, virtually no zooplankton is delivered to experimental tanks at HIMB due to scouring of the inflowing seawater by in-line invertebrates. Two experimental tanks were randomly assigned to each of the following four treatments: 1) Control (ambient seawater of pH 7.97 and unfed), 2) Fed (ambient seawater of pH 7.97 and fed), 3) OA (acidified seawater of pH 7.74 and unfed), and 4) OA+Fed (acidified seawater pH 7.74 and fed).
Coral holobiont physiology was assessed for parent colonies (i.e., in situ reef controls) and a subset of experimental corals in December 2014 (2 months after 2014 peak thermal stress; 1 month in experimental tanks), June 2015 (8 months after 2014 peak thermal stress; 7 months in experimental tanks), and November 2015 (one month after 2015 peak thermal stress; 13 months in experimental tanks). At each experimental time point, physiological trait measurements were conducted (calcification, photosynthesis, respiration, and zooplankton feeding rate capacity). Once all live measurements were completed, these ramets were frozen for further analyses (endosymbiont cell density, tissue biomass, and total lipids).
The seawater pH was reduced in the simulated OA treatments by bubbling CO gas into the tanks. To ensure tanks remained within target pH range, all tanks were spot-checked with a calibrated pH probe every 1–2 days throughout the experiment. This data was recorded during periods when physiological measurements were being made. Small adjustments to the CO2 bubbling rate were made as needed to maintain the desired 0.2 pH offset between ambient and simulated OA treatments.
Weekly, pH and total alkalinity measurements were made on seawater samples collected from each tank at18:00 h. Total alkalinity was measured using a modified Gran titration and checked for accuracy and precision (~5 μmol kg) using certified reference materials obtained from A. Dickson (Scripps Institution of Oceanography). pH was measured spectrophotometrically at 25 °C with m-cresol purple (precision ~0.002 pH units).
These parameters were then entered into CO2Sys to calculate pH in situ. The CO bubbling rate was then adjusted as necessary, pH was measured again using a calibrated pH probe, and any further adjustments were made until the desired pH was consistently read. In December 2014, March 2015, and June2015, diel cycle chemistry was measured over a 24-hour period across all tanks using the same method.
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