Larvae were obtained from brooding colonies coral S. caliendrum collected from 5 to 7 m deep on Hobihu Reef, Nanwan Bay, in March and June of 2010, placed into individual flow-through seawater tanks. Overflow water from each tank passed through mesh-lined (110 mm) cups that captured larvae. Following collection, larvae were settled onto clean pre-weighed glass microscope coverslips. Coverslips with coral recruits (n=18: experiment I; n=36: experiment II) were assigned randomly to the pCO2 treatments.
In experiment I, treatments consisted of steady ambient pCO2, steady high pCO2 and diurnally oscillating pCO2 on a natural phase; this design was augmented in experiment II by including a diurnally oscillating pCO2 on a reverse phase.
The DIC content of seawater was manipulated by bubbling premixed gas of a known pCO2 or by bubbling unmodified air for the ambient treatment. To mix the gas for the high pCO2 treatments, a system employing a variable-timed solenoid valve was used, which controlled the flow of air and CO2 into a mixing chamber to reach the target pCO2 of 650 matm in experiment I. This target value was selected to provide a conservative estimate for the atmospheric pCO2 by 2100 following the business as usual emission scenario A1 [30]. In experiment II, pCO2 levels were increased to 800 matm to test the effects of diurnally oscillating pCO2 levels that exceeded what is found at Hobihu reef. The solenoid valve was connected to an infrared gas analyser (S151, Qubit Systems, Ontario Canada), which monitored the output gas and provided dynamic control of the duty cycle of the solenoid, thereby providing a consistent concentration of mixed gas to the treatment tanks. Refer to the electronic supplementary material for seawater chemistry and field sampling methods.
For detailed description of methods, see Dufault et al. (2012), Proc. R. Soc. B. doi:10.1098/rspb.2011.2545
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