SUPPLEMENTARY INFORMATION
Interactive effects of ocean acidification and rising sea temperatures alter predation rate
and predator selectivity in reef fish communities
METHODS
The experiment took place at Lizard Island Research Station (14°40’S, 145°28’E), on the Great
Barrier Reef, Australia, in Nov-Dec 2012.
CO2 treatments:
CO2 treatments were maintained by CO2 dosing to a set pHNBS. Seawater was pumped from the
ocean into 2 x 60 L header tanks where it was diffused with ambient air (control) or CO2 to
achieve a pH of approximately 8.15 (control) and 7.85. The reduced pH values were selected to
achieve the approximate CO2 conditions required, based on preliminary observations of total
alkalinity, salinity and temperature of seawater at Lizard Island. A pH-controller (Aqua Medic,
Germany) was attached to each of the CO2 treated header tanks to maintain pH at the desired
level. A solenoid injected a slow stream of CO2 into a powerhead at the bottom of the header
tank whenever the pH of the seawater rose above the set point. The powerhead rapidly dissolved
CO2 into the seawater and also served as a vigorous stirrer. Identical powerheads were included
in control and CO2 header tanks. Equilibrated seawater from each sump was supplied at a rate of
~500ml.min-1 to four replicate 35-L aquaria for each CO2 and temperature treatment, each
housing a group of larval fishes or predators. Seawater temperature (C22, Comark, Norwich,
UK) and pHNBS (HQ40d, Hach, Loveland, CO, USA) of each aquaria was measured daily. Total
alkalinity of seawater was estimated by Gran titration from water samples taken from each CO2
treatment. Total alkalinity standardizations performed before processing each batch achieved
accuracy within 1% of certified reference material from Dr. A. Dickson (Scripps Institution of
Oceanography). Average seawater pCO2 was calculated using these parameters in the program
CO2SYS (Pierrot et al., 2006) using the constants K1, K2 from Mehrbach et al. (1973) refit by
Dickson & Millero (1987) (see Table 1).
Respirometry
Fish were placed individually into one of four darkened 20-ml glass vials that served as
respirometry chambers that were submerged in an aquarium maintained at the same temperature
and CO2 levels at which the fish were being maintained. Water was continuously recirculated
within each respirometry chamber via a peristaltic pump, which ensured a homogeneous oxygen
tension throughout the chamber. Including the tubing circuit, the total volume of each system
was 31.6 ±0.6 ml. A submersible pump connected to a digital relay timer (MFRT-1 Multi
Function Recycling Timer, Xiamen SUPERPRO Technology Co., Ltd., Xiamen, Fujian, China)
was used to flush the respirometry chambers with clean, aerated seawater from the aquarium at a
flow rate of 5 ml.min-1 for a period of 5 min. During the flush cycle, excess water from each
respirometry chamber overflowed out of a small piece of tubing extending just above the water
surface in the aquarium. After each flush cycle, the pump was off for 10 min, during which time
fish consumed O2 from the chamber. These periods of interrupted water flow were short enough
to ensure oxygen within the chambers did not fall below 80% saturation at any time, and flush
periods were long enough to eliminate accumulation of metabolic CO2 and allow water oxygen
levels to return to 100% saturation (Steffensen, 1989).
Preliminary experiments indicated that 90 min was ample time to ensure O2 consumption rates
had reached the lowest possible values, beyond which O2 consumption rates did not significantly
vary (Rummer et al., 2013). Fish were maintained in the respirometry system for an additional
90 min following this initial habituation period. During the entire 180 min trial, temperaturecompensated O2 concentration of the water within each chamber was continuously recorded (0.5
Hz) using oxygen-sensitive REDFLASH dye on contactless spots (2 mm) adhered to the inside
of each chamber and linked to a Firesting Optical Oxygen Meter (Pyro Science e. K., Aachen,
Germany) via fibre-optic cables. At the end of each respirometry trial, all fish were removed
from chambers, blotted dry with a paper towel and weighed to 0.0001g. Raw text files from the
Firesting system were imported offline into LabChart version 6.1.3 (ADInstruments, Colorado
Springs, CO, USA), which was used to analyse data. A modified version of the equations from
(Schurmann & Steffensen, 1997) was used to calculate ṀO2 (mg kg-1 h-1) for each 10 min
measuring period throughout each 180 min trial. The ṀO2Routine for each fish was then calculated
as the mean of the final three measurements minus the background O2 consumption, which was
measured daily before and at the end of each trial (assumed linear) and did not exceed 5% of the
ṀO2Routine of the fish. All systems were cleaned with a 70% ethanol solution daily to keep
background respiration low.
References
Dickson AG, Millero FJ (1987) A comparison of the equilibrium constants for the dissociation of
carbonic acid in seawater media. Deep-Sea Research, 34, 1733-1743.
Mehrbach C, Culberson CH, Hawley JE, Pytkowicz RM (1973) Measurements of the apparent
dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnology
and Oceanography, 18, 897-907.
Pierrot D, Lewis E, Wallace DWR (2006) MS Excel Program Developed for CO2 System
Calculations. (ed Carbon Dioxide Information Analysis Center DOE), Oak Ridge,
Tennessee, U.S.A.
Rummer JL, Stecyk JA, Couturier CS, Watson S-A, Nilsson GE, Munday PL (2013) Elevated
CO2 enhances aerobic scope of a coral reef fish. Conservation Physiology, 1, cot023.
Schurmann H, Steffensen J (1997) Effects of temperature, hypoxia and activity on the
metabolism of juvenile Atlantic cod. Journal of Fish Biology, 50, 1166-1180.
Steffensen JF (1989) Some errors in respirometry of aquatic breathers: how to avoid and correct
for them. Fish Physiology and Biochemistry, 6, 49-59.
Supp Figure 1: Mean (±SE) temperature measured in the mesocosm pools during the experiment.
Each Mesocosm replicate was measured four times per day (every 4 hours, from 0600 to 1800).
Dark lines represent elevated temperature treatments, while light lines represent the ambient
temperature treatments. No statistical differences were found between ambient vs. elevated CO2
treatment pools sharing the same temperature treatments.