Sampling and analytical procedures:

Capture and husbandry: Antarctic krill (Euphausia superba) were captured during the austral summer of 2013/2014. Krill were collected by net tow (2 m diameter, 1000 m mesh, non-filtering cod end) off the R/V Laurence M. Gould near the Western Antarctic Peninsula and transported directly to the Palmer Station biological laboratory. One to two thousand krill were housed in one 4'w x 3'h circular holding tank and two 5’ x 2’ x 1’ rectangular tanks provided with aeration and flow-through seawater. Water was non-filtered and individuals were able to feed on plankton ad libitum throughout the season.

Experimental treatments: Three experimental treatments were targeted in this study: (1) ambient temperature and ambient pCO2/pH (400ppm/8.10), (2) ambient temperature and elevated pCO2 (800ppm)/reduced pH (7.7), and (3) elevated temperature (3 degrees C) and elevated pCO2 (800ppm)/reduced pH (7.7). Two replicate feeding experiments were conducted: Experiment 1 and 2. Temperature treatments were obtained using two separate recirculating systems. Two 800 L cylindrical polycarbonate carboys were attached to temperature controlled chillers (Delta Star) and inline pumps. The carboys were filled with non-filtered seawater acquired from the Palmer Station intake line, placed in a flow-through water bath, and maintained at 0 degrees C. Another 800 L carboy was set up without a chiller and placed in an environmental chamber set at bated for about 24 hours before sacrificed for sampling the end points. The samples collected in the bottles at T0 and Tfinal include: pH, total alkalinity, and fluorometric chlorophyll a.

The systems were replaced with new water daily and allowed to acclimate to temperature for a minimum of 24 hours before the start of a trial or water change. High CO2 conditions were obtained using a peristaltic pump to inject straight CO2 into the propeller of a pump submerged in seawater. Treated water was then gently siphoned with minimal disturbance into treatment bottles.

For each of the three treatments, 14, 4 L wide-mouth polycarbonate bottles were filled with the appropriate equilibrated seawater. Two bottles per treatment served as T0 controls (no krill added) and were sacrificed for an initial suite of samples. Two bottles served as Tfinal (24 h) controls, and one juvenile krill was added to each of the remaining 10 bottles per treatment (Tfinal treatments). The Tfinal bottles were capped to maintain target pCO2/pH, incubated in the appropriate location to maintain desired temperature (water bath for ambient, 0 degrees C; 3 degrees C environmental chamber for elevated temperature), and incubated for about 24 hours before sacrificed for sampling the end points. The samples collected in the bottles at T0 and Tfinal include: pH, total alkalinity, and fluorometric chlorophyll a.

Analyses: pH was determined spectrophotometrically using the indicator dye thymol blue (Dickson et al. 2007; Zhang and Byrne 1996). Total alkalinity was determined on 100 ml subsamples with an open-cell, potentiometric titration of seawater (Metrohm 888 Titrando) with 0.1 M HCl following the potential of a pH electrode (Dickson et al. 2007). Tiamo software (version 2.3) was used to process the alkalinity data. Measurements of pH and TA were quality controlled using certified reference materials (CRMs) obtained from Andrew Dickson at UCSD Scripps Institute of Oceanography. An aliquot of seawater from each incubation bottle was also filtered onto a GF/F filter, which was wrapped in foil and frozen for fluorometric chlorophyll a analysis (Parsons et al. 1984). Salinity was measured with a bench top conductivity meter (YSI 3100) calibrated daily with a conductivity standard (50,000 uS/cm; Ricca Chemical Company).

Expt 1	Expt 2
Salinity (ave +/- sterr; n = 30) = 32.43 +/- 0.02	Salinity (ave +/- sterr; n = 30) = 32.63 +/- 0.03
Ambient temperature = 0 C	Ambient temperature = 0 C
Elevated Temperature = 3 C	Elevated Temperature = 3 C

2014_krill_graze.csv