Sampling and analytical procedures:

Capture and husbandry: Antarctic krill (Euphausia superba) were captured during the austral summer of 2014/2015. 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: Six experimental treatments were targeted in this study: 1) Ambient temperature (0 degrees C) and ambient pH (8.00); 2) Ambient temperature (0 degrees C) and pH = 7.50; 3) Ambient temperature (0 degrees C) and pH = 7.10; 4) Elevated temperature (3 degrees C) and ambient pH (8.00); 5) Elevated temperature (3 degrees C) and pH = 7.50; 6) Elevated temperature (3 degrees C) and pH = 7.10. Temperature treatments were obtained using two separate recirculating systems. Three 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. Three other 800 L carboys were set up without a chiller and placed in an environmental chamber at 3 degrees C. 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.

Krill were picked and placed in individual 4 L wide-mouth polycarbonate bottles with airtight lids (n = 26 per experimental treatment). Bottles were filled with ambient seawater. Bottles were immediately closed and placed in environmental chambers at ambient temperature (0 degrees C). Every 24-48 hours, 80% of the water was siphoned out of each bottle and replaced with new ambient seawater to minimize excretory and respiratory effect. Bottles were checked for molts twice per day. If a molt was observed, the molt was removed for processing (measured for krill uropod and total length), and the krill was placed in another individual 4 L wide-mouth polycarbonate bottle containing one of the six pre-acclimated treatment waters as described above to begin the second phase of the experiment. The bottle was then immediately closed and placed in environmental chambers at either 0 or 3 degrees C. This process was repeated for all the krill after each molted for the first time, enabling the experiment to continue with a total of 26 krill per treatment. Water changes every 24-48 hours with either ambient seawater (for krill awaiting first molt) or with pre-acclimated treatment water (for krill awaiting second molt) and twice daily checks for molts continued for the duration of the experiment. If a second molt was observed, that bottle was pulled from the experiment and the molt and live krill processed. The experiment ended on March 7 due to the end of the Palmer Station field season, and a majority of the krill did not molt a second time. However, the remaining krill were processed for uropod and total length and wet weight.

Analyses: Upon the first molting event for each krill, the molt was gently removed from the bottle. The molt was measured for uropod and total length (mm) with digital calipers (Fowler).

Once a krill molted a second time, both the krill and molt were gently removed from the bottle. Both the molt and krill were measured for uropod and total length (mm) with digital calipers (Fowler), and the krill wet weight (mg) was measured. At the end of the experiment, the remaining krill that did not molt a second time were processed for uropod and total length (mm) and wet weight (mg).

pH was measured in each experimental bottle at the start of the experiment. Salinity, pH, and total alkalinity were measured during water changes. The water changes occurred every 24-48 hours, but we were unable to collect salinity, pH, and total alkalinity samples at every water change due to time constraints. Salinity was measured with a bench top conductivity meter (YSI 3100) calibrated daily with a conductivity standard (50,000 uS/cm; Ricca Chemical Company). 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