Capture and husbandry: Antarctic krill (Euphausia superba) were captured during austral summers 2013/2014 and 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 Palmer Research Station. One to two thousand krill were housed in one 4'wx3'h circular holding tank and two 5’x2’x1’ 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: Four experimental treatments were targeted in this study, (1) ambient temperature and ambient CO2, (2) ambient temperature and high CO2 (800ppm), (3) high temperature (3C) and ambient CO2 (4) high temperature and high CO2.Temperature treatments were obtained using two separate recirculating systems. One 800 L cylindrical polycarbonate carboy was attached to a temperature controlled chiller (Delta Star) and inline pump. The carboy was placed in a flow-through water bath and maintained at 0C. A similar system was set up without a chiller and placed in an environmental chamber set at 3C. The systems were replaced with new water and allowed to acclimate to temperature 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 pumped with minimal disturbance into treatment buckets.

Krill of comparable size (average 1 g) were picked and placed in 19 L plastic buckets with airtight lids up to n = 20. Buckets were filled with one of the four treatment waters as described above. Buckets were immediately closed and placed in environmental chambers set to 0C or 3C. Every 24 hours 80% of the water was siphoned out and replaced to minimize excretory and respiratory effect of the animals on treatment conditions. Time points were set at time = 0, 1, 6, 12, 24, 48 hours and 7, 14, 19 days (168, 336, 456 hours). Each bucket was run in duplicate for each treatment at each time point. During season one two 48 hour experiments and three 24 hour long experiments were run. During season two one 24-hour, one 48-hour and one 19-day experiment were completed. Separate buckets were run in duplicate at 0C and 3C for TMAO analysis. For endpoint respirometry, krill were placed one at a time in 300mL airtight glass jars filled with 2um filtered treatment water. Each jar was paired with a blank containing no individuals, placed in water baths, covered, and put in 0C or 3C environmental chambers for the duration of the experiment.

Analyses: At the end of an experimental duration, blood was immediately taken from 5-10 krill using a 20 gauge needle and pooled. Blood pH was determined using a temperature controlled pH microelectrode (Microelectrodes, Inc. Bedford, NH, USA). Blood lactate was also measured using a handheld lactate meter (Roche Accutrend). The remaining 10 krill were decapitated and their bodies flash frozen for later analysis. Individual tissue (up to n=5) was weighed and homogenized in 500uL homogenate buffer containing 150 mmol L-¹ Potassium fluoride and 5 mmol L-¹ nitrilotriacetic acid (Portner et al., 1990). 75uL homogenate was injected into the Corning 965 total CO2 analyzer, Midland, MI, USA for TCO2 determination. Remaining homogenate was then used to measure pH using the microelectrode as described above. Alternate individuals (up to n=5) were used to measure tissue lactate. Tissue was homogenized 1:1 in DI water using 3 mL glass homogenizers on ice. The supernatant was then measured for lactate using the Accutrend lactate meter. Individuals used for TMAO determination were homogenized 1:5 in 5% trichloroacetic acid then supernatant used with the ferrous sulfate-EDTA method of TMAO determination described by Wekell and Barnett (1991). Endpoint respirometry measurements were taken using a Strathkelvin oxygen electrode and blanks used to correct for residual bacterial respiration.

long_term_trial.csv