Sample collection: Mid-water trawls were conducted in the western Ross Sea, Antarctica on board the R/V Nathaniel B. Palmer in January-February 2018 (cruise number 18-01). The Issacs-Kidd Midwater Trawl (IKMT, 1.8 m frame, 500 um mesh, non-filtering cod end) was fitted with a calibrated General Oceanics flow meter. Four trawls were completed, each used to conduct a grazing experiment using the gut fluorescence/gut evacuation technique. Sampling stations for experiments 1-3 were located on the Continental Shelf Adjacent to the Ross Ice Shelf barrier near 76ºS, and experiment 4 was located on the continental shelf near 74ºS.

Experiment 1 date and location: 1/16/2018; -76 00.044S, 176 59.909E
Experiment 2 date and location: 1/18/2018; -76 45.024S, 172 01.733E
Experiment 3 date and location: 2/6/2018; -76 27.063S, 168 07.142E
Experiment 4 date and location: 2/16/2018; -74 49.955S, 164 34.177E

Experimental Procedure: Immediately after each trawl (for each experiment), a subset of crystal krill, Euphausia crystallorophias, were processed for initial gut fluorescence and the other krill were sorted into experimental buckets for a time series sample collection. The krill collected for initial gut fluorescence were immediately measured for length, wrapped in foil, flash frozen in liquid nitrogen, and placed in a -80ºC freezer for later chlorophyll gut content analysis (see below). To determine gut evacuation rates, an additional 5 krill were processed immediately after collection for the initial time point, T0. The remaining krill were quickly sorted into five buckets to achieve a total of 12 krill per bucket. Each bucket was filled with 15L of 0.2µm filtered seawater, and 4.0 mg of activated granular charcoal powder was added at a final concentration of 265 µg L-1. The filtered seawater was maintained at in situ temperature in a cold incubator room for the duration of the 24-hr experiment. One krill per bucket was removed at time points of 5, 10, 15, 20, 25, 30, 40, 50, 60, and 120 minutes, yielding a sample size of five krill per time point. Finally, 1-2 krill were removed after 24 hours to obtain background gut fluorescence which is to be subtracted from all the other krill fluorescence samples. All krill removed at each time point were measured for standard length, wrapped in foil, flash frozen in liquid nitrogen, and placed in the -80ºC freezer.

 Analyses: Fluorometric analysis was then conducted on the krill samples using a Turner 10-AU Fluorometer before and after acidification. The analysis was performed in the dark to ensure that chlorophyll did not degrade during sample preparation and processing. Each krill was placed without homogenization into a 20mL glass scintillation vial where 5ml of 90% acetone was added to extract chlorophyll pigments. The samples were covered in foil and placed into the -20 ºC freezer for a 48-hr extraction. After this extraction period, the samples along with 3 prepared blanks of 5ml acetone were transferred to individual sample cuvettes. Each sample was placed in the fluorometer to obtain fluorescence values before acidification. The sample was then removed from the fluorometer, 2 drops of 1.2M hydrochloric acid was added, and the sample was placed back into the fluorometer to obtain values after acidification.