Methodology:

Collection and husbandry of fish

Harpagifer antarcticus were collected by hand from sublittoral sites at Rothera Point, Adelaide Island, Antarctic Peninsula (67.56861 S, 68.125 W) by SCUBA divers and held in a through-flow aquarium until return to the UK in a refrigerated transport system. In the UK, fish were maintained in a recirculating flow aquarium, under an automatic 12h light : dark regime (water temperature 0°C ± 1.0°C, salinity 34 - 36 PSU). The fish were fed twice weekly to satiation on shelled krill (Euphausia superba). Lipophrys pholis were collected subtidally in Weymouth (UK) by a commercial supplier and fed on chopped white fish (New Zealand Hoki, Macruronus novaezelandiae) as they refused krill. The mean water temperature of the L. pholis stock tank was 14°C ± 1.0°C. With the exception of temperature and diet, husbandry conditions for both species were identical.

Temperature acclimation

Experimental animals were weighed in water (± 0.1g) and digitally photographed to allow individual identification. The fish were placed in pairs, in jacketed aquaria (18.8 x 20.3 x 22.0cm) containing clean, aerated seawater at their stock tank temperature. Water temperatures were maintained (± 0.1°C) using a thermocirculator (Grant Instruments, Cambridge, UK). Once daily the aquaria were divided in half using a perspex insert, thereby allowing fish to be fed individually to satiation and their food consumption measured (± 0.1g).

Adjustment of aquaria to the required experimental water temperatures (H. antarcticus, -1, 1 and 3 °C, L. pholis, 3, 8, 13 and 18 °C) was carried out at a rate of 0.1°C hour-1 (H. antarcticus) or 0.5°C hour-1 (L. pholis), up to a maximum of 0.8°C day-1 (H. antarcticus) or 4°C day-1 (L. pholis). Fish were acclimated to the experimental temperature for 28 days prior to the measurement of protein synthesis, as Antarctic fish can take 3-4 weeks to acclimate to 4 °C and are the slower of the two species to acclimate. The fish were weighed on the first and penultimate day of the acclimation. Specific growth rates (SGR) were calculated for each fish.

equation (1)

SGR = (ln(W2)-ln(W1)/t) x 100

where SGR is expressed as % body mass. day-1, W1 and W2 represent the mass at the start and end of the acclimatization period respectively and t is the time in days between mass measurements.

Protein synthesis measurement

Whole animal protein synthesis rates were measured using a modification of the flooding dose method. Groups of fish were injected in the peritoneum with a flooding dose of unlabelled and 3H labelled phenylalanine (10microl.g-1 fish wet mass of 135mmol.l-1 L-[2,6-3H] phenylalanine at 3.6MBq.ml-1 (GE Healthcare, Little Chalfont, UK)). Fish were killed and frozen in liquid nitrogen after 1, 2 and 4h to allow validation of the flooding dose time-course (H. antarcticus, 3°C; L. pholis, 3 and 18°C). In other experimental treatments fish were killed after 2h, H. antarcticus, -1 and 1°C; L. pholis, 8 and 13°C. Baseline, pre-injection phenylalanine concentrations were measured in both species (n=10) to allow the calculation of phenylalanine flooding levels. All samples were frozen in liquid nitrogen and stored at -80°C prior to analysis.

Sample analysis

Samples were analysed following Fraser et al., (2002). The frozen fish were homogenized in a known volume of ice-cold, 0.2M perchloric acid (PCA). The homogenate was mixed thoroughly and a 2ml aliquot removed for analysis. The aliquot was centrifuged (Eppendorf 5810R, swing bucket rotor, 3980 x g, 10 min, 4°C) to separate the protein precipitate from the intracellular free-pool. The supernatant, was decanted and the NaOH soluble protein in the pellet measured using bovine serum albumin (Sigma-Aldrich, Poole, UK) as the standard. Subsequently, the pellet was ...(34)

Data collection:

Thermocirculator - Grant Instruments Cambridge

Centrifuge - Eppendorf 5810R, swing bucket rotor

Rotary evaporator - Buchi

Scintillation counter - Wallac 1409 LSC

Data quality:

Data did not require cleaning and no data was lost.