These data were generated in a comparative study of protein metabolism (protein synthesis, protein growth and protein degradation) in the Antarctic plunderfish, Harpagifer antarcticus and the Northern European blenny, Lipophrys pholis. The study carried out an examination of protein metabolism in these species at a range over overlapping temperatures covering the environmental range of the species. Protein synthesis was measured using the flooding dose methodology in animals held at the British Antarctic Survey in Cambridge, UK. The experimental work was carried out by Andrew Bowgen and Keiron Fraser. The aim of the study was to examine the effect of ambient habitat temperature on protein metabolism in two ecologically similar, but phylogenetically distant fish species, including one that only inhabited polar latitudes.
Andrew Bowgen was funded by a NERC PhD studentship and the study was completed as part of the British Antarctic Survey, Biodiversity, Function, Limits and Adaptations from Molecules to Ecosystems (BIOFLAME) project, part of the NERC funded Biological Responses to Extreme Antarctic Conditions and Hyperextremes (BIOREACH) programme.
Protein metabolism, RNA, fish growth, polar fishes, protein synthesis
Fraser, K. (2021). Harpagifer antarcticus and Lipophrys pholis protein metabolism (Version 1.0) [Data set]. NERC EDS UK Polar Data Centre. https://doi.org/10.5285/53737fbe-8cb8-4e3d-bc48-2b21e7c9d543
|Access Constraints:||The dataset is under embargo until publication of a related manuscript.|
|Use Constraints:||Data is released under Open Government Licence V3.0: http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/.|
|ISO Topic Categories:||
|Organisation||British Antarctic Survey|
|Name||Dr Keiron P P Fraser|
|Organisation||University of Plymouth|
|Reference:||Fraser KPP, Peck LS, Clark MS,Clarke A, Hill SL. (2022) Life in the freezer: protein metabolism in Antarctic fish. R. Soc. Open Sci. 9: 211272. https://doi.org/10.1098/rsos.211272.
Fraser, K.P.P., Clarke A., Peck L.S. (2002). Low-temperature protein metabolism: seasonal changes in protein synthesis and RNA dynamics in the Antarctic limpet Nacella concinna Strebel 1908. J. Exp. Biol. 205, 3077-3086. https://doi.org/10.1242/jeb.205.19.3077.
Fraser, K.P.P. and Rogers, A.D. (2007). Protein metabolism in marine animals: the underlying mechanism of growth. Adv. Mar. Bio. 52, 267-362. https://doi.org/10.1016/S0065-2881(06)52003-6.
|Quality:||Data did not require cleaning and no data was lost.|
|Lineage:||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.
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.
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.
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 washed twice with 0.2M PCA and hydrolysed in 6M HCl for 18h at 110°C. The acid was removed from the hydrolysed protein residue using repeated washes of distilled water and rotary evaporation to dryness between washes, before the residue was re-suspended in 0.5M sodium citrate buffer (pH 6.3). The phenylalanine concentration of the hydrolysed protein residue, injection solution and the intracellular free-pools was measured flourometrically, after enzymatic conversion of the phenylalanine to beta-phenylethylamine (PEA). The specific radioactivities of the free-pools, protein and injection solutions were measured using scintillation counting (Wallac 1409 LSC, Packard Bioscience Hionic-Fluor scintillant, 34% 3H counting efficiency) and expressed as disintegrations per minute (d.p.m.) nmol-1 phenylalanine.
The following equation was used to calculate the fractional (ks) rate of protein synthesis:
ks = (Sb/Sa) x (100/t) x 1440
where ks = % protein mass synthesized day-1 (%.day-1), Sb = specific radioactivity of protein-incorporated radiolabel (d.p.m. nmol-1 phe), Sa = specific radioactivity of the intracellular free-pool (d.p.m. nmol-1 phe), t = incorporation time from injection of radiolabel to death in minutes and 1440, the number of minutes in a day.
Protein growth rates (kg, %.day-1) were calculated using equation (1), but with W1 and W2 representing the protein mass at the beginning and end of the growth period respectively. Initial protein masses of the experimental fish were estimated from a sub-group of animals of similar mass. The protein synthesis retention efficiency (PSRE) is the fraction of synthesised protein that is retained as protein growth, and is a key factor in determining the growth efficiency of an organism. The PSRE was calculated as:
PSRE = (kg/ks) x 100
Further details are in
Fraser, K.P.P., Clarke A., Peck L.S. (2002a). Low-temperature protein metabolism: seasonal changes in protein synthesis and RNA dynamics in the Antarctic limpet Nacella concinna Strebel 1908. J. Exp. Biol. 205, 3077-3086.
All data are expressed as means ± standard error (SEM). Statistical analysis was carried out using Minitab version 19 (Minitab, Coventry, UK). Prior to statistical analysis, data were checked for normality and homogeneity of variances using the Anderson-Darling and Levene's tests. Parametric data were analysed using ANOVA and Tukey HSD tests, while non-parametric data were analysed using the Kruskal-Wallis test. Student's t-test was used to assess differences in metrics between species. Significance was accepted at p<0.05.
|Detailed Location||Antarctic Peninsula, Adelaide Island, Rothera Point|
|Data Collection:||Thermocirculator - Grant Instruments Cambridge
Centrifuge - Eppendorf 5810R, swing bucket rotor
Rotary evaporator - Buchi
Scintillation counter - Wallac 1409 LSC
|Data Storage:||The dataset consists of three CSV files.|