Dataset: Modern Seal Amino Acid Isotopes

Sampling Sites and Sample Collection:
Tissue samples from Ross, Weddell, and crabeater seals were collected along western Antarctica from the West Antarctic Peninsula (WAP) to the Ross Sea during multiple field seasons and, in most cases, body mass, age class (juvenile, subadult, and adult), gender, and location were recorded for each sampled seal. Seals were sampled during the austral summers of 2008/09 and 2010/11 on RV Oden cruises along the western Antarctic coast. Mostly whole blood samples were obtained. In some cases, clot (blood with serum removed), red blood cells (RBCs, whole blood exposed to an anticoagulant, heparin, before having plasma removed), and hair samples (body fur or whiskers) were also taken. The sampling protocol is described in Aubail et al. (2011); all animal captures were conducted in accordance with the regulations of the Swedish Polar Research Secretariat (Registration No. 2010-112).

All other samples were obtained from animal captures conducted under National Marine Fisheries Service permit No. 87-1851-00. Additionally, the Institutional Animal Care and Use Committee (IACUC) at the University of Santa Cruz (UC Santa Cruz) approved all protocols for the following samples. Whiskers were taken from crabeater seals during multiple cruises on the RV Lawrence M. Gould along the WAP.  Plasma was also obtained from a few of the fall 2007 individuals (G105, G110, and G112). In addition, serum or plasma was obtained from two Weddell seals during the fall 2007 sampling in this region, and whiskers were taken from two WAP Weddell seals in the austral summer of 2009/10. Hückstädt et al. (2012b) describe the procedure for sampling the whiskers, and Goetz et al. (2017) describe the protocol used for collecting the seal serum and plasma.

Several blood samples were obtained from Weddell seals in the McMurdo Sound region, Ross Sea, Antarctica over multiple field seasons. Twelve whole blood samples were taken from juvenile Weddell seals near Inexpressible Island (74.9 °S, 163.7 °E) during the austral summer of 2010/11. Whole blood samples were taken from Weddell seals in the austral summer of 2010/11 and austral spring of 2012. RBCs were sampled in the austral summer of 2009/10, austral summer of 2011/12, and austral spring of 2012. Whole blood, plasma, and serum were obtained from five Weddell seals sampled in the austral spring of 2015, and whole blood from an additional seven Weddell seals was also acquired during this time. Goetz et al. (2017) describe the sampling protocol for these Weddell seals.

Lastly, a few samples were obtained from crabeater seals in McMurdo Sound. Hair samples were taken from three recently deceased juvenile crabeater seals that were found on the seasonal pack ice around Cape Royds in the austral summer of 2009/10. Whole blood was sampled, using the protocol of Goetz et al. (2017), from a male adult crabeater seal found in Erebus Bay during the austral summer of 2010/11.

Taxonomic Groups:
Pinnipedia
     Phocidae
            Lobodon carcinophaga - crabeater seal
            Ommatophoca rossii - Ross seal
            Leptonychotes weddellii - Weddell seal

Sample Preparation:
After sample collection, all samples were kept frozen at -20 °C. Blood samples were freeze-dried with a Labconco Freeze Dry System (Lyph Lock 4.5) and homogenized manually prior to analysis. Lipid extraction was not performed on the blood samples. Blood has a relatively low lipid content and a test set of blood samples with and without lipid extraction revealed no significant effect of lipid extraction on blood values. Hair samples, which have higher lipid contents, were lipid extracted. Hair samples were washed with Milli-Q water (Thermo Fisher Scientific, Inc.) and then rinsed 3 times in an ultrasonic bath with petroleum ether for 15 minutes. Hückstädt et al. (2012a) used a similar protocol to lipid-extract the crabeater seal whisker samples.

Compound Specific Isotope Analysis (CSIA):
CSIA was performed at UC Santa Cruz via gas chromatography-isotope ratio mass spectrometry (GC-IRMS). All samples were prepared for GC-IRMS analysis using the method described in McCarthy et al. (2007) and McCarthy et al. (2013). In brief, samples were hydrolyzed (6 N HCl for 20 hr at 110 °C) and converted to trifluoroacetic anhydride (TFAA) derivatives. Samples were stored in a -20 °C freezer in a 1:3 TFAA:DCM (methylene chloride) mixture until the day of instrumental analysis. Immediately before the analysis, the TFAA:DCM mixture was evaporated under N2 and samples were diluted in ethyl acetate.

Amino acid δ15N values were measured on a Thermo Trace GC coupled to a Thermo-Finnigan DeltaPlus XP isotope-ratio-monitoring mass spectrometer (oxidation furnace at 980 ºC and reduction furnace at 650 ºC). The column for the N isotope analysis was a SGE Analytical Science BPX5 column 60 m by 0.32 mm with a 1 µm film thickness. The injector temperature was 250 ºC with a split He flow of 2 mL/min. The GC temperature program for δ15N analysis was: initial temp = 70 ºC hold for 1 min; ramp 1 = 10 ºC /min to 185 ºC, hold for 2 min; ramp 2 = 2 ºC/min to 200 ºC, hold for 10 min; ramp 3 = 30 ºC/min to 300 ºC, hold for 6 min. Directly measured amino acid δ15N values were corrected based on bracketing external standards, as described in McCarthy et al. (2013).

Samples were injected and analyzed 3 to 4 times, and the mean value, standard deviation, and number of injections are reported. The δ15N values of 11 amino acids could be quantified. These were alanine (Ala), glycine (Gly), threonine (Thr), serine (Ser), valine (Val), leucine (Leu), Pro, aspartic acid + asparagine (Asp), Glu, Phe, and lysine (Lys). In all samples, isoleucine (Ile) was either not detectable or had low peak areas (< 70) and, thus, Ile δ15N values should be considered with caution. Lysine was not detected in one sample (R101) and not in all injections of a few samples (R114, W006, and W013), but peak areas were sufficient (>70) when it was detectable.

Cited references:
Aubail A, Teilmann J, Dietz R, Rigét F, Harkonen T, Karlsson O, Rosing-Asvid A, Caurant F (2011) Investigation of mercury concentrations in fur of phocid seals using stable isotopes as tracers of trophic levels and geographic regions. Polar Biology 34:1411-1420. doi:10.1007/s00300-011-0996-z

Goetz KT, Burns JM, Hückstädt LA, Shero MR, Costa DP (2017) Temporal variation in isotopic composition and diet of Weddell seals in the western Ross Sea. Deep Sea Res II 140:36-44. doi:10.1016/j.dsr2.2016.05.017

Hückstädt LA, Burns JM, Koch PL, McDonald BI, Crocker DE, Costa DP (2012a) Diet of a specialist in a changing environment: the crabeater seal along the western Antarctic Peninsula. Marine Ecology Progress Series 455:287-301. doi:10.3354/meps09601

Hückstädt LA, Koch PL, McDonald BI, Goebel ME, Crocker DE, Costa DP (2012b) Stable isotope analyses reveal individual variability in the trophic ecology of a top marine predator, the southern elephant seal. Oecologia 169:395-406. doi:10.1007/s00442-011-2202-y

McCarthy MD, Benner R, Lee C, Fogel ML (2007) Amino acid nitrogen isotopic fractionation patterns as indicators of heterotrophy in plankton, particulate, and dissolved organic matter. Geochimica et Cosmochimica Acta 71:4727-4744. doi:10.1016/j.gca.2007.06.061

McCarthy MD, Lehman J, Kudela R (2013) Compound-specific amino acid δ15N patterns in marine algae: Tracer potential for cyanobacterial vs. eukaryotic organic nitrogen sources in the ocean. Geochimica et Cosmochimica Acta 103:104-120. doi:10.1016/j.gca.2012.10.037