Acetanilide

Moisture content was analyzed using a forced-air oven (UF 110, Memmert, Schwabach, Germany) at 105 °C for 24 h according to [22 (link)].
Crude protein content was determined by the Kjeldahl method [23 ] using a steam distillation apparatus (Vapodest 20, Gerhardt, Königswinter, Germany). The method was verified using acetanilide as the reference standard; the method blank was also included.
Protein content = nitrogen × 6.25 (where 6.25 = protein conversion factor).
Crude fat content was obtained by the Soxhlet method of Nielsen [24 ] using petroleum ether as the solvent. The solvent was then removed using a rotary evaporator (Büchi, R-200) at 50 °C.
Ash content of the insect samples was determined gravimetrically [25 ] using a muffle furnace (B 180, Nabertherm, Lilienthal, Germany) overnight at 550 °C. After the determination of the ash content, the ashes were collected in a low-density polyethylene container and stored for mineral analysis.
Chitin content was measured gravimetrically after deproteinization using 1 M NaOH and subsequent demineralization with 1 M HCl using the procedures outlined by Liu et al. [26 (link)].
Nitrogen-free extract (NFE) was calculated as:
100 − (crude proteins + crude lipids + ash + fibers) [27 ].
Energy content was estimated using the formula:
[(crude proteins × 17) + (crude lipids × 37) + (NFE × 17)] [28 (link)].

The study was conducted at La Mare aux Elephants (46°22′S, 51°40′E) located on Possession Island, Crozet Archipelago. Antarctic fur seals (Arctocephalus gazella) and subantarctic fur seals (A. tropicalis) breed sympatrically at this site, with a pup production of 164 Antarctic fur seals and 80 subantarctic fur seals during the study period [27] . Randomly selected breeding males and lactating females of unknown age were captured during December 2001 and January 2002, respectively. Male seals were sedated by intramuscular injection of a tiletamine-zolazepam mixture (Zoletil), while females were captured using a hoop net and placed on a restraint board. From each individual, a single whisker was collected by cutting with a pair of scissors as close to the skin as possible.
Prior to isotopic analysis, whiskers were hand-washed in 100% ethanol and then cleaned in distilled water for 5 min in an ultrasonic bath. Whiskers were measured, dried and cut into 3 mm-long consecutive sections starting from the proximal (facial) end. Sections were weighed on a microbalance within a range (0.03–2.06 mg) that produced meaningful isotopic measurements. Samples were then packed in tin containers, and carbon and nitrogen isotope ratios were determined by a continuous flow mass spectrometer (Thermo Scientific, Delta V Advantage) coupled to an elemental analyser (Thermo Scientific, Flash EA 1112). Results are presented in the conventional δ notation relative to PeeDee belemnite marine fossil limestone and atmospheric N₂ for δ¹³C and δ¹⁵N, respectively. Replicate measurements of internal laboratory standards (acetanilide) indicate measurement errors of <0.15‰ for both δ¹³C and δ¹⁵N. Isotopic data on male A. gazella whiskers, previously reported in [26] (link)), have been incorporated in the present study to investigate intra-and inter-specific variations in fur seal foraging strategies.
Five additional lactating female A. gazella were sampled at Bird Island (South Georgia) in 1992 as part of other studies [28] . They were aged by counting growth layers of tooth dentin. Seal whiskers were cut into 5 mm long consecutive sections from the root. Isotopic analyses were performed in the laboratory of Donald Schell at Fairbanks (Alaska) in 1994.
Keratinous tissues (including whiskers) are approximately 3‰ ¹³C enriched in pinnipeds compared to their diet [29] . Taking into account both the keratinous effect and the latitudinal gradient in blood δ¹³C values of top predators in the Southern Ocean [14] , [30] , the isotopic positions of the polar front (PF) and the subtropical front (STF) for fur seal whiskers were estimated at approximately −19 and −16‰, respectively. The subtropical zone (STZ) is defined as the area north of the STF, the subantarctic zone (SAZ, where the Crozet Islands are located) as the area between the STF and the PF, and the Antarctic zone (AZ) as the area south of PF.

At the Smithsonian Marine Station in Fort Pierce, Florida, USA, sponge samples were placed in a 60 °C oven to remove any residual moisture and ground to a fine powder using a mortar and pestle. Homogenized sponge tissue was acidified to remove carbonate and weighed into tared silver capsules for δ¹³C and δ¹⁵N as in Freeman & Thacker (2011) (link). Sponge and POM samples were analyzed in the Stable Isotope Ratio Mass Spectrometry laboratory (SIRMS) at the University of Hong Kong via combustion in a Eurovector EA3028 coupled to a Perspective IRMS (Nu Instruments). Analytical precision was determined by repeated analysis of an internal acetanilide standard (‘acet 6’; 70% C). Mean (±SE) precision during analysis was 0.2 ± 0.04 and 0.1 ± 0.01 for δ¹⁵N and δ¹³C, respectively. To assess photosymbiont abundance (chl a), analyses were carried out on dried tissue as in Freeman & Thacker (2011) (link) and Freeman et al. (2013) (link).

For pieces of Sargassum, approximately 2 g of sample dried powder was weighed in tin capsules for isotopic analysis. For POM, samples were duplicated: Only the subsamples that were not treated were analysed for δ¹⁵N. Stable isotope analyses were performed by continuous flow on a Flash EA 2000 elemental analyser (Thermo Scientific, Milan, Italy) coupled to a Delta V Plus, isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) at the Pôle Spectrométrie Océan (IUEM-UBO, Plouzané, France). The reference materials (USGS-61, USGS-62, and USGS-63) and an in house standard Thermo-Acetanilide were used for isotopic corrections and to assign the data to the appropriate isotopic scale: USGS-61 (certified values: δ¹⁵N = −2.87‰ ± 0.04‰ and measured values: δ¹⁵N = −2.89‰ ± 0.06‰), USGS-62 (certified values: δ¹⁵N = +20.17‰ ± 0.06‰ and measured values: δ¹⁵N = +20.17‰ ± 0.08‰), and USGS-63 (certified values: δ¹⁵N = +37.83‰ ± 0.06‰ and measured values: δ¹⁵N = +37.84‰ ± 0.10‰). Results were reported in the δ unit notation and expressed as parts per thousand relative to the international standards atmospheric N₂ for nitrogen. Analytical precision based on replicate measurements (after every five samples) of Thermo-Acetanilide was <0.1 for δ¹⁵N values.