Conflo 4

Stable isotope ratios were measured to provide evidence of inorganic carbon uptake strategies and C and N content were measured to provide indications on the nutrient status of the kelp. Samples for determination of δ¹³C isotopic ratios and carbon and nitrogen content were destructively sampled and frozen at –20°C until all treatments had been measured. Following this, all samples were freeze dried (FreezeZone 4.5, Labconco) and kept at –20°C until later analysis. δ¹³C isotopic ratios and carbon and nitrogen content were determined by weighing approximately 5 mg of dried tissue into tin cups (Sercon, UK) and analysed using an elemental analyser (NA1500, Fisons Instruments) coupled to an isotope ratio mass spectrometer (Delta V Plus, ThermoFisher Scientific) via a Universal Continuous Flow Interface (Conflo IV, ThermoFisher Scientific). Combustion and reduction were achieved at 1020°C and 650°C, respectively. Isotope values were normalized to the Vienna Pee Dee Belemnite scale via a three-point calibration using certified reference material and both precision and accuracy were ±0.1% (1 s.d.).

The δ¹³C values of DNA samples before centrifugation and the fractionated DNA of at least one sample of each triplicate were measured through isotope ratio mass spectrometry (IRMS; Delta V advantage) in conjunction with a Flash 2000 HT elemental analyzer (EA) connected to a ConFlo IV (all Thermo Fisher Scientific, USA). Two microliters of the total DNA or 5 μL of the fractionated DNA was placed in a 48-μL tin capsule and dried for 3 h at 55 °C. Then, the tin capsules were placed in an autosampler linked with an EA-IRMS (Thermo Fisher Scientific, USA). Urea was the control sample, and it was determined in triplicate before the DNA samples and after every ten samples, and the δ¹³C value of urea was − 40 ± 0.32‰. The corrected CO₂ was adopted as a reference, and the δ¹³C content (‰) was determined using the following equation: $${\mathrm{\delta }}^{13}\mathrm{C}(‱)=\left[\left({\text{R}}_{\text{sample}},-,{\text{R}}_{\mathrm{standard}}\right),/,{\text{R}}_{\mathrm{standard}}\right]\times 1000$$ where R = ¹³C/¹²C. R_standard is the Vienna Pee Dee Belemnite standard (V-PDB = 0.0111802).

We sent feather samples for δ²H analysis at the Cornell Stable Isotope Laboratory in Ithaca, NY. After washing in a 2:1 chloroform:methanol solution, feather samples were air dried under a fume hood and a subsample (0.35 mg) of vane material was loaded into silver capsules, crushed, and placed with internal lab standards into a desiccator for a minimum of three days prior to analysis. Samples were then loaded into a Zero Blank carousel under helium flow. Pyrolysis combustion on glassy carbon was at 1350°C in a Thermo Scientific Temperature Conversion Elemental Analyzer (TC/EA; Bremen, Germany) coupled via a Conflo IV (Thermo Scientific) to a Thermo Scientific Delta V Advantage isotope ratio mass spectrometer. Analysis of δ²H was conducted using the comparative equilibration method of Wassenaar and Hobson [52 (link)] with 2 calibrated keratin reference materials (CBS, δ²H = -197‰; KHS, δ²H = -54.1‰; SPK, δ²H = -121.6‰) corrected for linear instrumental drift. Based on within-run analyses of a third keratin standard, measurement error was approximately ± 3‰ for hydrogen isotopes in feather (δ²H_f). All δ²H values are reported relative to the Vienna Standard Mean Ocean Water–Standard Light Antarctic Precipitation scale.

Automated sampling of amino acid derivatives was conducted using a Trace 1310 GC (Thermo Scientific) in combination with a TriPlus RSH autosampler (Thermo Scientific). A sample volume of 0.1 μl was injected into an injection port held at 240°C in splitless mode onto a capillary column (DB‐35, 30 m × 0.320 mm ID × 0.50 μm film thickness, Agilent J&W GC Columns). The GC oven temperature program for a single injection is detailed in Appendix S2.
All eluting compounds off the column were oxidized inside a GC Isolink II (Thermo Scientific) combustion interface containing a reactor held at 1,000°C. Water was removed through a Nafion membrane downstream of the reactor while CO₂ was cryogenically trapped in tubing submerged in liquid nitrogen before transfer to the IRMS (DELTA V; Thermo Scientific) through a Conflo IV (Thermo Scientific) universal interface.
High purity N₂ (>99.9997% N₂, Airgas) was used as reference gas to initially calculate the isotopic composition. Raw data were drift‐corrected (for drift correction procedure see Appendix S3). Average precision across all amino acids was 0.33‰. Individual precision of each amino acid is given in Appendix S4.

Bulk carbon and nitrogen stable isotope ratios were obtained using an automated Elementar vario PYRO cube analyser interfaced with a continuous flow IsoPrime100 isotope ratio mass spectrometer for pteropods, and a Thermo Scientific Flash 2000HT analyser coupled with a Thermo Fisher Delta V Plus mass spectrometer through a Thermo Fisher Conflo IV for POM. For pteropods, SIA was performed at the Central Science Laboratory (CSL), University of Tasmania (Sandy Bay, Tasmania), and for POM, at the Australian Nuclear Science and Technology Organisation (ANSTO, Lucas Heights, Sydney). Isotopic ratios were expressed in delta (δ) notation and reported as parts per thousand (‰) deviations from conventional certified isotopic reference standards, Vienna Pee Dee Belemnite (for carbon) and atmospheric air (for nitrogen) (DeNiro & Epstein, 1978). At CSL, laboratory working standards of sulfanilamide were repeated every 6th sample for both isotopes to measure instrument stability and precision. Average standard deviations on triplicate measurements were 0.15‰ and 0.19‰ for δ¹³C and δ¹⁵N values of pteropods, respectively. At ANSTO, POM isotopic data are reported relative to IAEA secondary certified standards with a standard error of analysis to 1 standard deviation (SD) measured at ±0.3 mil. The carbon and nitrogen percentages, by weight, were converted to atomic C:N ratios.

Plasma samples were thawed at 4 °C overnight and their protein fraction was isolated by precipitation with sulfosalicylic acid (200 µL of 1 g/mL into 2 mL of sample). After 1 h of incubation at 4 °C and centrifugation (4500× g for 20 min at 4 °C), the supernatant and pellet were separated [19 (link)]. The pellet was rinsed three times with MilliQ water and then freeze-dried. The N stable isotopic composition (δ¹⁵N, i.e., natural relative abundance of the rare stable isotope of N) of plasma protein was determined using a Carlo Erba NA1500 elemental analyzer coupled to a Delta V plus isotope-ratio mass spectrometer via a Conflo IV (Thermo Scientific, Waltham, MA, USA). Stable isotope results were corrected via a three-point calibration using international primary reference standards. A secondary reference was included after every 12 samples to monitor any instrument drift. Nitrogen was quantified by comparison of peak areas against a response calibration curve.
Results are expressed using the delta notation according to the following equation:
where R_sample and R_standard are the N isotope ratio between the heavier isotope and the lighter isotope (¹⁵N:¹⁴N) for the sample being analyzed and the internationally defined standard (atmospheric N₂, R_standard = 0.0036765), respectively, and δ is the delta notation in parts per 1000 (‰) relative to the standard.