Mat delta plus

Aliquots (1 ml) were filtered onto precombusted GF filters (0.45 µm pore size), which were frozen at −20 °C until EA-IRMS analysis. Carbon isotopic ratios (R = ¹³C/¹²C) were determined on filters folded into tin capsules, using a CHN analyzer (ThermoFinnigan 1112 Series) interfaced with a mass spectrometer (ThermoFinnigan MAT Delta Plus) via a Conflow III open split interface. Abundances were calculated in relation to Vienna Pee Dee Belemnite-limestone (V-PDB), using in-house casein standards calibrated against IAEA-600 and IAEA-CH-6 international standards.
Bulk carbon assimilation was calculated as atom percent excess (APE) as follows: $APE=100\times \left(\frac{R_f}{R_f+1}-\frac{R_i}{R_i+1}\right)$ where R_f and R_i are the ¹³C isotope ratio at the final and initial sampling time, respectively.
Bulk carbon incorporation rates (F_bulk) were calculated as follows: $F_{bulk}=\frac{K_A\times {\rho }_C\times A}{t}$ where K_A is the fraction of assimilated carbon (calculated as described in Supplementary Methods), ρ_C is the amount of carbon per cell, A is the final cell abundance estimated through flow cytometry and t is the incubation time. We considered a range of ρ_C = 10–280 fg C cell⁻¹ [50 (link)].

Stable isotope sampling procedures and quantification followed Matley et al. (2017). Briefly, three tissues (plasma, red blood cells, and muscle) were collected from Plectropomus individuals and frozen (−20°C) until processing. Muscle tissue (no skin) was sampled from the dorsal musculature using sterile forceps and scalpel, and blood components were sampled from the 2nd or 3rd gill arch with a sterile needle/syringe. Frozen samples were freeze‐dried for 48 hr and ground into a powder, then samples were lipid‐extracted using a 2:1 chloroform:methanol solvent. Stable isotope values (δ¹³C and δ¹⁵N) were calculated using a continuous flow isotope ratio mass spectrometer (Finnigan MAT Deltaplus, Thermo—Finnigan) equipped with a Costech Elemental Analyzer (Costech Analytical Technologies). Stable isotope analysis exceeded accepted precision and accuracy standards (Matley et al., 2017).