Tricaine methanesulfonate

Rainbow trout were sacrificed by overexposure to tricaine methanesulfonate (Sigma-Aldrich, Madrid, Spain) at 0.2 g/L. Peripheral blood was sampled from the caudal vein using insulin syringes (NIPRO Bridgewater, NJ). Approximately 100 µL of blood was diluted in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA) supplemented with 10% FBS (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 µg/mL gentamicin (Gibco), 2 µg/mL fungizone (Gibco) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). Then, RBCs were purified by two consecutive density gradient centrifugations with Histopaque 1077 (7206g, Ficoll 1.007; Sigma-Aldrich). Finally, RBCs were washed twice with RPMI 2% FBS. Purity of RBCs of 99.9% was estimated by optical microscopy evaluation. Then, purified RBCs were cultured in the above indicated medium at a density of 10
⁷ cells/mL, in cell culture flasks, at 14°C, overnight.

All A. akindynos specimens were either collected on reefs surrounding Lizard Island, Australia, or obtained from an aquarium supplier (Cairns Marine) collecting fishes from the Northern Great Barrier Reef. Fish were anaesthetized with Tricaine methanesulfonate (Sigma-Aldrich) or an overdose of clove oil, and killed by decapitation. Specimens were classified as subdominant males, dominant males (as defined by the presence of testes and/or being the largest male of the group), and females (as defined by the presence of ovaries and/or being largest individual of the group), and size (total length) as well as affiliation to the same family (as defined as specimens living in the same anemone) were noted. A summary of the number of individuals used with their sex, size and family, where they were sourced from, and for which type of analysis they were used is provided in Table S5. A detailed description of the methods used in this study is provided in the Supplementary Information and move the whole sentence and place it directly under Methods (before Specimen Collection).

The study protocols were approved by the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences (Wuxi, China). The GIFT juveniles were maintained in well-aerated water and anesthetized by injecting 0.01% tricaine methanesulfonate (Sigma, St. Louis, MO, United States). Liver tissue and blood were collected based on the Guide for the Care and Use of Laboratory Animals in China.