Experiment Materials and Sample Preparation

Before sampling, experimental M. amblycephala were anesthetized in well-aerated water containing the 100 mg/L concentration of tricaine methanesulfonate. IB samples of three 1-year-old (IB_I, IBs growing rapidly at this stage) and three 3-year-old (IB_III, IBs growing quite slowly at this stage) fishes were collected and pooled, respectively, with three biological replicates. Total RNA was isolated using RNAiso Plus Reagent (Takara, Japan) following the manufacturer's protocol. RNA degradation and contamination were monitored on 1% agarose gels. RNA concentration was measured using the NanoDrop 2000 spectrophotometer. Qualified RNA samples are stored at −80°C and then used for RNA sequencing. All animals and experiments were conducted in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China). The study was approved by the Institutional Animal Care and Use Ethics Committee of Huazhong Agricultural University. All efforts were made to minimize animal suffering.