*2.1. Ethics Statement*

All trials in this study were authorized by the Animal Care and Use Committee of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (No. SCSFRI96-253) and were performed according to the regulations and guidelines formulated by this committee.

## *2.2. C. irritans Challenge and Sampling*

Healthy *T. ovatus* (body weight = 98 ± 15 g) were purchased from Linshui Marine Fish Farm in Hainan Province, China. Before the experiment, the fish were put into a circulating marine pool at 28 ± 2 ◦C with 25‰ salinity and acclimated for two weeks with commercial feed (Hengxin, Zhanjiang, China, crude protein > 37%, crude fat > 7%). The *C. irritans* strain used in this study was originally isolated from infected *T. ovatus* and artificially propagated in the laboratory according to the methods described in previous research [21].

Fifty healthy fish were randomly selected as the control group. Then, one hundred and twenty healthy fish were challenged with *C. irritans* at a dose of 600 theronts/fish. After infection for 0 h, 3 h, 6 h, 12 h, 1 d, 2 d and 3 d, five tissue samples (skin, gill, liver, spleen and head kidney) were collected from six challenged fish to examine enzyme activities and gene expression. Blood, brain, muscle, heart, gonad, spleen, kidney, liver, skin, gill, and intestine was detected via qRT-PCR. Before dissection, the fish were anaesthetized using MS222 (0.1 g/L; Sigma, Alcobendas, Spain). In addition, the second branchial arch and skins were taken and stored in 4% paraformaldehyde for tissue section samples. The same tissues from uninfected fish were regarded as a negative control at each time point. All samples were immediately frozen in liquid nitrogen and then stored at −80 ◦C until use.
