*3.2. Immune-Related Enzyme Activity Analysis*

To investigate the antioxidant activity and non-specific immune response of *T. ovatus* after infection with *C. irritans*, the enzyme activity of ACP, AKP, SOD and LZM in the defence against parasite infection was determined at local infection sites (skin and gills) and system immune tissues (liver, spleen and head kidney) (Figure 3).

**Figure 3.** Enzyme activities of ACP (**A**), AKP (**B**), SOD (**C**) and LZM (**D**) were detected in gill, skin, liver, spleen and head kidney after infection with *C. irritans* (0 h, 3 h, 6 h, 12 h, 1 d, 2 d and 3 d). The heatmap was constructed using Graphpad Prism 5.0 software.

The results of the ACP activity assay are shown in Figure 3A. In the gill, ACP was upregulated to a peak at 6 h, then decreased and had a second peak at 1 d. In the skin, ACP was upregulated to its maximum (3.14-fold relative to the control) from 3 h to 6 h and then downregulated. In the liver, ACP was upregulated to a maximum (2.39-fold relative to the control) from 3 h to 6 h and then it decreased to normal levels. In the liver, ACP was upregulated at 6 h and then downregulated. In the head kidney, ACP was upregulated to a peak (1.97-fold relative to the control) from 3 h to 6 h and then downregulated to normal levels. ACP had the smallest change in the gill and the largest change in the head kidney. The ACP activity in the skin was the lowest, with an average of 0.15 (U/gprot), and the ACP activity in the spleen was the highest, with an average of 0.93 (U/gprot).

The results of the AKP activity assay are shown in Figure 3B. In the gill, AKP showed no significant changes between the challenge group and the control group. In the skin, AKP was upregulated to a peak (3.80-fold relative to the control); then, it decreased and had a second peak (4.65-fold relative to the control) at 2 d. In the liver, AKP was upregulated at 3 h and 6 h, and it reached a peak (3.55-fold relative to the control) at 12 h and then was maintained to 2 d. In the spleen, AKP had a peak (4.96-fold relative to the control) at 6 h. In the head kidney, AKP reached a peak (6.01-fold relative to the control) at 3 h. AKP had the smallest change in the gill and the largest change in the head kidney. The activity of AKP in the gills was the lowest, with an average of 0.24 (U/gprot), and the activity of AKP in the liver was the highest, with an average of 1.94 (U/gprot).

The results regarding SOD activity are shown in Figure 3C. In the gill, SOD was upregulated to a peak (2.98-fold relative to the control) from 3 h to 1 d. In the skin, SOD was upregulated to a peak (4.70-fold relative to the control) from 3 h to 1 d. In the liver, SOD was upregulated at 3 h and 12 h and then maintained at a certain level. In the spleen, SOD was upregulated at 6 h and 12 h, and then it decreased, with a second peak at 3 d. In the head kidney, SOD was upregulated to a maximum from 3 h to 1 d and then downregulated. SOD displayed the smallest change in the spleen and the largest change in the skin. The activity of SOD in the spleen was the lowest, with an average of 1.06 U/mg prot, and the activity of SOD in the skin was the highest, with an average of 2.52 U/mg prot.

The results of the LZM activity assays are shown in Figure 3D. In the gill, LZM was upregulated to a maximum (3.27-fold relative to the control) from 3 h to 1 d. In the skin, LZM reached a peak at 3 h and was then maintained at a certain level. In the liver, no significant change was observed in the challenge group. In the spleen, LZM had a peak (1.41-fold relative to the control) on day 1. In the head kidney, LZM was upregulated at 6 h and had a peak (8.49-fold relative to the control) on day 3. LZM displayed the smallest

change in the liver and the largest change in the skin. The activity of LZM in the gills was the lowest, with an average of 43.30 U/mL, and the activity of LZM in the liver was the highest, with an average of 160.13 U/mL.

### *3.3. NEMO Bioinformatic Analysis*

The *NEMO* ORF of *T. ovatus* was 1650 bp in length and encoded 548 amino acids (GenBank accession number: MW076540) (Figure 4). The predicted molecular weight of NEMO was 62.68 kDa, and the predicted isoelectric point was 5.52. It included a 68 amino acid Pfam NEMO domain (28–95 aa); a 222 amino acid coiled coil domain (138–359 aa); a 95 amino acid Pfam CC2-LZ (coiled coil region–leucine zipper domain) (365–459 aa) and a 19 amino acid ZnF C2H2 (zinc finger domain) (526–545 aa).

**Figure 4.** *NEMO* ORF sequence and predicted amino acid sequence analysis. NEMO domain is marked by light grey shadows. The low-complexity region is underlined. The CC2-LZ (coiled coil region–leucine zipper) domain is marked by dark grey shadows. The ZnF C2H2 (Zinc finger) domain is marked by a box. \* stands for termination codon.

To study protein structure conservation, SMART was used to predict and align the NEMO protein structure of *T. ovatus* with other species (Figure 5A). Multiple alignment analysis of the NEMO amino acid sequence showed that NEMO has three conserved domains.

Multiple sequence alignment results revealed that the amino acid sequences of ToNEMO were highly conserved with the corresponding sequences of other species. To further study the homology of ToNEMO with other species, the amino acid sequence of ToNEMO was aligned with other species via MEGA 7.0 with the neighbour-joining (NJ) method (Figure 5B). In teleosts, ToNEMO had the highest homology with *Seriola dumerili* NEMO and *Seriola lalandi dorsalis* NEMO isoform X3 and the lowest homology with *Acipenser ruthenus* NEMO. Compared with Mammalia, Lepidosauria, Aves and Amphibia, ToNEMO had the highest homology with Mammalia and the lowest homology with Amphibia.

**Figure 5.** Protein structure (**A**) and phylogenetic tree (**B**) of comparing ToNEMO with other NEMO vertebrates. SMART was used to predict and aligned NEMO protein structure of *T. ovatus* and other species. MEGA 7.0 with neighbour-joining (NJ) method was used to structure phylogenetic tree. *T. ovatus* is marked by green shading (**A**) and red asterisk (**B**). The accession numbers are listed in Table S1.
