*2.4. Determination of Immunity-Related Enzyme Activities*

The acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and lysozyme (LZM) activities and total protein content (TP) were measured in five tissues from infected fish (skin, gill, liver, spleen and head kidney). ACP, AKP, SOD and LZM enzyme activity and total protein content were measured with assay kits according to the protocol supplied by the manufacturer of the kits. Colorimetry was used to determine ACP and AKP activity, and enzyme activity that produced 1 mg of phenol by 1 g of tissue protein at 37 ◦C in 15 min was defined as 1 U (U/g prot). To measure the SOD activity, 50 mL of supernatant was mixed with 1.3 mL of reaction solution containing. 75 mM nitroblue tetrazolium (NTB) and 20 mM riboflavin. The mixture was incubated at 37 for 40 min, and then, the appearance of NTB-diformazan was measured with a Mindray BS-420 automatic biochemical instrument (Shenzhen Mindray Biological Medical Electronics Co., Ltd., Shenzhen, China). The method of determining LZM activity has been described in a previous study [22].

### *2.5. Cloning of the NEMO Gene*

The tissues were homogenized with a tissue homogenizer (JXFSTPRP-24, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) after adding magnetic beads. Total RNA (1 μg) was isolated from each tissue using the HiPure Universal RNA Kit (Magen, Guangzhou, China) and reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). Moreover, the quantity and quality of the extracted RNA were detected using a NanoDrop 2000 spectrometer (Thermo Scientific, Waltham, MA, USA) and 1% agarose gels. The synthetic cDNA samples were stored at −20 ◦C until use. The NEMO predicted sequences were obtained from *T. ovatus* genomic data [23]. To determine the accuracy of the encoding sequence of NEMO, gene-specific primers were designed (Table 1) based on the proposed sequence.

#### *2.6. Bioinformatic Analysis of the NEMO Gene*

To learn about the bioinformatic features of the *NEMO* gene, the Compute pl/Mw online tool on ExPASy (https://web.expasy.org/compute\_pi/ (accessed on 11 December 2019)) was used to predict the molecular weight and isoelectric point of the protein encoded by *NEMO*. Showorf, an online tool from EMBOSS (http://www.bioinformatics.nl/embossexplorer/ (accessed on 11 December 2019)), showed the open reading frame and predicted amino acid sequence. Other species' *NEMO* genes were searched for and downloaded using

the NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 11 December 2019)). The structure and domains of the *NEMO* gene of *T. ovatus* were predicted using the SMART online tool (http://smart.embl-heidelberg.de/ (accessed on 11 December 2019)). The amino acid sequences of the conserved domains of the *NEMO* gene *of T. ovatus* were analysed using the Clustal Omega online tool in EMBL (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 11 December 2019)). The phylogenetic trees of NEMO were constructed using MEGA 7.0 software with the neighbour-joining (NJ) method.

**Primer Sequences (5** →**3 )** NEMO-ORF-R ATGGTGCAGCCACAACCTG NEMO-ORF-L CTGTATACAGTCCATGA NEMO-qRT-R GGCTCACTGGAGACTGTT NEMO-qRT-L GAGGAAATCTGCCTTGTA EF-1α-qRT-R CCCCTTGGTCGTTTTGCC EF-1α-qRT-L GCCTTGGTTGTCTTTCCGCTA

**Table 1.** Information regarding the *NEMO* primers used in this study.
