*2.7. Quantitative Real-Time PCR and Statistical Analysis*

To determine the relative mRNA expression level of *T. ovatus NEMO* in normal tissues under normal conditions and after infection with *C. irritans*, qRT-PCR was used to analyse the expression pattern of *NEMO* in five infected tissues (skin, gill, liver, spleen and head kidney) from *T. ovatus*. Based on the *NEMO* cDNA sequences, specific primers for *NEMO* were designed with Primer 5.0 software, and the housekeeping gene *EF-1α* (elongation factor 1, alpha) was used as a reference in a previously reported study [24]. The qRT-PCR procedure was executed as previously described. To evaluate the relative expression of these genes, the 2–ΔΔCT method was used [25].

#### *2.8. Statistical Analysis*

The data are shown as the means ± SE (n = 3). The one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was used in the present study at a significance level of *p* < 0.05. Statistical analyses were implemented using SPSS 20.0 (SPSS, Chicago, IL, USA).

#### **3. Result**

#### *3.1. Histological Analysis*

HE staining of the gill is shown in Figure 1. In the control group, the gill filaments and gill lamellae of healthy *T. ovatus* were completed and arranged in a comb shape, and the mitochondrial-rich cells, blood cells and epithelial cells were clearly identified. After challenge with *C. irritans* for 3–6 h, round or elliptical trophonts floated in the water and then attached to the gill lamellae of *T. ovatus*, with a size of 20.36 ± 5.00 μm. Vesiculateand horseshoe-shaped nuclei could be observed. After challenge for 12 h–1 d, the *C. irritans* trophont developed into an elliptical solid protomont with a size of 84.24 ± 4.00 μm; the gill lamellae were swollen, curved and hyperplastic. MRC and other structures were not observed. After challenge for 2–3 d, tomonts developed and asexually reproduced as theronts. Part of the tomonts split, and theronts were released into the water. Hyperplasia and curves were observed in the gill filaments, and a large number of tissues swelled, split, shed cells and exhibited tissue necrosis.

HE staining of the skin is shown in Figure 2. The structure of the epidermis and muscle layer of healthy *T. ovatus* was complete. After challenge for 3–6 h, there was no obvious change in the skin surface structure. After challenge for 12 h–2 d, the glandular parts of the epidermal layer thickened, and the mucous cells and secretion fluid increased. After challenge for 3 d, the skin tissue was destroyed, the epidermis layer was exposed, the cells fell off and tissue necrosis occurred.

**Figure 1.** HE staining of the gill tissue of *T. ovatus* infected by *C. irritans.* Abbreviations: GF: gill filaments; GL: gill lamellae; EC: epithelial cell; MRC: mitochondrial-rich cell; BC: blood cell; -: split; : swell; : hyperplasia; •: necrosis. The white arrow shows white blood cells migrating and aggregating at the parasitic site. \*: *C.irritans* (life cycle: 3–6 h: trophont; 12–24 h: protomont; 48 h: tomont; 72 h: theronts). Scale bar = 100 μm.

**Figure 2.** HE staining of the skin tissue of *T. ovatus* infected by *C. irritans.* Abbreviations: EP: epidermis layer; CO: dermis layer; MU: muscle layer; -: break; : flat; : thickening; the black arrow shows the distribution of mucous cells in the gland of the epidermis layer. Scale bar = 100 μm.
