*3.4. NEMO Gene Expression Pattern Analysis*

To study the relative mRNA level in various tissues, the constitutive expression of *NEMO* in blood, brain, muscle, heart, gonad, spleen, kidney, liver, skin, gill and intestine was detected via qRT-PCR (Figure 6A). The *NEMO* gene was highly expressed in the blood, brain and muscle, while low expression was observed in the intestine, gill and skin (*p* < 0.05).

To further study the expression pattern of *NEMO* in the defence against parasite infection, the mRNA levels of *NEMO* were determined in local infection sites (skin and gills) and systemic immune tissues (liver, spleen and head kidney) after *C. irritans* challenge (Figure 6B). The relative expression level of *NEMO* in gills was upregulated from 3 h to 12 h, and a peak (1.55-fold relative to the control) was observed at 12 h, and then, it was generally downregulated. In the skin, *NEMO* was upregulated to a peak from 3 h to 6 h, and then, it returned to normal levels. In the liver, *NEMO* was upregulated to its maximum (3.12-fold relative to the control) from 3 h to 12 h, then downregulated on 1 d, and it had a second peak (2.06-fold relative to the control) at 2 d. In the spleen, *NEMO* was upregulated at 6 h, 12 h, and 1 d and then returned to normal levels. In the head kidney, *NEMO* was upregulated to a peak from 6 h to 12 h and then generally downregulated to normal levels. According to these results, the difference in the relative expression of the *NEMO* gene in the gills and spleen was the smallest, and the difference in the relative expression in the liver was the largest. The highest peak of *NEMO* was observed at 6 h and 12 h after challenge with *C. irritans*.

**Figure 6.** *ToNEMO* transcriptions in various tissues. (**A**) qRT-PCR was used to test relative *NEMO* mRNA levels in healthy fish, containing blood (Bl), brain (Br), muscle (Mu), heart (He), gonad (Go), spleen (Sp), kidney (Ki), liver (Li), skin (Sk), gill (Gi) and intestine (In). Significant differences at *p* < 0.05 are labelled with different letters, and mean ± SEM of each mRNA quantity is shown for each tissue tested. (**B**) Temporal mRNA expression analyses of *ToNEMO* in different tissues (gill, skin, liver, spleen and head kidney) after PBS (control), *C. irritans* challenges (0 h, 3 h, 6 h, 12 h, 1 d, 2 d and 3 d). EF-1a is used as the internal control to calibrate the cDNA templates for all the samples. The heatmap was constructed using Graphpad Prism 5.0 software.

### **4. Discussion**

In the present study, *T. ovatus* was infected by stimulating *C. irritans*. HE staining was used to observe the life history of *C. irritans* and the histopathological characteristics of the infected *T. ovatus*. The enzyme activity of ACP, AKP, SOD and LZM was detected via colorimetry to study the histological and immune stress response of *T. ovatus* after challenge with *C. irritans*. Moreover, qRT-PCR was used to detect the relative expression levels of *NEMO* in response to *C. irritans* infection.

The infection of *T. ovatus* by *C. irritans* will result in direct mechanical damage to the parasitized tissue, internal tissue haemorrhage and necrosis, as well as the migration and proliferation of a large number of immune cells and the upregulation of chemokines [4,26]. Histopathological results showed hyperplasia in the gill filaments, cell shedding and tissue necrosis. The gill lamellae were swollen, separated and split seriously, and a large number of white blood cells had migrated and gathered in the parasitized part. Mucous cells in the skin had thickened, and mucous cells and secreted fluid quantity increased significantly. The skin was destroyed, the dermis layer was exposed, and cell shedding and tissue necrosis were observed. The pathological tissue morphology was similar to that in a previous report [4,26].

Immuno-related enzymes, such as ACP, AKP, SOD and LZM, are important immune indicators for aquaculture animals when they undergo pathogen invasion. ACP plays a role in killing and digesting pathogens in immune responses [13]. AKP is also a multifunctional enzyme involved in immune responses [13]. Research has shown that the serum ACP activity of snails (*Biomphalaria glabrata*) will increase significantly in response to the pathogen infection [27]. Studies have shown that when infected by acute viral necrosis virus (AVNV), the blood AKP level of *Chlmys frreri* was higher than that of the control group [28]. Increases in ACP and AKP activities indicate that defence against foreign materials is enhanced in *Apostichopus japonicus* [29]. According to these results, AKP activity in the head kidney and ACP activity in the skin were changed the most after

*C. irritans* challenge, suggesting that 6–12 h after infection, ACP and AKP may mainly participate in detoxification in these tissues.

Among antioxidant responses in fish, SOD production is a first line of defence against oxidative stress, converting superoxide anions to hydrogen peroxide and oxygen [30]. SOD showed significant upregulation in the immune tissues, suggesting that SOD may clear superoxide free radicals and enhance the antioxidant capacity of cells instead of directly acting on the pathogen at the site of *C. irritans* infection. LZM is also an important lysosomal enzyme that can lyse and digest pathogenic microorganisms [31]. The results of LZM showed significant upregulation in all five tissues. The upregulation of lysozymes on the skin may be due to the secretion of mucosal immune tissue as a barrier against the stimulation of cryptosporidium, and a high level of its expression in the immune system indicates the initiation of a non-specific immune response in the body.

Bioinformatic analysis showed that the NEMO domain, coiled coil region–leucine zipper domain and zinc finger domain were highly conserved in different species. It has been previously reported that orange-spotted grouper *TLR2* and its downstream pathway genes were significantly upregulated after challenge with *C. irritans* [32]. The challenge of *C. irritans* may activate the innate immune inflammatory response through the TLR2 pathway [6]. MyD88 and TIRAP are recruited by TLR2 and form the IRAK-TRAF6 complex. TRAF ubiquitination leads to the formation of the IKK complex via NEMO, IKKα and IKKβ, which results in the phosphorylation of IκB. Next, NF-κB is transferred to the nucleus to activate the target genes and induce the transcription of TNF, Pro-IL-1b, IkBz, ATF3, Zc3 h12a, TTP, etc. [6]. The NEMO domain is an important structure for recognizing and binding IKKβ [33]. The coiled coil domain ensures the integrity of the NEMO structure. Conservation among different species suggests that *T. ovatus* NEMO may play the same role in innate immunity as other species [34].

The infection of *T. ovatus* by *C. irritans* activates the innate immune signalling pathway of *TLR2,* and its downstream factors are significantly upregulated in infected sites and systemic immune tissues [35,36]. NEMO is a necessary regulator of NF-κB and plays a crucial role in the innate immunity response to pathogens, including bacteria and viruses. *NEMO* receives upstream signals from the TLR family [6].

The *NEMO* gene was upregulated in the skin, liver, spleen, head and kidney after infection, which is similar to the results found for the *TLR2* signalling pathway in orangespotted groupers infected by *C. irritans* reported in a previous study [32]. After infection, *NEMO* in the gill, skin, spleen and head kidney reached a peak at 6–12 h after infection. Compared with *TLR2*, the time at which *NEMO* reached its peak was significantly delayed. qRT-PCR results suggested that the pathogen recognition reaction was initiated at 3–6 h, and the innate immune *NEMO* downstream pathway was upregulated to its peak at 6–12 h.

In conclusion, the histological sections showed that there was considerable metamorphosis and hyperplasia in the parasitized sites (skin) with leukocyte aggregation and mucous cell increases after *C. irritans* infection. Moreover, the activities of four enzymes were significantly increased in different tissues after *C. irritans* infection. The *ToNEMO* transcripts were universally expressed in all of the examined tissues, with higher levels being observed in the immune-relevant and central nervous tissues. The mRNA levels of *ToNEMO* after *C. irritans* infection were significantly increased in the gill, skin, liver, spleen and head kidney. This study could help deepen the understanding of the poisoning mechanism of *C. irritans* to golden pompano at the molecular level. It could provide some basic data for the study of the healthy culture and physiological function regulation of golden pompano.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jmse11020262/s1, Table S1: Information for NEMO amino acid sequence alignment and constructing phylogenetic tree.

**Author Contributions:** K.-C.Z., D.-C.Z. conceived and designed the experiments; K.-C.Z. performed the experiments; B.-S.L. and N.Z. contributed to sample collection; H.-Y.G. and W.-F.L. analysed the data and wrote the paper; B.L., J.-W.Y. and D.-C.Z. assisted with writing and proofreading. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (U20A2064), the Central Public-interest Scientific Institution Basal Research Fund CAFS (NO.2020TD29), the China Agriculture Research System (CARS-47) and the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (2019KJ143).

**Institutional Review Board Statement:** All applicable international, national and institutional guidelines for the care were followed by the authors.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

**Data Availability Statement:** All data generated or analysed during this study are included in this published article.

**Conflicts of Interest:** The authors declare no conflict of interest.
