**1. Introduction**

*Cryptocaryon irritans* is an obligate ciliated parasite of fish. There are four stages in its life cycle: trophozoite, cyst precursor, cyst and larva [1]. *C. irritans* can cause severely fatal "white spot disease" in tropical or subtropical marine fish [1,2]. In recent years, as the density of marine aquaculture has increased, "white spot disease" has frequently broken out in the marine aquaculture areas of the South China Sea, which seriously threatens marine aquaculture fishes, especially *Trachinotus ovatus* [3]. However, there are still few studies on the histology of *T. ovatus* infection with *C. irritans* and its immune mechanism.

Moreover, *C. irritants* mainly invades the superficial epithelium of host fish, including the skin, gills and eyes, and it damages the physiological functions of these organs with pathological features of white spots on the fins, skin or gills. Fish may rub against the wall of the pool, swim irregularly, float on the surface or sink to the bottom or show lethargy or shortness of breath as signs of distress. Symptoms include pinhead-sized white nodules on the gills and body, mucus hyperproduction, skin discoloration, corneal cloudiness,

**Citation:** Guo, H.-Y.; Li, W.-F.; Zhu, K.-C.; Liu, B.-S.; Zhang, N.; Liu, B.; Yang, J.-W.; Zhang, D.-C. Pathology, Enzyme Activity and Immune Responses after *Cryptocaryon irritans* Infection of Golden Pompano *Trachinotus ovatus* (Linnaeus 1758). *J. Mar. Sci. Eng.* **2023**, *11*, 262. https:// doi.org/10.3390/jmse11020262

Academic Editor: Dariusz Kucharczyk

Received: 8 November 2022 Revised: 7 January 2023 Accepted: 16 January 2023 Published: 23 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ragged fins and pale gills. *C. irritans* infection results in the serious mortality of fish due to asphyxiation, osmotic imbalance and/or secondary bacterial infections. In fish stocks, mortality may increase rapidly within a few days, depending on the parasite strains, fish species and the water temperature [1].

In previous histological studies of *T. ovatus* challenged by *C. irritant*, their surface was directly mechanically injured, and the better-defined systemic immune tissue was indirectly damaged, with degeneration and necrosis [4]. The migration and aggregation of leukocytes, including lymphocytes, eosinophils, macrophages and neutrophils, was observed in gills, and IL-8 was significantly upregulated in skin [3].

Tumour-necrosis-factor-receptor-associated factor (TRAF) is a kind of intracellular junction protein previously discovered using yeast two-hybrid technology and glutathione transferase fusion technology [5]. TRAF participates in signal transduction pathways of Toll-like receptors (TLRs) and other families and regulates nuclear factor κB (NF-κB), which are important signal transduction proteins in cells [5,6]. NEMO is one of the most important components of the IKK (IκB kinase) complex, a key protein in the NF-κB pathway [7,8]. TRAF ubiquitination activation was shown to lead to the activation of the IκB kinase complex and the phosphorylation of NEMO (NF-kappa-B Essential Modulator) [5], which caused the released NF-κB to be transferred to the nucleus and initiate transcription of the TNF, Pro-IL-1b, IkBz, ATF3, Zc3 h12a and TTP genes [6]. The NF-κB pathway is essential for the immune response and inflammatory response to pathogens [9]. NEMO plays an essential role in the activation of the IKK kinase in the NF-κB signalling pathway [10]. NEMO is highly conserved. Structure prediction shows that it has an α-helix with three coiled coil regions, a leucine zipper domain and a C-terminal zinc finger structure [11].

The specific immunity of fish is lower than that of mammals, while the non-specific immunity of fish plays a major role in the body's immunity [12]. The antioxidant system of fish is the first line of defence in the innate immunity of fish. In addition to the intracellular activation of innate immune signalling pathways and the release of extracellular inflammatory factors, immune-related enzymes in the body are also important immune indicators when the body undergoes pathogen invasion. ACP plays a role in killing and digesting pathogens in immune responses. AKP is also a multi-functional enzyme involved in immune responses [13]. Superoxide dismutase (SOD) is the main antioxidant enzyme in the antioxidant system to remove excess active free radical ROS [14]. As a humoral immune factor, Lysozyme (LZM) activity can reflect the non-specific immune ability of fish to a certain extent [3,15].

Golden pompano is one of the most important farmed fish in the South China Sea [16]. Similar to other species, golden pompons are also affected by *C. irritans*. Although environmental studies have been reported [17–20], the infective characteristics and mechanisms of *C. irritans* in *T. ovatus* have not been adequately studied. In this study, the histopathology and gene expression of *NEMO* and immune-related enzyme activity were detected after *C. irritans* infection. The influence of the infection on the immune response of *T. ovatus* provides basic theoretical data and guidance for later studies of disease resistance breeding and blocking the effects of *C. irritans* on *T. ovatus*.
