**4. Discussion**

The gill is a key organ for osmoregulation in fish and plays an important role in ion regulation [25]. When salinity changes, gills not only need to carry out osmotic regulation in time, but also need to bear certain oxidative stress pressure [26]. In the present study, the change of salinity within 48 hours increased the antioxidant enzyme activity in the gills of the juvenile yellowfin tuna. The impacts of salinity on the activity of antioxidant enzymes in gills have been intensively studied. As SOD is the first part of antioxidant defense, it will encounter superoxide anion free radicals. During stress, SOD has a faster reaction speed and higher sensitivity, which is a good indicator for monitoring the aquatic ecosystem [27]. Ghanavatinasab et al. evaluated the impact of salinity on *Sparus flavipectus* when transferring fish from 20 to 5, 12, and 34. They found no significant differences in SOD activity between the groups after 14 days of the experiment [28]. The responses of SOD activity in fish to the salinity changes are species-dependent. In black porgy *Acanthopagrus schlegeli*, when environmental salinities decreased from 35 to 10 ppt, SOD activity of fish upregulated nearly twofold [29]. Moreover, the activity of SOD in *Scapharca broughtonii* showed similar responses after exposure to low environmental salinities [30]. It is speculated that when fish are subjected to salinity stress, different tissues in the fish may undergo various levels of oxidative stress and different resistance strategies. When the salinity dropped suddenly, the SOD in the gills of the juvenile yellowfin tuna participated in scavenging free radicals. Generally speaking, the H2O2 produced in most animals is mainly eliminated by GSH-Px [31]. At the same time, GSH-Px can also remove lipid peroxides such as fatty acids, and maintain the normal function of the cell membrane [32]. In the present study, the GSH-Px activities in stressed fish gills reached the highest level at 24 h and were reduced to similar levels to those observed in the control group. Such changes in the GSH-Px activities of fish gills may suggest that fish may fully recover from salinity shock after a 24 h adaption. In vivo, MDA is the final product of oxidation when free radicals act on lipid peroxidation, which can cause the cross-linking and polymerization of life macromolecules such as proteins and nucleic acids and has cytotoxicity [33]. At the same time, the MDA content also shows the degree of cell plasma membrane damage [34]. When the salinity decreases, the MDA in the gills of juvenile yellowfin tuna remains stable within a certain range in the whole process of antioxidation in a low-salt environment, which also proves that the cytoplasmic membrane is not damaged.

The activity of antioxidant enzymes in different tissues of the same fish is also different. The liver is a multifunctional organ that integrates metabolism, immunity, digestion, and other functions [25]. The liver is the tissue with more oxidation reactions, so the activity of antioxidant enzymes is higher [33]. The results of this study showed that the SOD activity in the liver of juvenile yellowfin tuna had no significant impact after the sudden drop in ambient salinity, which was contrary to the research results of *Epinephelus moora* [35], *Takifugu obscurus* [36], and *Oryzias melastigma* [37]. The cultivation salinity of *Dicentrarchus labrax* decreased from 37 to 5, and the SOD activity increased significantly after 12 h of stress [38,39]. The alternations in the SOD activity were relatively low, possibly due to the low response mechanism in the liver of migratory fish in the deep sea [40]. The increased content of GSH-Px and MDA in the first 24 h may relate to the elimination of excessive reactive oxygen free radicals in the body. Afterward, the content of GSH-Px and MDA in the fish liver decreased to a level similar to the control group, possibly as a result of the reaction between glutathione peroxidase and a variety of antioxidant enzymes [41,42]. In the present study, the adaptation processes of SOD, GSH-Px, and MDA were stable after 24 h in the liver, indicating that the antioxidant system in the fish liver had adapted to the salinity reduction. In contrast, however, antioxidant changes in the gills of the yellowfin tuna were different from those of the liver. The inhalation and excretion of fish gills are closely related to the gills. In the acute low-salt environment, the yellowfin tuna is required to maintain osmotic balance, and it also bears a certain amount of oxidative stress pressure [43]. The branchial vascular system is innervated and autonomously controlled by its nerves [44]. When the antioxidation data of their own tissues are stable, the gill tissue behaves differently from the liver and muscle in response to the oxidative stress caused by the sudden change of salinity. It is speculated that different tissues are subject to different degrees of oxidative stress, and there are also differences in the resistance strategies.

The red muscle and white muscle in fish are both part of the greater lateral muscle. As the yellowfin tuna needs to rely on the gills to obtain oxygen, the red muscle and white muscle are clearly distinguished. The red muscle has a high fat content, is rich in myoglobin and has a large amount of blood, and is dark red. The yellowfin tuna has strong endurance and developed red muscle. The white muscle contains no fat and is light

white [26]. Muscle is widely distributed in the fish body and has a large proportion [26]. Determining antioxidant enzyme activity in fish muscle can also reflect the stress status of the fish. In *Acipenser schrencki,* the muscles are sensitive tissues to osmotic pressure perception and can respond positively when salinity changes [45]. A previous study has demonstrated that the SOD activity in *Rachycentron canadum* muscle increased with the decrease in salinity [46]. However, the responses of SOD activity to the salinity stress in the muscles of yellowfin tuna were different from previous studies [47]. The SOD activity in the muscle of the juvenile yellowfin tuna decreased first and then increased and tended to be stable at the end. Such responses may suggest the antioxidant system in fish muscle has fully adapted to the ambient salinity of 29‰ at 48 h.

The malondialdehyde content in the muscle increased with time, and the overall expression level of the yellowfin tuna red muscle was higher than that of the white muscle [46,47]. The higher SOD activity in the yellowfin tuna red muscle reflected the low potential mechanism in the antioxidant process of yellowfin tuna red muscle [40]. In the present study, the content of the total antioxidant capacity reached the highest value at 24 h and decreased at 48 h, which may be related to the adaptation process of muscle tissue to the salinity stress. However, there are few reports about the effect of salinity stress on antioxidant enzymes in the white and red muscles of fish, and this subject may be worthy of further investigation.
