Potamogeton maackianus was collected from the Taihu Lake Basin in Jiangsu Province, China. Plants growing to a basically uniform height of 15–20 cm were selected and transplanted into a mud substrate in a laboratory culture tank. Ten strains per culture tank and three replicates per experimental concentration were set up. The experiment used tap water, and chemically pure Na₂SeO₃ was added to adjust Se in the water medium to three concentrations: 0, 20.94, and 67.68 mg/L. Before the experiment, 1 mg/mL Na₂SeO₃ mother liquor was prepared. During the experiment, the light intensity in the water column of the cultivation tank was 110–240 μE/m²/s, the water pH was 6.9–7.4, the water temperature was 25–29°C, and the water was changed once a day. After cultivation for 12 days, the plant stems and leaves were collected, washed, shredded, placed in a cold air dryer at −40°C, and dried for 48 h. We used a pulverizer to powder the dried material, then passed it through a 40-mesh sieve, and stored it in a refrigerator at 4°C for future use. The nutritional composition of each P. maackianus group is shown in the Supplementary Tables S1, S2. Inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700, United States) was used to determine the Se content in the P. maackianus powder, and this was 0.02, 8.83, and 16.92 mg/kg.

The feed formulae and nutritional composition are shown in Table 1. Casein was the main protein source and fish oil was the main fat source. The three P. maackianus powders with different Se contents were added to the basic feed to prepare three groups of isonitrogenous and isolipidic experimental diets (crude protein 40.17%, crude fat 9.74%). When preparing the feed, all diet ingredients were passed through an 80-mesh sieve, weighed, and mixed according to the specified proportions, then fish oil and an appropriate amount of water were added. The mixture was processed by a screw extruder into particles with a diameter of 1 mm. The particles were dried at 4°C and then stored at −20°C.

The crabs were farmed in a monoculture. Juvenile crabs weighing 2.41 ± 0.25 g were randomly divided into three groups and placed in plastic boxes (34 cm × 22 cm × 18 cm). Each crab was placed in a separate plastic box and acclimated for 10 days. The crabs were fed commercial feed during acclimation. We selected crabs with a similar size and complete appendages for the formal experiment. Each experimental group consisted of 80 crabs in 80 independent rearing plastic boxes. During the experiment, each plastic box was cleaned once a day at 15:10. The crabs were supplied with experimental feed twice a day (at 9:00 and 17:00) at 5% of body weight, and the residual feed after 2 h was collected, dried, and weighed. One-third of the water was changed every 2 days. During the rearing period, the dissolved oxygen was maintained above 5.5 mg/L, the water temperature was 26.8–29.0°C, the ammonia nitrogen was <0.01 mg/L, and the pH was 7.5 ± 0.2. The crabs were farmed under a natural light cycle, and the whole experimental period was 60 days. At the end of the rearing experiment, feeding was stopped for 1 day, and each crab was weighed separately. We removed the carapace, cut the internal bones and feet from the base of the breastplate, and removed the hepatopancreas and abdominal muscles from 20 crabs per treatment group. These were quick-frozen in liquid nitrogen and then stored at −80°C until analysis.

After the rearing experiment, to reduce the sampling stress, the juvenile crabs were kept in plastic boxes for 10 days. Cu²⁺ was then added to the water in the form of CuSO₄.5H₂O (analytical product). A stock solution was prepared with double-distilled water and then diluted to the experimental concentration. Based on the results of preliminary experiments, the Cu²⁺ concentration at which death occurred by 96 h (0.24 mg/L) was selected as the exposure concentration for this study. The Cu²⁺ concentration in water was measured by ICP-MS. Juvenile crabs in the different treatment groups were exposed to 0.24 ± 0.02 mg/L Cu²⁺ for 96 h. During the experiment, the Cu²⁺ concentrations in the water were adjusted every 12 h. At 12, 24, 48, and 96 h of Cu²⁺ stress, nine crabs in each experimental group were randomly selected, and their hepatopancreases were removed and washed with ice-cold saline. The samples were divided into two portions, which were immediately frozen in liquid nitrogen and stored at −80°C until analyses of physiological and biochemical indexes and gene expression had been performed.

The results are expressed as mean ± standard deviation (mean ± SD). Experimental data were subjected to analysis of variance using SPSS 21.0 (SPSS Inc., Chicago, IL, United States). The data were tested for a normal distribution and homogeneity of variance. Paired-samples t-test was used to compare different treatment times within the same experimental group, and Duncan’s multiple comparison was used to compare different treatment groups at the same time. The significance level was P < 0.05.

The actual Se contents in experimental diets as measured by ICP-MS were 0.05, 0.43, and 0.82 mg/kg (Table 3). After 60 days of rearing, there were significantly higher final weights in both the 0.43 and 0.82 mg/kg groups than in the 0.05 mg/kg group (Table 3). We found higher SGR and lower FCR in the 0.43 mg/kg group, compared with the 0.05 mg/kg group, but SGR and FCR did not differ significantly among the three groups (P > 0.05). The survival of juvenile crabs was not significantly different among the three experimental groups (91.5–92.9%; P > 0.05). In addition, there was no significant difference in the HSI among the experimental groups. When the Se content in the diet increased from 0.05 to 0.82 mg/kg, the Se content in the hepatopancreas of juvenile crabs increased from 0.42 to 1.87 mg/kg. Similar trends in concentrations of Se were found in muscle (0.21, 0.42, and 0.55 mg/kg in the 0.05, 0.43, and 0.82 mg/kg group, respectively).

The activities of amylase and trypsin in the hepatopancreas were not significantly different between the 0.82 mg/kg group and the 0.05 mg/kg group, but the activities of pepsin and lipase were significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group (Table 4). Lipase activity was also significantly higher in the 0.43 mg/kg group than in the 0.05 mg/kg group; but pepsin and trypsin activities did not differ significantly between these two groups. There was no significant difference in ALP and γ-GT activities between the 0.05 mg/kg group and the 0.43 mg/kg group; but their activities were significantly lower in those two groups than in the 0.82 mg/kg group (P < 0.05) (Table 5). As the Se content in the diet rose, the ACP activity and NO content in the hepatopancreas of Chinese mitten crab increased gradually. The ACP activity and NO content in the hepatopancreas of Chinese mitten crab differed significantly among the three experimental groups (P < 0.05). The hepatopancreas TC and TG contents were significantly higher in the 0.82 mg/kg group than in the 0.43 mg/kg group and the 0.05 mg/kg group. There was no significant difference in TG content between the 0.43 mg/kg group and the 0.05 mg/kg group (P > 0.05).

After 60 days of an Se-containing diet, the SOD activity in the hepatopancreas was significantly higher in the 0.43 mg/kg group than in the 0.05 mg/kg group and the 0.82 mg/kg group (Figure 1A). The activity of CAT in the hepatopancreas was significantly lower in the 0.05 mg/kg group than in the 0.43 mg/kg group and the 0.82 mg/kg group (P < 0.05) (Figure 1C), but did not differ significantly between the 0.43 mg/kg group and the 0.82 mg/kg group (P > 0.05). The MDA content was significantly higher in the 0.05 mg/kg group than in the 0.43 mg/kg group and the 0.82 mg/kg group (P < 0.05) (Figure 1E), and the POD (Figure 1B), GR (Figure 1D), and GSH-Px (Figure 1F) activities were significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group (P < 0.05).

At the end of the rearing experiment, the crabs were subjected to acute Cu²⁺ stress conditions for 96 h. Three crabs in the 0.05 mg/kg group died, but none died in the other experimental groups. The activities of SOD and CAT in each experimental group tended to increase in the first 24 h of Cu²⁺ stress. From 24 to 48 h, SOD and CAT activities in each experimental group were relatively stable, but were significantly higher than their pre-stress activities. In all groups, the MDA content in the hepatopancreas first increased, then decreased, then increased by 96 h of Cu²⁺ stress. All groups showed a sharp increase in MDA content in the first 12–24 h of Cu²⁺ stress. At 48 h, the MDA content was not significantly different from the pre-stress level in the 0.05 mg/kg group, but was significantly higher than pre-stress levels in the 0.43 mg/kg group and the 0.82 mg/kg group (P < 0.05). At 96 h of Cu²⁺ stress, the MDA content in the hepatopancreas was significantly lower in the 0.82 mg/kg group than in the 0.05 mg/kg group and the 0.43 mg/kg group. At 12 h of Cu²⁺ stress, the POD activities in all experimental groups were not significantly different from pre-stress activities, and POD activity was significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group at 24, 48, and 96 h of Cu²⁺ stress. There was no significant difference in POD activity in the 0.05 mg/kg group during the 96-h Cu²⁺ stress period (P > 0.05). At 12 h and 24 h of Cu²⁺ exposure, GR activity in the 0.82 mg/kg group was significantly higher than pre-stress activity, and significantly higher in the 0.82 mg/kg group than in the other experimental groups. At 48 h of Cu²⁺ exposure, there was no significant difference in GR activity among the three experimental groups (P > 0.05). At 48 h and 96 h of Cu²⁺ stress, the GSH-Px activities were significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group and the 0.43 mg/kg group (P < 0.05).

At the end of the rearing experiment the transcript level of MT, encoding metallothionein (Figure 2A), was significantly higher in the 0.82 mg/kg group than in the other groups. The transcript levels of LYZ (Figure 2B) and HSP70 (Figure 2C) were not significantly different between the 0.43 mg/kg group and the 0.05 mg/kg group, but the transcript level of LYZ was significantly higher in the 0.05 mg/kg group than in the 0.82 mg/kg group (P < 0.05).

Under Cu²⁺ stress, MT transcript levels in mitten crabs in each experimental group increased significantly. Within 12–96 h of Cu²⁺ stress, the MT transcript levels in each treatment group were significantly higher than the pre-stress levels. The transcript level of MT was significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group at all sampling times (P < 0.05). The transcript levels of LYZ in each experimental group increased first and then decreased, with peak levels at 24 h in the 0.05 mg/kg group and at 48 h in the 0.43 mg/kg group and the 0.82 mg/kg group. The LYZ transcript levels were significantly higher in the 0.82 mg/kg group than in the 0.05 mg/kg group at 12, 48, and 96 h of Cu²⁺ stress (P < 0.05). The transcript levels of HSP70 continuously increased in the 0.82 mg/kg group during the 96 h of Cu²⁺ stress; those in the 0.05 mg/kg group and the 0.43 mg/kg group increased to peak at 48 h of Cu²⁺ stress and then decreased. In addition, the HSP70 transcript levels were significantly higher in the 0.05 mg/kg group than in the 0.82 mg/kg group at 48 and 96 h (P < 0.05).

The growth performance of Chinese mitten crab significantly increased when the diets were supplemented with 0.43 mg/kg Se. The positive growth responses to Se supplementation of this result are consistent with those reported for Tian et al. (2014); Qin et al. (2016), and Kong et al. (2017). Se mainly improves animal growth performance because (1) Se is an important component of GSH-Px, which can reduce the body’s stress response, improve antioxidant capacity, enhance the body’s immune function, reduce mortality, and then promote growth (Tian et al., 2014); and (2) Se is a component of the enzyme 5′-deiodinase. An appropriate amount of Se in the diet can activate the synthesis and metabolism of thyroxine and catalyze the conversion of T4 to T3, which controls the gene expression and synthesis of growth hormone, and is beneficial for animal growth (Ibrahim et al., 2011). Thus, the improvement in growth performance and feed utilization in the 0.43 mg/kg Se-supplemented groups might be associated with the function of Se.

However, our study indicated that, as the Se content in the diet increased, the weight of Chinese mitten crab did not continue to increase. Adding Se to the diet may have both nutritional and toxic effects. Excess dietary Se may cause Se deficiency symptoms or slow or acute toxicity in aquatic animals. Kong et al. (2017) found that excess Se may generate free radicals and cause oxidative damage, thereby affecting normal growth and development in oriental river prawn. However, Wang et al. (2019) found that excess Se-yeast in the diet of Chinese mitten crab reduced oxidative stress in the hepatopancreas, and did not limit growth. Similarly, the organic Se we added to the diets came from plants, which may have fewer toxic effects on mitten crab. Yuan et al. (2018) also found that Se-biofortified corn fed to mitten crab was more readily biologically active while being less toxic.

The Se content in the hepatopancreas and muscle of juvenile crabs increased with rising levels of Se in the diet, indicating that the crab body was able to become enriched with Se from the diet. Organic Se can increase the body’s total Se accumulation (Yuan et al., 2018). Differences in metabolic pathways between organic Se and inorganic Se can affect Se accumulation (Kouba et al., 2014). Organic Se is similar to sulfur-containing amino acids. In protein synthesis, seleno-amino acids often replace sulfur-containing amino acids and are incorporated into proteins, which can increase the Se storage capacity (Schram et al., 2008). Therefore, adding Se-enriched plants to increase the content of organic Se in diets can effectively promote the ready accumulation of Se, which also provides a new strategy for the addition of organic Se in future.

In this study, pepsin and lipase activities were significantly lower in the 0.05 mg/kg group than in the 0.82 mg/kg group. Similar results have been found in studies on aquatic animals. For example, dietary supplementation with 0.6% Se-yeast increased protease activity in the hepatopancreas of Gibel carp (Liu et al., 2002). When 0.6 mg/kg Se was added to the diet, the activities of protease, amylase, and lipase were significantly increased in the hepatopancreas of grass carp (Ctenopharyngodon idellus) (Su et al., 2007). Proteases and lipases produced by the hepatopancreas determine digestive ability, which directly affects the digestion and absorption of nutrients such as proteins and fats (Qiang et al., 2017). In mammals, Se deficiency can impair the hepatic and pancreatic acinar and islet function and reduce acinar secretion, thereby inhibiting amylase and lipase activity (Zhang et al., 1997). The organic Se content in the diets of the 0.43 mg/kg group and the 0.82 mg/kg group was considered to facilitate adequate digestibility and consequent enzyme activity in the Chinese mitten crab hepatopancreas, enhance fat intake and metabolic capacity, and increase the accumulation of TG and TC.

The two important phosphatases ACP and ALP have important effects on the body’s metabolism and immunity. In mitten crab, feeding with 0.5 mg/kg Se-yeast stimulated ACP activity, suggesting that the immune system was activated in vivo (Wang et al., 2019). In addition, mitten crab fed on a fermented diet showed increased ACP phosphatase activity, and increased total protein and globulin contents in the hemolymph (Xu et al., 2019). In this study, we detected higher ACP activity in the hepatopancreas in the 0.43 mg/kg group, and this may have been related to the activated immune capacity of juvenile crabs. However, as levels of Se in feed increased (0.82 mg/kg), ACP activity was not inhibited, and this differed from the results of Wang et al. (2019). Different Se sources and differences in farming environments may affect crab immune response regulation. The addition of 0.82 mg/kg Se feed did not exceed the upper limit of addition, so there was no inhibitory effect. The biologically active compound NO can act as an antioxidant and anti-apoptotic substance to inhibit programmed cell death (Beligni and Lamattina, 1999). In our study, the inclusion of 0.05 mg/kg Se in the diet led to significantly inhibited activities of ACP, ALP, and γ-GT in juvenile crabs, and suppressed their immune function. The lower NO content in the body of 0.05 mg/kg group may be related to inhibition of the antioxidant system.

Our study found that the MDA content in juvenile crab was significantly lower in the 0.82 mg/kg group than in the 0.43 mg/kg group. The product of lipid peroxidation, MDA, is often used as an important indicator of oxidative damage. Activation of GSH-Px activity by Se has been shown to prevent lipid peroxidative damage in the hepatopancreas (Kouba et al., 2014). Similar results were found in Yuan et al. (2018) and Kong et al. (2017). The deposition of Se in the body acts as a reservoir for rapid utilization under oxidative stress, and promotes the biological activity of antioxidant enzymes (Kim and Kang, 2014).

Chinese mitten crabs inhabit the bottom of ponds, where contamination by heavy metals is more likely to occur. Therefore, at the end of the rearing experiment, the mitten crabs in the three treatment groups were subjected to Cu²⁺ stress for 96 h. The activities of GSH-Px and SOD, and their balance, play an important role in resistance to oxidative damage in the body. Together with CAT, they are the first line of cellular defense (Liu et al., 2018). The higher SOD, POD, GR, and GSH-Px activities in the 0.82 mg/kg group than in the other groups may have been more effective in removing free radicals under Cu²⁺ stress, leading to lower MDA contents and protection of the cell membrane. Our results show that oxidative stress caused by high Cu²⁺ can be alleviated by feeding Chinese mitten crab with a diet containing increased levels of Se. Wang et al. (2019) also found that the addition of 1 mg/kg Se-yeast in the diet increased activities of SOD and GSH-Px in Chinese mitten crab, reduced lipid peroxides, and enhanced resistance against nitrite stress compared with a 0.5 mg/kg Se-yeast group. Dose correction can balance Se and Cu²⁺ in animal bodies. Cu²⁺ is the structural center of SOD, and uptake of exogenous Cu²⁺ may activate SOD activity (Qiang et al., 2019). Therefore, at 48 h of Cu²⁺ stress, increased SOD and CAT activities in the 0.05 mg/kg group may reduce MDA accumulation in the hepatopancreas tissue of Chinese mitten crab. However, as the stress time increases, the SOD, CAT, GSH-Px, and GR activities of the 0.05 mg/kg group were significantly inhibited, and the MDA content was increased. Lack of Se in the body may result in limited resistance to the free radicals caused by Cu²⁺ stress, thereby leading to increased oxidative stress.

Metallothionein is a low-molecular-weight, cysteine-rich protein that is ubiquitous in organisms. It has a highly conserved structure, and plays an important role in maintaining homeostasis and detoxification (Felix-Portillo et al., 2014). In this study, juvenile crabs fed a diet containing 0.82 mg/kg Se showed a more rapid and continued response of MT expression under Cu²⁺ stress. The oxidative stress induced by Cu²⁺ exposure may be alleviated by combining active oxygen radicals with reduced sulfhydryl-enriched MT. In addition, a significant feature of MT is that it contains many cysteine residues, so Se enrichment of the hepatopancreas may help to stimulate MT expression (Hao et al., 2019). Supplementation with Se (2–4 mg/kg) can promote MT gene expression in the liver of turbot (Scophthalmus maximus) fed a diet rich in copper (1,000 mg/kg), leading to the relief of oxidative of stress and the maintenance of homeostasis (Hao et al., 2019). However, further research is required to explore the mechanism by which Se induces MT, and to determine whether it preferentially induces the synthesis of metal selenoproteins or demetallic MTs with different characteristics.

The activity of LYZ is directly related to the immunity and health of aquatic animals. In the present study, the transcript level of LYZ in the hepatopancreas was 17% lower in the 0.82 mg/kg group than in the 0.05 mg/kg group at the end of the rearing experiment. LYZ exists in lysosomes before it is secreted into body fluids. Se reduces LYZ activity in aquatic animals in the absence of stimulation by an antigen. This has been attributed to the ability of Se to protect the structural integrity of lysosomes, thereby reducing the amount of LYZ secreted (Low and Sin, 1999). However, when crabs were stimulated by exogenous Cu²⁺, Se facilitated the release of LYZ, suggesting that it can directly improve the activity of non-specific immune factors and enhance resistance to stress. The increased LZM activity of serum in mitten crab fed with increased levels of Se can improve immune response under nitrite stress (Wang et al., 2019). Alina et al. (2009) also found that Se in the diet can be assimilated into enzymes and proteins, which help to improve immunity. Interestingly, we found that the transcript level of LYZ in juvenile crabs increased sharply in the 0.05 mg/kg group within 24 h of Cu²⁺ stress, suggesting that Cu²⁺ caused severe oxidative stress in this group. The accumulation of lipid peroxides destroys cell membranes and promotes the release of a large amount of LYZ to relieve oxidative damage.

HSP70 plays an important role in protecting cells from damage and/or restores function to damaged cells, and can be used as an indicator of the physiological state of crustaceans (Han et al., 2018). At the end of rearing experiment, the transcript levels of HSP70 were significantly higher in the 0.05 mg/kg group than in the other groups, indicating that a lack of Se may regulate the expression of HSP70. This is consistent with the findings in turbot (Hao et al., 2019). After adding different levels of Se to the feed containing 1,000 mg/kg Cu²⁺, the expression of hepatic HSP70 in the 0 mg/kg Se group was found to be significantly higher than that of the groups with 2 and 4 mg/kg Se (Hao et al., 2019), suggesting that the addition of Se in the diet can regulate the expression of HSP70 gene to protect liver tissue. However, higher Se content in hepatopancreas tissue in juvenile crabs led to rapid up-regulation of HSP70 under Cu²⁺ stress; higher expression of HSP70 may help to relieve oxidative stress damage.