Effects of copper supplement on the immune function and blood-chemistry in adult Chinese horseshoe crab Tachypleus tridentatus

Aquaculture 515 (2020) 734576

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Effects of copper supplement on the immune function and blood-chemistry in adult Chinese horseshoe crab Tachypleus tridentatus

T

Zhen Xua,b,c, Youji Wanga,b,d, Yasmeen Gule, Qiongzhen Lif, Jie Songg, Menghong Hua,b,c,∗ a

National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, China c Shanghai Engineering Research Center of Aquaculture, China d International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, China e Department of Zoology, Government College Women University Faisalabad, Pakistan f Guangxi Institute of Fisheries, Nanning, 530021, China g Tianjin Era Biology Technology Co., Ltd, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tachypleus tridentatus Copper amino acid chelate Non-specific immune enzyme Hemolymph biochemical indicator Hemocyanin

Oxygen in hemocyanin binds to copper ions and turns the hemolymph of horseshoe crabs blue. Copper ions not only transport oxygen but also enhance the immune function of horseshoe crabs. A 28-day indoor culture experiment was conducted to study the effects of adding different levels of copper amino acid chelate (0, 50, or 100 mg kg−1) into semi-finished feed on the nonspecific immune enzyme activity and hemolymph biochemical parameters of Tachypleus tridentatus. Results revealed that the activities of serum antioxidant enzyme indicators, namely, copper–zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPX), total antioxidant capacity (T-AOC), lysozyme (LZM), alkaline phosphatase (AKP), and acid phosphatase (ACP), of copper-fed adult horseshoe crabs increased; on the other hand, malondialdehyde (MDA) activity decreased. Hemolymph biochemical indicators, triglyceride (TG) and cholesterol (CHO) decreased in the copper-fed group, but hemocyanin concentration (HC) levels increased during the experiment. At the end of experiment, all antioxidant enzymes and blood biochemical indicators in the copper-fed group were significantly different from those without copper supplement. Hemolymph T-AOC, MDA, AKP, ACP values in the low-level copper group (50 mg kg−1) were significantly different from those in the high-level copper group (100 mg kg−1).

1. Introduction Horseshoe crabs belong to arthropods of the family Limulidae, suborder Xiphosurida, and order Xiphosura. Only four species belong to three genera that exist in the world, namely, Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda, and are considered as “living fossils”. Only T. tridentatus and C. rotundicauda exist in China (Hu, Kwan, Wang, Cheung and Shin, 2015a; Obst et al., 2012). Horseshoe crabs have high scientific and economic value in bionics, medicine, and pharmacology (Botton et al., 2010). Tachypleus amebocyte lysate (TAL) is the only industrial chain formed by the development and utilization of horseshoe crab resources in China. In TAL industrial chain, starting from hemolymph collection, it not only produces diagnostic kits for class II medical devices with superior performance and high market potential, but also manufactures vitro diagnostic instruments. TAL plays important role in the development of fungal identification for early detection, diagnosis, and

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treatment. The increasing use of amebocyte lysate test method, the demand for horseshoe crabs has been increased. Development and utilization of biological resources requires large amount of the resource. Currently, TAL industrial chain reflects resource-driven characteristics in China (Yan et al., 2019). Considering the sharp decline of T. tridentatus population (Chen et al., 2004; Hu et al., 2009; Li and Hu, 2011; Shin et al., 2009), explorations of a large-scale culture mode of adult T. tridentatus are important for the sustainable use of horseshoe crab resources. Feed is not only a basis for aquaculture, but also the driving force behind the industrial development. One of the key factors required for large scale factory based on recovery culture of blood collected horseshoe crab is the optimal artificial feed formulation designs. Although several research studies on the nutritional requirements and feeds of T. tridentatus have been conducted (Hu et al., 2018; Hu, Wang, Cheung, & Shin, 2013, 2014), these works were generally carried out on juvenile horseshoe crabs; research on the artificial feed for adult horseshoe crabs

Corresponding author. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China. E-mail address: [email protected] (M. Hu).

https://doi.org/10.1016/j.aquaculture.2019.734576 Received 2 August 2019; Received in revised form 4 October 2019; Accepted 5 October 2019 Available online 05 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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has not been reported yet. Artificial feed formula strives to accelerate the recovery rate of hemolymph cells without damaging the life of T. tridentatus, and help to achieve a second hemolymph collection after the recovery of first collection crabs. Despite the continuous improvement of a potential breeding model, however, the third and fourth hemolymph collections may cause an urgent problem for the sustainable use of horseshoe crab resources. Horseshoe crabs lack an acquired immune system but have multiple defense systems. These defense systems are divided into cellular immunity and humoral immunity, that includes hemolymph coagulation, phenoloxidase activation, cell agglutination, release of antibacterial substances for antibacterial reactions, active oxygen formation, and phagocytosis (Iwanaga, 2002; Kawabata, 2002). Antioxidant enzymes are considered to be biomarkers for health and immune functions assessment (Mohseni et al., 2014; Roch, 1999; Rodrıguez and Moullac, 2000). Knowledge of dynamic trends of biochemical and health-related indicators of T. tridentatus after hemolymph collection and screening for the best feed additives can lay a foundation for developing the most suitable artificial feed formula. Hemocyanin in the hemolymph of T. tridentatus accounts for 90%–95% of the plasma protein. Oxygen-bound copper ions in hemocyanin turns crab hemolymph blue, and the copper ion content of this hemolymph is as high as 23.13 mg L−1. Prophenoloxidase activation system requires the presence of copper (Söderhäll and Cerenius, 1998). Therefore, copper ions not only transport oxygen but also enhance the immune function of Copper is key element of horseshoe crab hemolymph. In the hemolymph recovery process of horseshoe crabs, copper requirement is likely to be high, and copper-added feed may contribute to the hemolymph recovery of horseshoe crabs (Liu et al., 2007). Copper and organic trace minerals are combined with ligands such as amino acids, peptides and proteins, which are more easily absorbed by the body than inorganic trace minerals (copper sulfate) and more stable in the digestive tract, so organic forms of copper may have higher bioavailability, resulting in higher absorption or additional biological effects. Some studies have proved that dietary copper has a vital role in the innate immune response in crustaceans such as, L. vannamei (Davis et al., 1993) and P. monodon (Lee and Shiau, 2002). Lin et al. (2010) showed that the utilization of organic chelate copper is better than that of copper sulfate, this may be because organic chelating minerals inhibit the binding of compounds in the feed that cannot be absorbed by the body, such as phytic acid, thereby avoiding the inhibitory effect of the body on mineral absorption (Clearwater et al., 2002). The study evaluated the effects of dietary copper amino acid chelate on the nonspecific immune enzyme activity and hemolymph biochemical parameters of T. tridentatus. It could provide first-hand information for their optimal dietary copper supplementation level assessment and related hemolymph recovery technology.

Table 1 Chemical composition (%) of three experimental diet grops. Crude protein, crude lipid, ash, and carbohydrate content are expressed on a wet weight basis.

Moisture Crude protein Crude lipid Ash Analyzed copper (dry matter, mg kg−1)

Control

Low-level copper

High-level copper

41.71 ± 0.006 50.39 ± 0.029 4.99 ± 0.010 7.93 ± 0.002 5.588

39.95 ± 0.005 47.12 ± 0.016 3.99 ± 0.008 7.55 ± 0.005 53.176

40.62 ± 0.012 45.71 ± 0.003 4.03 ± 0.007 7.43 ± 0.009 109.663

Fig. 1. Effects of different copper supplement levels on plasma Cu/Zn superoxide dismutase (Cu/Zn-SOD) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

cylindrical-length: 5–6 cm; diameter: 0.5 cm) based on a pre-experiment, was adopted as basic diet. Copper amino acid chelate Availa®Cu 100 (Cu content, 10%; Zinpro Corp, USA) was added to such semifinished feed. Three treatment groups were set up: 0 (control group), low-level copper (copper content, 50 mg kg−1), and high-level copper (copper content, 100 mg kg−1) (Table 1). Horseshoe crabs were fed to satiation once daily (17:00) during the experimental period. The experimental conditions were: water temperature, 29–31 °C; salinity, 30–32‰; pH, 8.0–8.2; dissolved oxygen, 6–8 mg L−1; and photoperiod, 12 h (natural light 6:00 a.m.−6:00 PM).

2. Materials and methods 2.1. Experimental animals, feed and experimental conditions The experimental adult horseshoe crabs were purchased from local fishermen in Beihai City, Guangxi Province. Before the experiment, T. tridentatus was stored for 1 month. The adults were fed the same amount of Philippine clam meat (Ruditapes philippinarum) at 5:00 p.m. daily. Their breeding season lasts from June to September (Hong, 2011). Only males were selected as our experimental animals, in order to avoid potential effects of reproduction on tested parameters. After one month of temporary feeding, 27 males with intact appendages, no parasites and similar body weights were randomLy selected (initial body weight, 1454.71 ± 24.47 g; precursor width, 26.69 ± 0.58 cm). Males were divided into nine groups of three replicates and housed in nine circular circulating aquaculture tanks (diameter, 2 m; water depth, 0.9 m). In this experiment, a semi-finished feed (mixture of wheat flour and Philippine clam meat with 1:1.8 ratio, then manually made into

2.2. Sample collection Hemolymph was collected from T. tridentatus at 9:00 a.m. on the 0th, 14th and 28th day of the experiment. The carapace and sternite of horseshoe crabs were bent inward. Horseshoe crabs were repeatedly scrubbed with 70% alcohol before hemolymph samples were collected. A 2 mL syringe with a 22 G needle was inserted into the heart from the joint for hemolymph extraction, and 1 mL of hemolymph was extracted and transferred to a 2 mL sterile centrifuge tube containing 1 mL of 2

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Fig. 2. Effects of different copper supplement levels on plasma catalase (CAT) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Fig. 3. Effects of different copper supplement levels on plasma glutathione peroxidase (GPX) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

precooled heparin anticoagulant (Sigma, USA). After centrifugation at 4 °C and 10,000 rpm for 10 min, the supernatant was extracted. Three hemolymph samples were collected from each horseshoe crab to determine serum nonspecific immune enzymes and hemolymph biochemical indicators.

SOD activity was calculated by measuring the light absorption values of the control tube and measuring tube at 550 nm. One Cu/Zn-SOD activity unit (U) refers to the amount of Cu/Zn-SOD corresponding to 1 mg of tissue protein in a 1 mL reaction solution with 50% reduction in SOD inhibition efficiency. Vitality test of CAT: Addition of ammonium molybdate terminates the reaction of CAT, by decomposing H2O2. The remaining light absorption value of H2O2 and ammonium molybdate at 405 nm denotes the CAT activity of sample. One CAT activity unit (U) refers to the amount of 1 μmol of H2O2 decomposed per second per milliliter of serum. Vitality test of GPX: GPX activity is determined by analyzing the reduction of glutathione (GSH) in reaction of GSH with H2O2 catalyzed by GPX (Kong et al., 2017). Each 0.1 mL of serum was reacted at 37 °C for 5 min to reduce the GSH concentration in the reaction system by 1 mol L−1 as an enzyme activity unit. Vitality test of T-AOC: The ferrous ions produced by the reduction of iron ions under antioxidant oxidation can form a stable complex with morphine. The antioxidant capacity of T-AOC was measured by the change in light absorption at 520 nm (Tang et al., 2005). At 37 °C, the absorbance of the reaction system increased by 0.01 through 1 mg min−1 of tissue protein, denoted as one unit of T-AOC. Vitality test of MDA: MDA, which is the product of thiobarbituric acid and lipid peroxidation, condenses to form a red product. Colorimetry was performed at 532 nm (Buege and Aust, 1978). Vitality test of LZM: LZM hydrolyzes peptidoglycans on the cell wall of bacteria, which causes bacterial lysis, reduces bacterial concentrations, and decreases light absorption. One LZM activity unit (U) refers to the amount of enzyme required to decrease light absorption at 450 nm by 0.001 min−1. Vitality test of AKP: AKP can decompose to form sodium phenyl phosphate, which further produces free phenol and phosphoric acid.

2.3. Assay of experimental diet composition The determination of crude lipid, crude protein, ash and moisture content in diets was referred to Association of Official Analytical assistants Chemists. Moisture was determined by drying the samples to a constant weight at 105 °C. Crude lipid was extracted by the chloroformmethanol method. Crude protein was determined by automatic kjeldahl nitrogen apparatus (Kjeltec-2300, Foss, Sweden). The ash content was determined by high temperature burning in muffle furnace (SX2-410 N, Jiangsu, China) at 550 °C for 6 h. The content of copper in diets was determined by atomic absorption spectrometer (ICAP Q ICP-MS, Thermo Fisher, USA). 2.4. Determination of nonspecific immune enzyme activity Copper–zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPX), total antioxidant capacity (T-AOC), malondialdehyde (MDA), lysozyme (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), triglyceride (TG) and cholesterol (CHO) levels were measured using kits produced by Nanjing Jiancheng Bioengineering Institute, all enzymes and hemocyanin determination using visible spectrophotometer (722s, Shanghai, China). Vitality test of Cu/Zn-SOD: Xanthine oxidase method (Elstner and Heupel, 1976) and xanthine oxidase reaction system were used to generate O2−. O2− produces nitrite by oxidizing hydroxylamine, that shows a purplish red solution under the action of a colorant. Cu/Zn3

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Fig. 4. Effects of different copper supplement levels on plasma total antioxidant capacity (T-AOC) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Fig. 5. Effects of different copper supplement levels on plasma malondialdehyde (MDA) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Under alkaline conditions, the phenol could be reacted with 4-aminoantipyrine to form a red hydrazine derivative by oxidation with potassium ferricyanide. The level of enzyme activity was analyzed on the basis of differences in light absorption at 520 nm. One AKP activity unit (U) refers to the amount of enzyme required to produce 1 mg of phenol when 100 mL of serum is reacted with the substrate for 15 min at 37 °C. Vitality test of ACP: ACP was determined by the same principle employed for AKP determination. One ACP activity unit (U) was defined as the amount of enzyme required to produce 1 mg of phenol when 100 mL of serum is reacted with the substrate for 30 min at 37 °C.

2.6. Data analysis All the data was analyzed with SPSS 22.0 and represented as mean ± standard deviation (SD). Prior to the analysis, normality of the data was evaluated by using the Shapiro–Wilk W test, and homogeneity of variances was checked using Levene's test. Significant differences between groups were tested by one-way analysis of variance (ANOVA). Significant differences were designated at p < 0.05.

3. Results After the four weeks experiment, horseshoe crabs final weight were 1453.06 ± 24.60 g. No T. tridentatus died during the whole experiment, and no significant difference of final body weight among different treatments (p > 0.05). Dry matter, crude lipid, crude protein and ash contents showed no significant differences among different feeds (p > 0.05) (Table .1).

2.5. Determination of blood biochemical indicators Determination of TG content: TG produces glycerol under action of lipase. Glycerol produces hydrogen peroxide under the action of glycerol kinase and phosphoglycerol oxidase and then reacts with aminoantipyrine and phenol to form a red terpenoid under the action of peroxidase. Light absorption at 500 nm was measured as the TG content in sample. Determination of CHO content: CHO is oxidized by CHO oxidase to form hydrogen peroxide, which is then reacted with 4-aminoantipyrine and phenol that constitute red quinone imine. Red quinone imine light absorption value at 500 nm was measured as the CHO content in the sample. Hemocyanin determination: Hemocyanin content was checked by the method of Nickerson and Van Hold (1971). In this method, 10 μL of serum sample was collected and mixed with 990 μL of a diluent (10 mmol L−1 of CaCl2, 50 mmol L−1 of Tris-HCl, pH = 8.0). The optical density (OD) was measured at 334 nm, and the hemocyanin concentration was calculated using the following formula: Hemocyanin concentration (HC, mg mL−1) = 2.33 × OD 334 × 100.

3.1. Cu/Zn-SOD The Cu/Zn-SOD activity of the copper-fed groups increased as time passed by (Fig. 1). The Cu/Zn-SOD activities of the low- and high-level copper groups on day 28 were higher than those on days 0 and 14 (p < 0.05). On day 28, the Cu/Zn-SOD activity of the copper-fed groups was higher than the control group (p < 0.05); among the copper-fed groups, the Cu/Zn-SOD activity of the high-level copper group was the highest (62.22 ± 6.64 U mL−1). No change in Cu/ZnSOD activity was found between the low- and high-level copper groups during the experiment (p > 0.05).

4

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Fig. 6. Effects of different copper supplement levels on plasma lysozyme (LZM) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Fig. 7. Effects of different copper supplement levels on plasma alkaline phosphatase (AKP) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

3.2. CAT

(p < 0.05).

The CAT activity of the copper-fed group increased throughout the experiment (Fig. 2). The CAT activities of the low- and high-level copper groups on day 28 were higher than those on days 0 and 14 (p < 0.05). On day 28, the CAT activity of the copper-fed groups was higher than the control group (p < 0.05). Among those groups at the end of the experiment (day 28), the CAT activity of the low-level copper group was the highest (2.03 ± 0.34 U mL−1); the CAT activity of this group was higher than the control and high-level copper groups (p < 0.05).

3.5. MDA The MDA activity of the low-level copper group decreased throughout the experiment (Fig. 5). On day 28, it reaches a minimum value of 0.50 ± 0.25 nmol mL−1, which was lower than the two other groups. During experiment, MDA activity of the high-level copper group initially decreased and then increased. At the end of the experiment the MDA activity (day 28) was not different as compared with that on day 0 (p > 0.05). On days 14 and 28, MDA activity in the lowand high-level copper groups was lower than the control group (p < 0.05).

3.3. GPX The GPX activity of the copper-fed groups increased during the whole experiment (Fig. 3). On day 14, the GPX activity of the low-level copper group was significantly difference than the control group (p < 0.05). On day 28, the GPX activity of the low-level copper group peaks (94.84 ± 10.57 U mL−1), but no difference in GPX activity was found between the low- and high-level copper groups (p > 0.05). The GPX activity of the two groups was significantly higher than the control group (p < 0.05).

3.6. LZM The LZM content of the copper-fed group increased with time (Fig. 6). On day 14, no significantly differences were found in the LZM content among three groups (p > 0.05). On day 28, LZM content of the low-level copper group was the highest (110.75 ± 10.47 U mL−1), but no significant difference in LZM content was found between the lowand high-level copper groups (p > 0.05). LZM content of two groups was higher than control group (p < 0.05).

3.4. T-AOC 3.7. AKP The T-AOC of the copper-fed group gradually increased during the experiment (Fig. 4). On day 14, the T-AOC of the low-level copper group was significantly difference than the control group (p < 0.05). On day 28, the T-AOC of the low-level copper group peaks (0.30 ± 0.04 mmol L−1), significantly difference in T-AOC activity was found between the low- and high-level copper groups (p < 0.05). The T-AOC of the two groups was significantly higher than control group

The AKP of the copper-fed group gradually increased during the whole experiment (Fig. 7). On days 14 and 28, the AKP content of the high-level copper group was higher than that of the low-level copper and control groups (p < 0.05), peaking on day 28 (6.77 ± 1.00 U L−1). In the low-level copper group, AKP content was higher on day 28 than on days 0 (p < 0.05); whereas no significant difference was found 5

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Fig. 8. Effects of different copper supplement levels on plasma acid phosphatase (ACP) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Fig. 9. Effects of different copper supplement levels on plasma triglyceride (TG) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

between days 0 and 14 (p > 0.05).

3.11. HC

3.8. ACP

The HC of the copper-fed group increased, and the low-level copper group increased more extensively than high-level copper group (Fig. 11). During the experiment (days 14 and 28), the HC in the lowlevel copper group was high than the control group (p < 0.05), but no difference was found between the low- and high-level copper groups (p > 0.05). At the end of the experiment (day 28), the HC of the lowlevel copper group peaks at 1.17 ± 0.09 mg mL−1.

The ACP of the copper-fed group increased with time (Fig. 8). On days 14 and 28, the ACP content of the high-level copper group was higher than that of the low-level copper and control groups (p < 0.05), peaking on day 28 (12.42 ± 0.97U L−1). In the low-level copper group, the AKP content was also significantly higher on day 28 than on day 0 (p < 0.05).

4. Discussion 3.9. TG

SOD enzyme can effectively scavenge reactive oxygen species in living organism and the first and most important defense line in antioxidant systems (Tomanek et al., 2011). SOD is a mature metalloenzyme with different metal elements and are divided into the following three categories: Cu/Zn-SOD, Mn-SOD, and Fe-SOD. These three enzymes can catalyze superoxide anion radicals and disproportionate them into hydrogen peroxide and O2, but show different properties. In Cu/Zn-SOD, copper ions are located in the active center of the enzyme and related to its catalytic action, while zinc ions plays an important role in maintaining structural stability. The activity of Cu/ Zn-SOD enzyme is not determined by enzyme protein but also determined by Cu2+ and Zn2+ content (Sun et al., 2012). CAT activity is related to the elimination of H2O2, that mainly manifests when SOD increment reduces O2− to H2O2. CAT and GPX catalyze the conversion of H2O2 into water, by reducing the activity and inhibiting the increase of active oxygen and hydroxyl free radicals in the body; thus, the body's immune function improves (Bigorgne et al., 2011; Moreira et al., 2016; Sui et al., 2017). GPX uses GSH as cofactor to convert H2O2 into water and alcohol. Animal GPX can act as pro-oxidant that remove harmful peroxide metabolites in cells (Hu et al., 2015b; Peng et al., 2017). T-

The TG content of the copper-fed group gradually decreased with time (Fig. 9). This decrement in the low-level copper group was greater than the high-level copper group. On day 14, the TG content in the copper-fed group was lower than the control group, but no difference was found between the low- and high-level copper groups. On day 28, the TG content in the low-level copper group reaches a minimum value of 1.10 ± 0.18 mmol L−1, which was lower than the control and highlevel copper groups (p < 0.05). 3.10. CHO The CHO content of the copper-fed group decreased with time, and the high-level copper group decreased more extensively than the lowlevel copper group (Fig. 10). During the experiment (days 14 and 28), the CHO content in the high-level copper group was lower in the control group, but no difference was found between the low- and high-level copper groups (p > 0.05). On day 28, the TG content in the high-level copper group reaches a minimum value (0.33 ± 0.03 mmol L−1). 6

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Fig. 10. Effects of different copper supplement levels on plasma cholesterol (CHO) activity (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

Fig. 11. Effects of different copper supplement levels on plasma hemocyanin concentration (HC) (mean ± SD) of male Chinese horseshoe crab. Values with different small letters are significantly different among different treatments in the same sampling time point (p < 0.05). Values with different large letters are significantly different among different sampling time points for the same treatment group (p < 0.05).

AOC can be used as comprehensive index that measure the functional status of the body's antioxidant enzyme and nonenzymatic systems; the amount of T-AOC available represents the capacity of body's antioxidant enzyme and nonenzymatic systems for external stimulation and free radical metabolism state (Hong et al., 2018). LZM is an important regulator of innate immune responses; it attacks the peptidoglycan layer of bacterial cell wall by decomposing β-(1,4)-glycosidically linked N-acetyl succinate and N-acetylglucosamine, resulting in lysis of bacterial cells (Bayarri et al., 2014; Wei et al., 2012). LZM has functions for resisting bacteria, viruses, inflammation (Jash & Kumar, 2014). AKP and ACP participate in the degradation of exogenous proteins, carbohydrates, and lipids, and can catalyze the hydrolysis of various phosphorus-containing compounds as transphosphatase under alkaline and acidic conditions (Liu et al., 2004). AKP is an intrinsic plasma membrane enzyme present in the cell membrane of all animal cells (Blasco et al., 1993). ACP is often used to detect intracellular lysosomes, that kill and digest microbial pathogens during an immune response (Mazorra et al., 2002; Rajalakshmi and Mohandas, 2005; Zhang et al., 2016). In the present study, the serum activities of antioxidant enzymes, namely, Cu/Zn-SOD, CAT, GPX, T-AOC, LZM, AKP, and ACP, in adult horseshoe crab fed with copper-added feed increased, by the uptake of copper ions that promotes the production of these enzymes and improves the antioxidant function of adult horseshoe crab serum. At the end of the experiment, the activities of Cu/Zn-SOD, GPX, and LZM are not different between the low- and high-level groups, that indicate 50 mg kg−1 copper addition may increase the activity of these three antioxidant enzymes to maximum levels. At the end of the experiment, the serum activities of CAT and T-AOC in the low-level copper group are higher than those in the high-level copper group. Therefore, the optimal addition amount of copper in the feed of adult T. tridentatus is approximately 50 mg kg−1, similar to the optimal copper contents in the feeds of Chinese mitten crab Eriocheir sinensis at

20.78–40.34 mg kg−1 (Sun et al., 2012) and Chinese prawn Penaeus chinensis at 53 mg kg−1 (Liu et al., 1990). MDA is lipid peroxidative decomposition product produced by the free-radical damage of unsaturated fatty acids. It can cross-link with proteins and DNA to change protein activity, damage DNA, and induce gene mutations; hereby, its concentration could be used as an indicator of oxidative stress to measure the body's endogenous oxidative damage state (Li et al., 2016). Copper addition caused serum MDA decrease, which may reduce oxidative damage in the body. The MDA of the lowlevel copper group decreased more extensively than in the high-level copper group and continues to decline. Serum MDA in the high-level copper group decreased first and then increased, slightly different from the beginning of the experiment. Our result was consistent with Shao et al. (2012). Hereby, low levels of copper supplement (50 mg kg−1 copper) were helpful for improving antioxidant ability than that of high level copper supplement. Acid phosphatase and AKP are involved in rapid uptake and transport of biological substances. As an immune enzyme in the body, AKP is directly related to the absorption of phosphorus and calcium in seawater of crustaceans, the formation of calcium phosphate, and formation and secretion of chitin. At the same time, it forms an important detoxification system for crustaceans with other hydrolases and plays an important role in crustacean nonspecific immunity. ACP participates in the transfer and metabolism of phosphate groups, enhances the uptake and transport of substances, and forms the material basis for phagocytic sterilization with other enzymes, by destroying and eliminating foreign bodies invading the body. Previous studies on Megalobrama amblycephala (Shao et al., 2012), Macrobrachium nipponensis (Kong et al., 2014), Lateolabrax japonicus (Wang et al., 2015), and Litopenaeus vannamei (Yuan et al., 2019) showed that adding the appropriate amount of copper ions to feed could help for serum AKP and ACP activity improvement in aquatic animals. This supports our finding 7

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that 50 mg kg−1 dietary copper amino acid chelate supplement could increase AKP and ACP activity. Blood biochemical indicators such as TGs, CHO, and total protein are important indicators of animal health (Zhou et al., 2006). The blood biochemical indicators, TG and CHO contents, of the copper-fed group decreased with time, indicating copper addition may have an effect on the lipid metabolism of the body. Hemocyanin, a copper-containing respiratory protein found in the hemolymph lymphocytes of arthropods and mollusks, plays important role in oxygen transport, immune defense, protein storage, and osmotic pressure regulation (Coates et al., 2012; Coates et al., 2011; Engel et al., 2001). Engel et al. (2001) found that HC is high in Homarus americanus in industry areas with high copper content in sediments. Sun et al. (2012) and Kong et al. (2014) found that addition of the appropriate amount of copper to feed could improve hemocyanin levels in E. sinensis and the hepatopancreas of M. nipponensis. Sun et al. (2012) showed that appropriate copper addition also increased the HC in the hepatopancreas and hemolymph of E. sinensis. In present study, the serum HC in the copper-fed group increased with time as well. At the end of experiment, serum HC in the low-level copper group was higher than that in the control group. According to antioxidant enzyme activity and HC dynamic changes, low levels of copper (50 mg kg−1 copper) were more useful for adult T. tridentatus antioxidant capacity improvement than that of high levels (100 mg kg−1 copper).

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Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This research was supported by a grant from 2017 Beihai City 13th Five-Year Plan Marine Economic Innovation and Development Demonstration Project – Tachypleus Amebocyte Lysate and Chinese Horseshoe Crab Ecological Utilization Industry Chain Collaborative Innovation Project (Grant No.: Bhsfs006). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734576. Compliance with ethical standards The authors declare that they have no conflict of interests. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The number of collected animals in our study was as low as possible and the manipulation was fast and painless. During the experiment, the animals were kept alive as long as possible. After the experiment, crabs were fed clam meat twice daily to enhance their recovery. After two weeks all horseshoe crabs were released to the sea. Author contributions Jie Song and Menghong Hu conceived this project. Zhen Xu and Qiongzhen Li performed the research and analyzed the data. Youji Wang, Yasmeen Gul and Menghong Hu wrote the paper. All authors participated in the revision of this paper by providing comments and editing. 8

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