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Enhancing the immune response in the sea cucumber Apostichopus japonicus by addition of Chinese herbs Houttuynia cordata Thunb as a food supplement
Aquaculture and Fisheries 4 (2019) 114–121
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Enhancing the immune response in the sea cucumber Apostichopus japonicus by addition of Chinese herbs Houttuynia cordata Thunb as a food supplement Huifeng Danga,1, Teng Zhanga,1, Fan Yib, Shigen Yea, Juan Liua, Qiang Lic, Hua Lia, Ruijun Lia,∗ a
Agriculture and Rural Affairs Ministry of Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, College of Fisheries, Dalian Ocean University, Dalian 116023, China b Dalian Integrated Traditional Chinese and Western Medicine Hospital, Dalian 116000, China c Department of Ocean Technology, College of Marine and Biology Engineering, Yancheng Institute of Technology, Yancheng 224051, China
ARTICLE INFO
ABSTRACT
Keywords: Apostichopus japonicus Houttuynia cordata Thunb Humoral immunity Cellular immunity
The Chinese herb Houttuynia cordata Thunb (abbreviated as HCT hereafter) has been widely used in human and livestock medical research. However, few studies have tested the effects of HCT in aquaculture systems, particularly in echinoderms. In this study, the impact of HCT was evaluated when used as a feed additive to enhance the immune response of the sea cucumber, Apostichopus japonicus. Two concentrations, 1.5% and 3%, of HCT powder were tested by adding them to the feed. Feeding experiments had a four week duration and each week, the humoral and cellular immunity index of the intestine, tentacles, peristome, and coelomic fluid were analyzed. The results indicated that the percentage of A. japonicus coelomocytes significantly increased after supplementing feed with HCT, and reached a peak after one week of feeding with the 3% HCT supplementation. Analysis of reactive oxygen species (ROS) indicated that HCT in feed caused a notable increase in the coelomocyte ROS concentrations over the experimental period. In both the 1.5% and 3% HCT addition groups, the ROS peaked in the third week and then remained stable. In addition, alkaline phosphatase, acid phosphatase, superoxide dismutase, lysozyme activity of the intestine, tentacles, peristome and coelomic fluid were significantly improved during the four-week test period. In conclusion, 1.5% and 3% HCT added to feed boosts the immune responses of A. japonicus. HCT has potential as an immune enhancer for mariculture of sea cucumbers.
1. Introduction The sea cucumber, Apostichopus japonicus, has a high nutritional value thus it is one of the most important seafood products in northern China. In recent years, sea cucumber farming has increased substantially and this is associated with the increasing occurrence of sea cucumber diseases (Deng et al., 2009; Hua et al., 2010). The abuse of antibiotics for aquaculture of sea cucumber has increased environmental pollution, as well as induced severe food safety issues threatening human health (Goldman, 2004). To solve this problem, from the perspective of prevention and control, alternatives to antibiotics are being tested, such as animal and plant extracts, chemicals, bacterial extracts, polysaccharides, and vitamins. In recent years, the application of immune enhancers in aquaculture has become a widespread practice (Aftabuddin, Siddique, Romkey, & Shelton, 2017; Dawood, Koshio, Ishikawa, & Yokoyama, 2016; Tang et al., 2014). Houttuynia cordata Thunb (HCT) is derived from the grass and dried
roots of plants in the Family Saururaceae. Also known as Ceercao or Yuxingcao (in Chinese), the herb has a slightly cold taste and has detoxifying, diuretic, and other pharmaceutical properties (Kumar, Prasad, & Hemalatha, 2014). HCT as been shown to possess medicinally essential activities such as antileukemic (Chang et al., 2001), antitumor (Kim, Ryu, No, Choi, & Kim, 2001), antiviral (Hayashi, Kamiya, & Hayashi, 1995), antiallergy (Li et al., 2005), antioxidant (Cho et al., 2003) and can also be an adjuvant (Wang et al., 2002). Studies have shown that the active chemical ingredients in HCT are mainly divided into the following categories: volatile constituents (Liang et al., 2005), flavonoids (Xu, Ye, Wang, Yu, & Chen, 2006), organic acids (Yang et al., 2010), alkaloids (Ma et al., 2013), phenols (Meng et al., 2007, 2008; Wei et al., 2011), and others, such as inorganic salts, vitamins, and various metal elements. There are still many unknowns surrounding the different biological functions of HCT ingredients. Although human and livestock HCT research has already attracted more and more attention, there have been only a few studies on the use of HCT in aquaculture.
Corresponding author. 52 Heishijiao Street, Shahekou District, Dalian Ocean University, Dalian 116023, China. E-mail address: [email protected] (R. Li). 1 These authors contributed equally to this work. ∗
https://doi.org/10.1016/j.aaf.2018.12.004 Received 20 June 2018; Received in revised form 13 December 2018; Accepted 19 December 2018 Available online 27 December 2018 2468-550X/ © 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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HCT significantly improved the transformation rate of grass carp lymphocytes and enhanced immune activity in the fish Rachycentron canadum (Ding, Cheng, Mei, Yang, & Li, 2005; Wu, Chen, Ueng, & Nan, 2016). However, the effect of HCT as a supplement for sea cucumber (A. japonicus) immunity has not yet been reported. Thus, in the present study, HCT was added to sea cucumber feeds and the cellular and humoral immune indices determined in order to establish if it can contribute to maintain the health of sea cucumberin aquaculture.
pH = 7.4), and then transferred to a 1.5 mL centrifuge tube. Tissues were homogenized at 4 °C and centrifuged at 7000 ×g for 20 min. The supernatant collected and stored at −80 °C until used for immune-related enzyme activities. The activities of alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lysozyme (LSZ) were measured using a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2. Materials and methods
2.5. Cell immune index
2.1. Experimental animals Healthy sea cucumbers were purchased from Donggang City, Liaoning Province Jiu Chang Aquaculture Co., Ltd., with an average weight of approximately 5 g. A total of 150 sea cucumber were randomly allocated to three groups, maintained in 50 cm × 35 cm × 20 cm PVC tanks. The seawater temperature was 14 ± 2, pH 8 ± 0.12, salinity 30, dissolved oxygen > 6.5 mg/L. Water was changed every morning using filtered seawater from the Ai Ni Farm in Dalian Aquaculture Institute.
2.5.1. Phagocytic activity Coelomocyte phagocytosis experiments were conducted according to Jans et al. (Jans, Dubois, & Jangoux, 1995), with slight modifications. A 2 mg/mL yeast cell suspension was injected into the back of the sea cucumber, and after 4 h, coelomic fluid was drawn with a syringe. One-third of the coelomic fluid was extracted within a 1:1 mixture of anticoagulant fluid (heparin sodium). The cell suspension was observed under high magnification an 100 phagocytic cells were counted to calculate the phagocytic percentage (phagocytic percentage, PP) and phagocytic index (PI):
2.2. Feed preparation
Phagocytic percentage = number of phagocytic cells ingesting yeast cells / phagocytic cells × 100% Phagocytic index = number of yeast cells swallowed by phagocyte/ phagocytic cells ingesting yeast cell
Sea cucumber were fed with basal feed (purchased from the Dalian Fisheries Institute), the main ingredients are shown in Table 1. Experimental HCT was obtained from Dalian Integrated Traditional Chinese and Western Medicine Hospital, then, crushed and passed through a 160 mesh filter. 1.5% and 3% (w/w) HCT powder was added to the feed for sea cucumbers. A granulator was used to produce 0.1 cm diameter particles that were oven dried. The feed was stored in a refrigerator at −20 °C.
2.5.2. Reactive oxygen species of coelom fluid cells Reactive oxygen species were determined using the NBT reduction method with slight modifications (Chen et al., 2005). Weekly samples of coellemocytes were obtained once a week, and the collected coelomic fluid was mixed in a 1:1 ratio with anticoagulant. Coelomocytes were diluted to 2 × 106 cells/mL of the coelomic fluid and divided between a 96-well plates, and centrifuged at 2000×g for 5 min. The supernatant was removed, and 100 μL PMA was added to the coelomocytes. The mixture was placed in a 37 °C water bath for 30 min. After the incubation, 100 μL of 0.4% NBT was added followed by continuouse incubation at 37 °C for 30 min. The 96-well plate was removed and centrifuged at 2000×g for 5 min. The supernatant was poured off, and pure ethanol was added to each well. Plates were washed with 70% ethanol three times, air-dried, and 120 mL 2 M KOH lysis buffer, and 160 mL DMSO was added. After the reaction was complete, the absorbance was measured at a wavelength of 620 nm.
2.3. Experimental group The experiment was divided into three groups: the basal diet group (0%), the 1.5% HCT group and the 3% HCT group. Each group was divided into three parallel experiments, with 25 sea cucumbers randomly placed in each parallel trial. During the test, the water temperature was 14 ± 2 °C, pH was 8 ± 0.12, salinity was 30. Water was changed every morning using filtered seawater from the Ai Ni Farm in Dalian Aquaculture Institute. Each water change represented approximately one-third of the total tank water. Aquaria were continuously supplied with oxygen and feed provided at 9 a.m. and 8 p.m.
2.6. Data processing and analysis
2.4. Humoral immune index
The average mean ± standard deviation (Means ± SD) was obtained using SPSS17.0 for One-Way Analysis of Variance (One-Way ANOVA) and Duncan Multiple Comparison. P < 0.05 was considered to be the significance cut-off.
The feeding experiment with HCT supplemented feed was carried out for four weeks and sea cucumbers sampled once a week. For sampling three sea cucumbers were randomly selected, put on ice and the tentacles, body walls, muscles, and intestines removed. Coelomic fluid was also collected. Collected tissue samples were weighed and homogenized in 1:10 (W/V) of cold phosphate buffered saline (PBS,
3. Results 3.1. Phagocytic activity of A. japonicus coelomocytes
Table 1 Composition of the basal diet. Ingredients
Content
Macroalgae (Sargassum) meal Kelp meal Degummed kelp meal Yeast Fish meal Soybean meal Vitamin premix Mineral premix
20.00 30.00 34.00 8.00 3.00 3.0 1 1
The PP of the A. japonicus coelomocytes (Fig. 1) was significantly increased after supplementing the feed with HCT (P < 0.05) and the 3% HCT group had a peak in the first week. The PP was 17.91% in the control group, 26.09% in the 1.5% HCT group and 31.20% in the 3% HCT group. The PP of the 3% HCT group was generally higher than the 1.5% HCT group. The PI of coelomocytes of the experimental group fluctuated over time and the difference between the groups was not significant, indicating that the PI of the coelomocytes was not substantially changed after HCT feeding (Fig. 2). 115
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Fig. 1. Phagocytic percentage of coelomocytes in A. japonicus (400 × ). Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
3.2. Detection of ROS production in coelomocytes
Compared with these four detected tissues, The SOD activity in the coelomic fluid was higher than in the other tissues. Moreover, in coelomic fluid 3% HCT significantly enhanced the SOD activity relative to the 1.5% HCT supplementation.
The ROS detection results (Fig. 3) indicated that HCT increased the ROS concentration of A. japonicus coelomocytes during the experimental period. In both the 1.5% and 3% HCT groups ROS peaked in the third week and was stable thereafter.
3.4. ACP activity determination
3.3. Determination of SOD activity in A. japonicus
Fig. 5 showed the changing trends of ACP activity in various tissues of A. japonicus after HCT addition. Overall, ACP activity in the intestine, tentacles, peristome and coelomic fluid was significantly modified during the four-week dietary supplementation. Notably, in the intestine the ACP activity peaked after one-week dietary supplementation with
The SOD activity changed in various tissues of A. japonicus (Fig. 4). The activity of SOD in the intestine, tentacles, perisome, and coelomic fluid was significantly higher after HCT dietary supplementation.
Fig. 2. The influence of HCT on phagocytosis index of coelomocytes in A. japonicus. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 116
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Fig. 3. The influence of HCT on reactive oxygen species (ROS) production of coelomocytes in sea cucumber A. japonicus. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
Fig. 4. SOD activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 117
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Fig. 5. ACP activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
3% HCT. I In the tentacles, peristome, and coelomic fluid, ACP activity peaked after three weeks of dietary supplementation with 1.5% HCT.
intestine, tentacles, peristome and coelomic fluid was enhanced during the four-week experiment. The intestine AKP activity peaked after two weeks dietary supplementation with 1.5% HCT, and tentacle AKP activity peaked after two weeks dietary supplementation with 3% HCT. Peristome AKP activity reached a peak in the third week after 1.5% HCT supplementation. The coelomic fluid AKP activity reached a
3.5. AKP in various A. japonicus tissues The changes in AKP activity are shown in Fig. 6. AKP activity in the
Fig. 6. AKP activity in A. japonicus intestine. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 118
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Fig. 7. LSZ activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
In this study, we found that HCT could significantly improve the respiratory burst activity of the coelomocytes, and could change the trend of the reactive oxygen species. This is similar to that of coelomocyte phagocytosis since coelomocyte phagocytosis releases a large number of reactive oxygen species to eliminate foreign substances. It has been shown that neutrophils, multinucleated leukocytes, monocytes, and macrophages can produce large amounts of superoxide anion and reactive oxygen species in the activated state, which all have high reactivity. This cause damage and death of microorganisms (WitkoSarsat, Rieu, Descamps-Latscha, Lesavre, & Halbwachs-Mecarelli, 2000). In the respiratory burst process, the number of reactive oxygen species directly reflects the blood cell sterilization function (Wang et al., 2013). However, at present, we know little about the molecular mechanisms of how HCT improves the respiratory burst activity of Echinoderm coelomocytes. The results showed that HCT had significant effects on the activity of SOD in the intestine, tentacles, peristome, and coelomic fluid (P < 0.05). Increased production of reactive oxygen species can cause significant tissue damage in the body. An antioxidant system is needed to maintain the balance of free radicals. SOD is an essential antioxidant enzyme that can be used as an active oxygen scavenger for the removal of excess free radicals in the body. The modification in SOD is closely related to the change of reactive oxygen species in the body. The shift in SOD activity may be associated with the production of reactive oxygen species and the mechanism of antioxidant activity (Afonso, Champy, Mitrovic, Collin, & Lomri, 2007). HCT has excellent antioxidant activity in mammals in vitro and has noticeable scavenging effects on hydroxyl radicals and superoxide radicals (Lin et al., 2013). Moreover, HCT could improve the SOD activity of mouse serum, accompanied by decreasing the malondialdehyde (MDA) content in liver tissue (Tian et al., 2012). Taken together, these results may indicate that HCT improves antioxidant activity in vivo. LSZ is an alkaline enzyme capable of hydrolyzing viscous polysaccharides present in the pathogens and it has anti-inflammatory, antibacterial, and antiviral effects (Van et al., 2012). Lysosomal enzymes
maximum after three-weeks supplementation with 3% HCT. 3.6. LSZ activity assay Fig. 7 shows LSZ activity in various tissues after dietary supplementation with HCT. LSZ activity in the intestine, tentacles, peristome and coelomic fluid were significantly modified during the four-week experiment. LSZ activity in the intestine and tentacles peaked after twoweeks of dietary supplementation with 3% HCT. LSZ activity in the peristome peaked after one-week supplementation with 3% HCT. Coelomic fluid LSZ activity peaked in the first week after 1.5% HCT dietary supplementation. 4. Discussion A. japonicus, in common with other Echinoderms, does not possess adaptive immunity but relies primarily on non-specific immunity (Xue et al., 2015). Coelomocytes of A. japonicus, play a crucial role in nonspecific immunity as an essential defense system for phagocytosis and killing of foreign substances (Ramirez-Gomez, Aponte-Rivera, MendezCastaner, & Garcia-Arraras, 2010). The phagocytic activity of coelomic cells is a relevant index to evaluate the immunity of A. japonicus (Vazzana et al., 2017). In the present study, showed that four-week supplementation with HCT could significantly improve the PP of A. japonicus coelomocytes. The highest rate of PP in the first week was 17.91%, 26.09%, 31.20% in the control group and the 1.5% HCT group and 3% HCT group, respectively. The activity of PP increased approximately 1.5–2 times that of the control group. PP was 3% > 1.5% > control group and revealed that HCT addition increased coelomocyte phagocytic activity. Our experimental results are in good agreement with the effects of HCT reported in humans. Studies by Satthakarn, Chung, Promsong, and Nittayananta (2015) confirmed that HCT could significantly modulate oral innate immune mediators. Wan et al (2016) also found that HCT could down-regulate serum levels of interleukin-6 (IL-6) in oxaliplatin-treated rats. 119
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have been found in sea cucumber phagocytes, which are composed of LSZ, ACP, and AKP, which are involved in the degradation of foreign substances (Liu et al., 2012). The present study revealed that HCT had significant enhancement effects on LSZ in all tissues; all samples reached the highest levels in the first or second week and decreased in the third week. The fourth week showed no significant difference with the control group which was kept at a low level. In alignment with the literature, LSZ was shown to be the critical enzyme involved in dissolving the pathogen. Our results indicated that HCT might directly increase the capacity of A. japonicus to combat pathogens. HCT contains a number of active molecules with different biological functions. Studies have shown that the antibacterial action of HCT is due to Houttuynin (decanoyl acetaldehyde) in its volatile oil (Duan et al., 2008). Houttuynin can be used to synthesize Sodium Houttuyfonate with sodium bisulfite. Sodium Houttuyfonate has an excellent antibacterial properties on gram-positive bacteria (Guan et al., 2013; Zhao et al., 2005) and gram-negative bacteria (Cheng et al., 2012; Liu et al., 2013). In addition, the volatile oil of HCT is a novel and selective COX-2 inhibitor with anti-inflammatory activity (Li, Zhou, Zhang, & He, 2011). Likewise flavonoids exert anti-tumor effects (Fan, Qu, Li, & Sun, 2008; Xue et al., 2013), and chlorogenic acid and its derivatives have been known to exhibit antioxidation effects (Nuengchamnong, Krittasilp, & Ingkaninan, 2009). In the present study, whole dry HCT powder was used as a feed additive to enhance the immune response in A. japonicus. It remains to be established which active ingredient produced the effect of HCT in the sea cucumber. In conclusion, HCT can significantly improve the immunity of A. japonicus through modifying cellular immunity and antioxidant capacity. The response time and effect of HCT varied with tissue and this may be related to their function and the immune enzymes. The results of this study showed that HCT can be used as a potential immune enhancer in the process of sea cucumber culture.
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Enhancing the immune response in the sea cucumber Apostichopus japonicus by addition of Chinese herbs Houttuynia cordata Thunb as a food supplement Huifeng Danga,1, Teng Zhanga,1, Fan Yib, Shigen Yea, Juan Liua, Qiang Lic, Hua Lia, Ruijun Lia,∗ a
Agriculture and Rural Affairs Ministry of Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, College of Fisheries, Dalian Ocean University, Dalian 116023, China b Dalian Integrated Traditional Chinese and Western Medicine Hospital, Dalian 116000, China c Department of Ocean Technology, College of Marine and Biology Engineering, Yancheng Institute of Technology, Yancheng 224051, China
ARTICLE INFO
ABSTRACT
Keywords: Apostichopus japonicus Houttuynia cordata Thunb Humoral immunity Cellular immunity
The Chinese herb Houttuynia cordata Thunb (abbreviated as HCT hereafter) has been widely used in human and livestock medical research. However, few studies have tested the effects of HCT in aquaculture systems, particularly in echinoderms. In this study, the impact of HCT was evaluated when used as a feed additive to enhance the immune response of the sea cucumber, Apostichopus japonicus. Two concentrations, 1.5% and 3%, of HCT powder were tested by adding them to the feed. Feeding experiments had a four week duration and each week, the humoral and cellular immunity index of the intestine, tentacles, peristome, and coelomic fluid were analyzed. The results indicated that the percentage of A. japonicus coelomocytes significantly increased after supplementing feed with HCT, and reached a peak after one week of feeding with the 3% HCT supplementation. Analysis of reactive oxygen species (ROS) indicated that HCT in feed caused a notable increase in the coelomocyte ROS concentrations over the experimental period. In both the 1.5% and 3% HCT addition groups, the ROS peaked in the third week and then remained stable. In addition, alkaline phosphatase, acid phosphatase, superoxide dismutase, lysozyme activity of the intestine, tentacles, peristome and coelomic fluid were significantly improved during the four-week test period. In conclusion, 1.5% and 3% HCT added to feed boosts the immune responses of A. japonicus. HCT has potential as an immune enhancer for mariculture of sea cucumbers.
1. Introduction The sea cucumber, Apostichopus japonicus, has a high nutritional value thus it is one of the most important seafood products in northern China. In recent years, sea cucumber farming has increased substantially and this is associated with the increasing occurrence of sea cucumber diseases (Deng et al., 2009; Hua et al., 2010). The abuse of antibiotics for aquaculture of sea cucumber has increased environmental pollution, as well as induced severe food safety issues threatening human health (Goldman, 2004). To solve this problem, from the perspective of prevention and control, alternatives to antibiotics are being tested, such as animal and plant extracts, chemicals, bacterial extracts, polysaccharides, and vitamins. In recent years, the application of immune enhancers in aquaculture has become a widespread practice (Aftabuddin, Siddique, Romkey, & Shelton, 2017; Dawood, Koshio, Ishikawa, & Yokoyama, 2016; Tang et al., 2014). Houttuynia cordata Thunb (HCT) is derived from the grass and dried
roots of plants in the Family Saururaceae. Also known as Ceercao or Yuxingcao (in Chinese), the herb has a slightly cold taste and has detoxifying, diuretic, and other pharmaceutical properties (Kumar, Prasad, & Hemalatha, 2014). HCT as been shown to possess medicinally essential activities such as antileukemic (Chang et al., 2001), antitumor (Kim, Ryu, No, Choi, & Kim, 2001), antiviral (Hayashi, Kamiya, & Hayashi, 1995), antiallergy (Li et al., 2005), antioxidant (Cho et al., 2003) and can also be an adjuvant (Wang et al., 2002). Studies have shown that the active chemical ingredients in HCT are mainly divided into the following categories: volatile constituents (Liang et al., 2005), flavonoids (Xu, Ye, Wang, Yu, & Chen, 2006), organic acids (Yang et al., 2010), alkaloids (Ma et al., 2013), phenols (Meng et al., 2007, 2008; Wei et al., 2011), and others, such as inorganic salts, vitamins, and various metal elements. There are still many unknowns surrounding the different biological functions of HCT ingredients. Although human and livestock HCT research has already attracted more and more attention, there have been only a few studies on the use of HCT in aquaculture.
Corresponding author. 52 Heishijiao Street, Shahekou District, Dalian Ocean University, Dalian 116023, China. E-mail address: [email protected] (R. Li). 1 These authors contributed equally to this work. ∗
https://doi.org/10.1016/j.aaf.2018.12.004 Received 20 June 2018; Received in revised form 13 December 2018; Accepted 19 December 2018 Available online 27 December 2018 2468-550X/ © 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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HCT significantly improved the transformation rate of grass carp lymphocytes and enhanced immune activity in the fish Rachycentron canadum (Ding, Cheng, Mei, Yang, & Li, 2005; Wu, Chen, Ueng, & Nan, 2016). However, the effect of HCT as a supplement for sea cucumber (A. japonicus) immunity has not yet been reported. Thus, in the present study, HCT was added to sea cucumber feeds and the cellular and humoral immune indices determined in order to establish if it can contribute to maintain the health of sea cucumberin aquaculture.
pH = 7.4), and then transferred to a 1.5 mL centrifuge tube. Tissues were homogenized at 4 °C and centrifuged at 7000 ×g for 20 min. The supernatant collected and stored at −80 °C until used for immune-related enzyme activities. The activities of alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lysozyme (LSZ) were measured using a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2. Materials and methods
2.5. Cell immune index
2.1. Experimental animals Healthy sea cucumbers were purchased from Donggang City, Liaoning Province Jiu Chang Aquaculture Co., Ltd., with an average weight of approximately 5 g. A total of 150 sea cucumber were randomly allocated to three groups, maintained in 50 cm × 35 cm × 20 cm PVC tanks. The seawater temperature was 14 ± 2, pH 8 ± 0.12, salinity 30, dissolved oxygen > 6.5 mg/L. Water was changed every morning using filtered seawater from the Ai Ni Farm in Dalian Aquaculture Institute.
2.5.1. Phagocytic activity Coelomocyte phagocytosis experiments were conducted according to Jans et al. (Jans, Dubois, & Jangoux, 1995), with slight modifications. A 2 mg/mL yeast cell suspension was injected into the back of the sea cucumber, and after 4 h, coelomic fluid was drawn with a syringe. One-third of the coelomic fluid was extracted within a 1:1 mixture of anticoagulant fluid (heparin sodium). The cell suspension was observed under high magnification an 100 phagocytic cells were counted to calculate the phagocytic percentage (phagocytic percentage, PP) and phagocytic index (PI):
2.2. Feed preparation
Phagocytic percentage = number of phagocytic cells ingesting yeast cells / phagocytic cells × 100% Phagocytic index = number of yeast cells swallowed by phagocyte/ phagocytic cells ingesting yeast cell
Sea cucumber were fed with basal feed (purchased from the Dalian Fisheries Institute), the main ingredients are shown in Table 1. Experimental HCT was obtained from Dalian Integrated Traditional Chinese and Western Medicine Hospital, then, crushed and passed through a 160 mesh filter. 1.5% and 3% (w/w) HCT powder was added to the feed for sea cucumbers. A granulator was used to produce 0.1 cm diameter particles that were oven dried. The feed was stored in a refrigerator at −20 °C.
2.5.2. Reactive oxygen species of coelom fluid cells Reactive oxygen species were determined using the NBT reduction method with slight modifications (Chen et al., 2005). Weekly samples of coellemocytes were obtained once a week, and the collected coelomic fluid was mixed in a 1:1 ratio with anticoagulant. Coelomocytes were diluted to 2 × 106 cells/mL of the coelomic fluid and divided between a 96-well plates, and centrifuged at 2000×g for 5 min. The supernatant was removed, and 100 μL PMA was added to the coelomocytes. The mixture was placed in a 37 °C water bath for 30 min. After the incubation, 100 μL of 0.4% NBT was added followed by continuouse incubation at 37 °C for 30 min. The 96-well plate was removed and centrifuged at 2000×g for 5 min. The supernatant was poured off, and pure ethanol was added to each well. Plates were washed with 70% ethanol three times, air-dried, and 120 mL 2 M KOH lysis buffer, and 160 mL DMSO was added. After the reaction was complete, the absorbance was measured at a wavelength of 620 nm.
2.3. Experimental group The experiment was divided into three groups: the basal diet group (0%), the 1.5% HCT group and the 3% HCT group. Each group was divided into three parallel experiments, with 25 sea cucumbers randomly placed in each parallel trial. During the test, the water temperature was 14 ± 2 °C, pH was 8 ± 0.12, salinity was 30. Water was changed every morning using filtered seawater from the Ai Ni Farm in Dalian Aquaculture Institute. Each water change represented approximately one-third of the total tank water. Aquaria were continuously supplied with oxygen and feed provided at 9 a.m. and 8 p.m.
2.6. Data processing and analysis
2.4. Humoral immune index
The average mean ± standard deviation (Means ± SD) was obtained using SPSS17.0 for One-Way Analysis of Variance (One-Way ANOVA) and Duncan Multiple Comparison. P < 0.05 was considered to be the significance cut-off.
The feeding experiment with HCT supplemented feed was carried out for four weeks and sea cucumbers sampled once a week. For sampling three sea cucumbers were randomly selected, put on ice and the tentacles, body walls, muscles, and intestines removed. Coelomic fluid was also collected. Collected tissue samples were weighed and homogenized in 1:10 (W/V) of cold phosphate buffered saline (PBS,
3. Results 3.1. Phagocytic activity of A. japonicus coelomocytes
Table 1 Composition of the basal diet. Ingredients
Content
Macroalgae (Sargassum) meal Kelp meal Degummed kelp meal Yeast Fish meal Soybean meal Vitamin premix Mineral premix
20.00 30.00 34.00 8.00 3.00 3.0 1 1
The PP of the A. japonicus coelomocytes (Fig. 1) was significantly increased after supplementing the feed with HCT (P < 0.05) and the 3% HCT group had a peak in the first week. The PP was 17.91% in the control group, 26.09% in the 1.5% HCT group and 31.20% in the 3% HCT group. The PP of the 3% HCT group was generally higher than the 1.5% HCT group. The PI of coelomocytes of the experimental group fluctuated over time and the difference between the groups was not significant, indicating that the PI of the coelomocytes was not substantially changed after HCT feeding (Fig. 2). 115
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Fig. 1. Phagocytic percentage of coelomocytes in A. japonicus (400 × ). Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
3.2. Detection of ROS production in coelomocytes
Compared with these four detected tissues, The SOD activity in the coelomic fluid was higher than in the other tissues. Moreover, in coelomic fluid 3% HCT significantly enhanced the SOD activity relative to the 1.5% HCT supplementation.
The ROS detection results (Fig. 3) indicated that HCT increased the ROS concentration of A. japonicus coelomocytes during the experimental period. In both the 1.5% and 3% HCT groups ROS peaked in the third week and was stable thereafter.
3.4. ACP activity determination
3.3. Determination of SOD activity in A. japonicus
Fig. 5 showed the changing trends of ACP activity in various tissues of A. japonicus after HCT addition. Overall, ACP activity in the intestine, tentacles, peristome and coelomic fluid was significantly modified during the four-week dietary supplementation. Notably, in the intestine the ACP activity peaked after one-week dietary supplementation with
The SOD activity changed in various tissues of A. japonicus (Fig. 4). The activity of SOD in the intestine, tentacles, perisome, and coelomic fluid was significantly higher after HCT dietary supplementation.
Fig. 2. The influence of HCT on phagocytosis index of coelomocytes in A. japonicus. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 116
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Fig. 3. The influence of HCT on reactive oxygen species (ROS) production of coelomocytes in sea cucumber A. japonicus. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
Fig. 4. SOD activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 117
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Fig. 5. ACP activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
3% HCT. I In the tentacles, peristome, and coelomic fluid, ACP activity peaked after three weeks of dietary supplementation with 1.5% HCT.
intestine, tentacles, peristome and coelomic fluid was enhanced during the four-week experiment. The intestine AKP activity peaked after two weeks dietary supplementation with 1.5% HCT, and tentacle AKP activity peaked after two weeks dietary supplementation with 3% HCT. Peristome AKP activity reached a peak in the third week after 1.5% HCT supplementation. The coelomic fluid AKP activity reached a
3.5. AKP in various A. japonicus tissues The changes in AKP activity are shown in Fig. 6. AKP activity in the
Fig. 6. AKP activity in A. japonicus intestine. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05. 118
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Fig. 7. LSZ activity in various A. japonicus tissues. Note: I: control group; II: 1.5% HCT addition group; III: 3% HCT addition group; w: experiment week. Error bars represent SD; P < 0.05.
In this study, we found that HCT could significantly improve the respiratory burst activity of the coelomocytes, and could change the trend of the reactive oxygen species. This is similar to that of coelomocyte phagocytosis since coelomocyte phagocytosis releases a large number of reactive oxygen species to eliminate foreign substances. It has been shown that neutrophils, multinucleated leukocytes, monocytes, and macrophages can produce large amounts of superoxide anion and reactive oxygen species in the activated state, which all have high reactivity. This cause damage and death of microorganisms (WitkoSarsat, Rieu, Descamps-Latscha, Lesavre, & Halbwachs-Mecarelli, 2000). In the respiratory burst process, the number of reactive oxygen species directly reflects the blood cell sterilization function (Wang et al., 2013). However, at present, we know little about the molecular mechanisms of how HCT improves the respiratory burst activity of Echinoderm coelomocytes. The results showed that HCT had significant effects on the activity of SOD in the intestine, tentacles, peristome, and coelomic fluid (P < 0.05). Increased production of reactive oxygen species can cause significant tissue damage in the body. An antioxidant system is needed to maintain the balance of free radicals. SOD is an essential antioxidant enzyme that can be used as an active oxygen scavenger for the removal of excess free radicals in the body. The modification in SOD is closely related to the change of reactive oxygen species in the body. The shift in SOD activity may be associated with the production of reactive oxygen species and the mechanism of antioxidant activity (Afonso, Champy, Mitrovic, Collin, & Lomri, 2007). HCT has excellent antioxidant activity in mammals in vitro and has noticeable scavenging effects on hydroxyl radicals and superoxide radicals (Lin et al., 2013). Moreover, HCT could improve the SOD activity of mouse serum, accompanied by decreasing the malondialdehyde (MDA) content in liver tissue (Tian et al., 2012). Taken together, these results may indicate that HCT improves antioxidant activity in vivo. LSZ is an alkaline enzyme capable of hydrolyzing viscous polysaccharides present in the pathogens and it has anti-inflammatory, antibacterial, and antiviral effects (Van et al., 2012). Lysosomal enzymes
maximum after three-weeks supplementation with 3% HCT. 3.6. LSZ activity assay Fig. 7 shows LSZ activity in various tissues after dietary supplementation with HCT. LSZ activity in the intestine, tentacles, peristome and coelomic fluid were significantly modified during the four-week experiment. LSZ activity in the intestine and tentacles peaked after twoweeks of dietary supplementation with 3% HCT. LSZ activity in the peristome peaked after one-week supplementation with 3% HCT. Coelomic fluid LSZ activity peaked in the first week after 1.5% HCT dietary supplementation. 4. Discussion A. japonicus, in common with other Echinoderms, does not possess adaptive immunity but relies primarily on non-specific immunity (Xue et al., 2015). Coelomocytes of A. japonicus, play a crucial role in nonspecific immunity as an essential defense system for phagocytosis and killing of foreign substances (Ramirez-Gomez, Aponte-Rivera, MendezCastaner, & Garcia-Arraras, 2010). The phagocytic activity of coelomic cells is a relevant index to evaluate the immunity of A. japonicus (Vazzana et al., 2017). In the present study, showed that four-week supplementation with HCT could significantly improve the PP of A. japonicus coelomocytes. The highest rate of PP in the first week was 17.91%, 26.09%, 31.20% in the control group and the 1.5% HCT group and 3% HCT group, respectively. The activity of PP increased approximately 1.5–2 times that of the control group. PP was 3% > 1.5% > control group and revealed that HCT addition increased coelomocyte phagocytic activity. Our experimental results are in good agreement with the effects of HCT reported in humans. Studies by Satthakarn, Chung, Promsong, and Nittayananta (2015) confirmed that HCT could significantly modulate oral innate immune mediators. Wan et al (2016) also found that HCT could down-regulate serum levels of interleukin-6 (IL-6) in oxaliplatin-treated rats. 119
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have been found in sea cucumber phagocytes, which are composed of LSZ, ACP, and AKP, which are involved in the degradation of foreign substances (Liu et al., 2012). The present study revealed that HCT had significant enhancement effects on LSZ in all tissues; all samples reached the highest levels in the first or second week and decreased in the third week. The fourth week showed no significant difference with the control group which was kept at a low level. In alignment with the literature, LSZ was shown to be the critical enzyme involved in dissolving the pathogen. Our results indicated that HCT might directly increase the capacity of A. japonicus to combat pathogens. HCT contains a number of active molecules with different biological functions. Studies have shown that the antibacterial action of HCT is due to Houttuynin (decanoyl acetaldehyde) in its volatile oil (Duan et al., 2008). Houttuynin can be used to synthesize Sodium Houttuyfonate with sodium bisulfite. Sodium Houttuyfonate has an excellent antibacterial properties on gram-positive bacteria (Guan et al., 2013; Zhao et al., 2005) and gram-negative bacteria (Cheng et al., 2012; Liu et al., 2013). In addition, the volatile oil of HCT is a novel and selective COX-2 inhibitor with anti-inflammatory activity (Li, Zhou, Zhang, & He, 2011). Likewise flavonoids exert anti-tumor effects (Fan, Qu, Li, & Sun, 2008; Xue et al., 2013), and chlorogenic acid and its derivatives have been known to exhibit antioxidation effects (Nuengchamnong, Krittasilp, & Ingkaninan, 2009). In the present study, whole dry HCT powder was used as a feed additive to enhance the immune response in A. japonicus. It remains to be established which active ingredient produced the effect of HCT in the sea cucumber. In conclusion, HCT can significantly improve the immunity of A. japonicus through modifying cellular immunity and antioxidant capacity. The response time and effect of HCT varied with tissue and this may be related to their function and the immune enzymes. The results of this study showed that HCT can be used as a potential immune enhancer in the process of sea cucumber culture.
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