Enhancement of non-specific immune response in sea cucumber (Apostichopus japonicus) by Astragalus membranaceus and its polysaccharides

Fish & Shellfish Immunology 27 (2009) 757–762

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Enhancement of non-specific immune response in sea cucumber (Apostichopus japonicus) by Astragalus membranaceus and its polysaccharides Tingting Wang a, Yongxin Sun b, Liji Jin a, c, Yongping Xu a, c, *, Li Wang a, Tongjun Ren d, Kailai Wang a a

Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, People’s Republic of China Dalian Biotechnology Research Institute, Liaoning Academy of Agricultural Sciences, Dalian 116024, People’s Republic of China c Ministry of Education Center for Food Safety of Animal Origin, Dalian 116024, People’s Republic of China d Life Science College, Dalian Fishery University, Dalian 116023, People’s Republic of China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 June 2009 Received in revised form 1 September 2009 Accepted 3 September 2009 Available online 10 September 2009

In this study, the immunostimulatory effect of oral administration of different preparations (conventional fine powder [CP] and superfine powder [SP]) of Astragalus membranaceus root or its polysaccharides (APS) in sea cucumber (Apostichopus japonicus) was investigated. Sea cucumbers with an average initial weight of 49.3  5.65 g were fed with a diet containing 3% CP or SP or 0.3% APS over a period of 60 days. The non-specific humoral (phenoloxidase, lysozyme and agglutination titer) and cellular (phagocytic capacity and reactive oxygen species) responses were determined and compared with controls (no supplement) after 20, 40 and 60 days of feeding. Variation in the levels of responses was evident among different supplements. SP and APS significantly enhanced most of the immune parameters tested. Among the humoral responses, lysozyme activity significantly increased after feeding with SP-supplemented diet for 20, 40 or 60 days. Furthermore, lectin titer showed significant enhancement after 20 and 60 days of feeding with APS-supplemented diet. Significant increase in the production of reactive oxygen species was evident for all three supplements after 20 days of feeding, but no significant change in serum phenoloxidase activity was observed for any of the three supplements over the three different periods. Overall, significant modulation of the cellular responses was only noticed after 20 days of feeding with SP- or APS-supplemented diet. After 60 days, these two groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with Vibrio splendidus. These results indicated that dietary intake containing A. membranaceus root or its polysaccharides could enhance the immune responses of A. japonicus and improve its resistance to infection by V. splendidus. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Polysaccharide Astragalus membranaceus Apostichopus japonicus Non-specific immune response Disease resistance

1. Introduction Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China [1]. However, infectious diseases are becoming a severe problem with increasing culturing. Disease caused by Vibrio splendidus is most widespread in sea cucumber farming [2]. Antibiotics and chemotherapeutics used to control these diseases can result in the development of drug-resistant bacteria, environmental pollution and unwanted residues in aquaculture [3]. One of the most promising methods for controlling sea cucumber diseases in aquaculture is by strengthening their defense mechanisms through prophylactic administration of immunostimulants [4].

* Corresponding author at: Dalian University of Technology, Department of Bioscience and Biotechnology, No. 2 Linggong Road, Ganjingzi District, Dalian 116024, PR China. Tel.: þ86 411 8470 6359; fax: þ86 411 8470 6359. E-mail address: [email protected] (Y. Xu). 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.09.002

Chinese herbs have been used as immunostimulants in human for thousands of years in China. Recently, much attention has been paid to the immune stimulating function of some herbs in aquaculture. Shrimps and fishes fed with diets containing certain Chinese herbs were reported to show improved non-specific immunity, such as bacteriolytic activity and leukocyte function [5–8]. Among the many herbs used in Traditional Chinese Medicine, the root of Astragalus membranaceus has been used as an immune booster for nearly 2000 years, and it has been shown to have significant immunostimulatory effects [9–11]. A. membranaceus has been reported to significantly improve the non-specific immunity of fish [12,13]. A. membranaceus polysaccharide (APS) is one of the major active substances of A. membranaceus, and it plays an important role in the specific and non-specific immune responses [14]. It can activate mouse B cells and macrophages [15]. Although A. membranaceus and APS have been shown to enhance the nonspecific immunity of some animals, their immunostimulatory effects in sea cucumber have not been investigated.

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Our previous work has revealed that APS could significantly promote phagocytosis and superoxide anion (O 2 ) production in A. japonicus coelomocytes in vitro at 22 and 25  C [16]. In this study we investigated the effects of A. membranaceus root (prepared by conventional and superfine milling) or APS on the non-specific immune responses of A. japonicus as well as its resistance to disease caused by V. splendidus. 2. Materials and methods 2.1. Collection and maintenance of animals Sea cucumbers (49.3  5.65 g) were collected from an aquaculture farm in China, held in a recirculation system in Key Laboratory of Marine Culture and Biotechnology of Agriculture Ministry, Dalian Fisheries University (DLFU, Dalian, China), and kept in recirculating aquarium tanks (100 cm  30 cm  40 cm) at water temperature of 15–19  C. During the experiment, the pH of water was maintained at 7.8–8.2, and the salinity at 31–32&. 2.2. Immunostimulant preparation

Phagocytic activity was evaluated by the method of Barracco et al. [20] and Orda´s et al. [21] with slight modifications. Briefly, yeast cells (Saccharomyces cerevisiae) were washed in filtered seawater two times and harvested by centrifugation at 3000  g for 5 min, and then resuspended in filtered seawater. A calibrated concentration of 10.0  2.0 yeast/phagocytic amoebocytes for each animal was prepared in sterilized microcentrifuge tube. For the phagocytic activity assay, 0.1 ml of fresh coelomic fluid was added to 0.l ml of yeast suspension, and after thorough mixing, 0.15 ml aliquot was dispensed onto a glass slide. The slide was placed immediately into an aluminum box with wet filter paper and incubated at 17–20  C for 1 h. The slide was gently washed twice with filtered seawater, and the adherent phagocytic amoebocytes (PA) were fixed for 3 min with methanol for enumeration. Phagocytic capacity was determined as described by Silva and Peck [22]:

Phagocytic capacity ¼

No: of PA with yeast inside  100% Total no: of PA

2.5. Reactive oxygen species (ROS) production

A. membranaceus was purchased from the local Nepstar Chain Drugstore (Dalian, China). The roots were removed, washed with water, and then dried at 40  C. The dry roots were processed in a laboratory pulverizer and passed through a 0.25 mm aperture sieve. This product was designated as CP. CP was further processed with WZJ(BFM)-6J vibrational mill (Billionpower Tech & Engineering Co. Ltd. China) to yield a superfine form, designated as SP. The particle size of SP was about 48 mm. APS (purity: 70%) was commercially obtained from Xian, Tianyuan Biological Preparation Industry and Trade Ltd, China. Basal diet without supplement or supplemented with 3% (w/w) CP or SP or 0.3% (w/w) APS according to the common dose in aquaculture [17–19], and used as feed. The nutritional compositions of each diet supplement are shown in Table 1. 2.3. Experimental design and sampling procedure Water flow of the recirculating system was maintained at 625 ml/min. One third of the water volume of the recirculating system was replaced by fresh seawater once a day to maintain the water quality. Sea cucumbers were randomly divided into four treatments, with each treatment consisted of three tanks, with 15 animals per tank. The animals were fed with either basal diet only (control group), basal diet supplemented with CP (CP group), SP (SP group) or APS (APS group) at a rate of 3% body weight per day for 60 days. Coelomic fluid was collected from each individual sea cucumber by tail-cutting method. For serum separation, the collected coelomic fluid was spun down at 3000  g for 10 min at 4  C. The supernatant was stored in sterile microcentrifuge tubes at 70  C until use.

Intracellular ROS production was measured according to Chen et al. [23]. The coelmocytes collected from coelomic fluid by centrifugation were adjusted to 5  106 cells/ml with culture medium (5% foetal bovine serum, 0.5% penicillin/streptomycin solution in filtered (0.22 mm) seawater). Five hundred microliters of diluted coelmocytes was mixed with 89 ml of culture medium, and nitroblue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) were then added to a final concentration of 0.1% and 0.01 mM, respectively, and the sample was incubated at room temperature for 1 h. After incubation the supernatant was removed by centrifugation at 540  g for 10 min, and the cells were fixed with 35% of methanol. After washing twice with 70% methanol, the cells were resuspended in 0.92 M potassium hydroxide and 54% dimethyl sulphoxide in a final volume of 1.3 ml to dissolve the reduced-NBT (in the form of formazan), and the optical density of the sample was measured by absorbance at 625 nm. 2.6. Assay for serum phenoloxidase activity Phenoloxidase activity in the coelomic fluid was assayed as described by Ashida and So¨derha¨ll [24] using a 96-microtiter plate method. Fifty microliters of the coelomic fluid was mixed with 0.03 M phosphate buffer (pH ¼ 6.0) and 4.6 mM L-dihydroxyphenylalanine in a final volume of 240 ml in a 96-microtiter plate. For control, 50 ml of filtered seawater was used to substitute for the coelomic fluid. The OD value of the sample was measured at 1 min intervals for a total of 10 min at 490 nm. One unit of enzyme activity was defined as the amount of enzyme causing an increase in absorbance of 0.001 per min per ml serum. 2.7. Assay for serum lysozyme activity

Table 1 Nutritional component of different diets for A. japonicus (g kg1).

Crude protein Crude fat Total carbohydrate Ash Ca/P Lys

2.4. Phagocytic activity

Control

CP

SP

APS

152 39 31 535 7.9 7.73

158 41 31 530 7.5 7.91

162 40 35 515 7.6 7.90

151 39 38 498 8.0 7.38

Basal diet: soil 38%, seaweed 35%, fishmeal 15%, shrimp powder 5%, shell powder 3%, rice hull 2% and premix 2%.

Lysozyme activity was measured by a spectrophotometric method based on the lysis of Micrococcus lysodeikticus (No.1.0634, Institute of Microbiology, Chinese Academy of Sciences [Beijing, China]) [25]. Briefly, 3 ml of M lysodeikticus (OD about 0.3) in phosphate buffer (0.067 M, pH 6.4) was added to 50 ml of serum at 25  1  C. The reduction in the absorbance of the sample at 540 nm was determined after 0.5 and 4.5 min of incubation. One unit of lysozyme activity was defined as the amount of enzyme causing a reduction in absorbance of 0.001 per min.

T. Wang et al. / Fish & Shellfish Immunology 27 (2009) 757–762

1.2

Phagocytic capacity (%)

bA

0.8

aA bA

aB aB

aA

0.7 0.6

control CP SP APS

aB

aA

0.5 aA aA

aA

aB

0.3

1

control CP SP APS

cA bcA

0.8

aA

bA aB

aA

bB

0.4

bC

aB

aB

0.6

cB

bB

0.2

0.2 0.1 0

Optical density at 625 nm

1 0.9

0.4

759

0

20 days

40 days

20 days

Fig. 1. Phagocytic capacity of Apostichopus japonicus (Selenka) amoebocytes fed with different diets. Data are the means  SDs from six sea cucumbers. Bars indicate standard deviation. a,b: Significant difference (P < 0.05) among different groups within the same period. A,B: Significant difference (P < 0.05) within the same groups among different periods.

40 days

60 days

days after feeding

60 days

Fig. 2. Reactive oxygen species(ROS) production of Apostichopus japonicus (Selenka) amoebocytes fed with different diets. Data are the means  SDs from six sea cucumbers. Bars indicate standard deviation. a,b,c: Significant difference (P < 0.05) among different groups within the same period. A,B,C: Significant difference (P < 0.05) within the same group among different periods.

3. Results 2.8. Lectin titer assay Lectin titer assay was carried out in a 96-microtiter plate using whole rabbit blood. A 25-ml aliquot of coelomic fluid from each animal was serially diluted two-fold with 143.2 mM NaCl solution. The diluted coelomic fluid was added to 25 ml erythrocyte suspension (6.9  106 cells/ml) in the same buffer. The plates were incubated at room temperature for 1 h and then examined visually. A positive test showed the formation of a uniform layer over the surface of the well, whereas a negative test showed the formation of discrete aggregates at the bottom of the well. The lectin titer was recorded for the maximum dilution that still gave a positive agglutination. 2.9. Disease resistance test Disease resistance test was performed as described by Dong et al. [26]. At the end of the 60 day experiment, two sea cucumbers per tank (six animals per group) were sampled randomly and exposed to virulent V. splendidus (107 cells/ml) in a 20-l aeration tank for 60 h. The temperature of seawater was controlled at 18  C. No diet was given to the animals during the test. The sea cucumbers were observed for the presence of disease manifested as ulcer and/or peristome edema disease. In the case of ulcer, the lesions in the infected sea cucumber gradually expanded with increased mucus synthesis over large area of the body wall. The infected skin became eroded with deep ulceration, and assumed a bluish white colour. In the case of peristome edema, the criteria for the identification were swollen mouth that appeared like a translucent water bladder, as well as shrinking ambulacra that became flat resulting in the loss of function for attachment as reported previously [27]. 2.10. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA). When differences were found among different dietary supplements, Duncan’s multiple range tests and Fisher’s exact test were used to compare the mean differences and disease resistance rates. All statistical analyses were performed using SPSS program version 13.0 (SPSS, Chicago, IL, USA). The levels of significance were expressed as P-value less or greater than 0.05.

3.1. Phagocytic capacity The effects of different preparations of A. membranaceus root and its polysaccharide extract on the phagocytic capacity of A. japonicus are shown in Fig. 1. While CP group showed no difference in phagocytic capacity relative to that of the control after 20 days of feeding, phagocytic capacity of SP and APS groups increased significantly (P < 0.05) relative to that of the control. No significant difference in phagocytic capacity was detected between the control and each of the three tested groups after 40 and 60 days of feeding. Within groups, SP and APS groups showed significant decrease in phagocytic capacity after 40 days, but regained some increase after 60 days. Phagocytic capacity in the control and CP group remained relatively unchanged after 20 and 40 days of feeding, but almost doubled after 60 days. Benefit for phagocytic capacity was obtained with either SP- or APS-supplemented diet during the early stage (after 20 days) of the feeding. 3.2. ROS production ROS production in CP, SP and APS groups increased significantly (P < 0.05) relative to that of control after 20 day of feeding, with APS group showing the maximum increase (Fig. 2). However, after 40 days of feeding, the ROS production in the control was higher (P < 0.05) than that of CP and APS groups, but similar to that of SP group (Fig. 2). After 60 days of feeding, ROS production in CP group remained lower than that of control while SP group had similar ROS production to that of control. Decrease in ROS production was also obvious (P < 0.05) within CP, SP and APS groups over the first two feeding periods, with CP and APS groups showing at least a 50% decrease after 40 days. SP and APS groups regained some ROS production after 60 days. Less variation in ROS production was observed for the control over the three different feeding periods. None of the three test groups maintained a consistent increase in ROS production over that of control, but a diet supplemented with SP appeared to have the most effect on ROS production in sea cucumber. 3.3. Serum phenoloxidase activity There was no significant difference in serum phenoloxidase (PO) activity among the test groups during the whole experiment,

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12 control CP SP APS

0.35 0.3 0.25 0.2 0.15

bB bA

bA 10 abAB

Titer value

Phenoloxidase activity (Units ml-1)

0.4

8

abA

a a

bB

control CP SP APS

a a

a a

6 4

0.1

2

0.05 0

20 days

40 days

0

60 days

20 days

40 days

days after feeding

60 days

days after feeding

Fig. 3. Serum phenoloxidase activity of Apostichopus japonicus (Selenka) fed with different diets. Data are the means  SDs from six sea cucumbers.

although some increases relative to that of the control were observed for CP group after 20 and 40 days of feeding, and for APS group after 60 days (Fig. 3). There was also no significant difference in PO activity within groups over the different feeding periods. Thus all three supplements appeared to have no significant effect on serum phenoloxidase activity of sea cucumber. 3.4. Serum lysozyme activity After feeding for 20 and 40 days, SP and APS groups exhibited significant increase in lysozyme activity (P < 0.05) relative to that of the control and CP group (Fig. 4). After feeding for 60 days, the lysozyme activity of CP and SP groups were about 2-fold and 3-fold, respectively, higher than that of control, while no significant difference in lysozyme activity was observed between control and APS group (Fig. 4). Both control and test groups showed a significant reduction in lysozyme activity after 40 days, and while the control, CP and SP groups all regained significant increase in lysozyme activity after 60 days, the lysozyme activity of APS group remained relatively unchanged. The biggest effect on lysozyme activity was caused by SP-supplemented diet, whereby sea cucumbers fed with this diet consistently produced a significant increase in lysozyme activity over that of the control.

Fig. 5. Serum agglutination titer of A. japonicus (Selenka) fed with different diets. Data are the means  SDs from six sea cucumbers. Bars indicate standard deviation. a,b: Significant difference (P < 0.05) among different groups within the same period. A,B: Significant difference (P < 0.05) within the same groups among different periods.

3.5. Agglutination assay Only SP and APS groups showed noticeable increase in titer values relative to that of the control over the three different feeding periods. The increase in titer values for SP group became significant (P < 0.05) after 60 days of feeding (Fig. 5). Within groups, lectin titer in SP and APS groups appeared to decrease after 40 days and then increased to similar levels again after 60 days. In terms of enhancement effect on letin titer values, the biggest effect was therefore obtained with APS, and the least effect with CP. 3.6. Disease resistance test Two symptoms, ulcer and peristome edema, were used to assess the presence of diseases in sea cucumbers after exposure to V. splendidus. The occurrence of individual symptom and the cumulative symptom rates are shown in Table 2. Only sea cucumbers fed with SP- or APS-supplemented diet showed improved resistance (75% for SP group and 50% for APS group) to V. splendidus infection compared to control. There was no significant difference between treated and control groups (P > 0.05). 4. Discussion

Lysozyme activity (Units ml-1)

70 60

control CP SP APS

cC

bA bA

50

bC 40

aA aA

aB

30

bB bB

20

aB aB

10 0

aC

Medicinal herbs and polysaccharides from plants have been successfully used in aquaculture and were verified to have effects on the non-specific immunity such as phagocytic capacity, respiratory burst activity, lysozyme activity, and phenoloxidase activity [12,13,17]. However, little work has been undertaken to study the effect of plant substances on the immunity of A. japonicus. Recently, growing attention has been given to the immune mechanism of sea cucumber. Phagocytosis and ROS production were shown to play an important role in the immunity of echinoderms

Table 2 Susceptibility of A. japonicus to V. splendidus infection.

20 days

40 days

60 days

Treatment groupsa

Ulcer (%)

Peristome edema (%)

Cumulative symptom rate (%)

Control CP SP APS

33.34 16.67 0 16.67

33.34 50 16.67 16.67

66.67 66.67 16.67 33.34

days after feeding Fig. 4. Serum lysozyme activity of Apostichopus japonicus (Selenka) fed with different diets. Data are the means  SDs from six sea cucumbers. Bars indicate standard deviation. a,b,c: Significant difference (P < 0.05) among different groups within the same period. A,B,C: Significant difference (P < 0.05) within the same groups among different periods.

a

A total of six animals were used for each treatment group.

T. Wang et al. / Fish & Shellfish Immunology 27 (2009) 757–762

[28,29]. Furthermore, the activities of phenoloxidase [30], lysozyme [31], and lectins [32–34] have also been detected in some species of sea cucumber. In the present study, two different preparations of A. membranaceus root as well as an extract consisted of only A. membranaceus polysaccharides were administered to A. japonicus as a diet supplement, and the responses triggered by five immune parameters of the immune function were studied. The ingestion of foreign particles by sea cucumbers seems to be performed by the petaloid form of the phagocytic amoebocytes, and that most of the immune responses are actually induced by amoebocytes [35]. Since phagocytes act as dominant defense cells, an enhancement of phagocyte function is expected to be appropriate for resisting microbial infections. A. membranaceus only caused a significant increase in phagocytic activity during the early stage of feeding, as observed for SP and APS supplements (Fig. 1). Longer administering of these supplements seemed to provide no real benefit for phagocytic activity. Yin et al. [13] showed that phagocytic cells in tilapia (Oreochromis niloticus) fed with a diet supplemented with 0.1, 0.5 or 1.0% Astragalus radix for 3 weeks exhibited increased phagocytosis activity compared to control. But after 4 weeks of feeding the phagocytosis activity in groups fed with 0.5 and 1.0% dose showed no significant difference to those of control group. In echinodermata, stimulation of phagocytic amoebocytes results in increased uptake of oxygen and generation of superoxide anions (O 2 ), which in turn leads to the production of other highly reactive oxidants (e.g. hydrogen peroxide, hydroxyl radicals, singlet oxygen, and hypochlorite) that function as powerful microbiocidal and cytotoxic agents [29]. Since superoxide anion (O 2 ) is the first product to be released from the respiratory burst, the measurement of superoxide anion has been accepted as a precise way for measuring respiratory burst. Significant increase in ROS production was only observed for A. japonicus fed with A. membranaceus supplemented diet after 20 days (Fig. 2). Beyond this time, the ROS production either decreased or remained similar to that of the control, again, suggesting that the benefit of A. membranaceus appeared to occur during the early stage of feeding. Similar results in O 2 production were previously observed for shrimp hemocytes stimulated with Zymosan [36] or with b-glucan [37]. The decreased value of ROS production found after 40 and 60 days of feeding may be triggered by a self-protection mechanism of the organisms. It is known that high concentration of O 2 could be harmful or even lethal to the cells. In the sea cucumbers Holothuria tubulosa and Holothuria polii, phenoloxidase activities were detected in circulating coelomocytes, and the enzyme was shown to be responsible for melanin deposition during the processes of foreign-body encapsulation [30]. In the present study, we could not detect differences in phenoloxidase activity in serum of A. japonicus fed with a diet supplemented with different preparations of A. membranaceus root or its polysaccharides. Since phenoloxidase activity is detected in the coelomocytes and the encapsulating structures [31], the role of phenoloxidase in self-defense systems in sea cucumber may not be as critical as in crustaceans and insects. Lysosomal enzymes participate in the destruction of external substances in sea cucumber [38,39]. A 713 bp lysozyme cDNA has been cloned from the body wall of the sea cucumber Stichopus japonicus, and from the deduced amino acid sequence, the enzyme was speculated to have a role in bacterial lysis and fibrin hydrolysis [40]. Chinese medicine formulated with A. membranaceus or single medicine of A. membranaceus has been shown to increase lysozyme values in fish blood [6,12]. However, the regulation of the lysozyme activity in sea cucumber is rarely reported. Among the different preparations of A. membranaceus, SP had the biggest effect on serum lysozyme activity. SP group consistently maintained a higher

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level of serum lysozyme activity relative to the control. CP, which had larger particle size than SP, appeared to take much longer to exert its effect on increasing serum lysozyme activity (Fig. 4). Lectins are found in several species of sea cucumbers like S. japonicus [32,33], Cucumaria echinata [41] and Cucumaria japonica [34]. One of the roles of lectins in marine invertebrate is to act as humoral factors in the defense mechanism, as do immunoglobulins in vertebrates [42]. Titer value is considered as one of the methods for evaluating lectin activity. We have shown here that lectin activity increased significantly in sea cucumber fed with SPor APS-supplemented diet after 60 days of feeding. Unlike mollusk, where the lectin pathway and phenoloxidase system play an important role in polysaccharide-induced enhanced immunity [43], induction of immunity by polysaccharide in sea cucumber is less pronounced. Dong et al. [26] have shown that exposure of A. japonicus to V. splendidus at a concentration of 106 cells/ml for 6 days could result in the occurrence of diseases. To determine the efficacy of A. membranaceus as an immunostimulant, the extent of protection against pathogens for sea cucumbers fed with A. membranaceus supplemented diet was investigated. Only SP and APS offered protection against Vibrio spendidus infection. Jian and Wu [44] reported that tilapia (O. niloticus) fed with a diet containing 1.0% or 1.5% traditional Chinese medicine formulated from A. membranaceus have a survival rate of 93.3% after exposure to the pathogen, while cumulative mortality in the controls is 75%. It has been shown that the survival rate of shrimp and its resistance to bacteria could be enhanced by administration of b-glucan [45,46]. Our results showed that feeding sea cucumbers with SP- or APSsupplemented diet could increase their resistance to disease by more than 2-fold compared to control or those fed with CP-supplemented diet (Table 2). The lack of increase in resistance to disease observed for CP group was rather unexpected. Again, this may well be due to the ineffectiveness of the coarser form of root preparation in CP or to the potentially negative effects associated with long-term use of immunostimulants [47,48]. Thus, periodic administration is recommended. Itami et al. [49] reported that shrimp (P. monodon) periodically fed with peptidoglycan show significant improved resistance to Vibrio penaeicida. Many studies have confirmed the efficacy of superfine preparation of medical plants [50,51]. A possible mechanism could be that the plant cell walls in the superfine preparation were highly disrupted, which made the intracellular contents more accessible to the digestive enzymes of sea cucumber. With the exception of ROS production where significant increase was observed for the two different preparations (CP versus SP) of A. membranaceus root, SP appeared to produce a more rapid positive response than CP on all the immune parameters examined. This difference can be attributed to the smaller particle sizes of SP compared to CP. The fine particles and large surface area of medicinal plant powder may enhance its adhesive function leading to a longer retention time within the small intestinal lumen and hence, producing a larger and longer effect in the body of the animal that consumes it. In conclusion, the present study provided evidences that superfine powder of A. membranaceus root and extract of polysaccharides derived from this plant administered as feed supplement could significantly enhance the non-specific immunity of A. japonicus through improvement of various immune parameters such as phagocytic capacity, lysozyme activity, production of ROS and lectin titer. The benefit of this was demonstrated by a lower susceptibility to Vibrio infection. The low cost of A. membranaceus in China would make it a cost effective measure for controlling the diseases in sea cucumber in aquaculture. However, the effects of dosages and frequency of administering needed to produce an optimum outcome require further investigation.

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