Immune response of sea cucumber Apostichopus japonicus coelomocytes to several immunostimulants in vitro

Aquaculture 306 (2010) 49–56

Contents lists available at ScienceDirect

Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Immune response of sea cucumber Apostichopus japonicus coelomocytes to several immunostimulants in vitro Min Gu, Hongming Ma ⁎, Kangsen Mai, Wenbing Zhang, Qinghui Ai, Xiaojie Wang, Nan Bai The Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, PR China

a r t i c l e

i n f o

Article history: Received 20 January 2010 Received in revised form 23 March 2010 Accepted 22 May 2010 Keywords: Sea cucumber (Apostichopus japonicus) Immunostimulant Coelomocyte culture Immune response

a b s t r a c t The in vitro effects of β-glucan, mannan oligosaccharides (MOS), CpG oligodeoxynucleotide (CpG ODN), lactoferrin and vitamin C on the non-specific immune response of sea cucumber Apostichopus japonicus coelomocytes were studied. Coelomocytes were cultured in L-15 medium containing different concentrations of β-glucan (0, 5, 25 and 100 µg ml−1), CpG ODN (0, 0.5, 2.5 and 5 µM), MOS (0, 40, 80 and 120 µg ml−1), lactoferrin (0, 5, 25 and 100 µg ml−1) and vitamin C (0, 25, 100 and 250 µg ml−1). Coelomocytes were incubated with the above immunostimulants for 1 h, 3 h, 6 h, 12 h or 24 h except vitamin C for 3 h, 6 h, 12 h, 24 h or 48 h. Coelomocytes incubated in medium without immunostimulants were taken as controls at each incubation time. After exposure to these substances, coelomocytes were sampled and immune parameters viz., phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase (T-NOS) activity were determined. All immune parameters were significantly (P b 0.05) enhanced by β-glucan, MOS and CpG ODN. Lactoferrin induced significant (P b 0.05) increase in superoxide anion production and SOD activity of coelomocytes but did not affect phagocytosis and T-NOS activity. Vitamin C significantly (P b 0.05) enhanced the SOD and T-NOS activities of coelomocytes while no significant effects were observed in phagocytosis and superoxide anion production. The results suggest that β-glucan, MOS, CpG ODN, lactoferrin and vitamin C can enhance the non-specific immune response of sea cucumber in vitro. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Sea cucumber Apostichopus japonicus belongs to Echinodermata, Holothuroidea, Aspidochirotida. It is a traditional food and invigorant in China. Farming and sea ranching of A. japonicus have grown into a prosperous sector in northern China, where 2–3 billion juveniles and 60,000 t of sea cucumber (live weight) were produced each year (Deng et al., 2009). The rapid expansion and high farming density result in severe diseases, mainly skin ulceration syndrome, which is highly infectious and lethal to this animal (Dong et al., 2005). Therefore, it is urgent to find effective methods to control the diseases. The application of antibiotics or other chemicals against aquatic animal diseases is expensive and undesirable as it risks contamination of the environment and the final product (Capone et al., 1996; Gräslund and Bengtsson, 2001). The repeated application of antibiotics, in the long term, encourages the spread of drug resistant pathogens (Karunasagar et al., 1994; Smith et al., 1994). In contrast, use of immunostimulants has been proved to be an effective means to increase the immunocompetence and disease resistance of aquatic animals (Sakai, 1999). Several immunostimulants such as β-glucan (Dalmo and Bøgwald, 2008), chitosan (Wang and Chen, 2005), bovine lactoferrin

⁎ Corresponding author. Tel.: + 86 532 82031943; fax: + 86 532 82032038. E-mail address: [email protected] (H. Ma). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.05.024

(Esteban et al., 2005), vitamin C (Ai et al., 2004), nisin (Villamil et al., 2003) and CpG oligodeoxynucleotides (Meng et al., 2003) have been applied on aquatic animals and got promising results. β-glucans are homopolysaccharides that have β(1,3)-D-linkages in the backbone and may also possess β(1,6)-D-glucosidic linkages when branching. β-glucans are found in plants, algae, bacteria, yeast and mushrooms as the main constituent of cell wall (Dalmo and Bøgwald, 2008). Many in vitro tests and feeding trials have shown that β-glucans are able to enhance the resistance to infections as well as immune parameters such as phagocytosis, superoxide anion production and lysozyme activity in shrimps and fishes (Dalmo and Bøgwald, 2008). Synthetic oligodeoxynucleotides (ODNs) containing un-methylated CpG dinucleotides within the context of certain flanking bases have been shown to induce potent innate immune responses (Klinman, 1995). Studies have shown positive effects of CpG ODNs on several immune parameters such as respiratory burst, phagocytosis (Tassakka and Sakai, 2002; Meng et al., 2003; Lee et al., 2003) and resistance to bacterial infection in fish (Oumouna et al., 2002; Rhodes et al., 2004). CpG ODNs could also activate the prophenoloxidase (proPO) system of prawn Macrobrachium rosenbergii (Chuo et al., 2005) and enhanced lysozyme and phenoloxidase (PO) activity of mussel Hyriopsis cumingii (Hong et al., 2006). Mannan oligosaccharides (MOS) are derived from yeast cell walls, which have been used in poultry husbandry and in aquaculture as a feed additive (Torrecillas et al., 2007). In fish, feeding MOS promoted

50

M. Gu et al. / Aquaculture 306 (2010) 49–56

resistance to bacterial infection in rainbow trout Oncorhynchus mykiss (Staykov et al., 2007), channel catfish Ictalurus punctatus (Welker et al., 2007a) and sea bass Dicentrarchus labrax (Torrecillas et al., 2007). In crustacean, diet containing MOS could improve the growth, survival and immune response of prawn Penaeus latisulcatus (Hai and Fotedar, 2009) and lobster Panulirus ornatus (Sang and Fotedar, 2010). MOS could also improve survival, health status and immune response of marron Cherax tenuimanus under bacterial infection and stress conditions caused by air and NH3 exposures (Sang et al., 2009). Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein present in secretory fluids of mammals. In several fishes, its immunomodulating ability to promote immunity and disease resistance has been investigated (Kumari et al., 2003; Esteban et al., 2005; Ren et al., 2007; Welker et al., 2007b). It has also been reported that Lf can stimulate immunity and resistance against bacteria Aeromonas hydrophila infection in prawn M. rosenbergii (Chand et al., 2006). Vitamin C is an essential nutrient for many aquatic animals and closely related to fish and shrimp immunity (Lee and Shiau, 2002; Ai et al., 2004; Lin and Shiau, 2005). Beneficial effects of high exogenous level of vitamin C on different immune parameters such as serum bactericidal activity, phagocytosis, antibody levels, serum complement activity and lysozyme activity in fishes have been reported (Ortuno et al., 1999; Zhou et al., 2002; Lin and Shiau, 2005). Vitamin C could also increase total blood cells and ProPO activity in white shrimp Litopenaeus vannamei (López et al., 2003). The aim of the present work was to study the effects of the above mentioned immunostimulants on the immune response of A. japonicus coelomocytes in vitro and explore whether they could be used as potential immunostimulants for sea cucumber. 2. Materials and methods 2.1. Experimental animals Apparently healthy sea cucumbers (52.4 ± 9.2 g), namely those that did not undergo visceral ejection and head shaking and showed no symptoms of peristome oedema and skin ulceration as described by Deng et al. (2009) were obtained from Nanshan fish market, Qingdao, China. Then they were kept in cylindrical 150 l tanks with recirculating sea water. The water temperature was 14–16 °C, pH 7.5– 8.0, dissolved oxygen 6–7 mg l−1 and salinity 31–32. Sea cucumbers were left to acclimatize for 2 weeks and fed with dried seaweed Sargassum thunbergii meal at a rate of 1% of body weight per day and fasted for 24 h before sampling. 2.2. Isolation and primary culture of coelomocytes Sea cucumbers were sterilized in 7% benzalkonium bromide and 70% ethanol for about 2 min respectively. Then they were dissected by aseptic surgery technique as described by Xing et al. (1998). The coelomic fluids were collected and mixed with the anticoagulant solution (0.02 M EGTA, 0.48 M NaCl, 0.019 M KCl, 0.068 M Tri–HCl, pH 7.6, modified from Xing et al., 1998) in a 1:1(V:V) ratio. Cell suspension was filtered through a 100-µm nylon mesh to remove large tissue debris, and then was centrifuged at 2700 rpm, 18 °C for 10 min (Sorvall Legend RT, Germany). The cells were washed twice with isotonic buffer (0.001 M EGTA, 0.53 M NaCl, 0.01 M Tris–HCl, pH 7.6, modified from Xing et al., 1998) and resuspended in the Leiboviz's L-15 cell culture medium (Invitrogen Corporation, CA, USA) with antibiotics penicillin (100 U ml−1) and streptomycin sulfate (100 µg ml−1), and NaCl (0.39M) to adjust osmotic pressure. Cell suspension was adjusted to 107 cells ml−1. Cell viability was determined to be greater than 95% by trypan blue (0.4%) exclusion test. Aliquots of 200 µl cell suspension were dispensed into wells of 96-well culture microplates for the superoxide anion assay. Aliquots of 500 µl cell suspension were dispensed into wells of 48-well culture microplates for phagocytosis assay and enzyme

activity assay. The coelomocytes were incubated in 18 °C for 24 h in darkness prior to immunostimulant adding.

2.3. Incubation of coelomocytes with immunostimulants Five kinds of potential immunostimulants including β-glucan (Fluka, Germany), CpG ODN-1670 (Sangon, China), MOS (Sigma, Slovakia), lactoferrin (Sigma, United Kingdom) and vitamin C (Sigma, Japan) were dissolved in L-15 medium to prepare concentrations as follows: β-glucan: 0(control), 5, 25 and 100 µg ml−1; CpG ODN-1670: 0(control), 0.5, 2.5 and 5 µM; MOS: 0(control), 40, 80 and 120 µg ml−1; lactoferrin: 0 (control), 5, 25 and 100 µg ml−1; and vitamin C: 0(control), 25, 100 and 250 µg ml−1. After 24 h of cultivation, cells were centrifuged at 2700 rpm at 18 °C for 10 min (Sorvall Legend RT, Germany) and the supernatant was removed. Then each of the medium containing certain concentration of immunostimulants was added. The coelomocytes were incubated with vitamin C for 3 h, 6 h, 12 h, 24 h or 48 h, other immunostimulants for 1 h, 3 h, 6 h, 12 h or 24 h, and then the immune parameters of coelomocytes were assayed. Each concentration of these immunostimulants has four replicates. 2.4. Phagocytosis Phagocytosis of sea cucumber coelomocytes was measured by flow cytometry according to Esteban et al. (1998) with modifications. The bacteria strain Staphyloccocus aureus, bought from China General Microbiological Culture Collection Center (CGMCC), was used as target for phagocytosis. The strain was grown on trypticase soy broth (TSB) agar plates at 28 °C for 24 h. Then, an isolated colony was expanded in 100 ml TSB containing 50 μg ml−1 fluorescein isothiocyanate (FITC, Fluka) and grown at 28 °C for 12 h in dark with constant shaking to label the bacteria. After labeling, free FITC was removed by washing 3 times in PBS solution. The FITC-labeled bacteria were adjusted to 109 cells ml−1 in PBS and heat-killed at 60 °C for 15 min. The bacterial suspensions were stored at 4 °C until use. An aliquot of 50 μl FITC-labeled bacteria (109 cells ml−1) was added to each 500 µl coelomocytes, centrifuged at 2700 rpm at 18 °C for 5 min (Sorvall Legend RT, Germany), resuspended and incubated in dark at 18 °C for 1 h. Then, the micrplates were placed on ice to stop phagocytosis and 500 μl ice-cold L-15 medium was added into each well. The fluorescence of the extracellular bacteria was quenched by adding 80 μl ice-cold trypan blue (0.4% in PBS). The samples were analyzed in a flow cytometer (FC-500, BeckMan Coulter, USA) with an argon-ion laser at 488 nm. Each analysis is performed on 10,000 cells, which were acquired at the rate of 300 cells s−1. Phagocytosis was defined as the percentage of cells with one or more ingested bacteria, therefore showing high fluorescence, within the total cell population (10,000 cells). 2.5. Superoxide anion (O− 2 ) production Production of intracellular O− 2 by coelomocytes was evaluated by using the nitroblue tetrazolium (NBT) method (Song and Hsieh, 1994). Microplates were centrifuged at 2700 rpm at 18 °C for 10 min (Sorvall Legend RT, Germany) and the supernatant was removed. NBT (Sigma) was dissolved in L-15 medium to give a final concentration of 2 mg ml−1. Aliquots of 200 µl NBT L-15 solution were added to coelomocytes and incubated for 30 min at 18 °C. Supernatant was removed from each well and the cells were fixed by adding 200 µl 100% methanol and incubating for 10 min. Subsequently, the cells were washed twice with 70% methanol to remove unreduced NBT, air-dried. Reduced NBT was dissolved by adding 120 µl 2 M KOH, followed by 140 µl DMSO. The production of superoxide anion was expressed as the absorption value of wavelength of 630 nm (O.D.630).

M. Gu et al. / Aquaculture 306 (2010) 49–56

2.6. Superoxide dismutase activity The medium of coelomocytes was replaced by equal volume of icecold PBS. The coelomocytes were then homogenized on ice with a sonicator (Sonic, Vibra Cell, USA) for 10 s at 20% amplitude and centrifuged at 11,000 rpm for 5 min (Heraeus Biofuge Stratos, Germany). Supernatants were collected for enzymes assay. SOD was measured by its ability to inhibit superoxide anion generated by xanthine and xanthine oxidase reaction system according to Wang and Chen (2005) using an SOD detection kit (Nanjing Jiancheng Bioengineering Institute, China). The optical density was measured at 550 nm. One unit of SOD was defined as the amount required for inhibiting the rate of xanthine reduction by 50% in 1 ml reaction system. Specific activity was expressed as SOD unit per 107 cells.

51

− NO− 3 . NO2 was then detected by Griess assay reagents [sulphanilamide, H3PO4 and N-(1-naphthyl)-ethylendiamine (NED)]. The optical density was measured at 530 nm following the manufacturer's introduction. One unit of T-NOS activity was defined as the amount of T-NOS producing 1 nmol NO min−1. Specific activity was expressed as T-NOS unit per 107 cells.

2.8. Statistical analysis All data were subjected to one way ANOVA (analysis of variance) using SPSS 13.0 for Windows. Differences between the means were tested by Duncan's multiple range tests. The level of significance was chosen at P b 0.05 and the results are presented as means ± S.E. (standard error).

2.7. Total nitric oxide synthase activity

3. Results

Samples were prepared as described in Section 2.6. T-NOS activity was determined by its ability to convert L-Arginine to NO according to Marzinzig et al. (1997) using a T-NOS Kit (Nanjing Jiancheng Bioengineering Institute, China). NO could be oxidized to NO− 2 and

3.1. Phagocytosis Phagocytosis of coelomocytes incubated with 5 µg ml−1 β-glucan was significantly higher than that of control at 3 h, 6 h, 12 h and 24 h.

Fig. 1. Phagocytosis of sea cucumber coelomocytes incubated with β-glucan (A), CpG ODN (B), MOS (C) and Lf (D) or not (control). Data are expressed as mean ± S.E. Bars with different letters at a particular incubating time are significantly different (P b 0.05).

52

M. Gu et al. / Aquaculture 306 (2010) 49–56

However, high concentration of β-glucan (100 µg ml−1) significantly decreased the phagocytosis of coelomocytes at 1 h and 3 h (P b 0.05). CpG ODN significantly increased phagocytosis only at 5 µM, the highest concentration, at all the incubation time (P b 0.05). MOS significantly increased phagocytosis of coelomocytes at concentrations of 40 µg ml−1 and 80 µg ml−1 at 6 h, 12 h and 24 h, while the 120 µg ml−1 MOS only increased phagocytosis at 1 h (P b 0.05). Lf caused significant decrease in phagocytosis at 1 h and 3 h (P b 0.05) (Fig. 1). No significant difference was observed in phagocytosis of coelomocytes incubated with vitamin C for any assayed concentration or incubation time (data not shown).

3.2. Production of superoxide anion Superoxide anion production by coelomocytes incubated with all the three concentrations of β-glucan was significantly higher than that of control at 1 h and 3 h (P b 0.05). The increase of superoxide anion production lasted to 6 h in coelomocytes incubated with lower concentrations of β-glucan (5 and 25 µg ml−1). CpG ODN (2.5 µM) significantly enhanced the superoxide anion production of coelomocytes at 1 h, 3 h and 6 h (P b 0.05). MOS in concentration of 40 µg ml−1 and 80 µg ml−1 promoted superoxide anion production at 6 h and 12 h (P b 0.05). Lf with a concentration of 25 µg ml−1 significantly increased

Fig. 2. Superoxide anion production of sea cucumber coelomocytes incubated with β-glucan (A), CpG ODN (B), MOS (C) and Lf (D) or not (control). Data are expressed as mean ± S.E. Bars with different letters at a particular incubating time are significantly different (P b 0.05).

M. Gu et al. / Aquaculture 306 (2010) 49–56

superoxide anion production at 1 h and 3 h, and 5 µg ml−1 Lf significantly increased the superoxide anion production at 6 h (P b 0.05) (Fig. 2). No significant difference was observed in superoxide anion production of coelomocytes incubated with vitamin C (data not shown).

53

3.3. Superoxide dismutase activity The SOD activity of coelomocytes incubated with all the three concentrations of β-glucan was significantly higher than that of control at 1 h and 3 h (P b 0.05). The increase of SOD activity lasted to

Fig. 3. SOD activity of sea cucumber coelomocytes incubated with β-glucan (A), CpG ODN (B), MOS (C), Lf (D) and vitamin C (E) or not (control). Data are expressed as mean ± S.E. Bars with different letters at a particular incubating time are significantly different (P b 0.05).

54

M. Gu et al. / Aquaculture 306 (2010) 49–56

6 h in coelomocytes incubated with intermediate concentration (25 µg ml−1) of β-glucan. SOD activity of coelomocytes was significantly enhanced by 2.5 µM CpG ODN at 3 h, 6 h and 12 h, significantly enhanced by 5 µM CpG ODN (P b 0.05) at 12 h. MOS significantly increased the SOD activity of coelomocytes in all the concentrations at 12 h (P b 0.05). The SOD activity of coelomocytes was significantly increased by 25 µg ml−1 Lf at 1 h, 3 h and 6 h, significantly increased by 5 µg ml−1 Lf at 3 h, 6 h and 12 h (P b 0.05). SOD activity of coelomocytes incubated with higher concentrations of vitamin C was significantly higher than that of control at 6 h, 12 h and 24 h (P b 0.05) (Fig. 3).

6 h and 12 h, and the lower concentrations significantly increased T-NOS activity at 6 h and 12 h (P b 0.05). MOS significantly enhanced T-NOS activity at the concentration of 80 µg ml−1 at 3 h, 6 h and 12 h (P b 0.05). The T-NOS activity of coelomocytes was significantly increased under 25 µg ml−1 and 100 µg ml−1 vitamin C at 6 h, 12 h and 24 h, and 250 µg ml−1 vitamin C at 6 h and 12 h (P b 0.05) (Fig. 4). The T-NOS of coelomocytes incubated with Lf showed no statistically significant difference compared with control at any incubation time (data not shown).

3.4. Total nitric oxide synthase activity

Sea cucumbers lack acquired immune system. Coelomocytes are the major line of defense. Coelomocytes are involved in eliminating microbes through phagocytosis, encapsulation and synthesis of humoral protective factors (Eliseikina and Magarlamov, 2002; Dolmatova et al., 2004). During phagocytosis, phagocytes produce reactive oxygen intermediates (ROIs) and nitric oxide (NO) to attack

The T-NOS activity of coelomocytes incubated with 25 µg ml−1 and 100 µg ml−1 β-glucan was significantly higher than that of control at 1 h, 3 h, 6 h and 12 h (P b 0.05). The highest concentration (5 µM) of CpG ODN significantly increased T-NOS activity of coelomocytes at 1 h, 3 h,

4. Discussion

Fig. 4. T-NOS activity of sea cucumber coelomocytes incubated with β-glucan (A), CpG ODN (B), MOS (C) and vitamin C (D) or not (control). Data are expressed as mean ± S.E. Bars with different letters at a particular incubating time are significantly different (P b 0.05).

M. Gu et al. / Aquaculture 306 (2010) 49–56

microbes (Coteur et al., 2002; Chakravortty and Hensel, 2003). Here, we choose phagocytosis, super anion production, T-NOS activity and SOD activity to evaluate the immune capacity of sea cucumber coelomocytes. SOD was selected because of its protective effects against excess ROIs produced by activated coelomocytes (CampaCórdova et al., 2002). In innate immune system, pattern-recognition proteins (PRPs) recognize and bind pathogen-associated molecular patterns (PAMPs), which are highly conserved within microbial species but generally absent in the host (Janeway and Medzhitov, 2002). The recognition and binding in turn evoke subsequent internal defense. These PAMPs include β-glucans, lipopolysaccharides (LPS), peptidoglycans and some nucleic acid motifs such as CpG DNA (Yu et al., 2002; Janeway and Medzhitov, 2002; Sung et al., 2008). Some successfully used immunostimulants are basically this category of molecules. For example, β-glucan can trigger the activation of prophenoloxidase (proPO) system and enhance superoxide anion production, phagocytosis and SOD activity in shrimps (Chang et al., 2000; Campa-Córdova et al., 2002) by binding to β-glucan binding protein (βGBP) or LPS and β-glucan binding protein (LGBP) (Vargas-Albores et al., 1997; RomoFigueroa et al., 2004; Cheng et al., 2005). In our study, β-glucan, CpG ODNs and MOS are selected as PAMPs molecules. All immune parameters tested were significantly increased by β-glucan, suggesting that sea cucumber has similar recognition mechanism for β-glucan. Hartmann et al. (2000) have indicated that CpG ODNs are effective activators for the innate immune system of mammals. The present study showed that 5 μM CpG ODN enhanced phagocytosis ability of sea cucumber coelomocytes against S. aureus. CpG ODN have been shown to activate macrophages and to increase the levels of superoxide anion production in several aquatic animals including grass carp Ctenopharyngodon idellus (Meng et al., 2003), common carp C. carpio (Tassakka and Sakai, 2003), olive flounder Paralichthys olivaceus (Lee et al., 2003) and prawn M. rosenbergii (Sung et al., 2008) in vitro. Similarly, superoxide anion production of sea cucumber can also be enhanced in vitro by CpG ODN and the increase lasted to 6 h. However, coelomocytes incubated with high concentration (5 µM) of CpG ODN showed no significant difference in superoxide anion production at 3 h and 6 h. Studies on the immunomodulating effects of MOS on aquatic animals are scarce. In this study, MOS significantly increased the phagocytosis of coelomocytes incubated with lower concentrations of MOS (40 µg ml−1 and 80 µg ml−1). The enhancement of phagocytosis in macrophages has also been found in sea bass D. labrax fed with MOS (Torrecillas et al., 2007). Phagocytosis enhancement could be related to the activation of a mannose binding lectin (MBL) which has been isolated in sea cucumber A. japonicus and cloned (Bulgakov et al., 2007). This protein could mediate recognition and elimination of the pathogens expressing mannose-rich glycoconjugates on their surface (Neth et al., 2000). The present study showed that Lf directly stimulated the production of superoxide anion in sea cucumber coelomocytes in vitro. This was also reported in aquatic animals such as rainbow trout O. mykiss (Sakai et al., 1995), Indian major carp Catla catla (Kamilya et al., 2006) and European sea bass D. labrax (Henry and Alexis, 2009). The possible mechanism lies in that Lf may provide the leukocytes with the iron necessary to the respiratory burst system (Ambruso and Johnston, 1981). However, Lf failed to modulate phagocytosis in gilthead seabream S. aurata (Esteban et al., 2005). Similarly, Lf significantly increased superoxide anion production and SOD activity but failed to stimulate phagocytosis and T-NOS in sea cucumber in vitro. Available results regarding the effects of vitamin C on fish immune response are controversial. Some studies have shown beneficial effects of vitamin C on phagocytosis and respiratory burst (Mulero et al., 1998; Ortuno et al., 1999; Anbarasu and Chandran 2001; Hung et al., 2007), while some have not (Hardie et al., 1991; Thompson et al., 1993). In this study, the results were similar to the latter

55

situations. The present study showed T-NOS activity was increased significantly at 6 h, 12 h and 24 h except the highest concentration treatment, in which a reduction was observed at 24 h. Our results are similar to Hung et al. (2007), who reported that moderate dose (100 µg ml−1) of vitamin C activated the production of NO while the higher doses (200 µg ml−1 and 300 µg ml−1) decreased the NO production of tilapia O. hybrids macrophages. Beside the positive immune enhancing effects, high level of immunostimulants showed suppression of immune capacity of sea cucumber coelomocytes in vitro. Unlike chemotherapeutics, immunostimulants do not show a linear dose–effect relationship (Bliznakov and Adler, 1972). In fact they often show a distinct maximum at a certain intermediate concentration and even a complete absence of effect or an adverse toxic effect at higher concentrations (Floch et al., 1987). Overdose incubation with immunostimulants may result in immunosuppression, which had been observed in many aquatic animals. Sajeevan et al. (2009) reported that higher doses of glucan (0.3% and 0.4%) in feed resulted in reduced immune indices in Indian white shrimp Fenneropenaeus indicus. Sakai et al. (1995) noted that low concentrations of Lf were more effective than higher ones suggesting a potential inhibitory effect of high concentration of Lf in rainbow trout O. mykiss. On the other hand, long term immunostimulant exposure could also lead to negative results. In this respect, no significant difference was observed in superoxide anion production, SOD and T-NOS activities of sea cucumber coelomocytes incubated with all the five immunostimulants after 24 h or 48 h incubation. Immunity fatigue was also observed in black tiger shrimps P. monodon (Chang et al., 2000) and Indian white shrimp F. indicus fed with βglucan for 40 days (Sajeevan et al., 2009). The results of the present work indicate that β-glucan, CpG ODN, MOS, Lf and vitamin C can enhance immune capacity of sea cucumber A. japonicus coelomocytes in vitro. This provided basic information of these substances as potential immunostimulants in sea cucumber. Furthermore, this study indicated that the dose and duration of application of immunostimulants in sea cucumber culture should be considered to achieve the optimum result. Acknowledgements This work was supported by the National High-Tech Research and Development Program of China (863 project, 2006AA100313). We appreciate Mr. Mingzhuang Zhu for his technical help. References Ai, Q., Mai, K., Zhang, C., Wei, X., Duan, Q., Tan, B., Liufu, Z., 2004. Effects of dietary vitamin C on growth and immune response of Japanese seabass, Lateolabrax japonicus. Aquaculture 242, 489–500. Ambruso, D.R., Johnston Jr., R.B., 1981. Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system. J. Clin. Invest. 67, 352–360. Anbarasu, K., Chandran, M.R., 2001. Effects of ascorbic acid on the immune response of the catfish, Mystus gulio (Hamilton), to different bacterins of Ameromonas hydrophila. Fish Shellfish Immunol. 11, 347–355. Bliznakov, E.G., Adler, A.D., 1972. Non-linear response of the reticuloendothelial system upon stimulation. Pathol. Microbiol. 38, 393–410. Bulgakov, A.A., Eliseikina, M.G., Petrova, I.Y., Nazarenko, E.L., Kovalchuk, S.N., Kozhemyako, V.B., Rasskazov, V.A., 2007. Molecular and biological characterization of a mannanbinding lectin from the holothurian Apostichopus japonicus. Glycobiology 17, 1284–1288. Campa-Córdova, A.I., Hernández-Saavedra, N.Y., Philippis, R.D., Ascencio, F., 2002. Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to β-glucan and sulphated polysaccharide. Fish Shellfish Immunol. 12, 353–366. Capone, D.G., Weston, D.P., Miller, V., Shoemaker, C., 1996. Antibacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 145, 55–75. Chakravortty, D., Hensel, M., 2003. Inducible nitric oxide synthase and control of intracellular bacterial pathogens. Microbes Infect. 5, 621–627. Chand, R.K., Sahoo, P.K., Kumari, J., Pillai, B.R., Mishra, B.K., 2006. Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress. Fish Shellfish Immunol. 21, 119–129.

56

M. Gu et al. / Aquaculture 306 (2010) 49–56

Chang, C.F., Chen, H.Y., Su, M.S., Liao, I.C., 2000. Immunomodulation by dietary β-1, 3glucan in the brooders of the black tiger shrimp Penaeus monodon. Fish Shellfish Immunol. 10, 505–514. Cheng, W., Liu, C.H., Tsai, C.H., Chen, J.C., 2005. Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide- and β-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 18, 297–310. Chuo, C.P., Liang, S.M., Sung, H.H., 2005. Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides. Fish Shellfish Immunol. 18, 149–162. Coteur, G., Warnau, M., Jangoux, M., Dubois, P., 2002. Reactive oxygen species (ROS) production by amoebocytes of Asterias rubens (Echinodermata). Fish Shellfish Immunol. 12, 187–200. Dalmo, R.A., Bøgwald, J., 2008. β-glucans as conductors of immune symphonies. Fish Shellfish Immunol. 5, 384–396. Deng, H., He, C.B., Zhou, Z.C., Liu, C., Tan, K.F., Wang, N.B., Jiang, B., 2009. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. Aquaculture 287, 18–27. Dolmatova, L.S., Eliseikina, M.G., Romashina, V.V., 2004. Antioxidant enzymatic activity of coelomocytes of the far east sea cucumber Eupentacta fraudatrix. J. Evol. Biochem. Physiol. 40, 126–135. Dong, Y., Deng, H., Sui, X.L., Song, L., 2005. Ulcer disease of farmed sea cucumber (Apostichopus japonicus). Fish. Sci. 24, 4–6 (in Chinese with English abstract). Eliseikina, M.G., Magarlamov, T.Y., 2002. Coelomocyte morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae). Russ. J. Mar. Biol. 28, 197–202. Esteban, M.A., Mulero, V., Muñoz, J., Meseguer, J., 1998. Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res. 293, 133–141. Esteban, M.A., Rodríguez, A., Cuesta, A., Meseguer, J., 2005. Effects of lactoferrin on nonspecific immune responses of gilthead seabream (Sparus auratus L.). Fish Shellfish Immunol. 18, 109–124. Floch, F., Poirier, J., Fizames, C., Pimelautide, R.W., 1987. From experimental result to clinical trials: an illustration. In: Azuma, I., Jolles, G. (Eds.), Immunostimulants: Now and Tomorrow. Japan Science Society Press, Tokyo/Springer-Verlag, Berlin, pp. 183–204. Gräslund, S., Bengtsson, B., 2001. Chemicals and biological products used in south-east Asian shrimp farming, and their potential impact on the environment—a review. Sci. Total Environ. 280, 93–131. Hai, N.V., Fotedar, R., 2009. Comparison of the effects of the prebiotics (Bio-Mos® and β-1,3-D-glucan) and the customised probiotics (Pseudomonas synxantha and P. aeruginosa) on the culture of juvenile western king prawns (Penaeus latisulcatus Kishinouye, 1896). Aquaculture 289, 310–316. Hardie, L.J., Fletcher, T.C., Secombes, C.J., 1991. The effect of dietary vitamin C on the immune response of the Atlantic salmon (Salmo salar L.). Aquaculture 95, 201–214. Hartmann, G., Weeratna, R.D., Ballas, Z.K., Payette, P., Blackwell, S., Suparto, I., Rasmussen, W.L., Waldschmidt, M., Sajuthi, D., Purcell, R.H., Davis, H.L., Krieg, A.M., 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164, 1617–1624. Henry, M., Alexis, M.N., 2009. Effects of in vitro lactoferricin and lactoferrin on the head kidney cells of European sea bass (Dicentrarchus labrax, L.). Vet. Immunol. Immunopathol. 130, 236–242. Hong, X.T., Xiang, L.X., Shao, J.Z., 2006. The immunostimulating effect of bacterial genomic DNA on innate immune responses of bivalve mussel, Hyriopsis cumingii Lea. Fish Shellfish Immunol. 21, 357–364. Hung, S.W., Tu, C.Y., Wang, W.S., 2007. In vitro effects of singular or combined antioxidative vitamins and/or minerals on tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages. Fish Shellfish Immunol. 23, 1–15. Janeway, C.A., Medzhitov, R., 2002. Innate immune recognition: mechanisms and pathways. Annu. Rev. Immunol. 20, 197–216. Kamilya, D., Ghosh, D., Bandyopadhyay, S., Mal, B.C., Maiti, T.K., 2006. In vitro effects of bovine lactoferrin, mushroom glucan and Abrus agglutinin on Indian major carp, catla (Catla catla) head kidney leukocytes. Aquaculture 253, 130–139. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 28, 203–209. Klinman, D.M., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549. Kumari, J., Swain, T., Sahoo, P.K., 2003. Dietary bovine lactoferrin induces changes in immunity level and disease resistance in Asian catfish Clarias batrachus. Vet. Immunol. Immunopathol. 94, 1–9. Lee, M.H., Shiau, S.Y., 2002. Dietary vitamin C and its derivatives affect immune responses in grass shrimp, Penaeus monodon. Fish Shellfish Immunol. 12, 119–129. Lee, C.H., Jeong, H.D., Chung, J.K., Lee, H.H., Kim, K.H., 2003. CpG motif in synthetic ODN primes respiratory burst of olive flounder Paralichthys olivaceus phagocytes and enhances protection against Edwardsiella tarda. Dis. Aquat. Org. 56, 43–48. Lin, M.F., Shiau, S.Y., 2005. Dietary L-ascorbic acid affects growth, nonspecific immune responses and disease resistance in juvenile grouper, Epinephelus malabaricus. Aquaculture 244, 215–221. López, N., Cuzonb, G., Gaxiolac, G., Taboadac, G., Valenzuelac, M., Pascualc, C., Sánchez, A., Rosas, C., 2003. Physiological, nutritional, and immunological role of dietary β1,3-glucan and ascorbic acid 2-monophosphate in Litopenaeus vannamei juveniles. Aquaculture 224, 223–243. Marzinzig, M., Nussler, A.K., Stadler, J., Marzinzig, E., Barthlen, W.N., Nussler, C., Beger, H.G., Morris Jr., S.M., Brückner, U.B., 1997. Improved methods to measure end

products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric Oxide 1, 177–189. Meng, Z., Shao, J., Xiang, L., 2003. CpG oligodeoxynucleotides activate grass carp (Ctenopharyngodon idellus) macrophages. Dev. Comp. Immunol. 27, 313–321. Mulero, V., Esteban, M.A., Munoz, J., Meseguer, J., 1998. Effects of in vitro addition of exogenous vitamins C and E on gilthead seabream (Sparus aurata L.) phagocytes. Vet. Immunol. Immunopathol. 66, 185–199. Neth, O., Jack, D.L., Dodds, A.W., Holzel, H., Klein, N.J., Turner, M.W., 2000. Mannose binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect. Immun. 68, 688–693. Ortuno, J., Esteban, M.A., Meseguer, J., 1999. Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol. 9, 429–443. Oumouna, M., Jaso-Friedmann, L., Evans, D.L., 2002. Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: binding, specificity and identification of unique immunostimulatory motifs. Dev. Comp. Immunol. 26, 257–269. Ren, T., Koshio, S., Ishikawa, M., Yokoyama, S., Micheal, F.R., Uyan, O., Tung, H.T., 2007. Influence of dietary vitamin C and bovine lactoferrin on blood chemistry and nonspecific immune responses of Japanese eel, Anguilla japonica. Aquaculture 267, 31–37. Rhodes, L.D., Rathbone, C.K., Corbett, S.C., Harrell, L.W., Strom, M.S., 2004. Efficacy of cellular vaccines and genetic adjuvants against bacterial kidney disease in chinook salmon (Oncorhynchus tshawytscha). Fish Shellfish Immunol. 16, 461–474. Romo-Figueroa, M.G., Vargas-Requena, C., Sotelo-Mundo, R.R., Vargas-Albores, F., Higuera-Ciapara, I., Söderhäll, K., Yepiz-Plascencia, G., 2004. Molecular cloning of a β-glucan pattern-recognition lipoprotein from the white shrimp Penaeus (Litopenaeus) vannamei: correlations between the deduced amino acid sequence and the native protein structure. Dev. Comp. Immunol. 28, 713–726. Sajeevan, T.P., Philip, R., Singh, I.S., 2009. Dose/frequency: a critical factor in the administration of glucan as immunostimulant to Indian white shrimp Fenneropenaeus indicus. Aquaculture 287, 248–252. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63–92. Sakai, M., Kobayashi, M., Yoshida, T., 1995. Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin. Comp. Biochem. Physiol. B 110, 755–759. Sang, H.M., Fotedar, R., 2010. Effects of mannan oligosaccharide dietary supplementation on performances of the tropical spiny lobster juvenile (Panulirus ornatus, Fabricius 1798). Fish Shellfish Immunol. 28, 483–489. Sang, H.M., Ky, L.T., Fotedar, R., 2009. Dietary supplementation of mannan oligosaccharide improves the immune responses and survival of marron, Cherax tenuimanus (Smith, 1912) when challenged with different stressors. Fish Shellfish Immunol. 2, 341–348. Smith, P., Hiney, M.P., Samuelsen, O.B., 1994. Bacterial resistance to antimicrobial agents used in fish farming: a critical evaluation of method and meaning. Ann. Rev. Fish Dis. 4, 273–313. Song, Y.L., Hsieh, Y.T., 1994. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Dev. Comp. Immunol. 18, 201–209. Staykov, Y., Spring, P., Denev, S., Sweetman, J., 2007. Effect of a mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquac. Int. 15, 153–161. Sung, H.H., Chen, P.H., Liu, C.L., 2008. Initiation of signal transduction of the respiratory burst of prawn haemocytes triggered by CpG oligodeoxynucleotide. Fish Shellfish Immunol. 24, 693–700. Tassakka, A., Sakai, M., 2002. CpG oligodeoxynucleotides enhance the non-specific immune responses on carp, Cyprinus carpio. Aquaculture 209, 1–10. Tassakka, A., Sakai, M., 2003. The in vitro effect of CpG-ODNs on the innate immune response of common carp, Cyprinus carpio L. Aquaculture 220, 27–36. Thompson, I., White, A., Fletcher, T.C., Houlihan, D.F., Secombes, C.J., 1993. The effect of stress on the immune response of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin C. Aquaculture 114, 1–18. Torrecillas, S., Makol, A., Caballero, M.J., Montero, D., 2007. Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol. 23, 969–981. Vargas-Albores, F., Jimenez-Vega, F., Yepiz-Plascencia, G.M., 1997. Purification and comparison of β-1,3-glucan binding protein from white shrimp (Penaeus vannamei). Comp. Biochem. Physiol. B 116, 453–458. Villamil, L., Figueras, A., Novoa, B., 2003. Immunostimulatory effects of nisin in turbot (Scophthalmus maximus L.). Fish Shellfish Immunol. 14, 157–169. Wang, S., Chen, J.C., 2005. The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 19, 191–204. Welker, T.L., Lim, C., Yildirim-Aksoy, M., Shelby, R., Klesius, P.H., 2007a. Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catfish, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. J. World Aqua. Soc. 38, 24–35. Welker, T.L., Lim, C., Yildirim-Aksoy, M., Klesius, P.H., 2007b. Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine lactoferrin. Aquaculture 262, 156–162. Xing, J., Leung, M.F., Chia, F.S., 1998. Quantitative analysis of phagocytosis by amebocytes of a sea cucumber, Holothuria leucospilota. Invertebr. Biol. 117, 13–22. Yu, X.Q., Zhu, Y.F., Ma, C., Fabrick, J.A., Kanost, M.R., 2002. Pattern recognition proteins in Manduca sexta plasma. Insect Biochem. Mol. Biol. 32, 1287–1293. Zhou, X., Niu, C., Sun, R., Li, Q., 2002. The effect of vitamin C on the non-specific immune response of the juvenile soft-shelled turtle (Trionyx sinensis). Comp. Biochem. Physiol. 131, 917–922.