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Veterinary Immunology and Immunopathology 121 (2008) 206215 www.elsevier.com/locate/vetimm

Dietary administration of sodium alginate and k-carrageenan enhances the innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and its resistance against Vibrio alginolyticus
Ann-Chang Cheng b, Yu-Yuan Chen a, Jiann-Chu Chen a,*
a

Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC b Department of Aquaculture, National Penghu University, Makong, Penghu 880, Taiwan, ROC Received 6 May 2007; received in revised form 19 September 2007; accepted 26 September 2007

Abstract Brown-marbled grouper Epinephelus fuscoguttatus which had been fed diets containing sodium alginate and kappa (k)carrageenan at 5, 10 and 20 g kg1, respectively after 0, 2, 4, 6, 8 and 14 weeks were examined for survival, growth, innate cellular and humoral responses as compared to the sh that fed control non-supplemented diet. Survival was 100% for the sh that fed all diets after 14 weeks and no signicant difference in growth was observed among seven diets. The sh that fed a diet containing sodium alginate at 10 g kg1, and fed a diet containing k-carrageenan at 5 g kg1 over 28 weeks showed signicantly increased leucocyte count, respiratory burst, phagocytic activity and phagocytic index, The sh that fed a diet containing sodium alginate at 5 g kg1, and fed a diet containing k-carrageenan at 5 g kg1 over 28 weeks showed signicantly increased alternative complement activity (ACH50) and lysozyme activity. In another experiment, E. fuscoguttatus, which had been fed control diet, and all diets containing sodium alginate, and k-carrageenan after 14 weeks were challenged with Vibrio alginolyticus at 5.0 109 colony-forming units (cfu) sh1 and then placed in seawater of 34%. The survival of sh that fed a diet containing sodium alginate at 10 g kg1 and k-carrageenan at 5 g kg1 was signicantly higher than that of sh which fed the control diet over 96168 h. It was concluded that E. fuscoguttatus which fed a diet containing sodium alginate at 10 g kg1 or less, or fed a diet containing kcarrageenan at 5 g kg1 enhance the innate immunity, and increase the resistance from V. alginolyticus infection. # 2007 Elsevier B.V. All rights reserved.
Keywords: Epinephelus fuscoguttatus; Vibrio alginolyticus; Sodium alginate; k-Carrageenan; Respiratory burst; Phagocytic activity; Alternative complement activity; Lysozyme activity

1. Introduction Orange-spotted grouper Epinephelus coioides, brown-marbled grouper Epinephelus fuscoguttatus and malabia grouper Epinephelus malabaricus are

* Corresponding author. Tel.: +886 2 2462 0295; fax: +886 2 2462 0295. E-mail address: jcchen@mail.ntou.edu.tw (J.-C. Chen). 0165-2427/$ see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.09.011

the most commonly species currently being cultured in Southeast Asian countries. Several disease problems including vibriosis caused by Vibrio alginolyticus and Vibrio carchariae, viral diseases caused by nervous necrosis virus and iridovirus, and protozoan parasite like Crytocaryon irritans have threatened commercial grouper farming in these regions (Yii et al., 1997; Fukuda et al., 1999; Yambot and Song, 2006). Therefore, maintenance of sh in health, and enhancement of shs innate immunity are of the primary

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concern. Application of immunostimulants has been considered an effective means in increasing the level of hosts immunity and preventing disease outbreak (Anderson, 1992). Seaweed polysaccharides like carrageenan, fucoidan and sodium alginate have showed immunostimulatory effects on nsh. For example, intraperitoneal injection of kappa (k)-carrageenan and sodium alginate have been demonstrated to provide resistance against pathogens and increase the innate immune response in common carp Cyprinus carpio (Fujiki et al., 1994, 1997; Fujiki and Yano, 1997), snakehead Channa striata (Miles et al., 2001), rainbow trout Oncorhynchus mykiss (Peddie et al., 2002), sea bass Dicentrarchus labrax (Bagni et al., 2005), and grouper E. coioides (Cheng et al., 2007). Oral administration of chitosan or b-glucan has been considered to be a simple and non-stressful procedure on nsh (Siwicki et al., 1994; Ortuno et al., 2002; Misra et al., 2006). However, little is known on the protective effect against pathogen of sh and their innate response by using seaweed polysaccharide through the route of oral administration (Bagni et al., 2005). The aim of the present study was to examine the innate immune parameters including total leucocyte count, respiratory burst, phagocytic activity, phagocytic index, lysozyme activity and alternative complement activity (ACH50) in grouper E. fuscoguttatus when fed diets containing either sodium alginate or k-carrageenan at 5, 10 and 20 g kg1 after 0, 2, 4, 6 and 8 weeks, and examine the resistance of sh against V. alginolyticus after 14 weeks. 2. Materials and methods

control non-supplemented diet, three diets containing sodium alginate at 5, 10 and 20 g kg1, and three diets containing k-carrageenan at 5, 10 and 20 g kg1. They were prepared based on a diet that was described before (Chen and Tsai, 1994). The proximate analysis of basal diet was 46.5247.94%, 8.939.87%, 16.7417.62% and 5.416.01% for crude protein, crude lipid, ash and moisture, respectively. Grouper in similar size (80 3.0 g) were randomly distributed into 21 cages (55 cm 30 cm 30 cm), and each cage was stocked with 30 sh. There were triplicate cages for each diet. Twenty-one cages were placed in two recirculation system tanks (7000 l) that were connected to a biological lter system. Water was recirculated with a ow rate at 10.8 tonnes h1. Fish were fed to satiation on 8:00 and 20:00 daily. The feeding trial lasted for 14 weeks. During the experimental period, water temperature ranged from 28 to 30 8C, and dissolved oxygen (DO) maintained greater than 5.2 mg l1. Two studies were conducted; a study on the immune parameter, and a study on the resistance of grouper against V. alginolyticus. For the study of immune parameters assays, six sh from each cage were sampled. Fish were marked in tail to avoid repeat sampling for next time sampling. The body weight ranged from 76 to 85, 97 to 104, 119 to 128, 136 to 147, 157 to 168 and 186 to 199 g, averaging 80 3.0, 101 2.5, 122 2.9, 141 2.9, 162 3.3 and 194 3.7 g (mean S.D.) for the grouper that had been reared after 0, 2, 4, 6, 8 and 14 weeks with no signicant difference among seven diets. A total of 630 sh (30 7 3) were used for the study of growth. 2.3. Formalin-killed Escherichia coli

2.1. E. fuscoguttatus and culture of grouper Three thousand brown-marbled grouper E. fuscoguttatus fry (3.3 cm, 0.65 0.02 g) were obtained from a private farm at Kaohsiung, Taiwan. They were shipped to our laboratory, and kept in two 7000 l circular tanks with recirculating 5000 l seawater (34%) that was connected to a biological ltering system (1400 l water capacity) at 2830 8C. Fish were fed daily with a commercial diet (Grobest, Taoyuan, Taiwan) until they reached to the size of 80 g and used for the following experiment. 2.2. Preparation of diets and experimental design 2.4. Zymosan Sodium alginate obtained from Kimica Corporation (Tokyo, Japan), and k-carrageenan was obtained from MSC Co., Ltd. (Seoul, Korea). There were seven diets: a A suspension of 50 mg zymosan (Z4250, Sigma) in 5 ml PBS was prepared in capped glass culture tube, and Escherichia coli (DH5a) was grown overnight in 100 ml tryptic soy broth (TSB) at 37 8C. Formaldehyde (37%) was added to give 2% nal concentration and the culture was shaken at 22 8C overnight. Stock cultures were centrifuged at 700 g for 10 min at 4 8C. The supernatant uid was removed and the bacterial pellet was washed twice with 50 ml PBS (phosphate buffer solution) solution (NaCl, 8.0 g l1; KH2PO4, 200 mg l1; Na2HPO4, 1.15 g l1; KCl, 200 mg l1; CaCl22H2O, 133 mg l1; MgCl26H2O, 100 mg l1) and re-suspended in 50 ml PBS and kept at 4 8C for phagocytosis test (Ndong et al., 2007).

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placed in a boiling water bath for 30 min with frequent shaking. The suspension solution was centrifuged at 600 g for 5 min. The pellet was re-suspended in 10 ml chicken serum (C5405, Sigma), incubated for 30 min at 30 8C, and then centrifuged at 600 g for 5 min. The supernatant uid was removed and the zymosan pellet was washed twice with 10 ml PBS, resuspended in 50 ml PBS to give 1 mg ml1 and stored at 4 8C for respiratory burst assay. 2.5. Culture of V. alginolyticus V. alginolyticus (ATCC17749) obtained from Bioresources Collection and Research Center, Food Industry and Development Institute (Hinchu, Taiwan) was used for the study. It was cultured on tryptic soy agar (TSA supplemented with 3% NaCl, Difco) for 24 h at 28 8C before being transferred to 10 ml tryptic soy broth (TSB supplemented with 3% NaCl, Difco), where it remained for 24 h at 28 8C as stock culture for tests. The broth cultures were centrifuged at 7155 g for 15 min at 4 8C. The supernatant uids were removed and the bacterial pellets were re-suspended in saline at 2.6 109 cfu ml1 for the resistance test of grouper to V. alginolyticus. 2.6. Effect of sodium alginate and k-carrageenan on the innate immune parameters of grouper After 0, 2, 4, 6 and 8 weeks, 6 sh from each cage were sampled. Fish were anaesthetized in 0.02% benzocaine solution. Blood (1.01.5 ml) was sampled individually from the caudal vein using a heparinized syringe (25 g) tted with a needle. Total leucocyte count was measured using an automated hematology analyzer (KX-21, Sysmex, Japan). The remainder of blood was used for the subsequent tests. Plasma was obtained by centrifugation of the blood sample at 14,700 g (Model 5403, Eppendorf, Hamburg, German) for 5 min and then stored at 30 8C for later lysozyme activity and alternative complement pathway analyses. 2.7. Separation of leucocyte Blood (500 ml) was mixed with 500 ml of AL medium (AIM-V medium and Leibovitzs L 15 medium, GIBCO BRL, Gaithersburg, MD, USA), containing streptomycin and penicillin. Percoll (55%, P4937, Sigma) was layered under the mixed blood solution, and then centrifuged at 400 g (Model 5403, Eppendorf) for 15 min at 10 8C. The leucocytes were obtained from the interface and washed with AL medium by centrifugation at 600 g for

10 min at 10 8C. After centrifugation, the leucocytes were suspended in 1 ml AL medium with 5.5 mM glucose. The number of cell viability was analyzed by trypan blue (0.1%) with a haematocytometer (Miller et al., 1994). 2.8. Measurement of innate cellular response The respiratory burst of the leucocytes was quantied using the reduction NBT (nitroblue tetrazolium) to formazan as a measure of superoxide anion (O2) production (Chung and Secombes, 1988) The absorbance at 630 nm was measured spectrometrically in triplicates with a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA) using DMSO/KOH alone as a blank. Respiratory burst was expressed as NBT-reduction in 100 ml of leucocyte suspension. Three hundred microlitres of leucocyte suspension in L-15 medium were added in triplicate tubes. Three hundred microlitres of formalin-killed E. coli in PBS was added to each tube and incubated for 1 h at 17 8C. Then, 900 ml cold PBS was added, and the tubes were centrifuged at 300 g for 5 min. The supernatants were discarded and the pellets were taken up and smeared on slides. The slides were air-dried, then stained with Giemsa solution (Sigma, St. Louis, MO, USA). The cells were counted under a microscope (Leica DMIL, Leica Microsystem. GMSH, Wetzlar, Germany). The phagocytic activity was expressed as the number of phagocytic cells per 100 adherent cells. The phagocytic index (PI) was expressed as the average number of E. coli ingested by each phagocytic cell (Mathews et al., 1990). 2.9. Measurement of innate humoral response Lysozyme activity was measured based on the turbidimetric assay (Ellis, 1990) Briey, a standard suspension (0.2 mg ml1) of Micrococcus lysodeikticus (M3770, Sigma) was prepared in 0.05 M sodium phosphate buffer (pH 6.2). Test plasma (10 ml) was added to 200 ml of the bacterial suspension in a 96-well microplate, and the decrease in absorbance at 520 nm was recorded after 1 and 4 min at 22 8C. Standard solution containing 0, 10, 20, 30, 50 and 100 units ml1 of hen egg white lysozyme (L6876, Sigma) was used to construct a standard curve. A unit of lysozyme activity was dened as the amount of plasma causing a reduction in absorbance of 0.001 min1. The activity of alternative complement pathway was assayed using sheep red blood cells (SRBC, R3378, Sigma) as targets (Ortuno et al., 1998, 2001). Briey,

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SRBC were washed three times in phenol red-free Hanks balance salt solution (HBSS) supplemented with 0.5 mM Mg2+ and 10 mM EGTA, and re-suspended at 3% in HBSS. The plasma sample which was diluted (1:50) to different volumes ranging from 0.1 to 0.25 ml were dispensed into a series of test tubes and the total volume was made up to 0.25 ml with the same buffer and then was added to 0.1 ml of SRBC suspension. After incubation for 1 h at 25 8C, the mixture was centrifuged at 400 g for 5 min at 4 8C. The relative hemoglobin content of the supernatant was assessed by measuring the optical density (OD) at 414 nm using a spectrophotometer (Model U-2000 Hitachi, Tokyo, Japan). The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 100 ml of distilled water or HBSS to 100 ml samples of SRBC, respectively. Control samples in the test tubes without SRBC were also added in each assay. The degree of haemolysis (Y) (percentage of haemolytic activity with respect to the maximum) was estimated and the lysis curve for each specimen was obtained by plotting Y/(1 Y) against the volume of plasma added (ml) on a log10log10 scaled graph. The volume of plasma yielding 50% haemolysis (ACH50) was determined and the number of ACH50 units ml1 was obtained for each experimental group. 2.10. Effect of sodium alginate and k-carrageenan on the resistance of grouper to V. alginolyticus Groupers that had been fed control diet, diets containing sodium alginate and k-carrageenan at 5, 10 and 20 g kg1 after 14 weeks were sampled for the study of resistance of grouper to V. alginolyticus. The body weight ranged from 186 to 199 g averaging 194 3.7 g with no signicant different among seven diets. There were eight treatments (seven challenged test group and one unchallenged control group). Challenge tests were conducted in triplicate with 15 sh per replicate. Each sh was injected intraperitoneally with bacterial suspension (2.6 109 cfu ml1) at a rate of 1 ml per 100 g of body weight resulting in 5.0 109 cfu sh1. The sh that fed control diet, and then received V. alginolyticus at 5.0 109 cfu sh1 served as the challenged control. The sh that fed control diet, and then received saline solution (around 1 ml per 100 g of body weight) served as the unchallenged control (Table 1). Test and control groups were comprised of 15 sh and in triplicate cages. Each treatment was conducted with 45 sh. A total of 360 sh [(7 15 3) + (1 15 3)] were used for the study. The grouper (15 sh cage1) were then kept in separate

cages (55 cm 30 cm 30 cm) with volume of 50 l seawater (34%), which was placed in recirculation system tanks (7000 l). A total of 24 cages were placed in two tanks. Survival of sh in each cage was examined every 12 h before 96 h and every 24 h after 96 h, and the experiment lasted 168 h. 2.11. Statistical analysis Turkeys multiple range tests were used to determine the signicant differences among treatments using SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (resistance test) were normalised using an arc sin transformation before analysis. Differences were considered statistically signicant when p < 0.05. 3. Results 3.1. Survival and growth of grouper that fed different diets Survival was 100% for the sh that fed all diets after 14 weeks. Weight gain percentage was 21.731.8%, 44.663.3%, 68.491.0%, 92.9117.4% and 129.0 159.1% for the sh that fed all diets after 2, 4, 6, 8 and 14 weeks, respectively. No signicant difference in weight gain percentage was observed among the sh that fed seven diets. 3.2. Total leucocyte count (TLC) of E. fuscoguttatus that fed different diets The TLC of sh that fed a diet containing sodium alginate at 10 g kg1 was signicantly higher than that of sh that fed the control diet over 28 weeks. The TLC of sh that fed a diet containing sodium alginate at 5 g kg1, and fed a diet containing sodium alginate at 20 g kg1 was signicantly higher than that of sh that fed the control diet after 4, 6 and 8 weeks, and after 4, 6 weeks, respectively. The TLC of sh that had fed diets containing k-carrageenan at 5 and 10 g kg1 was signicantly higher than that of sh that fed the control diet over 28 weeks. The TLC of sh that fed a diet containing k-carrageenan at 20 g kg1 was signicantly higher than that of sh that fed the control after 4, 6 and 8 weeks (Fig. 1). 3.3. Effect of sodium alginate and k-carrageenan on the innate cellular response The respiratory burst of sh that fed a diet containing sodium alginate at 10 g kg1 was signicantly higher

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Table 1 Survival of brown-marbled grouper Epinephelus fuscoguttatus challenged with Vibrio alginolyticus when the groupers were fed control nonsupplemented diet, and diets containing sodium alginate and k-carrageenan at 5, 10 and 20 g kg1 after 14 weeks Bacterial dose (cfu grouper1) Saline 5.0 10 9 5.0 10 9 5.0 10 9 5.0 10 9 5.0 10 9 5.0 10 9 5.0 10 9 Diet (g kg1) No. grouper Survival (%) and surviving number of grouper (in each group), time after challenge (h) 72 Control Control Sodium alginate 5 Sodium alginate 10 Sodium alginate 20 k-Carrageenan 5 k-Carrageenan 10 k-Carrageenan 20 3 15 3 15 3 15 3 15 3 15 3 15 3 15 3 15 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 100 (15,15,15) 84 100 (15,15,15) 95.6 5.0 b (14,15,14) 100a (15,15,15) 100a (15,15,15) 100a (15,15,15) 100a (15,15,15) 100a (15,15,15) 100a (15,15,15) 96 100 (15,15,15) 86.7 0.0 c (13,13,13) 93.3 0.0 b (14,14,14) 100a (15,15,15) 100a (15,15,15) 100a (15,15,15) 95.6 5.0 b (14,15,14) 93.3 0.0 b (14,14,14) 120 100 (15,15,15) 77.8 5.0c (12,11,12) 86.7 0.0ab (13,13,13) 88.9 5.0a (13,14,13) 82.2 5.0bc (13,12,12) 86.7 0.0ab (13,13,13) 86.7 0.0ab (13,13,13) 82.2 5.0bc (12,12,13) 144 100 (15,15,15) 57.8 10.0c (8,8,10) 57.8 5.0c (8,9,9) 73.3 0.0a (11,11,11) 66.7 0.0ab (10,10,10) 68.9 5.0ab (10,11,10) 66.7 0.0ab (10,10,10) 64.4 5.0bc (9,10,10) 168 100 (15,15,15) 46.7 8.7 d (6,7,8) 57.8 5.0 c (8,9,9) 73.3 0.0 a (11,11,11) 62.2 5.0 bc (10,9,9) 66.7 0.0 ab (10,10,10) 62.2 5.0 bc (9,9,10) 55.6 5.0 c (8,8,9)

Data in the same column with different letters are signicantly different ( p < 0.05) among different treatments. Values are mean S.E. (n = 45 grouper in each treatment).

than that of sh that fed the control diet over 28 weeks. The respiratory burst of sh that fed a diet containing sodium alginate at 5 g kg1, and fed a diet containing sodium alginate at 20 g kg1 was signicantly higher than that of sh that fed the control diet after 4, 6 and 8 weeks. The respiratory burst of sh that fed all diets containing k-carrageenan was signicantly higher than that of sh that fed the control diet over 28 weeks (Fig. 2A). The phagocytic activity of sh that fed all diets containing sodium alginate was signicantly higher

than that of sh that fed the control diet over 28 weeks. The phagocytic activity of sh that fed a diet containing k-carrageenan at 5 g kg1 was signicantly higher than that of sh that fed the control diet over 28 weeks. The phagocytic activity of sh that fed diets containing kcarrageenan at 10 and 20 g kg1 was signicantly higher than that of sh that fed the control diet after 2, 4 and 6 weeks (Fig. 2B). The phagocytic index of sh that fed a diet containing sodium alginate at 10 g kg1 was signicantly higher than that of sh that fed the control diet over 28 weeks.

Fig. 1. Mean (S.E.) total leucocyte count of brown-marbled grouper Epinephelus fuscoguttatus that fed control diet, and diets containing sodium alginate and k-carrageenan at 5, 10, 20 g kg1 after 0, 2, 4, 6 and 8 weeks. Each bar represents mean value from 18 determinations with standard error. Data in the same time with different letters are signicant ( p < 0.05) among different diets.

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Fig. 2. Mean (S.E.) respiratory burst (A), phagocytic activity (B), and phagocytic index (C) of brown-marbled grouper E. fuscoguttatus that fed control diet, and diets containing sodium alginate and k-carrageenan at 5, 10, 20 g kg1 after 0, 2, 4, 6 and 8 weeks. See Fig. 1 for statistical information.

The phagocytic index of sh that fed a diet containing sodium alginate at 5 g kg1 was, and fed a diet containing sodium alginate at 20 g kg1 was signicantly higher than that of sh that fed the control diet after 4, 6 and 8 weeks, and after 2, 6 and 8 weeks, respectively. The phagocytic index of sh that fed a diet containing kcarrageenan at 5 g kg1 was signicantly higher than that of sh that fed the control diet over 28 weeks (Fig. 2C).

3.4. Effect of sodium alginate and k-carrageenan on the innate humoral response The ACH50 of sh that fed all diets containing sodium alginate was signicantly higher than that of sh that fed the control diet over 28 weeks. The ACH50 of sh that fed all diets containing k-carrageenan was signicantly higher than that

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Fig. 3. Mean (S.E.) ACH50 (A) and lysozyme activity (B) brown-marbled grouper E. fuscoguttatus that fed control diet, and diets containing sodium alginate and k-carrageenan at 5, 10, 20 g kg1 after 0, 2, 4, 6 and 8 weeks. See Fig. 1 for statistical information.

of sh that fed the control diet over 28 weeks (Fig. 3A). The lysozyme activity of sh that fed all diets containing sodium alginate was signicantly higher than that of sh that fed the control diet over 28 weeks. The lysozyme activity of sh that fed all diets containing k-carrageenan was signicantly higher than that of sh that fed the control diet over 28 weeks (Fig. 3B). 3.5. Effect of sodium alginate and k-carrageenan on the resistance of grouper E. fuscoguttatus to V. alginolyticus All the unchallenged control sh survived in 168 h. All the challenged sh that fed all diets containing sodium alginate and k-carrageenan survived after 84 h. By contrast, death began to occur after 96 h in the challenged sh that fed a diet containing sodium

alginate at 5 g kg1, and diets containing k-carrageenan at 10 and 20 g kg1. After 96168 h, survival of sh that fed a diet containing sodium alginate at 10 g kg1, and a diet containing k-carrageenan at 5 g kg1 were signicantly higher than that of sh that fed the control diet (Table 1). 4. Discussion In the present study, data of grouper that grew from initial weight of 80 3.0194 3.7 g after 14 weeks with 100% of survival, and with no signicant difference in weight gain among seven diets were very satisfactory as compared to the published data in juvenile (7.612.2 g) grouper E. malabaricus (Chen and Tsai, 1994; Lin and Shiau, 2005; Ye et al., 2006). The fact that the survival of grouper E. fuscoguttatus that fed all diets containing either sodium alginate or kcarrageenan, and then challenged with V. alginolyticus,

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did not show increase directly with amount of stimulant in diets suggests that higher dose of these polysaccharides did not increase the resistance against V. alginolyticus. The fact that the survival of grouper that fed a diet containing sodium alginate at 10 g kg1, was signicantly higher than that of grouper that fed diets containing sodium alginate at 5 and 20 g kg1, and the survival of grouper that fed a diet containing kcarrageenan at 5 g kg1 was signicantly higher than that of grouper that fed diets containing k-carrageenan at 10 and 20 g kg1 indicates that suitable dose of sodium alginate and k-carrageenan, and suggests that too much amount of these stimulants did not further increase the resistance against V. alginolyticus. Sea bass Dicentrarchus labrax that fed a diet containing sodium alginate after 15 days showed signicantly increased ACH50 and lysozyme activity (Bagni et al., 2005). In the present study, the fact that the grouper E. fuscoguttatus that fed diets containing sodium alginate or k-carrageenan at 5, 10 and 20 g kg1 all showed signicantly higher TLC, respiratory burst, phagocytic activity, phagocytic index, ACH50 and lysozyme activity after either 2 or 4 weeks of feeding suggests that these seaweed polysaccharides increase both humoral and cellular immune responses. It is very interesting to note that the grouper that fed a diet containing sodium alginate at 10 g kg1 showed signicantly higher immune parameters after 2 weeks of feeding, and these parameters still maintain signicantly higher after 8 weeks of feeding. Moreover, the grouper that fed a diet containing k-carrageenan at 5 g kg1 showed signicantly higher immune parameters after 2 weeks of feeding, and these parameters still maintain signicantly higher after 8 weeks of feeding. This fact indicates optimal amount of sodium alginate (10 g kg1) and optimal amount of k-carrageenan (5 g kg1). This fact also indicates that the data of TLC, humoral response and cellular response all correlate well with the challenge test: higher survival for the grouper that fed a diet containing sodium at 10 g kg1, and fed a diet containing k-carrageenan at 5 g kg1 after 168 h. The present study indicated an optimal dose of 10, and 5 g kg1 for sodium alginate and k-carrageenan, respectively. In vitro, the respiratory burst of glucantreated macrophages was maximal at glucan at 0.1 1.0 mg ml1, whereas the respiratory burst of glucantreated macrophages showed no effect or decrease at doses of 10 and 50 mg ml1 (Robertsen et al., 1994). The respiratory burst of yellow croaker Psudosciaena crocea that fed a diet containing glucan at 0.18% was signicantly lower than that of sh that fed a diet

containing glucan at 0.09% (Ai et al., 2007). The effects of stimulants are not directly dose-dependent. We like to make some comparisons between the effect of immune responses for the grouper that fed sodium alginate containing diets and fed k-carrageenan containing diets. The grouper that fed a diet containing sodium alginate at 10 g kg1 increased TLC by 43%, 48%, 70%, 40%, whereas the grouper that fed a diet containing k-carrageenan at 5 g kg1 increased TLC by 33%, 30%, 44% and 45%, as compared to sh that fed the control diet after 2, 4, 6 and 8 weeks, respectively. Similar humoral response and cellular response were obtained. For example, the grouper that fed a diet containing sodium alginate at 10 g kg1, and fed a diet containing k-carrageenan at 5 g kg1 increased the respiratory burst by 15% and 10%, phagocytic activity by 12% and 8%, phagocytic index by 35% and 19%, ACH50 by 21% and 10%, and lysozyme activity by 146% and 129% after 2 weeks of feeding. We do not know the exact mechanism why sodium alginate and k-carrageenan enhance immunity of grouper and its resistance against V. alginolyticus, and do not know why higher amount of sodium alginate and k-carrageenan may not further enhance the immunity. It is known that b-glucan is mainly composed of b-1,3-linked glucosyl residues with 95% of glucose (Engstad and Robertsen, 1993). Sodium alginate consists of D-mannuronate and L-gluronate, and the main component of k-carrageenan is a-D-galactose and b-D-galactose residue with sulfate ester groups (Renn, 1997). Glucan receptors exist in macrophages of both Atlantic salmon Salmo salar and channel catsh Ictalurus punctatus (Engstad and Robertsen, 1993; Ainsworth, 1994). Moreover, glucan enhances the innate immunity of sh through direct activation of macrophages (Robertsen, 1999). The present study indicated that the humoral response including respiratory burst, phagocytic activity and phagocytic index increased signicantly for the grouper that were fed diets containing sodium alginate and k-carrageenan. This supports the view that these seaweed polysaccharides have an effect on macrophage activation leading to enhanced immunity/resistance. Alginate and carrageenan have been reported to increase inammation and to elicit potent chemotactic activities when injected into peritoneal cavity (Corsini et al., 2005; Bylund et al., 2006). The pattern recognition molecules and receptors for these seaweed polysaccharides and how they trigger the innate immune response in teleosts are needed for further study. In conclusion, the present study documented that E. fuscoguttatus that fed all diets containing sodium

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alginate or k-carrageenan increases the immunity after 2 weeks. The grouper that fed a diet containing sodium alginate at 10 g kg1, and fed a diet containing kcarrageenan at 5 g kg1 increases TLC, innate humoral immune response, cellular immune response together with an increased resistance against V. alginolyticus. However, the grouper that fed diets containing either sodium alginate or k-carrageenan higher than above levels do not further increase immune response and increase resistance against V. alginolyticus suggesting optimal dose of these seaweed polysaccharides. Acknowledgements This research was supported partially by a grant from the Council of Agriculture (95-No-ker-14.2.3.-Yu-F14), and Center for the Marine Bioscience and Biotechnology, National Taiwan Ocean University. We appreciate Mr. S.A. Cheng for his technical assistance. References
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