- Email: [email protected]
The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus
Fish & Shellfish Immunology 22 (2007) 673e685 www.elsevier.com/locate/fsi
The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus Yu-Win Fu, Wen-Ying Hou, Su-Tuen Yeh, Chiu-Hsia Li, Jiann-Chu Chen* Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC Received 9 June 2006; revised 16 August 2006; accepted 16 August 2006 Available online 24 August 2006
Abstract The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when white shrimp Litopenaeus vannamei were immersed in seawater (34&) containing hot-water extract of red alga Gelidium amansii at 200, 400 and 600 mg l1, injected with hot-water extract at 4 and 6 mg g1 shrimp, and fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1. These parameters increased significantly when shrimp were immersed in seawater containing hot-water extract at 400 and 600 mg l1 after 1 h, when shrimp were injected with hot-water extract at 6 mg g1 shrimp after one day, and when shrimp were fed diets containing hot-water extract at 1.0 and 2.0 g kg1 after 14 days. Phagocytic activity and clearance efficiency were significantly higher for the shrimp that were fed diets containing hot-water extract at 1.0 and 2.0 g kg1 than those of shrimp that were fed diets containing hot-water extract at 0 and 0.5 g kg1 after 14 and 28 days. In a separate experiment, L. vannamei which had received hot-water extract via injection, or fed diets containing hot-water extract, were challenged after 3 h or 28 days with V. alginolyticus at 2 106 cfu shrimp1 and 1 106 cfu shrimp1, respectively, and then placed in seawater. The survival of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of control shrimp after 1 day, and the survival of shrimp fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 increased significantly after 3 days as well as at the end of the experiment (6 days after the challenge), respectively. It was concluded that L. vannamei that were immersed in hot-water extract at 400 mg l1, injected with hot-water extract at 6 mg g1 shrimp, and fed hot-water extract of G. amansii at 2.0 g kg1 or less showed increased immune ability as well as resistance to V. alginolyticus infection. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Litopenaeus vannamei; Vibrio alginolyticus; Hot-water extract of Gelidium amansii; Immersion; Injection; Diet; Immune parameter; Resistance
* Corresponding author. Tel./fax: þ886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.08.014
674
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
1. Introduction White shrimp Litopenaeus vannamei has become the primary species of penaeid shrimp currently being cultured in China, Indonesia, Thailand, and Taiwan. A bacterium Vibrio alginolyticus isolated from diseased L. vannamei is considered to be a secondary and opportunistic pathogen, and causes mortality of shrimp under stressful environments like low salinity and ammonia [1e3]. Disease outbreaks are associated with increases in the proportion of opportunistic bacteria like Vibrio spp. which become prevalent in cultured pond waters [4]. Therefore, the health of shrimp and enhancement of its immunity are of primary concern. Invertebrates lack an adaptive immune system and rely on innate immune responses against microbial invasion [5]. Circulating haemocytes are generally classified into hyaline, semi-granular and large granular cells [6]. Both semigranular and granular cells carry out the function of proPO (prophenoloxidase) activating system [7]. Phenoloxidase (PO) is the terminal enzyme in the proPO system and is activated by minute amounts of microbial cell wall components such as lipopolysaccharides (LPS) from Gram-negative bacteria, peptidoglycan from Gram-positive bacteria and b-1,3-glucan from fungal cell walls [8,9]. In decapod crustaceans, hyaline cells are generally involved in phagocytoisis, an important process of eliminating microorganisms or foreign particles [7,9]. A series of reactive oxygen species (ROS) are produced during phagocytosis. Starting this process, the membrane-bound enzyme complex, NADPH oxidase, assembles after binding of a foreign particle to the cell, and reduces molecular oxygen to superoxide anion (O 2 ), subsequently leading to the production of hydrogen peroxide (H2O2), singlet oxygen (1O2) and hydroxyl radicals (OH$) [10]. Superoxide anion is the first product released during this process known as respiratory burst, and plays an important role in anti-bacterial activity [11]. Several bacterial polysaccharides like schizophyllan, a b-1,3-glucan extracted from fungus Schizophyllum commune, a b-1,3-1,6-glucan extracted from yeast Saccharomycs cerevisiae, peptidoglycan extracted from Bifobacterium thermoplilum and LPS (lipopolysaccharide) extracted from Pantea agglomerans have been used as immunostimulants, and their immunostimulatory effects have been studied in fish [12,13] and crustaceans [14e17]. Some red algae contain antitumour polysaccharides, and have been reported to inhibit growth of Ehrlich carcinoma in mice [18]. The polysaccharides derived from red algae have also been used as immunostimulants in teleosts and shrimp. For example, intraperitoneal injection of hot-water extracts of Gloiopeltis complanata, Hypenea charoides and Chondrus ocellatus increased resistance of common carp Cyprinus carpio against Edwardsiella tarda, and yellowtail Seriola quinqueradiata against Streptococcus sp. infection [19]. White shrimp L. vannamei, receiving hot-water extract of Gracilaria tenuistipitata via injection, showed increased immune ability and resistance against V. alginolyticus [20]. Agars/agaroses and carrageenan which are the common polysaccharides obtained from Rhodophyta (red algae) and are polymers of 1,4-linked a-D-galactose and 3,6-anhydro-a-L-galactose backbone (agarobiose) and 1,3-linked a-D-galactose and 1,4-linked 3,6-anhydro-b-D-galactose backbone (carrabiose), respectively, are of significant commercial values in food, cosmetics and pharmaceuticals [21]. The principle sources of agars are Gelidinium and Gracilaria, and important sources of carrageenan are Eucheuma, Chondrus, Gigartina and Kappaphycus [21]. Gelidium amansii, an important and common red alga, inhabits the north east coast of Taiwan has been used for gel food and processed for agar, and is considered to be a potential source of an anti-bacterial agent and immunostimulant. This study examined the immune response of white shrimp L. vannamei and its resistance against V. alginolyticus following treatment with hot-water extract of G. amansii. The shrimp that were immersed in seawater containing hot-water extract, injected with hot-water extract, and fed diets containing hot-water extract were examined for immune parameters (total haemocyte count, phenoloxidase activity, respiratory burst, and superoxide dismutase activity). Phagocytic activity and clearance efficiency of L. vannamei against V. alginolyticus were studied when shrimp were fed diets containing hot-water extract after 14 and 28 days. In addition, L. vannamei that were injected with hotwater extract after 3 h, and fed diets containing hot-water extract after 28 days, were examined for resistance against V. alginolyticus. 2. Materials and methods 2.1. Culture of V. alginolyticus V. alginolyticus isolated from diseased L. vannamei was used in the study [4]. Broth cultures and preparation of bacterial suspension were conducted based on the method described previously [20]. The bacterial pellets were
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
675
suspended in saline at 1 108 and 5 107 cfu ml1, and 1 108 cfu ml1 as bacterial suspensions for the resistance test, and for the test of phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus, respectively. 2.2. Preparation of hot-water extract of G. amansii G. amansii was collected from the Keelung coast near the University. Hot-water extract of G. amansii was prepared based on the method described before [19,20]. The harvest weight of hot-water extract obtained from 10 g of the milled frond of G. amansii was 1.85 g. The hot-water extract contains 67% of sugar in weight, and the main components are galactose (55.1%), mannose (5.7%), xylose (6.2%) and fucose (3.9%) based on GC-MS after hydrolysis, reduction and acetylation of the sugar [22,23]. For the immersion test, the hot-water extract at 2, 4 and 6 g was first dissolved in 100 ml distilled water and then mixed in 10 l seawater (34&) to obtain final concentrations of 200, 400 and 600 mg l1, respectively. For the injection test, the hot-water extract was prepared with a phosphate buffered saline to make 2 and 3 mg ml1 as test solutions [20]. For the dietary administration test, there were four diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 which were prepared based on a diet described previously for L. vannamei [24]. The proximate analysis of basal diet was 39.2e40.6%, 7.2e7.5%, 11.2e11.6%, 13.0e13.2%, and 3.7e3.8% for crude protein, crude lipid, cellulose, ash and moisture, respectively. 2.3. L. vannamei and culture of shrimp About one thousand shrimp harvested from the University Marine Station adjacent to the coast of Keelung, Taiwan were shipped to the laboratory. Shrimp were placed in fiberglass tanks (8 m3), and acclimated to room temperature (25 1 C) for 2 weeks. During the acclimation period, shrimps were fed twice daily with a formulated shrimp diet (Tairou Feed Company, Tainan, Taiwan). There were three treatments: immersion, injection and dietary administration tests. Three studies were conducted: immune parameters, phagocytic activity and clearance efficiency, and resistance against V. alginolyticus. For the examinations of immune parameters (THC, phenoloxidase activity, respiratory burst and superoxide dismutase activity), test and control groups comprised 8 shrimps each. For the study of phagocytic activity and clearance efficiency to V. alginolyticus, test and control groups also comprised 8 shrimps each, and were conducted in the dietary administration test only. For the study of resistance of shrimp to V. alginolyticus, test and control groups comprised 10 shrimps each in triplicate, and were conducted in the injection and dietary administration tests. The shrimp ranged from 8.7 g to 13.2 g, averaging 10.6 2.1 g (mean SD) with no significant size differences among the treatments for the immersion and injection tests. The body weight of shrimp in the dietary administration test was 11.4, 12.4, 13.3, 14.5, 15.8, 19.0 and 21.0 g for the shrimp that had been fed diets containing hot-water extract after 0, 3, 6, 9, 14, 21 and 28 days with no significant difference among four treatments. Only shrimp in the intermoult stage were used for the subsequent tests [25]. For the dietary administration test, 8 tanks (0.9 m3) were stocked each with 80 shrimps (equivalent to density of 89 (shrimps) m3 with an average initial weight of 11.4 0.2 g. Tanks received continuous aeration, and water flow was 2 l min1. During the experimental period, water temperature ranged from 27 to 29 C, pH from 7.7 to 8.2, salinity from 33 to 35&, and dissolved oxygen concentration from 6.14e6.56 mg l1. Shrimps were fed their respective diets at a rate of 3% of body weight at 10:00 h and 18:00 h. Thirty shrimp were randomly sampled from each tank and weighed weekly, and the daily ration was adjusted accordingly. After 0, 3, 6, 9, 14, 21 and 28 days, 8 shrimps from each treatment were randomly sampled, and were examined for the THC, phenoloxidase (PO) activity, respiratory burst and SOD activity. After 14 and 28 days of rearing, 8 shrimps were sampled randomly from each treatment for the tests of phagocytic activity and clearance efficiency to V. alginolyticus. In addition, 30 shrimps from each treatment were sampled for the study of resistance against V. alginolyticus after 28 days of rearing. 2.4. The resistance of L. vannamei to V. alginolyticus For the injection test, L. vannamei was injected individually into the ventral sinus of the cephalothorax with hotwater extract of G. amansii at 2 and 3 mg ml1 solution at a rate of 20 ml per 10 g weight to reach doses of 4 and 6 mg g1 shrimp, respectively on the first day based on the same method described previously [20]. The challenge test was conducted on the second day by the injection of 20 ml of bacterial suspension (1 108 cfu ml1) resulting
676
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
in 2 106 cfu shrimp1 into the ventral sinus of the cephalothorax. Survival of shrimp was examined daily, and the experiment lasted 6 days (144 h). For the diet administration test, thirty shrimps which had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 were sampled after 28 days from each treatment and studied for the resistance of shrimp against V. alginolyticus. The challenge method used was the same as that described except the bacterial dose was 5 107 cfu ml1 resulting in 1 106 cfu shrimp1. The shrimp that were fed control diet, and then injected with saline (20 ml) served as the unchallenged control. 2.5. The immune parameters of L. vannamei that received hot-water extract of G. amansii For the immersion test, there were four concentrations (0, 200, 400, and 600 mg l1) and three exposure times (0.5, 1 and 3 h). Each treatment group was immersed in 20 l seawater containing hot-water extract at 0, 200, 400 and 600 mg l1, respectively. The amount of hot-water extract was 4, 8 and 12 g for the treatment of 200, 400 and 600 mg l1, respectively. For the injection test, there were four treatments (control, saline, 4 mg g1, and 6 mg g1) and five exposure times (0, 1, 2, 4 and 6 days). L. vannamei were injected individually in the ventral sinus of the cephalothorax with 20 ml of hot-water extract of G. amansii at 2 and 3 mg ml1 solution at a rate of 20 ml per 10 g weight to reach doses of 4 mg g1 and 6 mg g1, respectively. Shrimp injected with saline (20 ml) and shrimp with no injection served as the saline and control groups, respectively. For the dietary administration test, there were four treatments (0, 0.5, 1.0 and 2.0 g kg1) and seven sampling times (0, 3, 6, 9, 14, 21, 28 days). 2.6. Measurements of immune parameters Shrimp which been immersed in hot-water extract were sampled after 0.5, 1 and 3 h in the immersion test, injected with hot-water extract after 0, 1, 2, 4 and 6 days in the injection test, and fed diets containing hot-water extract after 0, 3, 6, 9, 14, 21 and 28 days were used for the studies. Haemolymph (100 ml) sampling, preparation of anticoagulant-haemolymph mixture, and counting of THC followed the procedures described previously [20]. Phenoloxidase (PO) activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) following the procedure of Herna´ndez-Lo´pez et al. [26]. The details of measurements were described previously [2]. The optical density at 490 nm for the shrimp’s PO activity was measured using a spectrophotometer (Model U-2000, Hitachi, Tokyo, Japan), and expressed as dopachrome formation in 50 ml of pure haemolymph. Respiratory burst of haemocytes was quantified using the reduction of NBT (nitroblue tetrazolium) to formazan as a measure of superoxide anion (O 2 ) as described previously [2]. The NBT solution was removed and the haemocytes were fixed with 100% methanol, and washed three times with 100 ml 70% methanol, and air-dried. Formazon was dissolved by the addition of 120 ml 2 M KOH and 140 ml dimethyl sulphoxide (DMSO). The optical density at 630 nm was measured using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA), and expressed as NBT-reduction in 10 ml of pure haemolymph. Superoxide dismutase (SOD) activity was examined for the dietary administration test only. SOD activity was measured by its ability to inhibit superoxide radical dependent reactions using the Ransod Kit (Randox, Crumlin, UK). The detail of the measurement was described previously [2]. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units ml1. 2.7. Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus L. vannamei that had been fed diets containing the hot-water extract for 14 and 28 days were used for the study. There were four treatments (0, 0.5, 1.0 and 2.0 g kg1) with two time periods (14 and 28 days). Tests were carried out in eight replicate test groups with one shrimp in each treatment and exposure time.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
677
Phagocytic activity and clearance efficiency tests were conducted by injecting 20 ml bacterial suspension (1 108 cfu ml1) resulting in 2 106 cfu shrimp1 based on the method described previously [20]. Phagocytic activity, defined as phagocytic rate (PR), was expressed as: PR ¼ fð phagocytic haemocytesÞ=ð total haemocytesÞg 100 The number of bacterial colonies for the shrimp that were fed control diet was expressed as the control group, and the number of colonies in the shrimp that were fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 served as the test group. Clearance efficiency, defined as percentage inhibition (PI) of V. alginolyticus, was calculated as follows: PI ¼ 100 fð cfu in test groupÞ=ðcfu in control groupÞg 100
2.8. Statistical analysis A multiple comparison (Tukey) test was used to examine the significant differences among treatments using the SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (resistance test) were normalised using an arc sin transformation before analysis. For statistically significant differences, it was required that p < 0.05. 3. Results 3.1. Survival and growth of L. vannamei fed diets containing hot-water extract Survival was 95%, 95%, 96% and 98% for the L. vannamei that had been fed diet containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1, respectively. Weight gain percentage was 83.4%, 83.4%, 84.3% and 84.5% for the shrimp that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1, respectively after 28 days. No significant differences in survival and growth were observed among the shrimp fed the four different diets. 3.2. The resistance of L. vannamei injected with hot-water extract All the unchallenged control shrimp survived. By contrast, death began to occur after 24 h in the challenged shrimp which had been injected with hot-water extract. After 24e144 h of challenge, survival of shrimp that received hotwater extract of G. amansii at 6 mg g1 was significantly higher than that of shrimp that received saline, and the control shrimp (Fig. 1A). 3.3. The resistance of L. vannamei fed diets containing hot-water extract All the unchallenged control shrimp survived. By contrast, death began to occur after 12 h in the challenged shrimp that had been fed diet containing hot-water extract at 0.5 g kg1 and the control diet. After 48 h of challenge, survival of shrimp that had been fed a diet containing hot-water extract at 2.0 g kg1 was significantly higher than that of shrimp that had been fed control diet. After 72e144 h of challenge, survival of shrimp that had been fed a diet containing hotwater extract at 0.5 g kg1 was significantly higher than that of shrimp that had been fed the control diet (Fig. 1B). 3.4. The immune parameters of L. vannamei that received hot-water extract of G. amansii The THC of L. vannamei that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of the control shrimp after 0.5, 1 and 3 h (Fig. 2A). The THC of L. vannamei that were injected with hot-water extract of G. amansii at 6 mg g1 was significantly higher than that of shrimp that were injected with hot-water extract at 4 mg g1, and the shrimp that were injected with saline, and the control shrimp after 1 day. The THC of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1 was significantly higher than that of control shrimp after 6 days (Fig. 2B). The THC of L. vannamei that had been fed a diet containing the hot-water extract at 2.0 g kg1 was significantly higher than that of shrimp fed the control diet after 6 days. The
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
678
A
100 90
Hot-water extract of Gelidium amansii (μg g-1) Challenged Unchallenged Control Saline 4 Control
80
6
Survival (%)
70 a
60
a
a
ab
ab
a
a
a
ab
ab
ab
50 ab 40 bc
bc
b
b
b
b
c
c
b
b
b
b
30 20 10 0 0
24
48
72
96
120
144
Time elapsed (h)
B
a a a a
100 90 80
a a a
a
a
ab
a
ab
b
b
Survival (%)
70
a
a
a
b
ab
ab
ab
c
b
b
b
c
c
c
60 50 40 30
Hot-water extract of Gelidium amansii (g kg-1)
20
Unchallenged
Challenged
Control
10
Control
0.5
1.0
2.0
0 0
24
48
72
96
120
144
Time elapsed (h) Fig. 1. Survival (%) of Litopenaeus vannamei that had been injected with hot-water extract of Gelidium amansii after one day, challenged with Vibrio alginolyticus at a dose of 2 106 cfu shrimp1, and placed in seawater at 34& and 25 1 C (A) and survival (%) of L. vannamei that had been fed diets containing hot-water extract after 28 days, challenged with V. alginolyticus at a dose of 1 106 cfu shrimp1, and placed in seawater at 34& and 25 1 C (B). Data (mean SE) in the same exposure time with different letters are significantly (p < 0.05) among treatments.
THC of L. vannamei that had been fed a diet containing hot-water extract at 1.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 14, 21 and 28 days (Fig. 2C). The PO activity of shrimp that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of shrimp that were immersed in 200 mg l1, and that of control shrimp after 0.5, 1 and 3 h (Fig. 3A). The PO activity of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of the control shrimp after 1e6 days (Fig. 3B). The PO activity of L. vannamei that had been fed diets containing
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Total haemocyte count (×105 ml-1)
A
Hot-water extract of Gelidium amansii (mg l-1) Control
350
200
400
679
a
600
a
a 300
a
250
a
200
b
b
bc
c
b
b
b
150 100 50 0 0.5
1
3
Time elapsed (h)
Total haemocyte count (×105 ml-1)
B
Hot-water extract of Gelidium amansii (µg g-1) Control
300
Saline
6
4 a
a
250
ab
200
b a a a a
b
150
ab
b
a
a a
b
b
b
ab
ab
a
100 50 0 0
1
2
4
6
Time elapsed (days)
Total haemocyte count (x105 ml-1)
C
Hot-water extract of Gelidium amansii in diet (g kg-1)
300
0
0.5
1.0
2.0
250 a a
150
a a a a
a a a a
b ab
ab a b
b
a a
a a
200 ab b
b
ab
ab
bc
b c
100 50 0 0
3
6
9
14
21
28
Time elapsed (days) Fig. 2. THC (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, THC (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and THC (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. Each bar represents mean value from eight shrimp with standard error. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
680
Phenoloxidase activity (O.D.490 nm)
A
Hot-water extract of Gelidium amansii (mg l-1)
0.60
Control
200
400
600
a
0.50
a a
0.40
a
0.30
a
b b
a
0.20
b
b
b
b
0.10 0.00 0.5
1
3
Time elapsed (h)
Phenoloxidase activity (O.D. 490 nm)
B
Hot-water extract of Gelidium amansii (μg g-1)
0.25
Control a
0.20
Saline
4
6 a
b 0.15
a ab
ab
b b
a a a a
a
a a
b b
ab
b b
0.10
0.05
0.00 0
1
2
4
6
Time elapsed (days)
Phenoloxidase activity (O.D.490 nm)
C Hot-water extract of Gelidium amansii in diet (g kg-1)
0.70
0
0.5
2.0 a
1.0
0.60
a
0.50 a a
a
0.40
a
ab a
a b
a a
0.30 a 0.20
a a a a
a
a
a
a a
a a b
b
b
c
0.10 0.00 0
3
6
9
14
21
28
Time elapsed (days) Fig. 3. Phenoloxidase activity (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, phenoloxidase activity (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and phenoloxidase activity (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Respiratory burst (O.D.630 nm)
A
0.25
681
Hot-water extract of Gelidium amansii (mg l-1) 200
Control
400
600
a
0.20 a
a
0.15
a
a
ab
b
b
b
b
a
b
0.10
0.05
0.00 0.5
1
3
Time elapsed (h)
Respiratory burst (O.D. 630 nm)
B
Hot-water extract of Gelidium amansii (μg g-1)
0.25
Control a
Saline
4
6
a
0.20
a
a
a ab
0.15 b a
a
b
b
a a
b
b
b
b
ab
ab a
0.10
0.05
0.00 0
1
2
4
6
Time elapsed (days)
Respiratory burst (O.D.630 nm)
C
Hot-water extract of Gelidium amansii in diet (g kg-1)
0.30
0.5
0
2.0
1.0
0.25 0.20
a
a
a a
a
c
b
a a a
b c
b
a
a
a a
b
a a a
a a a 0.15
a
a
d
c
0.10 0.05 0 0
3
6
9
14
21
28
Time elapsed (days)
Fig. 4. Respiratory burst (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, respiratory burst (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and respiratory burst (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Superoxide dismutase activity (units ml-1)
682 1.2
Hot-water extract of Gelidium amansii in diet (g kg-1) 0
0.5
1.0
2.0
1.0
0.8
a a a a
a a a a
a a a a
0
3
6
b
a a a
a a a b
a a a
a a a b
b
0.6
0.4
0.2
0 9
14
21
28
Time elapsed (days) Fig. 5. Superoxide dismutase activity of Litopenaeus vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 14 and 28 days. See Fig. 1 for statistical information.
hot-water extract at 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 9e28 days (Fig. 3C). The respiratory burst of shrimp that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of the control shrimp after 0.5, 1 and 3 h (Fig. 4A). The respiratory burst of shrimp that were injected with hot-water extract of G. amansii at 4 and 6 mg g1 was significantly higher than that of shrimp that were injected with saline as well as the control shrimp after 1 and 2 days. Respiratory burst of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of the control shrimp after 4 and 6 days (Fig. 4B). The respiratory burst of L. vannamei that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 9, 14, 21 and 28 days (Fig. 4C). No significant difference in SOD activity was observed for L. vannamei among four diets at the beginning, and after 3 and 6 days. However, SOD activity of shrimp fed diets containing 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of control shrimp after 9, 14, 21 and 28 days (Fig. 5). 3.5. Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus Phagocytic activity increased directly with the amount of hot-water extract in diet. Phagocytic activity of shrimp that had been fed diets containing hot-water extract at 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 14 days, and phagocytic activity of shrimp that had been fed diet containing hotwater at 0.5 g kg1 was significantly higher than that of shrimp that had been fed control diet after 28 days (Fig. 6A). Clearance efficiency increased directly with the amount of hot-water extract in diet. Clearance efficiency increased by 26%, 48% and 68% for the shrimp that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 as compared to the shrimp fed the control diet, after 14 days. Clearance efficiency increased by 28%, 50% and 72% for the shrimp that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 as compared to that of shrimp fed the control diet, after 28 days (Fig. 6B). 4. Discussion L. vannamei that received hot-water extract of G. tenuistipitata at 6 mg g1 via injection showed increased resistance against V. alginolyticus [20], and shrimp that received hot-water extract of G. amansiii via injection at 6 mg g1 and dietary administration at 0.5 g kg1 had increased resistance against V. alginolyticus after 1e6 days of challenge in
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
A
Hot-water extract of Gelidium amansii in diet (g kg -1)
50
0
Phgagocytic activity (%)
683
0.5
40
1.0
a
2.0 b
a b
30
c
c
d
c 20
10
0 14
28
Time elapsed (days)
B
Hot-water extract of Gelidium amansii in diet (g kg-1)
90
0
Clearance efficiency (%)
80
0.5
1.0
2.0
a
a
70 60
b
b
50 40
c
c 30 20 10
d
d 0 14
28
Time elapsed (days) Fig. 6. Phagocytic activity (A) and clearance efficiency (B) of Litopenaeus vannamei that had been fed diets containing hot-water extract of Gelidium amansii at 0, 0.5, 1.0 and 2.0 g kg1, after 14 and 28 days. See Fig. 1 for statistical information.
the present study. Common carp C. carpio that received intraperitoneal injection of kappa(k)-carrageenan extracted from C. ocellatus had increased resistance against E. tarda and A. hydrophila [27]. Therefore, hot-water extracts of G. amansii and G. tenuistipitatai, and k-carrageenan all showed positive effects on resistance of teleost fish and shrimp against pathogen infections [19,20,27]. Increased levels of THC, PO activity and respiratory burst were observed when L. vannamei were injected with hotwater extract of G. tenuistipitata [22], and also when they received hot-water extract of G. amansii via immersion, injection, and dietary administrations in the present study. An intraperitoneal injection with k-carrageenan extracted from C. ocellatus increased the migration of head kidney phagocytes to the peritoneal cavity of common carp C. carpio [28]. Both phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus increased significantly, and these levels correlated well with increased resistance against V. alginolyticus when shrimp were injected with hot-water extract of G. tenuistipitata [20], and when shrimp were received hot-water extract of G. amansii in the present study. L. vannamei that received hot-water extract of G. amansii via immersion at 400 mg l1 after 3 h, via injection at 6 mg g1 after 6 days, and via dietary administration at 1.0 g kg1 after 14 days still maintained a significantly higher THC, PO activity and respiratory burst indicating a persisting effect of the hot-water extract. The fact that SOD activity increased together with an increase of superoxide anion of shrimp that received diets containing hot-water extract indicated increases in both activities of NADPH oxidase and SOD. SOD is considered to scavenge superoxide anion which might cause damage to the host. Further research is needed to study the gene expression of immune related proteins like proPO, peroxinectin, serine proteinase, and protein inhibitors which are involved in the activation of the proPO system, and SOD, glutathione peroxidase and catalase which are involved in the antioxidative system [29e31].
684
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
In conclusion, the present study documented that administrations of hot-water extract of G. amansii, a galactose polymer, through immersion, injection or dietary uptakes increased the immune ability of white shrimp L. vannamei by increasing THC, PO activity, respiratory burst, and resistance against V. alginolyticus. The hot-water extract of G. amansii can be used as an immunostimulant through immersion, injection, and diet administrations. L. vannamei that had been fed diets containing hot-water extract of G. amansii at 1.0 g kg1 showed significantly increased immunity and resistance against V. alginolyticus infection after 14 days. Acknowledgements This research was supported by a grant from the Council of Agriculture (93-No-ker-9.2.2.-Yu-F1e6), and Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University. We thank Dr. K.P. Chao for his technical support, and Mr. S.H. Wang for his technical assistance in the experiment. References [1] Liu CH, Cheng W, Hsu JP, Chen JC. Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing. Diseases of Aquatic Organisms 2004;61:169e74. [2] Liu CH, Chen JC. Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus. Fish & Shellfish Immunology 2004;16:321e34. [3] Wang LU, Chen JC. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels. Fish & Shellfish Immunology 2005;18:269e78. [4] Lavilla-Pitogo CR, Lean˜o EM, Paner MG. Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent vibriosis in the rearing environment. Aquaculture 1998;164:337e49. [5] Hoffmann JA, Kafatoa FC, Janeway Jr CA, Ekekowitz RAB. Phylogenetic perspectives in innate immunity. Science 1999;284:1313e8. [6] Tsing A, Arcier JM, Brehe`lin M. Haemocytes of penaeids and palaemonid shrimps: morphology, cytochemistry and hemograms. Journal of Invertebrate Pathology 1989;53:64e77. [7] Johansson MW, So¨derha¨ll K. Cellular immunity in crustaceans and the proPO system. Parasitology Today 1989;5:171e6. [8] Smith VJ, So¨derha¨ll K, Hamilton M. b-1.3-glucan induced cellular defense reaction in the shore crab, Carcinus maenas. Comparative Biochemistry and Physiology 1984;77A:636e9. [9] So¨derha¨ll K, Cerenius L, Johansson MW. The prophenoloxidase activating system in invertebrates. In: So¨derha¨ll KS, Iwanaga GR, Vasta GR, editors. New directions in invertebrate immunology. Fair Haven (NJ, USA): SOS Publications; 1996. p. 229e53. [10] Mun˜oz M, Ceden˜o R, Rodriguez J, Van der Knaap WPW, Mialhe E, Bache`re E. Measurement of reactive oxygen intermediate production in haemocyte of the penaeid shrimp, Penaeus vannamei. Aquaculture 2000;191:89e107. [11] Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Developmental and Comparative Immunology 1993;17:211e9. [12] Robertsen B, Engstad RE, Jorensen JB. Beta-glucans as immunostimulants in fish. In: Stolen JS, Fletcher TC, editors. Modulators of fish immune responses: models for environmental toxicology, biomarkers, and immunostimulators. Fair Haven (NJ, USA): SOS Publications; 1994. p. 83e99. [13] Sakai M. Current research status of fish immunostimulants. Aquaculture 1999;172:63e92. [14] Song YL, Huang CC. Application of immunostimulants to prevent shrimp diseases. In: Fingerman M, Nagabhushanam R, editors. Recent advances in marine biotechnology. Immunobiology and pathology, Vol. 5. Enfield (New Hampshire, USA): Science Publishers, Inc.; 1999. p. 173e88. [15] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection? Fish & Shellfish Immunology 2003;15:71e90. [16] Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, et al. Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 1998;164:277e88. [17] Takahashi Y, Kondo M, Itami T, Honda T, Inagawa H, Nishizawa T, et al. Enhancement of disease resistance against penaeid acute viraemia and induction of virus-inactivation in haemolymph of kuruma shrimp, Penaeus japonicus by administration of Pantoea agglomerans lipopolysaccharide (LPS). Fish & Shellfish Immunology 2000;10:55e8. [18] Noda H, Amano H, Arahsima K, Hashimoto S, Nisizawa K. Antitumour activity of polysaccharides and lipids from marine algae. Nippon Suisan Gakkaishi 1989;55:1265e71. [19] Fujiki K, Matsuyama H, Yano T. Effect of hot-water extracts from marine algae on resistance of carp and yellowtail against bacterial infections. Science Bulletin of Faculty of Agriculture, Kyushu University 1992;45:137e41. [20] Hou WY, Chen JC. The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish & Shellfish Immunology 2005;19:127e38. [21] Renn D. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends in Biotechnology 1997;15:9e14. [22] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 1956;28:350e6.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
685
[23] Chao KP, Su YC, Chen CS. Chemical composition and potential for utilization of the marine alga Rhizoclonium sp. Journal of Applied Phycology 1999;11:525e33. [24] Cheng W, Liu CH, Kuo CM, Chen JC. Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish & Shellfish Immunology 2005;18:1e12. [25] Robertson L, Bray W, Leung-Truillo J, Lawrence A. Practical molt staging of Penaeus setiferus and Penaeus stylirostris. Journal of World Aquaculture Society 1987;18:180e5. [26] Herna´ndez-Lo´pez J, Gollas-Galva´n T, Vargas-Albores F. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comparative Biochemistry and Physiology 1996;113 C:61e6. [27] Fujiki K, Shin DH, Nakao M, Yano T. Protective effect of k-carrageenan against bacterial infections in carp Cyprinus carpio. Journal of Faculty of Agriculture, Kyushu University 1997;42:113e9. [28] Fujiki K, Shin DH, Nakao M, Yano T. Effects of k-carrageenan on the non-specific defense system of carp Cyprinus carpio. Fisheries Sciences 1997;63:934e8. [29] Holmblad T, So¨derha¨ll K. Cellular adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture 1999;172:111e23. [30] Cerenius L, So¨derha¨ll K. The prophenoloxidase-activating system in invertebrates. Immunological Reviews 2004;198:116e26. [31] Bell KL, Smith VJ. Occurrence and distribution of antioxidant enzymes in the haemolymph of the shore crab Carcinus maenas. Marine Biology 1995;123:829e36.
The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus Yu-Win Fu, Wen-Ying Hou, Su-Tuen Yeh, Chiu-Hsia Li, Jiann-Chu Chen* Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC Received 9 June 2006; revised 16 August 2006; accepted 16 August 2006 Available online 24 August 2006
Abstract The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when white shrimp Litopenaeus vannamei were immersed in seawater (34&) containing hot-water extract of red alga Gelidium amansii at 200, 400 and 600 mg l1, injected with hot-water extract at 4 and 6 mg g1 shrimp, and fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1. These parameters increased significantly when shrimp were immersed in seawater containing hot-water extract at 400 and 600 mg l1 after 1 h, when shrimp were injected with hot-water extract at 6 mg g1 shrimp after one day, and when shrimp were fed diets containing hot-water extract at 1.0 and 2.0 g kg1 after 14 days. Phagocytic activity and clearance efficiency were significantly higher for the shrimp that were fed diets containing hot-water extract at 1.0 and 2.0 g kg1 than those of shrimp that were fed diets containing hot-water extract at 0 and 0.5 g kg1 after 14 and 28 days. In a separate experiment, L. vannamei which had received hot-water extract via injection, or fed diets containing hot-water extract, were challenged after 3 h or 28 days with V. alginolyticus at 2 106 cfu shrimp1 and 1 106 cfu shrimp1, respectively, and then placed in seawater. The survival of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of control shrimp after 1 day, and the survival of shrimp fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 increased significantly after 3 days as well as at the end of the experiment (6 days after the challenge), respectively. It was concluded that L. vannamei that were immersed in hot-water extract at 400 mg l1, injected with hot-water extract at 6 mg g1 shrimp, and fed hot-water extract of G. amansii at 2.0 g kg1 or less showed increased immune ability as well as resistance to V. alginolyticus infection. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Litopenaeus vannamei; Vibrio alginolyticus; Hot-water extract of Gelidium amansii; Immersion; Injection; Diet; Immune parameter; Resistance
* Corresponding author. Tel./fax: þ886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.08.014
674
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
1. Introduction White shrimp Litopenaeus vannamei has become the primary species of penaeid shrimp currently being cultured in China, Indonesia, Thailand, and Taiwan. A bacterium Vibrio alginolyticus isolated from diseased L. vannamei is considered to be a secondary and opportunistic pathogen, and causes mortality of shrimp under stressful environments like low salinity and ammonia [1e3]. Disease outbreaks are associated with increases in the proportion of opportunistic bacteria like Vibrio spp. which become prevalent in cultured pond waters [4]. Therefore, the health of shrimp and enhancement of its immunity are of primary concern. Invertebrates lack an adaptive immune system and rely on innate immune responses against microbial invasion [5]. Circulating haemocytes are generally classified into hyaline, semi-granular and large granular cells [6]. Both semigranular and granular cells carry out the function of proPO (prophenoloxidase) activating system [7]. Phenoloxidase (PO) is the terminal enzyme in the proPO system and is activated by minute amounts of microbial cell wall components such as lipopolysaccharides (LPS) from Gram-negative bacteria, peptidoglycan from Gram-positive bacteria and b-1,3-glucan from fungal cell walls [8,9]. In decapod crustaceans, hyaline cells are generally involved in phagocytoisis, an important process of eliminating microorganisms or foreign particles [7,9]. A series of reactive oxygen species (ROS) are produced during phagocytosis. Starting this process, the membrane-bound enzyme complex, NADPH oxidase, assembles after binding of a foreign particle to the cell, and reduces molecular oxygen to superoxide anion (O 2 ), subsequently leading to the production of hydrogen peroxide (H2O2), singlet oxygen (1O2) and hydroxyl radicals (OH$) [10]. Superoxide anion is the first product released during this process known as respiratory burst, and plays an important role in anti-bacterial activity [11]. Several bacterial polysaccharides like schizophyllan, a b-1,3-glucan extracted from fungus Schizophyllum commune, a b-1,3-1,6-glucan extracted from yeast Saccharomycs cerevisiae, peptidoglycan extracted from Bifobacterium thermoplilum and LPS (lipopolysaccharide) extracted from Pantea agglomerans have been used as immunostimulants, and their immunostimulatory effects have been studied in fish [12,13] and crustaceans [14e17]. Some red algae contain antitumour polysaccharides, and have been reported to inhibit growth of Ehrlich carcinoma in mice [18]. The polysaccharides derived from red algae have also been used as immunostimulants in teleosts and shrimp. For example, intraperitoneal injection of hot-water extracts of Gloiopeltis complanata, Hypenea charoides and Chondrus ocellatus increased resistance of common carp Cyprinus carpio against Edwardsiella tarda, and yellowtail Seriola quinqueradiata against Streptococcus sp. infection [19]. White shrimp L. vannamei, receiving hot-water extract of Gracilaria tenuistipitata via injection, showed increased immune ability and resistance against V. alginolyticus [20]. Agars/agaroses and carrageenan which are the common polysaccharides obtained from Rhodophyta (red algae) and are polymers of 1,4-linked a-D-galactose and 3,6-anhydro-a-L-galactose backbone (agarobiose) and 1,3-linked a-D-galactose and 1,4-linked 3,6-anhydro-b-D-galactose backbone (carrabiose), respectively, are of significant commercial values in food, cosmetics and pharmaceuticals [21]. The principle sources of agars are Gelidinium and Gracilaria, and important sources of carrageenan are Eucheuma, Chondrus, Gigartina and Kappaphycus [21]. Gelidium amansii, an important and common red alga, inhabits the north east coast of Taiwan has been used for gel food and processed for agar, and is considered to be a potential source of an anti-bacterial agent and immunostimulant. This study examined the immune response of white shrimp L. vannamei and its resistance against V. alginolyticus following treatment with hot-water extract of G. amansii. The shrimp that were immersed in seawater containing hot-water extract, injected with hot-water extract, and fed diets containing hot-water extract were examined for immune parameters (total haemocyte count, phenoloxidase activity, respiratory burst, and superoxide dismutase activity). Phagocytic activity and clearance efficiency of L. vannamei against V. alginolyticus were studied when shrimp were fed diets containing hot-water extract after 14 and 28 days. In addition, L. vannamei that were injected with hotwater extract after 3 h, and fed diets containing hot-water extract after 28 days, were examined for resistance against V. alginolyticus. 2. Materials and methods 2.1. Culture of V. alginolyticus V. alginolyticus isolated from diseased L. vannamei was used in the study [4]. Broth cultures and preparation of bacterial suspension were conducted based on the method described previously [20]. The bacterial pellets were
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
675
suspended in saline at 1 108 and 5 107 cfu ml1, and 1 108 cfu ml1 as bacterial suspensions for the resistance test, and for the test of phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus, respectively. 2.2. Preparation of hot-water extract of G. amansii G. amansii was collected from the Keelung coast near the University. Hot-water extract of G. amansii was prepared based on the method described before [19,20]. The harvest weight of hot-water extract obtained from 10 g of the milled frond of G. amansii was 1.85 g. The hot-water extract contains 67% of sugar in weight, and the main components are galactose (55.1%), mannose (5.7%), xylose (6.2%) and fucose (3.9%) based on GC-MS after hydrolysis, reduction and acetylation of the sugar [22,23]. For the immersion test, the hot-water extract at 2, 4 and 6 g was first dissolved in 100 ml distilled water and then mixed in 10 l seawater (34&) to obtain final concentrations of 200, 400 and 600 mg l1, respectively. For the injection test, the hot-water extract was prepared with a phosphate buffered saline to make 2 and 3 mg ml1 as test solutions [20]. For the dietary administration test, there were four diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 which were prepared based on a diet described previously for L. vannamei [24]. The proximate analysis of basal diet was 39.2e40.6%, 7.2e7.5%, 11.2e11.6%, 13.0e13.2%, and 3.7e3.8% for crude protein, crude lipid, cellulose, ash and moisture, respectively. 2.3. L. vannamei and culture of shrimp About one thousand shrimp harvested from the University Marine Station adjacent to the coast of Keelung, Taiwan were shipped to the laboratory. Shrimp were placed in fiberglass tanks (8 m3), and acclimated to room temperature (25 1 C) for 2 weeks. During the acclimation period, shrimps were fed twice daily with a formulated shrimp diet (Tairou Feed Company, Tainan, Taiwan). There were three treatments: immersion, injection and dietary administration tests. Three studies were conducted: immune parameters, phagocytic activity and clearance efficiency, and resistance against V. alginolyticus. For the examinations of immune parameters (THC, phenoloxidase activity, respiratory burst and superoxide dismutase activity), test and control groups comprised 8 shrimps each. For the study of phagocytic activity and clearance efficiency to V. alginolyticus, test and control groups also comprised 8 shrimps each, and were conducted in the dietary administration test only. For the study of resistance of shrimp to V. alginolyticus, test and control groups comprised 10 shrimps each in triplicate, and were conducted in the injection and dietary administration tests. The shrimp ranged from 8.7 g to 13.2 g, averaging 10.6 2.1 g (mean SD) with no significant size differences among the treatments for the immersion and injection tests. The body weight of shrimp in the dietary administration test was 11.4, 12.4, 13.3, 14.5, 15.8, 19.0 and 21.0 g for the shrimp that had been fed diets containing hot-water extract after 0, 3, 6, 9, 14, 21 and 28 days with no significant difference among four treatments. Only shrimp in the intermoult stage were used for the subsequent tests [25]. For the dietary administration test, 8 tanks (0.9 m3) were stocked each with 80 shrimps (equivalent to density of 89 (shrimps) m3 with an average initial weight of 11.4 0.2 g. Tanks received continuous aeration, and water flow was 2 l min1. During the experimental period, water temperature ranged from 27 to 29 C, pH from 7.7 to 8.2, salinity from 33 to 35&, and dissolved oxygen concentration from 6.14e6.56 mg l1. Shrimps were fed their respective diets at a rate of 3% of body weight at 10:00 h and 18:00 h. Thirty shrimp were randomly sampled from each tank and weighed weekly, and the daily ration was adjusted accordingly. After 0, 3, 6, 9, 14, 21 and 28 days, 8 shrimps from each treatment were randomly sampled, and were examined for the THC, phenoloxidase (PO) activity, respiratory burst and SOD activity. After 14 and 28 days of rearing, 8 shrimps were sampled randomly from each treatment for the tests of phagocytic activity and clearance efficiency to V. alginolyticus. In addition, 30 shrimps from each treatment were sampled for the study of resistance against V. alginolyticus after 28 days of rearing. 2.4. The resistance of L. vannamei to V. alginolyticus For the injection test, L. vannamei was injected individually into the ventral sinus of the cephalothorax with hotwater extract of G. amansii at 2 and 3 mg ml1 solution at a rate of 20 ml per 10 g weight to reach doses of 4 and 6 mg g1 shrimp, respectively on the first day based on the same method described previously [20]. The challenge test was conducted on the second day by the injection of 20 ml of bacterial suspension (1 108 cfu ml1) resulting
676
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
in 2 106 cfu shrimp1 into the ventral sinus of the cephalothorax. Survival of shrimp was examined daily, and the experiment lasted 6 days (144 h). For the diet administration test, thirty shrimps which had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 were sampled after 28 days from each treatment and studied for the resistance of shrimp against V. alginolyticus. The challenge method used was the same as that described except the bacterial dose was 5 107 cfu ml1 resulting in 1 106 cfu shrimp1. The shrimp that were fed control diet, and then injected with saline (20 ml) served as the unchallenged control. 2.5. The immune parameters of L. vannamei that received hot-water extract of G. amansii For the immersion test, there were four concentrations (0, 200, 400, and 600 mg l1) and three exposure times (0.5, 1 and 3 h). Each treatment group was immersed in 20 l seawater containing hot-water extract at 0, 200, 400 and 600 mg l1, respectively. The amount of hot-water extract was 4, 8 and 12 g for the treatment of 200, 400 and 600 mg l1, respectively. For the injection test, there were four treatments (control, saline, 4 mg g1, and 6 mg g1) and five exposure times (0, 1, 2, 4 and 6 days). L. vannamei were injected individually in the ventral sinus of the cephalothorax with 20 ml of hot-water extract of G. amansii at 2 and 3 mg ml1 solution at a rate of 20 ml per 10 g weight to reach doses of 4 mg g1 and 6 mg g1, respectively. Shrimp injected with saline (20 ml) and shrimp with no injection served as the saline and control groups, respectively. For the dietary administration test, there were four treatments (0, 0.5, 1.0 and 2.0 g kg1) and seven sampling times (0, 3, 6, 9, 14, 21, 28 days). 2.6. Measurements of immune parameters Shrimp which been immersed in hot-water extract were sampled after 0.5, 1 and 3 h in the immersion test, injected with hot-water extract after 0, 1, 2, 4 and 6 days in the injection test, and fed diets containing hot-water extract after 0, 3, 6, 9, 14, 21 and 28 days were used for the studies. Haemolymph (100 ml) sampling, preparation of anticoagulant-haemolymph mixture, and counting of THC followed the procedures described previously [20]. Phenoloxidase (PO) activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) following the procedure of Herna´ndez-Lo´pez et al. [26]. The details of measurements were described previously [2]. The optical density at 490 nm for the shrimp’s PO activity was measured using a spectrophotometer (Model U-2000, Hitachi, Tokyo, Japan), and expressed as dopachrome formation in 50 ml of pure haemolymph. Respiratory burst of haemocytes was quantified using the reduction of NBT (nitroblue tetrazolium) to formazan as a measure of superoxide anion (O 2 ) as described previously [2]. The NBT solution was removed and the haemocytes were fixed with 100% methanol, and washed three times with 100 ml 70% methanol, and air-dried. Formazon was dissolved by the addition of 120 ml 2 M KOH and 140 ml dimethyl sulphoxide (DMSO). The optical density at 630 nm was measured using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA), and expressed as NBT-reduction in 10 ml of pure haemolymph. Superoxide dismutase (SOD) activity was examined for the dietary administration test only. SOD activity was measured by its ability to inhibit superoxide radical dependent reactions using the Ransod Kit (Randox, Crumlin, UK). The detail of the measurement was described previously [2]. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units ml1. 2.7. Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus L. vannamei that had been fed diets containing the hot-water extract for 14 and 28 days were used for the study. There were four treatments (0, 0.5, 1.0 and 2.0 g kg1) with two time periods (14 and 28 days). Tests were carried out in eight replicate test groups with one shrimp in each treatment and exposure time.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
677
Phagocytic activity and clearance efficiency tests were conducted by injecting 20 ml bacterial suspension (1 108 cfu ml1) resulting in 2 106 cfu shrimp1 based on the method described previously [20]. Phagocytic activity, defined as phagocytic rate (PR), was expressed as: PR ¼ fð phagocytic haemocytesÞ=ð total haemocytesÞg 100 The number of bacterial colonies for the shrimp that were fed control diet was expressed as the control group, and the number of colonies in the shrimp that were fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 served as the test group. Clearance efficiency, defined as percentage inhibition (PI) of V. alginolyticus, was calculated as follows: PI ¼ 100 fð cfu in test groupÞ=ðcfu in control groupÞg 100
2.8. Statistical analysis A multiple comparison (Tukey) test was used to examine the significant differences among treatments using the SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (resistance test) were normalised using an arc sin transformation before analysis. For statistically significant differences, it was required that p < 0.05. 3. Results 3.1. Survival and growth of L. vannamei fed diets containing hot-water extract Survival was 95%, 95%, 96% and 98% for the L. vannamei that had been fed diet containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1, respectively. Weight gain percentage was 83.4%, 83.4%, 84.3% and 84.5% for the shrimp that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1, respectively after 28 days. No significant differences in survival and growth were observed among the shrimp fed the four different diets. 3.2. The resistance of L. vannamei injected with hot-water extract All the unchallenged control shrimp survived. By contrast, death began to occur after 24 h in the challenged shrimp which had been injected with hot-water extract. After 24e144 h of challenge, survival of shrimp that received hotwater extract of G. amansii at 6 mg g1 was significantly higher than that of shrimp that received saline, and the control shrimp (Fig. 1A). 3.3. The resistance of L. vannamei fed diets containing hot-water extract All the unchallenged control shrimp survived. By contrast, death began to occur after 12 h in the challenged shrimp that had been fed diet containing hot-water extract at 0.5 g kg1 and the control diet. After 48 h of challenge, survival of shrimp that had been fed a diet containing hot-water extract at 2.0 g kg1 was significantly higher than that of shrimp that had been fed control diet. After 72e144 h of challenge, survival of shrimp that had been fed a diet containing hotwater extract at 0.5 g kg1 was significantly higher than that of shrimp that had been fed the control diet (Fig. 1B). 3.4. The immune parameters of L. vannamei that received hot-water extract of G. amansii The THC of L. vannamei that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of the control shrimp after 0.5, 1 and 3 h (Fig. 2A). The THC of L. vannamei that were injected with hot-water extract of G. amansii at 6 mg g1 was significantly higher than that of shrimp that were injected with hot-water extract at 4 mg g1, and the shrimp that were injected with saline, and the control shrimp after 1 day. The THC of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1 was significantly higher than that of control shrimp after 6 days (Fig. 2B). The THC of L. vannamei that had been fed a diet containing the hot-water extract at 2.0 g kg1 was significantly higher than that of shrimp fed the control diet after 6 days. The
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
678
A
100 90
Hot-water extract of Gelidium amansii (μg g-1) Challenged Unchallenged Control Saline 4 Control
80
6
Survival (%)
70 a
60
a
a
ab
ab
a
a
a
ab
ab
ab
50 ab 40 bc
bc
b
b
b
b
c
c
b
b
b
b
30 20 10 0 0
24
48
72
96
120
144
Time elapsed (h)
B
a a a a
100 90 80
a a a
a
a
ab
a
ab
b
b
Survival (%)
70
a
a
a
b
ab
ab
ab
c
b
b
b
c
c
c
60 50 40 30
Hot-water extract of Gelidium amansii (g kg-1)
20
Unchallenged
Challenged
Control
10
Control
0.5
1.0
2.0
0 0
24
48
72
96
120
144
Time elapsed (h) Fig. 1. Survival (%) of Litopenaeus vannamei that had been injected with hot-water extract of Gelidium amansii after one day, challenged with Vibrio alginolyticus at a dose of 2 106 cfu shrimp1, and placed in seawater at 34& and 25 1 C (A) and survival (%) of L. vannamei that had been fed diets containing hot-water extract after 28 days, challenged with V. alginolyticus at a dose of 1 106 cfu shrimp1, and placed in seawater at 34& and 25 1 C (B). Data (mean SE) in the same exposure time with different letters are significantly (p < 0.05) among treatments.
THC of L. vannamei that had been fed a diet containing hot-water extract at 1.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 14, 21 and 28 days (Fig. 2C). The PO activity of shrimp that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of shrimp that were immersed in 200 mg l1, and that of control shrimp after 0.5, 1 and 3 h (Fig. 3A). The PO activity of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of the control shrimp after 1e6 days (Fig. 3B). The PO activity of L. vannamei that had been fed diets containing
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Total haemocyte count (×105 ml-1)
A
Hot-water extract of Gelidium amansii (mg l-1) Control
350
200
400
679
a
600
a
a 300
a
250
a
200
b
b
bc
c
b
b
b
150 100 50 0 0.5
1
3
Time elapsed (h)
Total haemocyte count (×105 ml-1)
B
Hot-water extract of Gelidium amansii (µg g-1) Control
300
Saline
6
4 a
a
250
ab
200
b a a a a
b
150
ab
b
a
a a
b
b
b
ab
ab
a
100 50 0 0
1
2
4
6
Time elapsed (days)
Total haemocyte count (x105 ml-1)
C
Hot-water extract of Gelidium amansii in diet (g kg-1)
300
0
0.5
1.0
2.0
250 a a
150
a a a a
a a a a
b ab
ab a b
b
a a
a a
200 ab b
b
ab
ab
bc
b c
100 50 0 0
3
6
9
14
21
28
Time elapsed (days) Fig. 2. THC (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, THC (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and THC (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. Each bar represents mean value from eight shrimp with standard error. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
680
Phenoloxidase activity (O.D.490 nm)
A
Hot-water extract of Gelidium amansii (mg l-1)
0.60
Control
200
400
600
a
0.50
a a
0.40
a
0.30
a
b b
a
0.20
b
b
b
b
0.10 0.00 0.5
1
3
Time elapsed (h)
Phenoloxidase activity (O.D. 490 nm)
B
Hot-water extract of Gelidium amansii (μg g-1)
0.25
Control a
0.20
Saline
4
6 a
b 0.15
a ab
ab
b b
a a a a
a
a a
b b
ab
b b
0.10
0.05
0.00 0
1
2
4
6
Time elapsed (days)
Phenoloxidase activity (O.D.490 nm)
C Hot-water extract of Gelidium amansii in diet (g kg-1)
0.70
0
0.5
2.0 a
1.0
0.60
a
0.50 a a
a
0.40
a
ab a
a b
a a
0.30 a 0.20
a a a a
a
a
a
a a
a a b
b
b
c
0.10 0.00 0
3
6
9
14
21
28
Time elapsed (days) Fig. 3. Phenoloxidase activity (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, phenoloxidase activity (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and phenoloxidase activity (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Respiratory burst (O.D.630 nm)
A
0.25
681
Hot-water extract of Gelidium amansii (mg l-1) 200
Control
400
600
a
0.20 a
a
0.15
a
a
ab
b
b
b
b
a
b
0.10
0.05
0.00 0.5
1
3
Time elapsed (h)
Respiratory burst (O.D. 630 nm)
B
Hot-water extract of Gelidium amansii (μg g-1)
0.25
Control a
Saline
4
6
a
0.20
a
a
a ab
0.15 b a
a
b
b
a a
b
b
b
b
ab
ab a
0.10
0.05
0.00 0
1
2
4
6
Time elapsed (days)
Respiratory burst (O.D.630 nm)
C
Hot-water extract of Gelidium amansii in diet (g kg-1)
0.30
0.5
0
2.0
1.0
0.25 0.20
a
a
a a
a
c
b
a a a
b c
b
a
a
a a
b
a a a
a a a 0.15
a
a
d
c
0.10 0.05 0 0
3
6
9
14
21
28
Time elapsed (days)
Fig. 4. Respiratory burst (A) of Litopenaeus vannamei that were immersed in seawater containing hot-water extract of Gelidium amansii at 200, 400 and 600 mg l1, and the control shrimp, respiratory burst (B) of L. vannamei that were injected with hot-water extract at 4 and 6 mg g1, shrimp injected with saline, and the control shrimp, and respiratory burst (C) of L. vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 0, 3, 6, 9, 14, 21 and 28 days. See Fig. 1 for statistical information.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
Superoxide dismutase activity (units ml-1)
682 1.2
Hot-water extract of Gelidium amansii in diet (g kg-1) 0
0.5
1.0
2.0
1.0
0.8
a a a a
a a a a
a a a a
0
3
6
b
a a a
a a a b
a a a
a a a b
b
0.6
0.4
0.2
0 9
14
21
28
Time elapsed (days) Fig. 5. Superoxide dismutase activity of Litopenaeus vannamei that had been fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg1 after 14 and 28 days. See Fig. 1 for statistical information.
hot-water extract at 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 9e28 days (Fig. 3C). The respiratory burst of shrimp that were immersed in hot-water extract of G. amansii at 400 and 600 mg l1 was significantly higher than that of the control shrimp after 0.5, 1 and 3 h (Fig. 4A). The respiratory burst of shrimp that were injected with hot-water extract of G. amansii at 4 and 6 mg g1 was significantly higher than that of shrimp that were injected with saline as well as the control shrimp after 1 and 2 days. Respiratory burst of shrimp that were injected with hot-water extract at 6 mg g1 was significantly higher than that of the control shrimp after 4 and 6 days (Fig. 4B). The respiratory burst of L. vannamei that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 9, 14, 21 and 28 days (Fig. 4C). No significant difference in SOD activity was observed for L. vannamei among four diets at the beginning, and after 3 and 6 days. However, SOD activity of shrimp fed diets containing 0.5, 1.0 and 2.0 g kg1 was significantly higher than that of control shrimp after 9, 14, 21 and 28 days (Fig. 5). 3.5. Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus Phagocytic activity increased directly with the amount of hot-water extract in diet. Phagocytic activity of shrimp that had been fed diets containing hot-water extract at 1.0 and 2.0 g kg1 was significantly higher than that of shrimp that had been fed the control diet after 14 days, and phagocytic activity of shrimp that had been fed diet containing hotwater at 0.5 g kg1 was significantly higher than that of shrimp that had been fed control diet after 28 days (Fig. 6A). Clearance efficiency increased directly with the amount of hot-water extract in diet. Clearance efficiency increased by 26%, 48% and 68% for the shrimp that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 as compared to the shrimp fed the control diet, after 14 days. Clearance efficiency increased by 28%, 50% and 72% for the shrimp that had been fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg1 as compared to that of shrimp fed the control diet, after 28 days (Fig. 6B). 4. Discussion L. vannamei that received hot-water extract of G. tenuistipitata at 6 mg g1 via injection showed increased resistance against V. alginolyticus [20], and shrimp that received hot-water extract of G. amansiii via injection at 6 mg g1 and dietary administration at 0.5 g kg1 had increased resistance against V. alginolyticus after 1e6 days of challenge in
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
A
Hot-water extract of Gelidium amansii in diet (g kg -1)
50
0
Phgagocytic activity (%)
683
0.5
40
1.0
a
2.0 b
a b
30
c
c
d
c 20
10
0 14
28
Time elapsed (days)
B
Hot-water extract of Gelidium amansii in diet (g kg-1)
90
0
Clearance efficiency (%)
80
0.5
1.0
2.0
a
a
70 60
b
b
50 40
c
c 30 20 10
d
d 0 14
28
Time elapsed (days) Fig. 6. Phagocytic activity (A) and clearance efficiency (B) of Litopenaeus vannamei that had been fed diets containing hot-water extract of Gelidium amansii at 0, 0.5, 1.0 and 2.0 g kg1, after 14 and 28 days. See Fig. 1 for statistical information.
the present study. Common carp C. carpio that received intraperitoneal injection of kappa(k)-carrageenan extracted from C. ocellatus had increased resistance against E. tarda and A. hydrophila [27]. Therefore, hot-water extracts of G. amansii and G. tenuistipitatai, and k-carrageenan all showed positive effects on resistance of teleost fish and shrimp against pathogen infections [19,20,27]. Increased levels of THC, PO activity and respiratory burst were observed when L. vannamei were injected with hotwater extract of G. tenuistipitata [22], and also when they received hot-water extract of G. amansii via immersion, injection, and dietary administrations in the present study. An intraperitoneal injection with k-carrageenan extracted from C. ocellatus increased the migration of head kidney phagocytes to the peritoneal cavity of common carp C. carpio [28]. Both phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus increased significantly, and these levels correlated well with increased resistance against V. alginolyticus when shrimp were injected with hot-water extract of G. tenuistipitata [20], and when shrimp were received hot-water extract of G. amansii in the present study. L. vannamei that received hot-water extract of G. amansii via immersion at 400 mg l1 after 3 h, via injection at 6 mg g1 after 6 days, and via dietary administration at 1.0 g kg1 after 14 days still maintained a significantly higher THC, PO activity and respiratory burst indicating a persisting effect of the hot-water extract. The fact that SOD activity increased together with an increase of superoxide anion of shrimp that received diets containing hot-water extract indicated increases in both activities of NADPH oxidase and SOD. SOD is considered to scavenge superoxide anion which might cause damage to the host. Further research is needed to study the gene expression of immune related proteins like proPO, peroxinectin, serine proteinase, and protein inhibitors which are involved in the activation of the proPO system, and SOD, glutathione peroxidase and catalase which are involved in the antioxidative system [29e31].
684
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
In conclusion, the present study documented that administrations of hot-water extract of G. amansii, a galactose polymer, through immersion, injection or dietary uptakes increased the immune ability of white shrimp L. vannamei by increasing THC, PO activity, respiratory burst, and resistance against V. alginolyticus. The hot-water extract of G. amansii can be used as an immunostimulant through immersion, injection, and diet administrations. L. vannamei that had been fed diets containing hot-water extract of G. amansii at 1.0 g kg1 showed significantly increased immunity and resistance against V. alginolyticus infection after 14 days. Acknowledgements This research was supported by a grant from the Council of Agriculture (93-No-ker-9.2.2.-Yu-F1e6), and Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University. We thank Dr. K.P. Chao for his technical support, and Mr. S.H. Wang for his technical assistance in the experiment. References [1] Liu CH, Cheng W, Hsu JP, Chen JC. Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing. Diseases of Aquatic Organisms 2004;61:169e74. [2] Liu CH, Chen JC. Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus. Fish & Shellfish Immunology 2004;16:321e34. [3] Wang LU, Chen JC. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels. Fish & Shellfish Immunology 2005;18:269e78. [4] Lavilla-Pitogo CR, Lean˜o EM, Paner MG. Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent vibriosis in the rearing environment. Aquaculture 1998;164:337e49. [5] Hoffmann JA, Kafatoa FC, Janeway Jr CA, Ekekowitz RAB. Phylogenetic perspectives in innate immunity. Science 1999;284:1313e8. [6] Tsing A, Arcier JM, Brehe`lin M. Haemocytes of penaeids and palaemonid shrimps: morphology, cytochemistry and hemograms. Journal of Invertebrate Pathology 1989;53:64e77. [7] Johansson MW, So¨derha¨ll K. Cellular immunity in crustaceans and the proPO system. Parasitology Today 1989;5:171e6. [8] Smith VJ, So¨derha¨ll K, Hamilton M. b-1.3-glucan induced cellular defense reaction in the shore crab, Carcinus maenas. Comparative Biochemistry and Physiology 1984;77A:636e9. [9] So¨derha¨ll K, Cerenius L, Johansson MW. The prophenoloxidase activating system in invertebrates. In: So¨derha¨ll KS, Iwanaga GR, Vasta GR, editors. New directions in invertebrate immunology. Fair Haven (NJ, USA): SOS Publications; 1996. p. 229e53. [10] Mun˜oz M, Ceden˜o R, Rodriguez J, Van der Knaap WPW, Mialhe E, Bache`re E. Measurement of reactive oxygen intermediate production in haemocyte of the penaeid shrimp, Penaeus vannamei. Aquaculture 2000;191:89e107. [11] Bell KL, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Developmental and Comparative Immunology 1993;17:211e9. [12] Robertsen B, Engstad RE, Jorensen JB. Beta-glucans as immunostimulants in fish. In: Stolen JS, Fletcher TC, editors. Modulators of fish immune responses: models for environmental toxicology, biomarkers, and immunostimulators. Fair Haven (NJ, USA): SOS Publications; 1994. p. 83e99. [13] Sakai M. Current research status of fish immunostimulants. Aquaculture 1999;172:63e92. [14] Song YL, Huang CC. Application of immunostimulants to prevent shrimp diseases. In: Fingerman M, Nagabhushanam R, editors. Recent advances in marine biotechnology. Immunobiology and pathology, Vol. 5. Enfield (New Hampshire, USA): Science Publishers, Inc.; 1999. p. 173e88. [15] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection? Fish & Shellfish Immunology 2003;15:71e90. [16] Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, et al. Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 1998;164:277e88. [17] Takahashi Y, Kondo M, Itami T, Honda T, Inagawa H, Nishizawa T, et al. Enhancement of disease resistance against penaeid acute viraemia and induction of virus-inactivation in haemolymph of kuruma shrimp, Penaeus japonicus by administration of Pantoea agglomerans lipopolysaccharide (LPS). Fish & Shellfish Immunology 2000;10:55e8. [18] Noda H, Amano H, Arahsima K, Hashimoto S, Nisizawa K. Antitumour activity of polysaccharides and lipids from marine algae. Nippon Suisan Gakkaishi 1989;55:1265e71. [19] Fujiki K, Matsuyama H, Yano T. Effect of hot-water extracts from marine algae on resistance of carp and yellowtail against bacterial infections. Science Bulletin of Faculty of Agriculture, Kyushu University 1992;45:137e41. [20] Hou WY, Chen JC. The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish & Shellfish Immunology 2005;19:127e38. [21] Renn D. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends in Biotechnology 1997;15:9e14. [22] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 1956;28:350e6.
Y.-W. Fu et al. / Fish & Shellfish Immunology 22 (2007) 673e685
685
[23] Chao KP, Su YC, Chen CS. Chemical composition and potential for utilization of the marine alga Rhizoclonium sp. Journal of Applied Phycology 1999;11:525e33. [24] Cheng W, Liu CH, Kuo CM, Chen JC. Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus. Fish & Shellfish Immunology 2005;18:1e12. [25] Robertson L, Bray W, Leung-Truillo J, Lawrence A. Practical molt staging of Penaeus setiferus and Penaeus stylirostris. Journal of World Aquaculture Society 1987;18:180e5. [26] Herna´ndez-Lo´pez J, Gollas-Galva´n T, Vargas-Albores F. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comparative Biochemistry and Physiology 1996;113 C:61e6. [27] Fujiki K, Shin DH, Nakao M, Yano T. Protective effect of k-carrageenan against bacterial infections in carp Cyprinus carpio. Journal of Faculty of Agriculture, Kyushu University 1997;42:113e9. [28] Fujiki K, Shin DH, Nakao M, Yano T. Effects of k-carrageenan on the non-specific defense system of carp Cyprinus carpio. Fisheries Sciences 1997;63:934e8. [29] Holmblad T, So¨derha¨ll K. Cellular adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture 1999;172:111e23. [30] Cerenius L, So¨derha¨ll K. The prophenoloxidase-activating system in invertebrates. Immunological Reviews 2004;198:116e26. [31] Bell KL, Smith VJ. Occurrence and distribution of antioxidant enzymes in the haemolymph of the shore crab Carcinus maenas. Marine Biology 1995;123:829e36.