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Administration of the hot-water extract of Spirulina platensis enhanced the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

- Jan 09, 2018 -


Author

Carina Miranda Tayag, Yong-Chin Lin, Chang-Che Li, Chyng-Hwa Liou, Jiann-Chu Chen*

Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung 202, Taiwan, ROC

 

Abstract

White shrimp Litopenaeus vannamei which had been injected with the hot-water extract of Spirulina

platensis at 6, 10, and  20μg g-1, or immersed in aerated seawater containing extract at 200, 400, and

600 mg L-1 were challenged with Vibrio alginolyticus at 1.5×106 or 1.4×106 colony-forming units (cfu) shrimp-1, and then placed in seawater. Survival rates of shrimp that received the extract of 

S. platensis at 6e 20μg g-1, and those of shrimp immersed in seawater containing the extract at 400 and 600 mg L-1 were significantly higher than those of control shrimp after 24-96 and 48-96 h, respectively. In a separate experiment, the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and lysozyme activity were measured when shrimp were injected with the extract at 6, 10, and 20μg g-1, and immersed in seawater containing the extract at 200, 400, and 600 mg L-1. These parameters directly increased with the concentration, and significantly increased when shrimp were immersed in the seawater containing the extract at 0.5-4 h. L. vannamei that received all doses of the extract via injection or via immersion all had increased phagocytic activity and clearance efficiency to V. alginolyticus at 12-72 h and 3 - 4 h, respectively. It was concluded that L. vannamei that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.

 

Keywords: Litopenaeus vannamei, Vibrio alginolyticus, Hot-water extract, Spirulina platensis,

Innate immunity, Challenge test ,Phagocytic activity, Clearance efficiency

 

1. Introduction

Crustaceans have no immune memory, and rely on innate immunity to protect themselves against pathogen infections and other external factors that continually threaten their lives [1].

Haemocytes play important roles in the immune response and can be classified into 3 types: hyaline cells (HCs), semi-granular cell, and granular cells (GCs) based on the presence of cytoplasmic granules. HCs are involved in phagocytosis, whereas semi-gran The white shrimp Litopenaeus vannamei which is naturally distributed along the Pacific coasts of Central and South America has become the most important cultured species worldwide due to successful development of techniques for manipulating brood stocks, spawning, and seed production. However, shrimp farming has suffered great losses due to deteriorated and stressful environments that lead to the outbreaks of viral disease like white spot syndrome virus (WSSV), and bacterial disease caused by Vibrio alginolyticus and V. harveyi [3,4]. Therefore, maintaining good pond condition, preventing disease outbreaks, and enhancing immunity are of primary concern. Several polysaccharides derived from bacteria like

β-glucan, lipopolysaccharide (LPS), peptidoglycan, and those derived from seaweed like alginate and carrageenan are considered immunostimulants, and their abilities to activate the innate immunity of teleost and shrimp have been studied in vitro and in vivo [5-7].

Polysaccharides of the cyanobacterium, Spirulina platensis, a blue-green filamentous alga and its hot-water extract possess antioxidant abilities [8,9], and can inhibit viral replication, inhibit cancer development, and enhance antibody production [9,10]. 

Dietary administration of S. platensis was reported to increase the phagocytic activity of channel catfish Ictalurus punctatus, and common carp Cyprinus carpio [11,12], and increase the resistance against Vibrio and WSSV in penaeid shrimp [13,14]. However, little is known about the effect of S. platensis on the immune response and its possible mode of action in enhancing shrimp immunity.

Accordingly, this study was undertaken to examine the immune response of white shrimp L. vannamei and its resistance against V. alginolyticus when shrimp received the hot-water extract of S. platensis. The immune parameters including the HC count, the GC (including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, phagocytic activity, and clearance efficiency were examined, as well as resistance to infection by V. alginolyticus.

 

2.  Materials and methods

2.1.  Culture of V. alginolyticus

A known pathogenic strain of V. alginolyticus isolated from diseased L. vannamei, which displayed symptoms of anorexia, lethargy, poor growth, and whitish musculature was used for the study [4]. The broth culture of V. alginolyticus followed a previously reported method [15]. Bacterial pellets were resuspended in saline solutions at 7.5×107, 7.0×107, and 9.3×107 colony-forming unit (cfu) mL-1 as stock bacterial suspensions for the resistance, and phagocytic activity and clearance efficiency tests, respectively.

 

2.2.  Experimental design

S. platensis powder was purchased from Gih Hwa Enterprises Co., Ltd., Taipei, Taiwan. The hot-water extract of S. platensis was prepared based on a method described before [16]. Ten grams of S. platensis powder was added to 250 ml of deionized water and boiled for 3h. The suspension was filtered through a nylon mesh.

The filtrate was concentrated under reduced pressure. The harvest weight of the hot-water extract obtained from 10 g of S. platensis dry powder was 2.55 g. Carbohydrate and protein contents in the

extract were 15.8% as glucose and 35.6% as bovine serum albumin (BSA), respectively, measured by the phenolesulfuric acid method [17] and Bio-Rad Protein assay based on the Bradford method [18].

The hot-water extract was prepared with sterile saline to make solutions of 3, 5 and 10 mg mL-1, which were served as the test solutions.

White shrimp L. vannamei obtained from a farm in Ilan, Taiwan were shipped to the laboratory. Shrimp were placed in 3 fibreglass circular tanks for 2 weeks before the experiment. During the acclimation period, shrimp were fed with a commercial shrimp diet (Tairou Feed Co, Tainan, Taiwan). There were two tests; an immersion test, and an injection test. Three studies were conducted: immune parameters, phagocytic activity and clearance efficiency, and resistance against V. alginolyticus. For the study of resistance of shrimp to V. alginolyticus, tests and control groups comprised 10 shrimp each in triplicate. For the examination of immune parameters, test and control groups comprised 8 shrimp each. For the study of phagocytic activity and clearance efficiency to V. alginolyticus, tests and control groups also comprised 8 shrimp each. The weight of the shrimp used in this study ranged 9.2±11.9 g, averaging 10.5±1.4 g (mean  SD) with no significant size differences among treatments.

 

2.3.  Effects of extract injection and extract immersion on the resistance of L. vannamei to V. alginolyticus 

For the injection test, white shrimp L. vannamei were injected individually into the ventral sinus of the cephalothorax with the hot-water extract solutions of S. platensis at 3, 5 and 10 mg mL-1 solution at a rate of 20μl per 10 g weight to reach doses of 6,10, and  20μg g-1, respectively. Shrimp injected with saline (20 μl) and shrimp with no injection of hot-water extract respectively served as the saline and control groups. For the immersion test, L. vannamei (10 shrimp each) were immersed in 10 L of seawater containing the extract at 0 (control), 200, 400, and 600 mg L-1. Shrimp that were placed in the seawater without the addition of extract served as the control group. The amount of extract was 0, 2, 4, and 6 g for the treatments of 0, 200, 400, and 600 mg L-1, respectively.

 After 24 h of the injection test, and after 3 h of the immersion test, the challenge test was conducted by injecting 20 μl of a bacterial suspension (7.5×107 and 7.0×107 cfu mL-1) resulting in 1.5×106 and 1.4×106 cfu shrimp1 into the ventral sinus of the cephalothorax, respectively. Shrimp that received no hot-water extract, and then received V. alginolyticus at 1.5×106 and 1.4×106 cfu shrimp1 served as the challenged control in the injection test and immersion test, respectively. Shrimp that received no hot-water extract, and then received saline (20μl) served as the unchallenged control (Fig. 1). Experimental and control shrimp (10 shrimp aquarium1) were kept in 40-L glass aquaria containing 28 L of 35‰ seawater. There were 6 and 5 treatments in the injection test, and in the immersion test, respectively. Each treatment was conducted with 30 shrimp. Survival of shrimp was examined every 12 h during the first 12 h, then every 24 h after that until the end of the experiment at 96 h. 

 

2.4.  Immune parameters of L. vannamei that received the hot-water extract of S. platensis For the injection test, number of treatment, exposure time, and injection dose were the same as those described in the resistance test.

 For the immersion test, number of treatment, and immersion concentration were the same as those describe in the resistance test except for the four exposure times (0.5, 1, 3, and 4 h). Eight shrimp with no treatment were used as the initial control.  

FIG1. hot-water extract of Spirulina platensis.jpg 

Fig. 1. Relative percentage survival (RPS) of white shrimp Litopenaeus vannamei that had been injected with saline, and shrimp injected with different concentrations of the hot-water extract of Spirulina platensis at 6, 10, and 20μg g-1 after 1 day, and then challenged with Vibrio alginolyticus (A), and RPS of L. vannamei that were immersed in seawater containing the hot-water extract of S. platensis at 0, 200, 400 and 600 mg L-1 for 3 h, and then challenged with V. alginolyticus (B). Data in the same time with different letters significantly differ (p < 0.05) among different treatments. Values are presented as the mean ± standard error (SE)  

 

 

After 0, 12, 24, 48, 72, and 96 h of the injection test, and after 0, 0.5, 1, 3, and 4 h of the immersion test, haemolymph (250 μl) was individually withdrawn from the ventral sinus of each shrimp using a 

1-ml sterile syringe with 25 gauge needle, and placed in a tube containing 2250 μl of an anticoagulant solution (30 mM trisodium citrate, 0.34Msodium chloride, and 10mMEDTAatpH7.55, with the 

osmolality adjusted to 780m Osm kg-1 with 0.115M glucose). 

Adrop of the anticoagulantehaemolymph mixture was placed in a haemocytometer to measure HC, GC (including semi-granular cell), and the THC, using an inverted phase-contrast microscope (Leica DMIL, Leica Microsystems, Wetzlar, Germany). The remainder of the haemolymph mixture was used for subsequent tests. 

PO activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) [19]. The diluted haemolymph was centrifuged at 800×g at 4 C for 20 min. The supernatant was discarded, and the pellet was rinsed, resuspended gently in 1 ml cacodylate-citrate buffer (sodium cacodylate 0.01 M, sodium chloride 0.45 M, trisodium citrate 0.10 M; pH 7.0), and then centrifuged again. The optical density at 490 nm was measured using a Hitachi U-2000 spectrophotometer (Tokyo, Japan). The optical density of the background PO activity was 0.02. The optical density of the shrimp's PO activity was expressed as dopachrome formation per 50 μl haemolymph [20]. 

RB of haemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the superoxide anions produced, as described previously [20]. The optical density at 630 nm for the shrimp's RB was measured in triplicate using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). RB activity is expressed as NBT reduction in 10 μl of haemolymph. SOD activity was measured by its ability to inhibit superoxide radical-dependent reactions using a Ransod Kit (Randox, Crumlin, UK) based on a previously described method [21]. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. The activity was expressed as SOD units mL-1 [20]. 

GPx activity was measured by its activity to catalyze the oxidation of glutathione in the presence of cumene hydroperoxide using a Ransel RS-5004 kit (Randox) based on a previously described method [20]. Briefly, 20 μl of a diluted haemolymph mixture was added to the reaction mixture containing 40 ml cumene hydroperoxide and 10 mM buffer. The optical density of NADPH was measured at 340 nm and 37 C, and the rate of the reaction was estimated from the absorbance readings in the first 3 min after adding cumene hydroperoxide [22]. 

Additional experiment with similar design and haemolymph treatment was conducted for the measurement of lysozyme activity. A diluted haemolymph was mixed with Micrococcus lysodeikticus (Sigma, USA), and the lysozyme activity was conducted following previously described methods [23,24]. The reaction was carried out at room temperature and the absorbance at 530 nmwas measured after 30 s and 4.5 min. A unit (U) of lysozyme activitywas defined as the amount of sample causing decrease in absorbance of 0.01 per min. 

 

2.5.  Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus 

For the injection test and the immersion test, dose, concentration, number of treatment, number of shrimp, and exposure time were the same as those described in the immune parameters test except for the exposure time in the injection test. After 0, 12, 24, 48, and 72 h of the injection test, and after 0, 0.5, 1, 3, and 4 h of the immersion test, 20μl of a bacterial suspension (9.3×107 cfu mL-1) resulting in 1.86×106 cfu shrimp-1 was individually injected into the ventral sinus. After injection, the shrimp were kept for 1 h in a separate tank containing 40 L of water at 25±1 C. Then, 200 μl of haemolymph was collected from the ventral sinus and mixed with 200 μl of a sterile anticoagulant. This mixture was divided into 2 equal sub-samples, one to measure phagocytic activity and the other to measure the clearance efficiency. Phagocytic activity and clearance efficiency were measured following previously described methods [20] Two hundred haemocytes were counted. Phagocytic activity, defined as the phagocytic rate (PR in %) was expressed as follows: PR=[(phagocytic haemocytes)/(total haemocytes)]×100. 

The number of bacterial colonies in the control shrimp was expressed as the control group, and the colonies of shrimp that received saline as well as shrimp that received the hot-water extract of S. platensis at 6, 10, and  20μg g-1 served as the test groups. The clearance efficiency, defined as the percentage inhibition (PI) of V. alginolyticus was calculated as follows: PI=100 - [(cfu in test group)/(cfu in control group)]×100. 

 

2.6.  Statistical analysis

A multiple-comparison test (Tukey's) test was used to examine significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Percent data (resistance study) were normalized using an arcsine transformation before the analysis. Statistical significance of the difference required that p < 0.05. 

 

3.  Results 

3.1. Effects of extract injection and extract immersion on the resistance of L. vannamei to V. alginolyticus

In the injection test, all unchallenged control shrimp survived. By contrast, death began to occur after 12 h in challenged shrimp which received the hot-water extract at 6 mg g1 and saline, and in the challenged control shrimp. At 24-96 h of challenge, survival of shrimp that received the hot-water extract of S. platensis at 6, 10, and  20μg g-1 was significantly higher than that of shrimp received saline and of the control shrimp (Fig. 1A). In the immersion test, all unchallenged control shrimp survived. By contrast, death began to occur after 12 h in challenged shrimp that immersed in seawater containing extract at 0, 200, 400, and 600 mg L-1. At 48-96 h of challenge, survival of shrimp immersed in seawater containing extract at 400 and 600 mg L-1 was significantly higher than that of control shrimp (Fig. 1B). 

 

3.2.  Immune parameters of L. vannamei that had received the hot-water extract of S. platensis 

In the injection test, HC, GC and the THC of L. vannamei that received the hot-water extract at 

20μg g-1 were significantly higher than those of shrimp that received saline, and of control shrimp at 24-48 h (Fig. 2). In the immersion test, HC, GC, and the THC of L. vannamei immersed in seawater containing extract at 400 and 600 mg L-1 were significantly higher than those of control shrimp at 

0.5-4 h (Fig. 3).  

FIG2. hot-water extract of Spirulina platensis.jpg

 Fig. 2. Hyaline cell (A), granular cell (including semi-granular cell) (B), and the total haemocyte count (C) of white shrimp Litopenaeus vannamei that were injected with the hotwater extract of Spirulina platensis at 6, 10, and 20 μg g-1 shrimp injected with saline, and control shrimp after 0, 12, 24, 48, 72 and 96 h. Each bar represents the mean value from 8 shrimp with the standard error. Data at the same exposure time with different letters significantly differ (p < 0.05) among treatments.

   


FIG3. hot-water extract of Spirulina platensis.jpg 

Fig. 3. Hyaline cell (A), granular cell (including semi-granular cell) (B), and the total haemocyte count (C) of white shrimp Litopenaeus vannamei that were immersed in seawater containing the hot-water extract of Spirulina platensis at 0 (control), 200, 400 and 600 mg L-1 after 0, 0.5, 1, 3 and 4 h. See Fig. 2 for statistical information.  

 


In the injection test, the PO activity, RB, and SOD activity of shrimp that received the hot-water extract of S. platensis at 10 and  20μg g-1 were significantly higher than those of control shrimp at 12-96 h. The GPx activity of shrimp that received the hot-water extract at  20μg g-1 was significantly higher than that of control shrimp at 12-96 h (Fig. 4). In the immersion test, the PO activities and RBs of L. vannamei immersed in seawater containing extract at 400 and 600 mg L-1 were significantly higher than those of control shrimp at 0.5-4 h. The SOD activity of L. vannamei immersed in seawater containing extract at 600 mg L-1 was significantly higher than that of control shrimp at 0.5-4 h. The GPx activities of L. vannamei immersed in seawater containing extract at 200, 400 and 600 mg L-1 were significantly higher than those of control shrimp at 1-4 h (Fig. 5). The lysozyme activities of L. vannamei that received hot-water extract of S. platensis at 10 and  20μg g-1 were significantly higher than those of control shrimp at 12, 24, 48, and 96 h (Fig. 6A). In the immersion test, the lysozyme activities of L. vannamei immersed in seawater containing extract at 400 and 600 mg L-1 were significantly higher than those of control shrimp at 0.5-4 h (Fig. 6B).

 

3.3.  Phagocytic activity and clearance efficiency of L. vannamei to V. alginolyticus 

In the injection test, phagocytic activity and clearance efficiency directly increased with the concentration of the extract. Phagocytic activities and clearance efficiencies of shrimp that had received extract at 6, 10, and 20μg g-1 were all significantly higher than those of control shrimp at 12-72 h. Phagocytic activities of shrimp that had received extract at 6, 10, and 20μg g-1 significantly

increased to 35%, 51%, and 63%, respectively after 24 h (Fig. 7A). Clearance efficiencies of shrimp that had received extract at 6, 10 and  20μg g-1 significantly increased to 27%, 42%, and 53%, respectively, after 24 h (Fig. 7B). Similar trends were observed in the immersion test. Phagocytic activities of L. vannamei immersed in seawater containing extract at 200, 400, and 600 mg L-1 were 31%, 37%, and 39%, respectively which were significantly higher than that of control shrimp (20%) after 4 h (Fig. 8A). Clearance efficiencies significantly increased to 26%, 38%, and 43% for shrimp immersed in seawater containing extract at 200, 400, and 600 mg L-1 at 4 h (Fig. 8B). 

 

4.  Discussion 

Common carp C. carpio fed S. platensis showed decreased numbers of Aeromonas hydrophila in the liver and kidneys, together with increases of phagocytic activity and the production of superoxide anions in phagocytic cells [12]. Channel catfish I. punctatus fed S. platensis showed increased resistance against Edwardsiella ictaluri [11]. Banana shrimp Fenneropenaeus merguiensis fed S. platensis showed resistance against V. harveyi infection [13]. White shrimp L. vannamei fed a diet supplemented with S. platensis showed delayed onset of clinical signs of WSSV [14]. In the present study, L. vannamei that had received an extract of S. platensis showed increased resistance against V. alginolyticus together with increased phagocytic activity and clearance efficiency. Therefore, both S. platensis and its extract show positive responses in protecting against pathogen in fish and shrimp. 

With respect to the immune response, HC, GC, and the THC of white shrimp L. vannamei that received the extract at 20μg g-1 had increased by 29%, 28% and 29%, respectively at 48 h, and HC, GC, and the THC of L. vannamei immersed in seawater containing extract at 400 mg L-1 had increased by 53%, 53% and 53%, respectively, at 3 h. The fact that L. vannamei that received extract via immersion showed greater efficiency in the haemocyte count than shrimp that received extract via injection supports immersion as a practicalway of stimulating shrimp immunity [6]. 

An injection of β-glucan at 5 mg mL-1 was reported to cause upregulation of the Runt gene that is involved in haemocytes released in hematopoiesis, and acceleration of the maturation of haemocyte precursors in the crayfish Pacifastacus leniusculus [25]. An injection of LPS at 1.5 mg mL-1 caused proliferation of cells in the haematopoietic tissue of tiger shrimp Penaeus monodon 24 h postinjection [26], and an injection of LPS at 0.15 mg mL-1 caused a significantly increased percentage of proliferating haemocytes in Marsupenaeus japonicus 120 h post-injection [27]. In the present study, the fact that L. vannamei that received the extract via injection or immersion all increased haemocyte count suggests a proliferation of haemocyte in hematopoiesis. 

During phagocytosis, several reactive oxygen intermediates (ROIs) including superoxide anions are produced. SOD, an antioxidative enzyme, plays an important role in scavenging superoxide anions in crustaceans [28]. Both S. platensis and its hot-water extract have antioxidant activity [8]. It contains an important enzyme, SOD that acts directly by slowing down the rate of oxygen radical-generating reactions [9]. Significant increases in superoxide anion and SOD activities occurred in haemocyte of L. vannamei when immersed in seawater containing b-1,6-glucan at 0.5 mg mL-1 after 6 h [29]. In the present study, the fact that the activities of SOD and GPx increased together with an increase in superoxide anion suggests an increase in the activity of NADPH oxidase, which gives rise to superoxide anion and scavenging of superoxide anion. The increases in RB and SOD activity caused increased production of H2O2 and led to an increase in the activity of GPx, indicating the occurrence of antioxidant activity and induction of the SOD system in shrimp that had received the S. platensis extract [8,30]. 

In the present study, PO activity, RB, SOD activity, GPx activity, and lysozyme activity of shrimp that had received the hot-water extract at  20μg g-1 still maintained higher levels after 72 and 96 h of treatment, and those parameters of shrimp immersed in seawater containing extract at 

400-600 mg L-1 all still maintained significantly higher at 4 h. Further research is needed to evaluate the immune stimulatory effect and resistance against Vibrio infection when shrimp are fed diets containing extracts of S. platensis. In fish, β-glucan enhances the innate immunity through direct activation of macrophages [31]. The β-glucan receptor exists in Atlantic salmon Salmo salar and channel catfish I. punctatus [32,33]. The peritoneal phagocytes of fish fed S. platensis showed enhanced phagocytosis to zymosan [11]. Similarly, banana shrimp F. merguiensis fed S. platensis showed enhanced phagocytic activities to V. harveyi and Escherichia coli [13]. The present study indicated that both superoxide anion and phagocytic activity increased in L. vannamei following treatment with the S. platensis extract. This fact suggests that polysaccharides of S. platensis extract receptors exist in macrophages and haemocytes. This fact also suggests that the S. platensis polysaccharides stimulate the activation of innate immunity in fish and shrimp. 


FIG4. hot-water extract of Spirulina platensis.jpg  

Fig. 4. Phenoloxidase (PO) activity (A), respiratory burst (RB) (B), superoxide dismutase (SOD) activity (C) and glutathione peroxidise (GPx) activity (D) of white shrimp Litopenaeus vannamei that were injected with the hot-water extract of Spirulina platensis at 6, 10, and 20μg g-1, shrimp injected with saline, and control shrimp after 0, 12, 24, 48, 72, and 96 h. See Fig. 2 for statistical information.  



 

 FIG5. hot-water extract of Spirulina platensis.jpg  

Fig. 5. Phenoloxidase (PO) activity (A), respiratory burst (RB) (B), superoxide dismutase (SOD) activity (C) and glutathione peroxidise (GPx) activity (D) of white shrimp Litopenaeus vannamei that were immersed in seawater containing the hot-water extract of Spirulina platensis at 0 (control), 200, 400 and 600 mg L-1 after 0, 0.5, 1, 3 and 4 h. See Fig. 2 for statistical information.   

 

 

β-glucan is composed of 95% glucose [32], and the polysaccharides of the hot-water extract of S. platensis are mainly composed of rhamnose, fructose, galactose, and glucose [34,35]. In crustaceans, LPS and b-1,3-glucan-binding protein (LGBP) and bglucan- binding protein (bGBP) are common pattern recognition proteins (PRPs), and both contain a glucanase motif, 2 polysaccharide recognition motifs (a polysaccharide-binding motif and a β-glucan recognition motif), and 2 integrin-binding motifs, RGD and RGD [36]. In vitro, the binding mixture of LPS and LGBP can activate the proPO-activating system in Drosophila cells [37]. In crayfish P. leniusculus, the bGBP and β-glucan complex can bind to the surface of granular cells through its RGD motif which may indicate binding to integrin and then induce the spread and degradation of haemocytes [38]. In L. vannamei, the integrinbinding motifs, RGD and KGD were observed in LGBP and peroxinectin which are important proteins involved in the serine proteinase cascade [30,39,40]. Further study is needed to identify integrin and its function involved in the binding of LGBP and peroxinectin in L. vannamei. 

In conclusion, the present study documents that the hot-water extract of S. platensis, a rhamnose- and fructose-rich polymer, through injection (10 and 20μg g-1) or immersion (200 and 400 mg L-1) increased the innate immunity of white shrimp L. vannamei by increasing the haemocyte count, PO activity, release of superoxide anions, SODactivity, GPx activity, and lysoymeactivity of. L. vannamei that received extract at 10 and 20 μg g-1 via an injection, or received extract at 400 and 600 mg L-1 via immersion showed increased resistance against V. alginolyticus with a concomitant increase in phagocytic activity and clearance efficiency against V. alginolyticus. The extract of S. platensis is considered to be a suitable immunostimulant in L. vannamei.    


FIG6. hot-water extract of Spirulina platensis.jpg  

Fig. 6. Lysozyme activity (A) of white shrimp Litopenaeus vannamei that were injected with the hot-water extract of Spirulina platensis at 6, 10, and 20μg g-1, shrimp injected with saline, and control shrimp after 0, 12, 24, 48, 72 and 96 h, and lysozyme activity (B) of white shrimp Litopenaeus vannamei that were immersed in seawater containing the hot-water extract of Spirulina platensis at 0 (control), 200, 400 and 600 mg L-1 after 0, 0.5, 1, 3 and 4 h. See Fig. 2 for statistical information.  



FIG7. hot-water extract of Spirulina platensis.jpg 

Fig. 7. Phagocytic activity (A) and the clearance efficiency (B) of white shrimp Litopenaeus vannamei that were injected with the hot-water extract of Spirulina platensis at 6, 10, and 20μg g-1, shrimp injected with saline, and control shrimp after 0, 12, 24, 48, 72 and 96 h. See Fig. 2 for statistical 

information. 



 FIG8. hot-water extract of Spirulina platensis.jpg 

Fig. 8. Phagocytic activity (A) and clearance efficiency (B) of white shrimp Litopenaeus vannamei that were immersed in seawater containing the hot-water extract of Spirulina platensis at 0 (control), 200, 400 and 600 mg L-1  after 0, 0.5, 1, 3 and 4 h. See Fig. 2 for statistical information.   

 

 

Acknowledgements 

This research was supported by grants (NSC 95-2313-B-019-013 and NSC-98-2815-C-019-026-B) from the National Science Council,Taiwan and partial support from the Centre for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University. Heartfelt gratitude is also expressed to the Democratic Pacific Union, Taiwan for providing a scholarship to the first author.

  

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