The increase of immunity and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii by feeding with selenium enriched-diet

Fish & Shellfish Immunology 29 (2010) 623e629

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The increase of immunity and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii by feeding with selenium enriched-diet Shieh-Tsung Chiu a, Shu-Ling Hsieh b, Shinn-Pyng Yeh a, Shun-Ji Jian a, Winton Cheng a, **, Chun-Hung Liu a, * a b

Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan 912, ROC Department of Food Science, National Kaohsiung Marine University, Kaohsiung, Taiwan 811, ROC

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 February 2010 Received in revised form 21 May 2010 Accepted 8 June 2010 Available online 16 June 2010

The effects of inorganic selenium (Se) (sodium selenate, SSe) and organic selenium (seleno-L-methionine, MSe) supplementation on the immune response, antioxidant status, and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii, were studied. Five experimental diets, including a control diet (without Se enrichment), 0.5 mg (kg diet)
1 of MSe, 1 mg (kg diet)
1 of MSe, 0.5 mg (kg diet)
1 of SSe, and 1 mg (kg diet)
1 of SSe, were used. After 75 days of culture, prawn fed the Se-enriched diets had lower mortality compared to that of prawn fed the control diet after being challenged by the pathogen, Debaryomyces hansenii. No significant differences in the total hemocyte count, superoxide dismutase activity, or clearance efficiency of prawn were recorded among the control and treated groups. Significantly increased phenoloxidase and phagocytic activities in prawn fed the Se-enriched diets were found compared to the controls. Respiratory bursts of prawn fed both forms of 1 mg Se (kg diet)
1 significantly increased compared to control prawns. For the antioxidant status analysis, glutathione peroxidase, glutathione reductase, and glutathione s-transferase of prawn fed the SSe-enriched diet at 1 mg (kg diet)
1 were significantly increased. The results indicated that the cheaper selenium, SSe is recommended to be added in prawn feed at the concentration of 0.5 mg resulting in 1.5 mg SSe (kg diet)
1 increased prawn immunity and disease resistance against the pathogen, D. hansenii. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Macrobrachium rosenbergii Selenium Immune response Antioxidant status Debaryomyces hansenii

1. Introduction After the failure of tiger shrimp (Penaeus monodon) aquaculture industry caused by serious disease problem, giant freshwater prawn Macrobrachium rosenbergii became a popular and important aquaculture species in Taiwan with a production level of 10,059 tons in 2008. However, the prawn aquaculture industry also suffers serious problems of bacterial disease including from the yeast (Debaromyces hansenii) infections in the cool season [1,2] and Lactococcus garvieae in the hot season, that have resulted in declining production of farmed prawn in Taiwan [3]. Minerals were reported by several studies [4,5] to be involved in the health status of farmed crustaceans. Previous studies showed that adequate dietary mineral concentrations for shrimp growth performance were generally lower than adequate dietary mineral concentrations for physiologic and immune responses of shrimp. * Corresponding author. Tel.: þ886 8 7703202x6228; fax: þ886 8 7740401. ** Corresponding author. Tel.: þ886 8 7703202x6374; fax: þ886 8 7740401. E-mail addresses: [email protected] (W. Cheng), [email protected]. edu.tw (C.-H. Liu). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.06.012

For tiger shrimp, adequate dietary copper concentrations for growth and non-specific immune responses in diets were 15e21 and 10e30 mg Cu (kg diet)
1, respectively [5], and adequate dietary zinc concentrations for growth and non-specific immune responses in diets were 32e34 and 35e48 mg Zn (kg diet)
1 respectively [4]. Se, an essential trace element for normal life processes, was discovered in 1817, and its biologically active form was found by Rotruck et al. [6] in which glutathione peroxidase (GPx) was identified as a very potent antioxidant that protects the body from damage due to oxidation by free radicals. In addition, Se was also shown to be related to the growth, development, oncology, and immune functions of animals [7,8]. Dietary Se deficiency has a documented immunosuppressive effect upon humans [8], cattle [9], poultry [10], and lepidopteran larvae [11]. In addition, Se might also play an important role in cancer prevention [12,13]. Mechanisms of Se’s anticancer actions are not fully understood; however, several have been proposed: antioxidant protection, enhanced carcinogen detoxification, enhanced immune surveillance, modulation of cell proliferation, inhibition of tumor cell invasion, and inhibition of angiogenesis [13]. In insects, Se was shown to have negative effects on the larval growth and development of the

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cabbage looper, Trichoplusia ni; however, its larvae fed Se in the penultimate and ultimate instars were more resistant to a baculovirus infection (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) than were larvae not fed Se in the final instars [11], and a similar result was also shown in Heliothis virescens larvae against a baculovirus infection (Helicorerpa zea single nucleopolyhedrovirus, HzSNPV) [14]. In aquatic animal research, some papers demonstrated that high levels of Se might ultimately be toxic to fish [15,16], but most research showed that Se is an essential micronutrient for fish [17e20]. To our knowledge, there is no information on Se related to prawn immune systems and disease resistance. Therefore, the aims of this study were to evaluate the effects of different forms of selenium (seleno-L-methionine and sodium selenate) in the diet on the immune response and disease resistance of prawn. 2. Materials and methods 2.1. Experimental design The effects of Se on the immune response of the giant freshwater prawn, M. rosenbergii, and its disease resistance were evaluated in this study. A control diet (without Se addition), and four Se-enriched diets, including 0.5 mg (kg diet)
1 of seleno-L-methionine (MSe), 1 mg (kg diet)
1 of MSE, 0.5 mg (kg diet)
1 sodium selenate (SSe), and 1 mg (kg diet)
1 SSe was prepared and fed individually to prawn juveniles in triplicate for 75 days of growingout. After 75 days of feeding, prawn were randomly sampled for analysis of the total hemocyte count (THC), phenoloxidase (PO) activity, and respiratory bursts, as well as glutathione peroxidase (GPx), glutathione reductase (GR), glutathione s-transferase (GST), and superoxide dismutase (SOD) levels in hemocytes, and the phagocytic activity, clearance efficiency, and disease resistance to D. hansenii. For immune parameter analysis, 10 prawn were randomly sampled for THC, PO activity, respiratory burst, GPx, GR, GST and SOD, and other 10 prawn for phagocytic activity and clearance efficiency. 2.2. Diet preparation The formulations of the experimental diets are presented in Table 1. Organic Se (MSe, S3132, Sigma, St. Louis, MO, USA) and inorganic Se (SSe, S8295, Sigma) were individually incorporated into the test diets at the levels of 0.5 and 1 mg (kg diet)
1, respectively, and the diet without Se addition was used as the

Table 1 Ingredients of experimental diets (g kg
1) used for the giant freshwater prawn, Macrobrachium rosenbergii culture. Ingredient

Se-enriched diets (mg kg
1) Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe

Fish meal 430 Soybean meal 65 Yeast meal 25 Shrimp shell meal 70 Wheat flour 350 cellulose 1 MSe 0 SSe 0 Gluten 25 Fish oil 8 Mineral mixture 20 without selenium Vitamin mixture 6

430 65 25 70 350 0 1 0 25 8 20

430 65 25 70 350 0 0 1 25 8 20

6

6

430 65 25 70 350 0.5 0.5 0 25 8 20

430 65 25 70 350 0.5 0 0.5 25 8 20

6

6

Mineral mix without selenium and Vitamin mix were prepared according to the formula of Cheng and Hardy [50].

control. Se was added to the experimental diets with a corresponding decrease in the amount of cellulose. The ingredients were ground in a Hammer mill to pass through a 60-mesh screen. Experimental diets were prepared by mixing the dry ingredients with fish oil and then adding water until a stiff dough resulted. Each diet was then passed through a mincer with a die, and the resulting spaghetti-like strings were dried in a drying cabinet using an air blower at 40  C to a moisture level of 10%. After drying, the pellets were stored in plastic bins at
4  C until being used. Proximate analyses of the experimental diets showed no significant differences in the moisture (6.69% w 7.43%), ash (16.72% w 16.83%), crude protein (47.20% w 47.83%), or ether extract (14.39% w 14.59%) levels among all experimental diets. The inherent Se in the control diet was 1.18  0.07 mg kg
1, and in the Se-enriched diets were 2.21  0.45 mg kg
1 (1 mg MSe), 2.19  0.86 mg kg
1 (1 mg SSe), 1.58  0.21 mg kg
1 (0.5 mg MSe), and 1.54  0.14 mg kg
1 (0.5 mg SSe) analyzed by a flame atomic absorption spectrophotometer (Speact AA 240-FS, VARIAN, USA) equipped with a hydride generator (VGA-77, VARIAN). 2.3. Prawn rearing A 75 day growth trial was conducted in indoor fiberglassreinforced plastic (FRP) tanks at the Department of Aquaculture, National Pingtung University of Science and Technology. In total, 600 prawn were randomly assigned to five groups and fed individually with the control diet, and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 mg SSe. Each group consisted of three replicates. Each replicate consisted of 40 shrimp in a 1 ton circular FRP tank with 0.8 tons of fresh water (equivalent to 35 shrimp m
2). Aeration was supplied by a single air-stone to maintain the dissolved oxygen at 6 mg L
1. Prawn were fed twice daily at a ratio of 5% of the body weight at 08:00 and 15:00. Any uneaten portion was collected 1 h after feeding, and then immediately dried in an oven at 80  C. The amount of all test diets fed was calculated by subtracting the uneaten portion, and data were recorded on a daily basis. During the culture period, each tank was continually supplemented and allowed to overflow at a volume of w0.5 L h
1. To evaluate the water quality, the pH, water temperature, and dissolved oxygen (DO) were respectively determined using a pH meter, thermometer, and DO meter on a daily basis; ammonia-N and nitrite-N were measured according to the methods of Bower and Bidwell [21] and Bendschreider and Robinson [22], respectively, once every 2 weeks during the growing-out phase. After 75 days of culture, prawns were randomly sampled to evaluate the immune response and disease resistance. 2.4. Susceptibility of prawn to D. hansenii The yeast, D. hansenii, was cultured on tryptic soy agar (TSA, Difco, USA) for 24 h at 28  C before being transferred to 10 ml tryptic soy broth (TSB, Difco), where it remained for 24 h at 28  C as a stock culture for the tests. The broth cultures were centrifuged at 7155g for 20 min at 4  C. The supernatants were removed, and the bacterial pellets were resuspended in a saline solution at 1  107 colony-forming units (cfu) ml
1 as stock bacterial suspensions for the susceptibility study. The challenge test was conducted in triplicate by an injection of 20 ml of a bacterial suspension resulting in 2  105 cfu prawn
1 into the ventral sinus of the cephalothorax. The prawn that were fed 1 mg MSe or 1 mg SSe and then received saline (20 ml) served as the unchallenged control. Experimental prawn (10 prawn aquarium
1) were kept in 60 L glass aquaria containing 40 L of water at 28  C.

S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

There were therefore a total of seven treatments. Each treatment was conducted with 30 prawn. Water was renewed daily in the period of challenge test.

625

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. Specific activity was expressed as GPx U (mg protein)
1.

2.5. Immune response analysis 2.5.1. THC measurement Hemolymph (100 ml) was withdrawn and mixed with 900 ml of an anticoagulant solution (30 mM trisodium citrate, 0.34 M sodium chloride, 10 mM EDTA, at a pH of 7.55, and an osmolality adjusted with glucose to 490 mOsm kg
1). A drop of the diluted hemolymph was placed on a hemocytometer to measure the THC using an inverted phase-contrast microscope (Leica DMIL, Leica Microsystems, Wetzlar, Germany). 2.5.2. PO activity assay PO was spectrophotometrically measured by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) following the procedures of a previous study [23]. The optical density of the shrimp’s phenoloxidase activity was expressed as dopachrome formation in 50 ml of hemolymph. 2.5.3. Respiratory burst assay Respiratory bursts of hemocyte were quantified using the reduction of nitro blue tetrazolium (NBT) to formazan as a measure of superoxide anion (O
2 ) production [23]. Respiratory bursts were expressed as NBT-reduction in 10 ml of hemolymph. 2.5.4. Hemocyte lysate supernatants (HLSs) Diluted hemolymph was prepared as described above and then centrifuged at 500g at 4  C for 20 min, and the supernatant was removed and used as a plasma preparation. The hemocyte pellet was washed with the same buffer, and homogenized in phosphatebuffered solution (PBS) on ice. The hemocyte lysate supernatants were then centrifuged at 20,000g (Hitachi, CF16RX, Tokyo, Japan) for 60 min at 4  C. The resulting supernatant (inactive HLS) was used for the analysis of SOD, GPx, GR, and GST activities. 2.5.5. SOD analysis SOD activity was measured by its ability to inhibit superoxide radical-dependent reactions using a Ransod kit (Randox, Crumlin, UK) following the manufacturer’s instructions. Briefly, 10 ml of HLS was mixed with the reaction mixture (200 ml) that contained 0.05 mM xanthine and 0.025 mM 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) dissolved in 50 mM CAPS (at pH 10.2) and 0.94 mM EDTA, and then 30 ml xanthine oxidase (XO) was added. In the presence of 250 ml XO (80 U/L), superoxide and uric acid were produced from the xanthine. The superoxide radicals then reacted with INT to produce a red formazan dye. The optical density was measured at 505 nm and 37  C, and the rate of the reaction was estimated from the absorbance readings 0.5 and 3 min after adding the XO. A reference standard (SOD) was supplied with the Ransod kit. The specific activity was expressed as SOD U (g protein)
1. 2.5.6. GPx analysis GPx activity was measured using the Ransel RS-504 kit (Randox) following the manufacturer’s instructions. GPx catalyses the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized form of glutathione is immediately converted to the reduced form with the concomitant oxidation of NADPH to NADPþ. The decrease in absorbance at 340 nm is then measured. Briefly, 15 ml of a diluted hemolymph mixture was added to the reaction mixture containing 40 ml cumene hydroperoxide and 10 mM buffer. The optical density

2.5.7. GR analysis GR was assayed using a GR assay kit (Sigma, GRSA) following the manufacturer’s instructions. This assay is based on the reduction of glutathione (GSSG) by NADPH in the presence of GR. The reaction is measured by the decrease in absorbance at 340 nm using an extinction coefficient (3mM) of 6.22 for NADPH. Briefly, 100 ml of 2 mM oxidized glutathione, 80 ml assay buffer (100 mM potassium phosphate buffer (pH 7.5), with 1 mM EDTA and 1 mg ml
1 bovine serum albumin), and 20 ml of sample were mixed in three wells of a microplate. The reaction was initiated by the addition of 2 mM of an NADPH solution and then mixed by shaking. The absorbance was read once every 10 s at 340 nm using a plate reader to obtain at least 11 time points. A negative control with the assay buffer instead of an enzyme sample was also assayed. The specific activity was expressed as GR U (mg protein)
1. 2.5.8. GST analysis GST analysis was performed using a GST assay kit. (703302, Cayman Chemical Ann Arbor, MI, USA). The reagent measures the total GST activity, including cytosolic and microsomal portions by measuring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) by reduced glutathione. The conjugation is accompanied by an increase in absorbance at 340 nm. Briefly, 150 ml of assay buffer, 20 ml of glutathione, and 20 ml of a sample were added to three wells of a microplate. Thereafter, 10 ml of CDNB was mixed and carefully shaken for a few seconds. The absorbance was read once every minute at 340 nm using a plate reader to obtain at least 5 time points. In addition, non-enzymatic wells and a positive control (equine liver GST) were also used. The specific activity was expressed as GST nmol min
1 (mg protein)
1. 2.5.9. Analysis of protein concentration HLS protein was determined using a Bio-Rad Protein Assay Kit no. 500-0006 (Bio-Rad Laboratories, Richmond, CA, USA) using bovine albumin (with a molecular weight of 66,000) as a standard. 2.5.10. Phagocytic activity and clearance efficiency assays For the phagocytic activity and clearance efficiency tests, 20 ml of a bacterial suspension (1  109 cfu ml
1) resulting in 2  107 cfu prawn
1 were injected into the ventral sinus. After injection, the prawns were kept for 3 h in separate tanks containing 40 L of water. Then, 200 ml of hemolymph was collected from the ventral sinus and mixed with 200 ml of sterile anticoagulant. This mixture was divided into 2 equal subsamples, one to measure phagocytic activity and the other to measure the clearance efficiency. The analysis of phagocytic activity and clearance efficiency were conducted following the method described in our previous study [23]. The phagocytic activity and clearance efficiency were defined as phagocytic rate (PR) and the percentage inhibition (PI) of D. hansenii, respectively as follows: PR ¼ [(phagocytic hemocytes)/(total hemocytes)]  100%. PI ¼ 100 e [(cfu of the test group)/(cfu of the control group)]  100%.

2.6. Statistical analysis A multiple comparison (Tukey’s) test was conducted to examine significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Before the analysis, the

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S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

16 -1

T H C (x 1 0 c e ll m l )

percent data were normalized using an arcsine-transformation. Statistically significant differences required that p < 0.05.

8

3. Results

A

a a

a

12 a

10

a

8 6 4 2 0 Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe -1

Se-enriched diets (mg kg )

P O a c tiv ity (O.D .490n m )

0.5

B a

0.4

a

a ab

0.3 b

0.2 0.1 0

Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe -1

Se-enriched diets (mg kg )

R e s p ira to ry b u rs t (O.D .630n m )

During the 75 days of culture, water parameters were found to be within acceptable ranges for prawn growth in all treatments and the control, and no significant differences in total ammonia-N (0.02e0.22 mg L
1), nitrite-N (0.02e0.59 mg L
1), pH (6.9e7.3), temperature (24e27  C), or dissolved oxygen (6.23e6.89 mg L
1) were detected among the control and treatment groups. No significant differences in the growth or survival rates among all treatments and the control were found. The percentage of gain weight of prawn in the control and treatment groups of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were 312.56%  22.53%, 294.61%  1.76%, 319.84%  47.33%, 278.08%  27.04%, and 271.80%  37.39%, respectively. The survival rate of prawn in the control and treatment groups of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were 80.83%  0.83%, 78.75%  3.75%, 76.25%  8.75%, 80.00%  2.04%, and 86.67%  2.20%, respectively. After 75 days of culture, prawn from the treatment and control groups were randomly selected for pathogen (D. hansenii) challenge. No dead prawns were recorded in either of the unchallenged control groups. The mortality occurred at 24 h after the injection in the challenge control and 1 mg MSe treatment groups. However, dead prawn in the 1 mg SSe, 0.5 mg MSe, and 0.5 mg SSe treatment groups were recorded at 48 h after the injection. At the end of the challenge trial, the cumulative mortalities of the control and 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe treatment groups were 80%  5.8%, 56.7%  6.7%, 43.3%  3.3%, 30%  5.8%, and 30%  14.5%, respectively. The cumulative mortality of prawn in the control group was significant higher than those in the 1 mg SSe, 0.5 mg MSe, and 0.5 mg SSe groups (Table 2). The THC, PO activity, and respiratory bursts of prawn fed the Se-enriched diets and control diet for 75 days are shown in Fig. 1. No significant differences in the THCs of prawn were recorded, and the THCs of prawn fed the control and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were (11.67  2.18)  108, (11.44  1.55)  108, (8.87  0.95)  108, (8.02  0.56)  108, and (13.84  1.38)  108 cells ml
1, respectively (Fig. 1A). Prawn fed the Se-enriched diets had higher PO activities compared to control prawn (Fig. 1B). However, the PO activity of prawn fed the 0.5 mg MSe-enriched diet did not significantly differ from control prawn. The PO activity of prawn fed the control and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MS, and 0.5 SSe were 0.19  0.01, 0.33  0.02, 0.30  0.02, 0.27  0.02, and 0.34  0.05, respectively. Prawn fed the 1 mg MSe- and 1 mg SSe-enriched diets had significantly higher respiratory bursts compared to prawn fed the control, and 0.5 mg MSe- and 0.5 mg SSe-enriched diets. Respiratory bursts of 0.18  0.02, 0.31  0.04, 0.29  0.04, 0.18  0.03, and 0.12  0.01 were recorded in prawn fed the control, 1 mg MSe-,

14

0.4

C

a a

0.3 0.2

b

b

b 0.1 0 Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe -1

Se-enriched diets (mg kg ) Fig. 1. Total hemocyte count (A), phenoloxidase activity (B), and respiratory bursts (C) of giant freshwater prawn, Macrobrachium rosenbergii, fed a diet without selenium (Se) as the control, and diets with different forms of Se including inorganic SE (sodium selenate, SSe) at the concentrations of 0.5 and 1 mg (kg diet)
1, and organic Se (seleno
1 L-methionine, MSe) at the concentrations of 0.5 and 1 mg (kg diet) for 75 days. Data (mean  SE) with different letters significantly differ (p < 0.05) among treatments.

1 mg SSe-, 0.5 mg MSe-, and 0.5 SSe-enriched diets, respectively (Fig. 1C). The effects of the Se-enriched diets on the prawn’s antioxidant status are shown in Fig. 2. No significant differences in SOD

Table 2 Cumulative mortality (Mean  S.D.) of giant freshwater prawn, Macrobrachium rosenbergii fed with Se-enriched diets, after being challenged by pathogen, Debaryomyces hansenii. Data with differ markers among different treatment are significant difference at the same time. Treatments

Bacterial injection (cfu prawn
1)

No. of shrimp

Cumulative mortality (%), time post injection (h) 24

48

72

96

120

144

1 mg MSe 1 mg SSe Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe

Saline Saline 105 105 105 105 105

30 30 30 30 30 30 30

0 0 10  5.8a 3.3  3.3a 0 0 0

0 0 40  5.8a 23.3  3.3ab 6.7  6.7b 10  5.8b 10  5.8b

0 0 53.3  8.8a 43.3  6.7ab 10  10c 20  5.8bc 16.7  12bc

0 0 76.7  8.8a 53.3  8.8ab 36.7  3.3b 23.3  8.8b 23  14.5b

0 0 80  5.8a 56.7  6.7ab 43.3  3.3b 30  5.8b 30  17.3b

0 0 80  5.8a 56.7  6.7ab 43.3  3.3b 30  5.8b 30  14.5b

S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

SOD activity (U g protein-1)

6 5

A

a

4 a 3

a

a

a

2 1 0 Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe

-1

GPx activity (U mg protein )

Se-enriched diets (mg kg-1) 0.4

B

a

0.3 ab

ab 0.2 b

b

0.1

0 Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe

Se-enriched diets (mg kg -1 )

-1

GR activity (U mg protein )

5

C

a

4 3 ab ab

2

b

1

significant raised after being fed the 1 mg SSe-enriched diet (Fig. 2B). However, GPx activities of prawn fed the 1 mg MSe-, 0.5 mg SSe-, and 0.5 mg MSe-enriched diets did not significantly differ from that of prawns fed the control diet. GPx activities of 0.15  0.01, 0.18  0.04, 0.27  0.06 U, 0.13  0.01 U, and 0.22  0.02 U (mg protein)
1 were recorded in prawns fed the control and 1 mg MSe-, 1 mg SSe-, 0.5 mg MSe-, and 0.5 SSeenriched diets, respectively. The highest GR activity of prawn was detected in the 1 mg SSe treatment group, and it was significantly higher than that of control prawn. No significant differences in GR activity were recorded among prawn fed the control and Se-enriched diets of 1 mg MSe, 0.5 mg SSe, and 0.5 mg MSe. GR activities of 0.50  0.13, 1.83  0.55, 3.25  1.20, 1.00  0.53, and 1.75  0.23 U (mg protein)
1 were recorded in prawn fed the control and 1 mg MSe-, 1 mg SSe-, 0.5 mg MSe-, and 0.5 SSeenriched diets, respectively (Fig. 2C). The GST activity of prawn fed the 1 mg SSe-enriched diet (4.30  0.57 nmol
1 min
1 (mg protein)
1) was significantly higher than those of prawn fed the control diet (2.29  0.22 nmol
1 min
1 (mg protein)
1), and Seenriched diets of 1 mg MSe (1.90  0.30 nmol
1 min
1 (mg protein)
1) and 0.5 mg SSe (2.35  0.74 nmol
1 min
1 (mg protein)
1). No significant difference in GST activity was recorded between prawn fed the Se-enriched diets of 1 mg SSe and 0.5 mg MSe (Fig. 2D). The effects of Se on phagocytic activity and clearance efficiency of prawn to D. hansenii are shown in Fig. 3. Phagocytic activities of prawn fed the Se-enriched diets, including 1 mg MSe, 1 mg SSe, and 0.5 mg MSe were significantly higher compared to that of prawn fed the control diet (Fig. 3A). The phagocytic activities of prawn fed the control and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were 11.4%  0.93%, 16.4%  0.91%, 18.33%  1.69%, 14.67%  0.60%, and

b

0 1 mg MSe

1 mg SSe

-1

Se-enriched diets (mg kg )

-1 -1

GST activity

-1

(nmol min mg protein )

D a

5 4 3

ab b b

24

0.5 mg MSe 0.5 mg SSe

Phagocytic activity (%)

Control

6

627

b

A

a

20 ab

b

16

bc

c

12 8 4 0

2

Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe -1

1

Se-enriched diets (mg kg )

0 Control

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe

-1

Se-enriched diets (mg kg )

-1

Se-enriched diets (mg kg )

were recorded among the control and various treatment groups after 75 days of experimental diet consumption. SOD levels in hemocytes of prawn were 2.21  0.21, 2.32  0.40, 3.65  1.16, 2.78  0.38, and 2.93  0.29 U (g protein)
1 after being fed the control and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe, respectively (Fig. 2A). The GPx activity of prawn was

Control Clearance efficiency (%)

Fig. 2. Superoxide dismutase (SOD) (A), glutathione peroxidase (GPx) (B), glutathione reductase (GR) (C), and glutathione s-transferase (GST) (D) of giant freshwater prawn, Macrobrachium rosenbergii, fed a diet without selenium (Se) as the control, and diets with different forms of Se including inorganic SE (sodium selenate, SSe) at the concentrations of 0.5 and 1 mg (kg diet)
1, and organic Se (seleno-L-methionine, MSe) at the concentrations of 0.5 and 1 mg (kg diet)
1 for 75 days. The statistical analysis is as described in the legend to Fig. 1.

0

1 mg MSe

1 mg SSe

0.5 mg MSe 0.5 mg SSe

a

-50 -100 a -150 -200 -250

a

B

a

a

Fig. 3. Phagocytic activity (A) and clearance efficiency (B) of giant freshwater prawn, Macrobrachium rosenbergii, fed a diet without selenium (Se) as the control, and diets with different forms of Se including inorganic SE (sodium selenate, SSe) at the concentrations of 0.5 and 1 mg (kg diet)
1, and organic Se (seleno-L-methionine, MSe) at the concentrations of 0.5 and 1 mg (kg diet)
1 for 75 days. The statistical analysis is as described in the legend to Fig. 1.

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S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

13.75%  0.75%, respectively. There were no significant differences in clearance efficiency of prawn against D. hansenii among the control and various treatments in this study (Fig. 3B). The clearance efficiency of prawn fed the 1 mg MSe-, 1 mg SSe-, 0.5 mg MSe-, and 0.5 SSe-enriched diets were
96.46%  59.70%,
98.54%  104.89%,
124.48%  99.00%, and
63.76%  47.95, respectively, compared to control prawn.

4. Discussion Microelements, such as minerals, play important roles in growth performance and health of aquatic animals [4,5,17,24]. In addition, minerals are essential for the antioxidant defense system [24,25], e.g., Se is involved in the catalytic site of GPx, a selenoprotein. Se, a microelement is essential for normal organisms, despite some research having shown that a high concentration of Se has adverse effects on aquatic animals, even leading to death [17,26]. However, there is little research evaluating Se requirements of aquaculture animals, especially decapod crustaceans, because Se abounds in materials for aqua-feed, such as soybeans and fishmeal [27]. A high level of 1.18  0.07 mg Se (kg diet)
1 was determined in the control diet (without Se enrichment) used in this study. The growth and survival rates of prawn fed the control diet did not significantly differ from those of prawn fed the Se-enriched diets. Therefore, it is thought that around 1.1e1.2 mg (kg diet)
1 of Se in the diet (control diet) is sufficient for prawn growth. In humans, the health implications of a decline in the Se status were studied. Although the mechanisms involved have not yet to be fully elucidated, it is well established that dietary Se is important for a healthy immune response [28,29]. The effects of a Se deficiency can include reduced T-cell counts, and impaired lymphocyte proliferation and responsiveness [30]. Se plays an important role as an anticancer nutrient in modulating cell proliferation and inhibiting tumor cell invasion as reviewed by Zeng and Combs [13]. Therefore, Se is of fundamental importance to human health. Similarly, Se improved the growth rate, humoral immune response, and antioxidant status as found in a study of lambs [31]. In insects, Se may be relevant to microbial biological control in the cabbage looper, Trichoplusia ni, larvae fed a Se-containing diet resulting in improved resistance to a baculovirus infection [11]. Dietary Se levels were directly correlated with plasma Se levels, and plasma Se levels were in turn correlated with baculovirus resistance [14]. In the present study, prawn fed the Se-enriched diets had significantly better immunity, including PO activity, respiratory bursts, and phagocytic activity of hemocytes, and antioxidant status, including GPx, GR, and GST activities of hemocytes, which led to an increased disease resistant against the pathogen, D. hansenii. It is suggested that Se supplementation in the diet of prawn may contribute to a more-robust antimicrobial defense. Plants, like micro- and macroalgae in water, assimilate Se from the soil or water as the inorganic forms, selenite (SeO3) and selenate (SeO4), and subsequently convert Se into several organic forms and selenoproteins. In most cases, organic Se was shown to have greater bioavailability than the inorganic forms and thus to be superior as supplements [32]. In the present study, almost bioavailability of both organic Se and inorganic Se was shown in this study according to the results of PO activity, respiratory burst, phagocytic activity and cumulative mortality after pathogen challenge. However, inorganic Se exhibited higher antioxidant status, including GPx, GR, and GST activity in hemocyte of prawn compared to organic Se. Aquatic animals simultaneously obtain both organic and inorganic Se from their food, but they may also directly absorb inorganic Se from the water. In aquaculture, aquafeed price is going up and it is the highest cost for aquatic animal

production. Thus, the cheaper inorganic Se used as additive in aqua-feed is recommended compared to organic Se. PO activity of prawn significant increased which may have been caused by either form of dietary Se at levels of up to 1 mg (kg diet)
1 in this study. The proPO system is acknowledged to be the most important immune system in crustaceans [33,34]. The activated proPO system is involved with some important molecules that are released to perform crucial immune responses, including non-self recognition, melanin formation, adhesion, and cellecell communication [35e39]. The terminal enzyme of the proPO system, PO, activity can reflect the status of proPO system activation. Therefore, higher PO activities in prawn fed the Se-enriched diets are considered to reflect higher immunity and to directly contribute to microbicidal actions against the pathogen, D. hansenii. An increase in oxygen radical secretion in response to pathogen infections was demonstrated in mammals [40] and crustaceans [41,42]. Sarathi et al. [42] showed that increased superoxide anion (respiratory bursts) and decreased SOD levels were detected in Fenneropenaeus indicus infected with Vibrio alginolyticus and whitespot syndrome virus (WSSV). The increase in superoxide anions is considered to be beneficial in terms of enhancing immunity against pathogen infection as the results showed in our previous study: respiratory bursts per cell increased in viral-infected white shrimp, Litopenaeus vannamei, compared to healthy shrimp [41]. In this study, prawn fed the 1 mg MSe- and 1 mg SSe-enriched diets showed increases in respiratory bursts compared to control prawn and prawn fed the 0.5 mg MSe- and 0.5 mg SSe-enriched diets for 75 days. This increase in respiratory bursts of prawn fed the Se-enriched diets reflects an increased antimicrobial ability. In the immune response, reactive oxygen species (ROS) are formed by phagocytic cells during the process of phagocytosis. However, an excess of ROS production during defense may damage the host itself and lead to loss of cell function, and ultimately apoptosis or necrosis [43]. Therefore, an increase in antioxidants is needed to protect cells after an immune response to a pathogen. Antioxidative enzymes are SOD, GPx, GR, GST, etc. In the present study, an insignificant difference in SOD was recorded among the control and treatment groups, but GPx, GR, and GST significantly increased after prawn had been fed the Se-enriched diets for 75 days. It is thought that dietary Se can increase the ability to defend against oxidative damage by ROS, especially GPx, a selenoprotein containing the 21st amino acid, selenocysteine (Sec), converted from dietary Se. Therefore, determination of the GPx activity can be an effective way to estimate the bioavailability of Se [17,44]. In the present study, prawn fed the SSe-enriched diets at the levels of 0.5 and 1 mg kg
1 had better GPx activities compared to prawn fed the control diet and MSe-enriched diets. This indicates that inorganic Se added to the diet for prawn is more effective in improving the immune response, disease resistance, and antioxidant status. Phagocytosis is an important cellular defense mechanism, and the clearance efficiency is also an important defense mechanism performed by hemocytes, the lymphoid organ, and the hepatopancreas in crustaceans [45e47]. These two immune parameters are also widely used to evaluate a decapod crustacean’s health status under different treatments of probiotic, immunostimulants, and environmental stress [48,49]. Most studies showed that animals with better phagocytic activity and clearance efficiency have better disease resistance [48,49]. In this study, the capability to engulf an invasive pathogen, D. hansenii, by prawn significantly increased following feeding of the Se-enriched diets, which led to increased resistance against D. hansenii by the prawn. However, an insignificant difference in efficiency of clearing D. hansenii by prawn among the control and treatments did not correspond to the mortalities of prawn in the control and treatment groups. It is thought that the amounts of visible colonies on the medium do not

S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

reflect the true virulence of the pathogen. Thus, low mortalities of prawn in Se-enriched diet treatments after being challenged by D. hansenii may have been caused by improvements in the immune status and also a decrease in the virulence of D. hansenii. In conclusion, Se is a potent nutritional immune and antioxidant enhancer that possesses biological effects of increasing PO activity, respiratory bursts, GPx activity, GR activity, GST activity, phagocytic activity and disease resistance against the pathogen, D. hansenii, in this study. The control diet without the addition of Se (but containing around 1 mg Se (kg diet)
1) would meet the requirement for prawn growth. Based on the result of challenge test, enrichment of 0.5 mg Se in the prawn diet resulting in 1.5 mg Se (kg diet)
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