Fish & Shellfish Immunology 29 (2010) 623e629

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

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
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan 912, ROC
b
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: The effects of inorganic selenium (Se) (sodium selenate, SSe) and organic selenium (seleno-L-methionine,
Received 6 February 2010 MSe) supplementation on the immune response, antioxidant status, and disease resistance of the giant
Received in revised form freshwater prawn, Macrobrachium rosenbergii, were studied. Five experimental diets, including a control
21 May 2010
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,
Accepted 8 June 2010
Available online 16 June 2010
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, Debar-
yomyces hansenii. No significant differences in the total hemocyte count, superoxide dismutase activity, or
Keywords:
Macrobrachium rosenbergii
clearance efficiency of prawn were recorded among the control and treated groups. Significantly increased
Selenium phenoloxidase and phagocytic activities in prawn fed the Se-enriched diets were found compared to the
Immune response controls. Respiratory bursts of prawn fed both forms of 1 mg Se (kg diet)
1 significantly increased
Antioxidant status compared to control prawns. For the antioxidant status analysis, glutathione peroxidase, glutathione
Debaryomyces hansenii 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.

1. Introduction For tiger shrimp, adequate dietary copper concentrations for

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: winton@mail.npust.edu.tw (W. Cheng), chliu@mail.npust.
edu.tw (C.-H. Liu).
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]. Mecha-
nisms of Se’s anticancer actions are not fully understood; however,
several have been proposed: antioxidant protection, enhanced
carcinogen detoxification, enhanced immune surveillance, modu-
lation 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

1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2010.06.012
624 S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

cabbage looper, Trichoplusia ni; however, its larvae fed Se in the
penultimate and ultimate instars were more resistant to a baculo-
virus infection (Autographa californica multiple nucleopolyhe-
drovirus, 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 fresh-
water prawn, M. rosenbergii, and its disease resistance were eval-
uated in this study. A control diet (without Se addition), and four
Se-enriched diets, including 0.5 mg (kg diet)
1 of seleno-L-methi-
onine (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 growing-
out. 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. Se was added to the experimental diets with a corre-
sponding 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 spectro-
photometer (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 fiberglass-
reinforced 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 indi-
vidually 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 over-
flow 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 Bend-
schreider 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

Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe The yeast, D. hansenii, was cultured on tryptic soy agar (TSA,
Fish meal 430 430 430 430 430 Difco, USA) for 24 h at 28  C before being transferred to 10 ml
Soybean meal 65 65 65 65 65 tryptic soy broth (TSB, Difco), where it remained for 24 h at 28  C as
Yeast meal 25 25 25 25 25 a stock culture for the tests. The broth cultures were centrifuged at
Shrimp shell meal 70 70 70 70 70
Wheat flour 350 350 350 350 350
7155g for 20 min at 4  C. The supernatants were removed, and the
cellulose 1 0 0 0.5 0.5 bacterial pellets were resuspended in a saline solution at 1  107
MSe 0 1 0 0.5 0 colony-forming units (cfu) ml
1 as stock bacterial suspensions for
SSe 0 0 1 0 0.5 the susceptibility study.
Gluten 25 25 25 25 25
The challenge test was conducted in triplicate by an injection of
Fish oil 8 8 8 8 8
Mineral mixture 20 20 20 20 20 20 ml of a bacterial suspension resulting in 2  105 cfu prawn
1 into
without selenium the ventral sinus of the cephalothorax. The prawn that were fed
Vitamin mixture 6 6 6 6 6 1 mg MSe or 1 mg SSe and then received saline (20 ml) served as the
Mineral mix without selenium and Vitamin mix were prepared according to the unchallenged control. Experimental prawn (10 prawn aquarium
1)
formula of Cheng and Hardy [50]. 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.
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 Micro-
systems, 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 phosphate-
buffered 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-nitro-
phenol)-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 for-
mazan 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 gluta-
thione 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
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.
2.5.7. 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
626 S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629

percent data were normalized using an arcsine-transformation. 16

Statistically significant differences required that p < 0.05.
A a
a
14 a

T H C (x 1 0 c e ll m l )
-1
12
3. Results a
10
a

8
8
During the 75 days of culture, water parameters were found to
be within acceptable ranges for prawn growth in all treatments and 6
the control, and no significant differences in total ammonia-N 4
(0.02e0.22 mg L
1), nitrite-N (0.02e0.59 mg L
1), pH (6.9e7.3), 2
temperature (24e27  C), or dissolved oxygen (6.23e6.89 mg L
1)
0
were detected among the control and treatment groups. Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
No significant differences in the growth or survival rates among Se-enriched diets (mg kg )
-1

all treatments and the control were found. The percentage of gain
weight of prawn in the control and treatment groups of 1 mg MSe,
0.5
1 mg SSe, 0.5 mg MSe, and 0.5 SSe were 312.56%  22.53%, B
294.61%  1.76%, 319.84%  47.33%, 278.08%  27.04%, and

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

a
0.4
271.80%  37.39%, respectively. The survival rate of prawn in the a
a
control and treatment groups of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, 0.3 ab
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. 0.2
b
After 75 days of culture, prawn from the treatment and
control groups were randomly selected for pathogen (D. hansenii) 0.1
challenge. No dead prawns were recorded in either of the
unchallenged control groups. The mortality occurred at 24 h after 0
the injection in the challenge control and 1 mg MSe treatment Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
groups. However, dead prawn in the 1 mg SSe, 0.5 mg MSe, and Se-enriched diets (mg kg )
-1

0.5 mg SSe treatment groups were recorded at 48 h after the

injection. At the end of the challenge trial, the cumulative 0.4
C
R e s p ira to ry b u rs t (O.D .630n m )

mortalities of the control and 1 mg MSe, 1 mg SSe, 0.5 mg MSe, a

a
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 0.3
cumulative mortality of prawn in the control group was signifi-
b b
cant higher than those in the 1 mg SSe, 0.5 mg MSe, and 0.5 mg 0.2
SSe groups (Table 2). b
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. 0.1
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 0
MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were (11.67  2.18)  108, Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
(11.44  1.55)  108, (8.87  0.95)  108, (8.02  0.56)  108, and Se-enriched diets (mg kg )
-1

(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. Respira-
tory 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-,
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-
L-methionine, MSe) at the concentrations of 0.5 and 1 mg (kg diet)

1
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 antioxi-
dant 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 No. of Cumulative mortality (%), time post injection (h)
(cfu prawn
1) shrimp
24 48 72 96 120 144
1 mg MSe Saline 30 0 0 0 0 0 0
1 mg SSe Saline 30 0 0 0 0 0 0
Control 105 30 10  5.8a 40  5.8a 53.3  8.8a 76.7  8.8a 80  5.8a 80  5.8a
1 mg MSe 105 30 3.3  3.3a 23.3  3.3ab 43.3  6.7ab 53.3  8.8ab 56.7  6.7ab 56.7  6.7ab
1 mg SSe 105 30 0 6.7  6.7b 10  10c 36.7  3.3b 43.3  3.3b 43.3  3.3b
0.5 mg MSe 105 30 0 10  5.8b 20  5.8bc 23.3  8.8b 30  5.8b 30  5.8b
0.5 mg SSe 105 30 0 10  5.8b 16.7  12bc 23  14.5b 30  17.3b 30  14.5b
 S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629 627

6 significant raised after being fed the 1 mg SSe-enriched diet

A
SOD activity (U g protein-1)

a (Fig. 2B). However, GPx activities of prawn fed the 1 mg MSe-,

5
0.5 mg SSe-, and 0.5 mg MSe-enriched diets did not significantly
4 differ from that of prawns fed the control diet. GPx activities of
a a
3 a 0.15  0.01, 0.18  0.04, 0.27  0.06 U, 0.13  0.01 U, and
a 0.22  0.02 U (mg protein)
1 were recorded in prawns fed the
2 control and 1 mg MSe-, 1 mg SSe-, 0.5 mg MSe-, and 0.5 SSe-
1 enriched diets, respectively. The highest GR activity of prawn was
detected in the 1 mg SSe treatment group, and it was signifi-
0
cantly higher than that of control prawn. No significant differ-
Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
ences in GR activity were recorded among prawn fed the control
Se-enriched diets (mg kg-1)
and Se-enriched diets of 1 mg MSe, 0.5 mg SSe, and 0.5 mg MSe.
0.4
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
GPx activity (U mg protein )

B
-1

a
control and 1 mg MSe-, 1 mg SSe-, 0.5 mg MSe-, and 0.5 SSe-
0.3
enriched diets, respectively (Fig. 2C). The GST activity of prawn
ab
ab
fed the 1 mg SSe-enriched diet (4.30  0.57 nmol
1 min
1 (mg
0.2 protein)
1) was significantly higher than those of prawn fed the
b
b control diet (2.29  0.22 nmol
1 min
1 (mg protein)
1), and Se-
0.1 enriched 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
0
Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe between prawn fed the Se-enriched diets of 1 mg SSe and 0.5 mg
Se-enriched diets (mg kg -1 ) MSe (Fig. 2D).
The effects of Se on phagocytic activity and clearance effi-
5 ciency of prawn to D. hansenii are shown in Fig. 3. Phagocytic
C a
GR activity (U mg protein )
-1

activities of prawn fed the Se-enriched diets, including 1 mg MSe,

4 1 mg SSe, and 0.5 mg MSe were significantly higher compared to
that of prawn fed the control diet (Fig. 3A). The phagocytic
3 activities of prawn fed the control and Se-enriched diets of 1 mg
ab
ab MSe, 1 mg SSe, 0.5 mg MSe, and 0.5 SSe were 11.4%  0.93%,
2
b 16.4%  0.91%, 18.33%  1.69%, 14.67%  0.60%, and
1 b

0
24
Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
-1
A a
Phagocytic activity (%)

Se-enriched diets (mg kg ) 20

ab
16 b
6 bc
D c
12
(nmol min mg protein )

a
-1

5
8
4
GST activity

ab
b
3 4
b
-1

b
2 0
-1

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 ) Control 1 mg MSe 1 mg SSe 0.5 mg MSe 0.5 mg SSe
0
Fig. 2. Superoxide dismutase (SOD) (A), glutathione peroxidase (GPx) (B), glutathione a
Clearance efficiency (%)

reductase (GR) (C), and glutathione s-transferase (GST) (D) of giant freshwater prawn,
-50
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 -100
concentrations of 0.5 and 1 mg (kg diet)
1, and organic Se (seleno-L-methionine, MSe) a
at the concentrations of 0.5 and 1 mg (kg diet)
1 for 75 days. The statistical analysis is -150
as described in the legend to Fig. 1. a
-200
B a
a
-250
were recorded among the control and various treatment groups
after 75 days of experimental diet consumption. SOD levels in Fig. 3. Phagocytic activity (A) and clearance efficiency (B) of giant freshwater prawn,
hemocytes of prawn were 2.21  0.21, 2.32  0.40, 3.65  1.16, 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
2.78  0.38, and 2.93  0.29 U (g protein)
1 after being fed the concentrations of 0.5 and 1 mg (kg diet)
1, and organic Se (seleno-L-methionine, MSe)
control and Se-enriched diets of 1 mg MSe, 1 mg SSe, 0.5 mg MSe, at the concentrations of 0.5 and 1 mg (kg diet)
1 for 75 days. The statistical analysis is
and 0.5 SSe, respectively (Fig. 2A). The GPx activity of prawn was as described in the legend to Fig. 1.
628 S.-T. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 623e629
13.75%  0.75%, respectively. There were no significant differ-
ences 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 defi-
ciency 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 inhib-
iting 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 sele-
nate (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 chal-
lenge. 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, aqua-
feed 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 acti-
vated 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 activa-
tion. 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 white-
spot 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 hepato-
pancreas 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)
1 would benefit the immune status and antioxidant
status of prawn and their disease resistance to D. hansenii. In
addition, the use of the cheaper inorganic Se in prawn feed is
suggested.
Acknowledgements
This study was supported by a grant from the National Science
Council of the ROC (NSC96-2313-B-020-008-MY3).
References
[1] Hsu JP, Liu CI. Studies on yeast infection in cultured giant freshwater prawn
(Macrobrachium rosenbergii). Fish Dis Res 1994;15:55e68.
[2] Liu CI, Hsu JP, Chien MS. Studies on pathogenicity of yeast in cultured shrimp.
COA Fish Ser 1996;54:5e24.
[3] Cheng W, Chen JC. Isolation and characterization of Enterococcus-like bacte-
rium causing muscle necrosis and mortality with Macrobrachium rosenbergii
in Taiwan. Dis Aquat Organ 1998;34:93e101.
[4] Shiau SY, Jiang LC. Dietary zinc requirements of grass shrimp, Penaeus mon-
odon, and effects on immune responses. Aquaculture 2006;254:476e82.
[5] Lee MH, Shiau SY. Dietary copper requirement of juvenile grass shrimp,
Penaeus monodon, and effects on non-specific immune responses. Fish Shell-
fish Immunol 2002;13:259e70.
[6] Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG.
Selenium: biochemical role as a component of glutathione peroxidase. Science
1973;179:585e90.
[7] Ellis DR, Salt DE. Plants, Se and human health. Curr Opin Plant Biol
2003;6:273e9.
[8] Beck MA, Handy J, Levander OA. Host nutritional status: the neglected viru-
lence factor. Trends Microbiol 2004;12:417e23.
[9] Hintze KJ, Lardy GP, Marchello MJ, Finley JW. Areas with high concentrations
of Se in the soil and forge produce beef with enhanced concentrations of Se.
J Agric Food Chem 2001;49:1062e7.
[10] Payne RL, Southern LL. Comparison of inorganic and organic Se sources for
broilers. Poult Sci 2005;84:898e902.
[11] Popham HJR, Shelby KS, Popham TW. Effect of dietary Se supplementation on
resistance to baculovirus infection. Biol Control 2005;32:419e26.
[12] Whanger PD. Selenium and its relationship to cancer: an update. Br J Nutr
2004;91:11e28.
[13] Zeng H, Combs Jr GF. Selenium as an anticancer nutrient: roles in cell
proliferation and tumor cell invasion. J Nutr Biochem 2008;19:1e7.
[14] Shelby KS, Popham HJR. Increased plasma selenium levels correlated with
elevated resistance of Heliothis virescens larvae against baculovirus infection.
J Invert Pathol 2007;95:77e83.
[15] Hilton JW, Hodson PV, Slinger SJ. The requirement and toxicity of selenium in
rainbow trout (Salmo gairdneri). J Nutr 1980;110:2527e35.
[16] Hamilton SJ. Review of residue-based selenium toxicity thresholds for fresh-
water fish. Ecotoxicol Environ Safe 2003;56:201e10.
[17] Lin YH, Shiau SY. Dietary selenium requirements of juvenile grouper
Epinephelus malabaricus. Aquaculture 2005;250:356e63.
[18] Wang Y, Han J, Li W, Xu Z. Effect of different selenium source on growth
performances, glutathione peroxidase activities, muscle composition and
selenium concentration of allogynogenetic crucian carp (Carassius auratus
gibelio). Anim Feed Sci Technol 2007;134:243e51.
[19] Monteiro DA, Rantin FT, Kalinin AL. The effects of selenium on oxidative stress
biomarkers in the freshwater characid fish matrinxã, Brycon cephalus
(Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BRÒ
(methyl parathion). Comp Biochem Physiol C 2009;149:40e9.
[20] Lorentzen M, Maage A, Julshamn K. Effects of dietary selenite or selenome-
thioneine on tissue selenium levels of Atlantic salmon (Salmo salar). Aqua-
culture 1994;121:359e67.
[21] Bower CE, Bidwell JP. Ionization of ammonia in seawater: effects of temper-
ature, pH and salinity. J Fish Res Board Can 1978;35:1012e6.
629
[22] Bendschneider K, Robinson RJ. A new spectrometric method for the deter-
mination of nitrite in the sea water. J Mar Res 1952;11:87e96.
[23] 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 Immunol 2004;18:1e12.
[24] Lin YH, Shaiu SY. The effects of dietary selenium on the oxidative stress of
grouper, Epinephelus malabaricus, fed high copper. Aquaculture 2007;267:
38e43.
[25] Martinez-Alvarez RM, Morales AE, Sanz A. Antioxidant defenses in fish: biotic
and abiotic factors. Rev Fish Biol Fish 2005;15:75e88.
[26] Wang WN, Wang AL, Zhang YJ. Effect of dietary higher level of selenium and
nitrite concentration on the cellular defense response of Penaeus vannamei.
Aquaculture 2006;256:558e63.
[27] Gabrielsen BO, Opstvedt O. Availability of selenium in fish meal in comparison
with soybean meal, corn gluten meal and selenomethionine relative to sele-
nium in sodium selenite for restoring glutathione peroxidase activity in
selenium-depleted chicks. J Nutr 1980;22:1096e100.
[28] Taylor EW. Selenium and cellular immunity e evidence that selenoproteins
may be encoded in the þ1 reading frame overlapping the human CD4, CD8,
and HLA-DR genes. Biol Trace Elem Res 1995;49:85e95.
[29] Hoffmann PR, Berry MJ. The influence of selenium on immune response. Mol
Nutr Food Res 2008;52:1273e80.
[30] Kiremidjian-Schumacher L, Roy M, Wishe HI, Cohen MW, Strotzky G.
Supplementation with selenium and human immune cell functions. 2. Effect
on cytotoxic lymphocytes and natural killer cells. Biol Trace Elem Res
1994;41:115e27.
[31] Kumar N, Garg AK, Dass RS, Chaturvedi VK, Mudgal V, Varshney VP. Selenium
supplementation influences growth performance, antioxidant status and
immune response in lambs. Anim Feed Sci Technol 2009;153:77e87.
[32] Finley JW. Bioavailability of Se from foods. Nutr Rev 2006;64:146e51.
[33] Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrate
animals. J Biochem Mol Biol 2005;38:128e50.
[34] Söderhäll K, Cerenius L. Role of the prophenoloxidase-activating system in
invertebrate immunity. Curr Opin Immunol 1998;10:23e8.
[35] Söderhäll K, Cerenius L, Johansson MW. The prophenoloxidase activating
system and its role in invertebrate defence. Primordial immunity: foundations
for the vertebrate immune system. Ann NY Acad Sci 1994;712:155e61.
[36] Johansson MW. Cell adhesion molecules in invertebrate immunity. Develop
Comp Immunol 1999;23:303e15.
[37] Lee SY, Wang R, Söderhäll K. A lipopolysaccharide and b-1,3-glucan-binding
protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus.
J Biol Chem 2000;275:1337e43.
[38] Liu CH, Chen W, Kuo CM, Chen JC. Molecular cloning and characterisation of
a cell adhesion molecule, peroxinectin from the white shrimp Litopenaeus
vannamei. Fish Shellfish Immunol 2004;17:13e26.
[39] Liu CH, Cheng W, Chen JC. The peroxinectin of white shrimp Litopenaeus
vannamei is synthesized in the semi-granular and granular cells, and its
transcription is up-regulated with Vibrio alginolyticus infection. Fish Shellfish
Immunol 2005;18:431e44.
[40] Oda T, Akaike T, Hamamoto T, Suzuki F, Hirano T, Maeda H. Oxygen radicals in
influenza-induced pathogenesis and treatment with pyran polymer-conju-
gated SOD. Science 1989;244:974e6.
[41] Yeh SP, Chen YN, Hsieh SL, Cheng W, Liu CH. Immune response of white
shrimp, Litopenaeus vannamei, after a concurrent infection with white spot
syndrome virus and infectious hypodermal and hematopoietic necrosis virus.
Fish Shellfish Immunol 2009;26:582e8.
[42] Sarathi M, Ishaq Ahmed VP, Venkatesan C, Balasubramanian G, Prabavathy J,
Sahul Hameed AS. Comparative study on immune response of Fenneropenaeus
indicus to Vibrio alginolyticus and white spot syndrome virus. Aquaculture
2007;217:8e20.
[43] Nordberg J, Arnér ESJ. Reactive oxygen species, antioxidants, and the
mammalian thioredoxin system. Free Radic Biol Med 2001;31:1287e312.
[44] Wang YB, Xu BH. Effect of different selenium source (sodium selenite and
selenium yeast) on broiler chickens. Anim Feed Sci Technol 2008;114:
309e14.
[45] Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP. Invertebrate immunity:
basic concepts and recent advances. Int Rev Cytol 1985;97:183e350.
[46] Alday-Sanz V, Roque A, Turnbull JF. Clearing mechanisms of Vibrio vulnificus
biotype I in the black tiger shrimp Penaeus monodon. Dis Aquat Organ
2002;48:91e9.
[47] van de Braak CBT, Botterblom MHA, Huisman EA, Rombout JHWM, van der
Knaap WPW. Preliminary study on haemocyte response to white spot
syndrome virus infection in black tiger shrimp Penaeus monodon. Dis Aquat
Organ 2002;51:149e55.
[48] Rengpipat S, Rukpratanporn S, Piyatiratitivorakul S, Menasaveta P. Immunity
enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacte-
rium (Bacillus S11). Aquaculture 2000;191:271e88.
[49] Chiu CH, Guu YK, Liu CH, Pan TM, Cheng W. Immune responses and gene
expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus
plantarum. Fish Shellfish Immunol 2007;23:364e77.
[50] Cheng ZJ, Hardy RW. Protein and lipid sources affect cholesterol concentra-
tions of juvenile Pacific white shrimp, Litopenaeus vannamei (Boone). J Anim
Sci 2004;82:1136e45.