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Antioxidant status of Penaeus monodon fed
with Dunaliella salina supplemented diet
and resistance against WSSV
M. MADHUMATHI
*
Research scholar, Center for advanced studies in Botany
University of Madras, Maraimalai campus, Chennai-65
Tamil Nadu, India.
Mobile No: +91 9841111534
madhumathi83@gmail.com
R.RENGASAMY
Director &Professor, Center for advanced studies in Botany
University of Madras, Maraimalai campus, Chennai-65
Tamil Nadu, India.
Mobile No: +91 9176295052
profrrengasamy@yahoo.com
Abstract
The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus
(WSSV) using Dunaliella salina algal cells which contains an antioxidant betacarotene for the shrimp non-
specific immunity. To determine the antioxidant activity, the shrimp were treated in vivo (orally with feed)
methods at the concentration of 0.5 %, 1.0 % and 2 % D. salina incorporated with pellet feed, respectively. In
the present study, anti-WSSV activity of D. salina incorporated diet by in vivo methods showed strong
antioxidant activity and the immunological parameters such as proPO, SOD,catalase were higher in the WSSV-
infected shrimp treated with D. salina incorporated diet when compared to control groups. These results
strongly indicate that in vivo of D. salina incorporated diet enhances immunity of the shrimp. Based on the
present data and the advantages of harvesting D. salina at low price, we believe that oral administration of D.
salina live cells along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp to
some extent.
Keywords: Penaeus monodon; Dunaliella salina; antioxidant activity.
1. Introduction
The White Spot Syndrome Virus (WSSV) continues to be one of the most serious disease problems
faced by the shrimp farming industry worldwide (Flegel, 1997; Karunasagar et al., 1997; Hsu et al., 1999).
White spot syndrome virus (WSSV) is spread by cannibalism of sick or dying shrimp or via contaminated water.
Many other animals (crabs, lobsters, shellfish) may harbor and spread the disease (Chang et al,1998 Sahul
Hameed et al., 2003).The quick spread of the disease among the shrimp population and the broad host range,
make the control of this virus almost impossible, thus novel strategies to control WSSV are needed. Various
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studies have been carried out to obtain the performance of immunostimulants for enhancing immune response
and reduction of disease impacts. Several diseases were controlled by oral administration of immunostimulation.
Immunostimulants are suitable for boosting immature immune system and effective against a number
of opportunistic and secondary pathogens (Felix et al., 2004). Thus modern shrimp hatchers are now trying to
resolve this problem of WSSV infection by using immunostimulant incorporated shrimp feed. Studies have been
carried out to obtain the performance of immunostimulants for enhancing immune response and reduction of
disease impacts. Certain carotenoids obtained from algal sources like astaxanthin and β-carotene induces
immune system in shrimp and will certainly continue to play an important role in disease control in intensive
shrimp culture (Karin van de, 2002). In order to know the role of this immunostimulant of carotenoids obtained
from algae on resistance of shrimp against WSSV and its consequences on the immune condition of shrimp, the
present study was carried out to determine the effect of betacarotene obtained from Dunaliella salina
incorporated with different concentrations on prophenol oxidase, superoxide dismutase, catalase (SOD)
production during the course of the WSSV challenge to assess the non-specific immune status of the shrimp.
2. Materials and Methods
2.1. Preparation of betacarotene from D. salina
Ten litre of Dewalnes medium were prepared with 35 ppt and inoculated with 100 ml of pure Dunaliella
salina culture. The cultures were kept in out door condition for a period of 20 days. On 15
th
day orange stage is
attained. Then the cultures were harvested and centrifuged at 10,000 rpm for 10 min. The obtained biomass
were lyophilised and stored in the refrigerator until they were used for the bioassay test.
2.2. Preparation of test diets
One litre of fresh water was taken in a vessel and boiled on a heater. The ingredients were cooked as
described below. The rice bran was added at first and mixed thoroughly, followed by the addition of fishmeal,
coconut cake, squid meal, wheat flour powder and mixed thoroughly, until the feed became a paste and cooled
to room temperature and added the ingredients such as fish oil, vitamin mix and tapioca. To this different
percentages of harvested biomass of D. salina cells (dry wt) such as 0.5 %, 1.0 % and 2 % were added
separately and mixed thoroughly. Then the paste was taken pressed and squeezed through 2 mm
2
pores and
collected the shreds on a polythene sheet. The shreads without D. salina served as control. The shreds were
allowed to dry at room temperature for 2 days kept in plastic bags and sealed properly until given to shrimp.
2.3. Collection and maintenance of experimental animals
A total of 100 Penaeus monodon of 4-5 g body weight, were obtained from the shrimp pond at
Meenjur, near chennai and maintained in 1000 L fibre glass tank with air- lift biological filters at room
temperature (27 - 30°C) with salinity between 20 and 25 %. The experiment was conducted at the Aqua Nova
Hatcheries, Kanathur, Chennai. The seawater was pumped from the Bay of Bengal and kept for 24 h and
removed the sand and other suspended particles at the bottom. Then the seawater was initially chlorinated with
25 ppm sodium hypochlorite followed by dechlorination by vigorous aeration, passed through sand filter and
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used for the experiment. Temperature, pH and salinity (Aqua fauna, Japan) were recorded on the spot. This
experiment was carried out for a period of 30 days.
2.4. Feeding trial on Penaeus monodon
The experimental shrimp, Penaeus monodon was fed with normal diet (control) for a period of 2 days
and then taken for the experimental study. The animals were divided into four groups of each 10 shrimps in 100
L seawater in fibre tanks at room temperature (27-30°C) with salinity between 20 and 25 ppt. Group I animals
were fed with the normal pellet feed without D. salina (control). In Group II, the shrimp were fed with 0.5 %
D. salina incorporated feed, Group III fed with 1.0 % D. salina incorporated feed, and Group IV fed with 2 %
D. salina incorporated feed for a period of one month. The animals were fed twice daily at 09:00: and 17:00 h
for a period of 30 days. The amount of feed given was 5 % of animal body weight. Every alternate days, 50 %
volume of the water along with the excreta of animals and other waste were removed and compensated with
fresh seawater.
2.5. Determination of immunological and antioxidant activity
The uninfected shrimp fed with different concentrations of D. salina incorporated diet were sacrificed weekly
for a period of one month for immunological assay such as prophenoloxidase assay (proPO), superoxide anion
assay, superoxide dismutase activity and catalase assay were analyzed for different groups.
2.6. Haemolymph collection
Haemolymph of 300 μl per shrimp was collected directly from the heart of animal every week for a
period of one month. The haemolymph was collected by using a 23-guage needle and 1.0 mL syringe contained
300 μl (4°C) precooled 10 % sodium citrate solution as anticoagulant in glass distilled water.
2.7. Estimation of protein
The estimation of protein was carried out by following the method of Lowery et al., 1951. One hundred
µL of haemolymph samples were made up to 1 mL separately with glass distilled water. To this 4.5 mL of
alkaline copper reagent was added shaken well and allowed to stand for 10 minutes. To this 0.5 mL of Folin’s
reagent was added and kept at room temperature at 37°C for 20 minutes. The absorbance was read at 660 nm
using UV visible spectrophotometer (UV- 1601 Shimadzu). Total protein expressed as μg/mL in haemolymph.
2.8. Phenoloxidase activity
The Prophenol Oxidase assay was carried out by following the method of Pascual et al., 2003. The 0.1
mL Haemolymph sample was made up to 1.0 mL with glass distilled water and centrifuged at 3000 rpm at 4° C
for 10 minutes. The supernatant was discarded and the pellet was collected, rinsed and resuspended in sodium
cacodylate buffer and centrifuged again. The pellet was once again resuspended with 200 µL of sodium
cacodylate buffer. To 100 µL of the sample 50 µL trypsin (1 mg/mL) was added and kept for 10 minutes at 25°
C, then 50 µL of L- DOPA was added followed by the addition of 800 µL cacodylate buffer after 5 minutes.
Phenoloxidase activity was measured under ELISA reader by recording the formation of dopachrome from L-
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DOPA (Leonard et al., 1985). The data were recorded every one minute interval for a period of 5 minutes at 490
nm.
2.9. Assay of Super oxide dismutase
SOD assay was carried out in Haemolymph protein samples of the experimental animals as described
by Misra and Fridovich , 1972. To 0.1 mL of haemolymph protein 0.75 mL ethanol and 0.15 mL
chloroform (chilled in ice) were added separately and centrifuged at 10,000 rpm at 4°C for 10 minutes. To 0.5
mL of supernatant 0.5 mL of EDTA (0.6 mM) solution and 1mL of carbonate bicarbonate buffer (0.1 M) pH
10.2 were added. The reaction was initiated by the addition of 0.5 mL of substrate (Epinephrine 1.8 mM) and
the increase in absorbance was recorded at 480 nm at every 30 seconds for 3 minutes. The values are expressed
as 50 % inhibition of epinephrine auto oxidation /min /mg protein.
2.10. Catalase Assay
The Catalase assay was carried out by following the method of Takaharat et al., 1960. To 1.2 mL of
phosphate buffer (0.05 M, pH 7), 0.2 mL haemolymph of protein samples was added separately and the catalase
enzyme reaction was initiated by addition of 1 mL of substrate H
2
O
2
(0.03 M in phosphate buffer). The decrease
in OD at 240 nm was recorded at every 30 seconds for 3 minutes. The enzyme blank was run simultaneously
with 1mL of glass distilled water instead of H
2
O
2
. A standard contained catalase was carried out simultaneously
and expressed as µmoles of H
2
O
2
decomposed /min/mg protein.
2.11. In vivo determination of antioxidant activity of healthy shrimp challenged with WSSV infected
shrimp
The shrimps fed with normal pellet feed and three different concentrations of D. salina incorporated
feed were challenged by WSSV infected tissues (200 mg) of P. monodon by oral route obtained from the
Central Institute of Brackish Aquaculture, Chennai, were divided into four groups of 10 shrimp per group and
each trial was conducted in triplicates. In Group I, shrimp fed with normal feed for 2 days and then on 3
rd
day
alone it was fed twice a day orally with WSSV infected shrimp treated as positive control showed 100%
mortalitily within 48 hrs p.i. with gross signs including lethargy, reduced feed consumption, reddishness on
dorsal side and appearance of white spots in cephalothoracic region. In Group II , shrimp fed with D. salina
incorporated diet without any challenge towards WSSV (negative control) showed 100 % survival till the end
of the study period (up to 10
th
day). In Group III, shrimp fed with 0.5% D. salina incorporated diet for 2 days
and fed with WSSV infected shrimp on third day alone twice a day and from fourth day fed with normal pellet
feed, similarly feed was given in Group IV, shrimp fed with 1.0 % D. salina incorporated diet challenged with
WSSV infection and Group V, 2.0 % D. salina incorporated diet challenged with WSSV infection. In all the
groups, the shrimps were fed twice daily. Initially 5–7% of body weight of feed was fed to shrimp and thereafter
it was adjusted according to the feeding response of the shrimp in each tank. Uneaten food and waste matter
were removed before feeding. The experimental animals were examined twice per day for gross signs of
disease, and the number of deaths was recorded. For the analysis of immunological and hematological
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parameters, samples of haemolymph were taken from live infected animals on 1, 3, 6 and 9 days. The
procedures for the estimation of the assays were described earlier. The immunological results were compared
with the experimental animals. All the live and dead animal samples were analyzed for PCR using the primer
meant for WSSV.
Statistical analyses
The experimental data were tabulated and analyzed using one-way ANOVA by the Agres statistical
software package (Agres, 1994). The least significant difference (LSD) analysis was performed to group the
treatment mean values.
3. Results
3.1. Immunostimulant and antioxidant activity of healthy P. monodon fed with D. salina test diets
3.1.1 Prophenoloxidase assay
The shrimp fed with the diet without D. salina (control) showed the proPO levels of 0.11, 0.11, 0.11,
and 0.11 U/min/mg of protein on 1
st
, 2
nd
, 3
rd
and 4
th
weeks (Fig. 1), respectively. The above values were less
than 64 %, 66 %, 68 % and 70 % to the animal fed with 1.0 % D. salina incorporated diet. Among the three
different concentrations of D. salina incorporated diets, the animal fed with 1.0 % diet showed maximum proPO
of 0.38 U/min/mg of protein on 4
th
week. Whereas the shrimp fed with 0.5 % and 2.0 % D. salina incorporated
diet showed 0.3 and 0.28 U/min/mg of protein, respectively on 4
th
week.
c
d
d
d
b
b
b
b
a
a
a
a
b
c
c
c
0
0.1
0.2
0.3
0.4
0.5
Iweek II week III week IV week
No. of weeks
OD 490 nm units/min/mg protein
control
o.5 % D. salina
1.0 % D. salina
2 % D. salina
Fig. 1. Prophenol oxidase assay of P. monodon fed with D. salina incorporated diets
3.2. Superoxide dismutase assay
3.2.1. Superoxide dismutase assay of Haemolymph
The SOD levels of the haemolymph of animals when fed with the diet incorporated D. salina showed
enhanced values to that of control (diet without D. salina). The shrimp fed with 1.0 % D. salina incorporated
diet showed 1.72, 1.9, 1.4 and 1.14 Rapid auto oxidation production (RAP) (Fig. 2), which were more than 28
%, 35 %, 12 % and 7 % to that of the control on 1
st
, 2
nd
, 3
rd
and 4
th
weeks respectively. Whereas the shrimp fed
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Vol. 3 No. 10 October 2011
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with 0.5 % D. salina incorporated diet showed 1.2, 1.32, 1.2 and 1.04 (RAP) and 2 % D. salina incorporated
diet showed 1.6, 1.21, 1.2 and 1.07 (RAP) on 1
st
, 2
nd
, 3
rd
and 4
th
weeks, respectively.
abcc
d
c
b
d
b
a
a
a
c
c
d
b
0
0.5
1
1.5
2
2.5
I week II week III week IV week
No. of weeks
Relative superoxide anion
production
Control
0.5 % D. salina
1.0 % D. salina
2 % D. salina
Fig. 2. Superoxide dismutase assay of hemolymph of P. monodon fed with D. salina incorporated diets
3.3. Catalase assay
3.3.1. Catalase assay of haemolymph
The catalase activity of haemolymph in control ranged from 15.46 to 19.23 µmoles H
2
O
2
decomposed -
/min/mg of protein. The shrimp fed with 1.0 % D. salina incorporated diet showed the maximum catalase
activity of 31.69, 36.69, 25.31 and 15.34 µmoles H
2
O
2
decomposed /min/mg of protein (Fig. 3) which were
more than 51 %, 55 %, 28 % and 20 % to that of the control on 1
st
, 2
nd
, 3
rd
and 4
th
weeks respectively.
b
d
d
d
a
b
c
c
d
a
a
a
c
c
b
b
0
5
10
15
20
25
30
35
40
Iweek II week III week IV week
Days of post infection
µ moles of H2O2 decomposed/min/mg of
Protein
Control
0.5 % D. salina
1.0 % D. salina
2 % D. salina
Fig. 3. Catalase assay of hemolymph of P. monodon fed with D. salina incorporated diets
3.4. Challenge test
The shrimp, P. monodon fed with three different percentages (0.5 %, 1.0 % and 2.0 %) D. salina
incorporated diets for a period of 30 days were challenged with WSSV. The animal P. monodon fed with
D. salina incorporated diet without any challenge towards WSSV (negative control) showed 100 % survival till
the end of the study period (up to 10
th
day). Whereas the animal challenged with WSSV infection (positive
control) fed without the diet D. salina showed 100 % mortality within 48 h. However, the animals fed with
1.0 % D. salina incorporated diet showed 40 % mortality on 5
th
day and 100 % mortality on 10
th
day. Similarly,
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ISSN : 0975-5462
Vol. 3 No. 10 October 2011
7254
the animals fed with 0.5 % D. salina incorporated diet showed 50 % mortality on 5
th
day and 100 % mortality
on 10
th
day and 2 % D. salina incorporated diet showed 55 % mortality on 5
th
day and 100 % mortality on 10
th
day.
3.5. PCR amplification of genomic DNA of WSSV P. monodon
The live animals in treated and negative control groups were WSSV negative by PCR whereas
moribund animals from positive group showed WSSV positive by PCR. The animals kept in the tank without
WSSV survived during the study period (Data not shown).
3.6. Immunostimulant and antioxidant activity of WSSV infected P. monodon fed with D. salina
incorporated diets
3.6.1. Prophenoloxidase assay
The WSSV infected shrimp fed with 1.0 % D. salina incorporated diet showed proPO levels of 0.26,
0.31, 0.33 and 0.28 (unit/min/mg of protein) on 1
st
, 3
rd
, 6
th
and 9
th
day of post infection (Fig. 4) respectively. The
above values were less than 16 %, 6 %, 5 % and 26 % respectively, to that of the negative control (without
infection). Whereas the infected animals fed with 2 % showed less than 26 %, 36 % and 35 % compared to the
animals fed with 1.0 % D. salina incorporated diets.
a
a
a
a
c
dee
c
d
c
d
b
b
b
b
c
d
d
c
0:00
0:07
0:14
0:21
0:28
0:36
0:43
1369
Days
OD 490 nm units/min/mg protein
Negative control
Positive control
WSSV+0.5 % D. salina
WSSV+1.0 %D. salina
WSSV+2.0 % D. salina
Fig. 4. Prophenol oxidase assay of WSSV infected P. monodon fed with D. salina incorporated diets
3.7. Superoxide dismutase assay
3.7.1. Superoxide dismutase assay of haemolymph
The SOD levels in the haemolymph of WSSV infected shrimp fed D. salina at different concentrations
revealed that the shrimp fed with 1.0 % D. salina incorporated diet showed the SOD activity of 2.46 RAP on 3
rd
day (Fig. 5), which was less than 12 % to that of negative control (without WSSV infection) (2.82 RAP).
Whereas the SOD levels of the infected shrimp fed with 0.5 % and 2 % D. salina incorporated diet showed less
than 29 % and 56 % when compared to negative control.
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ISSN : 0975-5462
Vol. 3 No. 10 October 2011
7255
a
a
a
a
e
eee
c
d
d
c
b
b
b
bd
c
c
b
0
0.5
1
1.5
2
2.5
3
1369
Days
Relative superoxide anion
production
Negative control
Positive control
WSSV+0.5 % D. salina
WSSV+1.0 % D. salina
WSSV+2.0 % D. salina
c
Fig. 5. Superoxide dismutase assay of haemolymph of WSSV infected P. monodon fed with D. salina incorporated diet
3.8. Catalase assay
3.8.1. Catalase assay of haemolymph
The catalase activity in haemolymph of WSSV infected shrimp fed with 1.0 % D. salina incorporated diet
showed the catalase level of 25.46 µmoles H
2
O
2
decomposed /min/mg of protein on 6
th
day (Fig. 6) which was
more than 35 % and 43 % of the animals fed with 0.5 % and 2 % D. salina incorporated diets, respectively.
Whereas the animals without WSSV infection (negative control) fed with D. salina incorporated diet showed
31.69, 36.69, 25.31 and 15.34 µmoles H
2
O
2
decomposed /min/mg of protein on 1
st
, 3
rd
, 6
th
and 9
th
day,
respectively.
a
a
a
a
e
eee
d
d
d
d
b
b
b
b
c
c
c
c
0
5
10
15
20
25
30
35
40
1369
Days
µ moles of H2O2
decomposed/min/mg of Protein
Negative control
Positive control
WSSV+0.5 % D. salina
WSSV+1.0 % D. salina
WSSV+2.0 % D. salina
Fig. 6. Catalase assay of haemolymph of WSSV infected P. monodon fed with D. salina incorporated diet
4. Discussion
The color desired by the consumer for shrimp in aquaculture is by feeding the diet supplemented with
carotenoids. Irrespective of whether the shrimp is fed with astaxanthin or β-carotene, main carotenoids
accumulated is astaxanthin in free and esterified form. Boonyaratpalin et al., (2001) stated that P. monodon has
the metabolic ability to convert β-carotene into astaxanthin. Since β-carotene acts as an antioxidant, it is used in
the feed supplement of shrimps and fishes in aquaculture. Hence in this study, Dunaliella salina of three
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Vol. 3 No. 10 October 2011
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different concentrations viz: 0.5 %, 1.0 % and 2.0 % when incorporated in the diet and fed to shrimp showed
that the growth of P. monodon was found similar among the three concentrations. When compared to the control
(without D. salina diet) 1.0 % diet showed better growth. Similarly, Boonyaratpalin et al., 2001 reported a
higher survival rate and growth in P. japonicus fed with astaxanthin-supplemented diets than supplement of β-
carotene or algal meal.
Carotenoids pigments are involved in antioxidants activities in aquatic animals and moreover
carotenoids are known to enhance immune function and disease resistance in higher animals as stated by
(Hunter, 2000, Supamattaya et al., 2005). Phenoloxidase (PO), the key enzyme in the synthesis of melanin,
occurs in haemolymph as an inactive proenzyme prophenoloxidase (proPO). ProPO is activated to form PO
(phenol oxidase) when it reacts with zymosan (carbohydrates from yeast cell walls), bacterial
lipopolysaccharide (LPS), urea, calcium ions, trypsin, or heat (Soderhall et al., 1986). Hence an attempt was
made on the present study with P. monodon fed with β-carotene producing D. salina incorporated diets for a
period of 30 days, and also challenged against WSSV infection. The animal without WSSV infection but fed
with D. salina incorporated diet showed 100 % survival till the experimental study indicated its non-toxic
effects. It is worthwhile to mention that the control animals (fed without D. salina but with WSSV infection)
showed 100 % mortality within 48 h. However, the animal fed with 1.0 % D. salina incorporated diet
challenged with WSSV showed 40 % mortality on 5
th
day followed by 100 % mortality at the end of 10
th
day.
Thus indicating that the possible role of D. salina for protecting the animals to WSSV to certain extent.
Screening of Penaeus monodon shrimp for WSSV is one of the effective ways to check the vertical
transmission of disease through the hatcheries (Hsu et al., 1999). Henceforth, efforts were initiated to check the
presence of WSSV through PCR amplification in P. monodon. In the present study all the dead animals showed
amplification with the primer corresponded to WSSV confirmed that their mortality was due to WSSV
infection. Instead the animals kept in the tank without WSSV survived well during the study period.
In the present study, WSSV treated with three concentration of D. salina incorporated diet by in vivo
methods showed that proPO, O2 _ were all significantly (P < 0.05) higher than those of the WSSV infected and
control groups. This was especially true for the treated shrimp group where values were greater than those of the
control groups. Albores et al., (1993) stated that phenoloxidase (PO) is the terminal enzyme in the proPO
system of the arthropod defence system and acts as both recognition and effector component, by promoting cell-
to-cell communication and subsequently eliminating pathogens. The active materials formed during the
activation of proPO stimulate several cellular defence reactions, including phagocytosis, nodule formation,
hemocyte locomotion, non-self recognition and other immune reactions (Johanson et al., 2000). Activated
phenoloxidases generate high cytotoxic quinines that can inactivate viral pathogens (Ourth et al., 1993). In vivo
administration of 1.0 % D. salina live cells incorporated diet enhances immunity of tiger prawns which showed
a significantly (P < 0.05) higher proPO concentration than the control and WSSV infected groups. After viral
challenging, the shrimp with 1.0 % feed showed value initially increased and reached the maximum level at 24 h
p.i. and 6
th
day of in vivo experiment respectively. WSSV infected shrimp groups showed increased levels of
proPO levels; 48 h p.i. the group showed increased levels of proPO which were high in the level seen in healthy
shrimp. These results indicate that a high level of proPO enhances resistance in shrimp against WSSV. Our
experiment, D. salina live cells incorporated diet treated group PO seems to act as a promoter of the shrimp
immune system by enhancing the pigmentation, increasing the O2 _ production and SOD activity, and then
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Vol. 3 No. 10 October 2011
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postponing the dead of WSSV Shrimp to some extent. Similar findings were observed by Felix et al., 2004
noticed highest proPO activity was observed in WSSV infected P. monodon fed with Sargassum wightii
(seaweed). A gradual increase of proPO activity was observed up to 6
th
day after challenging with Vibrio
parahemolyticus thereafter there was a gradual decrease in the activity. Balasubramanian et al., (2008) reported
that increased levels of proPO assay were observed in shrimp fed with plant derived antiviral compound from
Cyanodon dactylon.
Super oxide dismutase (SOD) is one of the main antioxidant defence enzymes generated in response to
oxidative stress. Sarathi et al., (2007) and Mohankumar and Ramasamy (2007) observed the activity of SOD
was significantly lowered in WSSV-infected F. indicus. Also in the present study, the activity of SOD was
significantly lowered in the WSSV-infected hemolymph of P. monodon, whereas D. salina incorporated diet -
treated in vivo shrimp significantly recovered when compared with control animals. These results concur with
the findings of Lin and Chang et al., 2003 who have found that SOD decreases in WSSV infected P. monodon.
In the present study, SOD level in the Haemolymph of shrimp fed with three different concentrations of
D. salina incorporated diet showed high levels of SOD when compared to control (without D. salina). Nakano
et al., (1999) observed for the first time that astaxanthin (algal pigment) supplementation in diet fed to rainbow
trout influenced liver function and increased SOD defensive potential against oxidative stress. Similarly, Chang
et al., 2003 observed that the shrimp fed with b-glucan (BG) diets showed significantly higher levels of O
2
concentration than the BG free group as observed in shrimp treated with C. dactylon plant extract. Holmblad
and Soderhall (1999) observed that SOD is related to immunity in crustacean. The high level of O
2
in
P. monodon fed with D. salina incorporated diets indicated that the alga may be the potential immunostimulant.
Mohankumar and Ramasamy, (2007) observed that hydrogen peroxide is toxic to cells and catalase is
a major primary antioxidant defense component that catalyses the decomposition of H
2
O
2
which is produced by
the action of superoxide dismutase to H
2
O The present study revealed that the catalase assay of haemolymph of
P. monodon fed with three different concentrations of D. salina incorporated diets showed increased levels of
catalase when compared to control. In the present study, the activity of catalase in haemolymph of WSSV
infected P. monodon fed with 1.0 % D. salina incorporated diet showed less than 30 % to that of the negative
control.
In conclusion, this study suggests that increasing proPO activity, the superoxide anion production and
catalase production of WSSV infected P. monodon showed lower activities than the healthy shrimp (without
WSSV infection). Whereas the SOD of Haemolymph of WSSV infected P. monodon fed with D. salina
incorporated diet showed a slightly less activity when compared to the shrimp fed with D. salina without WSSV
infection. The above results indicated that the shrimp fed with D. salina incorporated diet showed a slightly
enhancement in immune resistance towards WSSV infected P. monodon
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