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Fish & Shellfish Immunology 32 (2012) 551e564

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Fish & Shellfish Immunology

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The effect of fucoidan from brown seaweed Sargassum wightii on WSSV resistanceand immune activity in shrimp Penaeus monodon (Fab)

Grasian Immanuel a,*, Madasamy Sivagnanavelmurugan a, Thangapandi Marudhupandi a,Srinivasan Radhakrishnan b, Arunachalam Palavesam a

aMarine Biotechnology Division, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam 629 502, Kanyakumari District,Tamilnadu, IndiabCentral Electrochemical Research Institute, Karaikudi 630 003, Sivagangai District, Tamilnadu, India

a r t i c l e i n f o

Article history:Received 14 July 2011Received in revised form10 December 2011Accepted 4 January 2012Available online 9 January 2012

Keywords:FucoidanWSSVSargassum wightiiImmunostimulantRT-PCR

* Corresponding author. Tel.: þ91 4652 253078.E-mail address: gimmas@gmail.com (G. Immanue

1050-4648/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.fsi.2012.01.003

a b s t r a c t

The polysaccharide-fucoidan was extracted from brown seaweed Sargassum wightii and characterizedthrough FT-IR and 13C & 1H NMR analysis. The extracted fucoidan was supplemented with pellet diets atthree different concentrations (0.1, 0.2 and 0.3%). The fucoidan supplemented diets were fed to Penaeusmonodon for 45 days, then challenged with WSSV and the mortality percentage was recorded daily up to21 days. During the challenge test, the control group showed 100% mortality within 10 days, but in theexperimental groups, the mortality percentage (51e72% within 21 days) was decreased considerably(P < 0.05) with respect to the concentrations of fucoidan. The reduction in mortality percentage ofexperimental groups over control group was ranged from 50.81 to 68.06%. During challenge experiment,the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity,superoxide dismutase activity and phagocytic activity were measured before injection of WSSV (0 day)and after the injection of WSSV on 10th and 21st days, respectively. All the immunological parameters ofexperimental groups were significantly (P < 0.05) increased than control group. RT-PCR analysisconfirmed the considerable reduction of WSSV DNA copy numbers with respect to the concentration offucoidan. It was concluded that P. monodon fed with fucoidan of S. wightii supplemented diet hadenhanced the innate immunity and increased resistance against WSSV infection.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

White spot syndrome virus (WSSV) is one of the dreadfuldiseases among the common shrimp diseases [1]. It can easilyspread and cause infection to a broad host range. Several potentialWSSV carriers or vectors live within or around shrimp farms, theseinclude many wild decapod crustaceans [2], rotifers [3,4], cope-pods, insects [5] and polychaete worms [6]. The principal clinicalsign of WSSV is the presence of white spots in the exoskeleton ofthe diseased shrimp. Other signs include a rapid reduction in thefood consumption, lethargy and reddening of appendages.Mortality rates are usually very high and cumulative mortality canreach 100% within 3e10 days from the onset of visible gross signs[7,8].

The study of using immunostimulants may be an effectivemeans for increasing the immunocompetency and disease

l).

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resistance in shrimp [9]. The immune defense system of shrimp hasfound to be induced by feeding with several dietary poly-saccharides such as peptidoglycan [10], lipopolysaccharide [11],glucan [12], sodium alginate [13] and fucoidan [14]. Hot waterextract of seaweed Sargassum sp. have been reported for the inhi-bition of WSSV infection in shrimp Penaeus monodon post larvae[15]. Herbal immunostimulants were also found to be effectiveagainst WSSV infection [16e18]. Oral administration of antiviralplant extract of Cynodon dactylon againstWSSV in P. monodon givenexcellent support [19]. Administration of an acyclic nucleosidephosphonate, cidofovir ((S)-1-3-hydroxy-2-phosphonyl methoxypropyl cytosine) (HPMPC) supplemented with a marine blue greenalgae Spirulina platensis in shrimp diet has been shown to delay themortality of WSSV-infected shrimp [20].

In recent years, fucoidan has been demonstrated as the sulfatedpolysaccharide, exhibited antiviral activities both in vivo and in vitroof interest in view of their low cytotoxicity compared with othermedicines. Fucoidans from seaweeds like Undaria pinnatifida [21],Stoechospermum marginatum [22], Cystoseria indica [23] showedantiviral activities against HSV-1 and HSV-2 without cytotoxicity

Table 1Composition of basal diets (% by weight) for Penaeus monodon.

No. Materials (% by weight) Control Experimental diets (%)

0.1 0.2 0.3

1 Fish meal 56 56 56 562 Groundnut oil cake 21 20.7 20.7 20.73 Soybean powder 11 11 11 114 Wheat bran 6 6 6 65 Cellulose 0 0.2 0.1 06 Fucoidan 0 0.1 0.2 0.37 Vitamins and mineral mix 2 2 2 28 Cod liver oil 2 2 2 29 Binder 2 2 2 2

Source: Yeh et al. (2008) modified [34].

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564552

for Vero cell cultures [23]. Furthermore, fucoidans extracted fromAdenocystis utricularis showed inhibitory activities against thereplication of several enveloped virus such as human immunode-ficiency and human cytomegalo viruses [24]. Fucoidan has no directinactivating effect on virions in a virucidal assay. The mechanism ofantiviral activities of fucoidan is to inhibit viral sorption so as toinhibit viral-induced syncytium formation [23]. Oral intake of thefucoidan might take the protective effects through direct inhibitionof viral replication and stimulation of both innate and adaptiveimmune defense functions [25]. Oral administration of crudefucoidan extracted from Sargassum polycystum has been reportedto reduce the impact ofWSSV in black tiger shrimp P. monodon [14].The fucoidan has been used for the expression of PhagocytosisActivating Protein (PAP) gene in shrimp P. monodon to inactivateWSSV [26]. Takahashi et al. [27] have observed the ability offucoidan containing meals and extracts to control WSSV in Japa-nese kuruma shrimp. Considering the importance of the abovefindings, the present study was undertaken to determine theimmunostimulatory effect of fucoidan extracted from Indian brownseaweed Sargassum wightii on P. monodon against WSSV.

2. Materials and methods

2.1. Extraction of fucoidan from seaweed

The brown seaweed S. wightii was collected from the coastalvillages of Kanyakumari District, Tamilnadu, India. The collectedseaweed was washed thoroughly and dried under shade at roomtemperature. The dried seaweed was ground well by using mixergrinder and sieved using a nylon sieve in order to remove seaweedfiber [15,28]. The fucoidanwas extracted by themethod proposed byYanget al. [29]. 20gofmilled seaweedwas treatedwith1 L of ethanolwith constant mechanical stirring for 12 h at room temperature toremove pigments and proteins. Then washed with acetone andcentrifuged at 1800� g for 10 min. The residue was dried at roomtemperature. From this dried biomass, 5 g was taken and extractedwith 100 ml of distilled water at 65 �C with stirring for 1 h. Theextractionwas conducted twice and the extractswere combined. Theextract was centrifuged at 18500� g for 10 min and the supernatantwas collected. The supernatant was mixed with 1% CaCl2 and thesolutionwas kept at 4 �C for overnight to precipitate alginic acid. Thesolutionwas centrifuged at 18500� g for 10min and the supernatantwas collected. Ethanol (99%) was added in to the supernatant toobtain the final ethanol concentration of 30% and the solution wasplaced at 4 �C for 4 h. Then the solutionwas centrifuged at 18500� gfor 10 min and the supernatant was collected. More ethanol (99%)was added into the supernatant to obtain the final concentration of70% and the solutionwas placed at 4 �C overnight. The fucoidanwasobtained by the filtration of the solution with a nylon membrane(0.45 mmpore size) and the product was washed with ethanol (99%)and acetone. Then the fucoidan was dried at room temperatureovernight. The yield of the fucoidan obtained was 2.832 � 0.204%.The dried fucoidan was packed in an airtight container until furtheruse. The fucose content of fucoidanwas analyzed by phenol sulfuricacid method [30] and the sulfate content was analyzed by bariumchloride method [31]. The fucose and sulfate contents of fucoidanwere 70.61 � 2.18% and 45.06 � 1.30%, respectively.

2.1.1. Purification of fucoidan250 mg of extracted fucoidan was dissolved in 25 ml of distilled

water and heated at reflux with 0.75 ml of 3.0 M HCl for 3 h. Aftercooling, the mixture was centrifuged at 3000� g and the super-natant solution was neutralized with 1.0 M NaOH and poured over100 ml of ethanol. Then the precipitate was dissolved in distilledwater and freeze dried.

2.1.2. Hydrolysis of fucoidanIn order to convert the polysaccharide into monosaccharide, the

purified fucoidan was subjected for hydrolysis. For this, 20 mg ofpurified fucoidan was hydrolyzed with 2 M H2SO4 at 100 �C for30 min. The hydrolyzed material was neutralized with 6 M NaOH,then it was freeze dried and kept for further analysis.

2.1.3. FT-IR analysisThe qualitative analysis of the active principles of the fucoidan

was done by Fourier Transmission Infra Red (FT-IR; Shimadzu,Japan) method described by Kemp [32]. The frequency of thespectra set to analysis was between 4000 and 400 cm�1 wavenumber and the vibration spectrum was recorded as graphicalchart.

2.1.4. NMR analysisAfter hydrolysis, the purified fucoidan was dissolved in 0.5 ml

D2O (Deuterium oxide) and the proton number and carbon numberof fucoidan was identified and confirmed by 1H and 13C NMRexperiments using a Bruker Biospin Avance 400 NMR spectrometer(1H frequency¼ 400.13 MHz, 13C frequency ¼ 100.62 MHz) at 298 Kusing 5-mm broad band inverse probe head equippedwith shieldedz-gradient and XWIN-NMR software version 3.5 using TMS as aninternal reference. One-dimensional 1H and 13C spectra were ob-tained using one pulse sequence. One-dimensional 13C spectra usingSpin Echo Fourier Transform (SEFT) and Quaternary Carbon Detec-tion (QCD) 42 sequences were also performed to aid the structureidentification [33].

2.2. Preparation of pellet feed supplemented with fucoidan

Three diets containing different concentrations of fucoidanwereprepared by following the procedure described by Yeh et al. [34](Table 1). The basal diet contained 0.2% cellulose (without fucoi-dan) served as the control diet. The extracted fucoidan was addedindividually to the test diet at different concentrations such as 0.1,0.2 and 0.3% with a corresponding decrease in the amount ofcellulose. The ingredients were ground up in a Hammermill to passthrough a 60 mesh screen. The experimental diets were preparedby mixing the dry ingredients with fish oil and then adding wateruntil a still dough resulted. Then the dough was palletized by usinga laboratory model palletizer having 1 mm diameter. The palletizedfeed was dried by using hot air oven at 40 �C. After drying, thefinished pellet feeds were stored in plastic containers at 4 �C untiluse. Proximate analysis of the basal diet was 42.35e42.72% crudeprotein, 2.45e2.70% carbohydrate and 1.57e1.62% crude lipid.

2.3. Collection and maintenance of experimental shrimp

The shrimp P. monodon post larvae (PL 20) were obtained fromMATSYAFED Hatchery (Quilon, Kerala). Immediately after arrival in

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 553

the laboratory, the post larvaewere stocked in 100 L fiber glass tankat room temperature (28 � 1 �C) with the water salinity of32 � 1 ppt. Natural filtered seawater was used in all experiments.The tanks were well aerated to maintain the oxygen level above6 ppm. The animals were kept in the tanks for 10 days and fed withlive feed (Artemia franciscana nauplii) for acclimatization beforestart of the experiments.

2.4. Feeding experiment

After measuring the length and weight, uniform size ofP. monodon post larvae at the PL30 stage were selected from theacclimatized stock and transferred in to individual experimentaltanks (0% e control and 0.1, 0.2 and 0.3% fucoidan supplementedpellet feeds), each containing 750 L of filtered seawater in 1000 Lcapacity FRP tanks at ambient temperature (28 � 1 �C) and salinity(32 � 1 ppt). The shrimp post larvae were maintained at thestocking density of 1/5 L. Mild aeration was given continuously inorder to maintain the optimal oxygen level. An ad libitum feedingregimewas applied to all tanks throughout the experiment, and thefood amount was adjusted 3 times a day (6:00, 14:00 and 18:00 h)@ 30, 30, and 40% of pellet feed, respectively. The control groupwasfed with fucoidan unsupplemented pellet feed. The unfed wascollected after the respective hours of feeding and 25% water wasexchanged daily during the experimental period. The feedingexperiment was prolonged for 45 days. Simultaneously, triplicateswere maintained in each group.

2.5. Preparation of WSSV inoculum

The WSSV-infected P. monodon with prominent white spots onthe exoskeleton were collected from local shrimp farms. Head softtissue from cephalothorax including gills was homogenized andcentrifuged at 3000� g for 20 min at 4 �C. The supernatant wasrecentrifuged at 8000� g for 30 min at 4 �C and the final super-natant was filtered through a 0.4 mm membrane filter. The filtratewas then stored at �20 �C for infectivity studies. The presence ofWSSV in the tissue sample of infected shrimp was checked bynested PCR assay [15,35].

2.6. WSSV challenge experiment

After 45 days of feeding experiment, the shrimp were collectedand reared separately in 1000 L capacity sterile FRP tanks with750 L filter sterilized aerated seawater at the rate of 1/10 L. Airstones and air tubes were sterilized previously by immersing in2.6% sodium hypochlorite and then washed thoroughly with ster-ilized tap water before use. The tanks were covered properly toprevent contamination. Aseptic techniques were observedthroughout the experiment. The shrimp were fed on the samefucoidan supplemented diets and control group was fed withunsupplemented diet. The shrimp were injected with WSSV inoc-ulum (stored fluid filtrate) intramuscularly at a dose of 0.01 ml pershrimp. Simultaneously, a negative control group of shrimp werealso maintained after injected with 0.01 ml saline per shrimp. Theimmunological parameters such as total hemocyte count (THC),prophenoloxidase activity (PO), superoxide anion activity (NBT),superoxide dismutase activity (SOD) and phagocytic activity wereanalyzed in the haemolymph samples of shrimps before injectionof WSSV (0 day) and after 10th and 21st days of challenge experi-ment [12]. During the challenge experiment, 50% water wasexchanged daily in order to remove uneaten food materials andshrimp waste materials in the experimental tanks. After WSSVinjection, the survival of P. monodon was monitored at regularintervals of 8 h until all the shrimp had succumbed. The dead

shrimp were removed from the respective tanks during eachobservation intervals. The results obtained in every 8 h intervalswere pooled and presented as per day interval up to 21 days.

2.7. Cumulative mortality index (CMI)

The cumulative mortality index (CMI) was calculated bysumming the number of dead shrimps at each day interval over theexperimental period as follows:

CMI ¼ Dx1 þ Dx2 þ Dx3 þ.Dxnðfinal dayÞwhere, D is the number of dead shrimps at the respective day. Thehigher the CMI value, the lower the resistance to WSSV. Using thisCMI value, the reduction in mortality percentage was calculated[15,28,36,37].

2.8. Analysis of immunological parameters

2.8.1. Haemolymph collectionHaemolymph was withdrawn from the ventral sinus of each

group of shrimp in to a 1 ml sterile syringe (25 gauge needle)containing precooled (4 �C) anticoagulation solution (trisodiumcitrate 30 mM, sodium chloride 0.34 M, EDTA 10 mM, pH 7.55 andwith an osmolality of 780 mOsm kg�1).

2.8.2. Total hemocyte countAfter withdrawal of haemolymph, the hemocytes were imme-

diately diluted with 0.5% trypan blue in 2.6% NaCl and the hemo-cytes were counted using a hemocytometer and calculated thenumber of cells (Total hemocytes/mm3).

2.8.3. Prophenoloxidase assayAfter haemolymph withdrawal, it was immediately transferred

in to a microtube and hemocytes were broken by sonication for10 s. Phenoloxidase activity in haemolymph samples was deter-mined using L-Dihydroxyphenylalanine (L-DOPA) as a substrate.Tris buffered saline (TBS) at 30 ml was added to the cuvette con-taining 30 ml of haemolymph sample. Then, 60 ml of L-DOPA solu-tion (1.6 mg/ml in TBS) was added and mixed. Then, 200 ml of TBSwas added as diluent and enzyme activity was determined bymeasuring the absorbance of dopachrome at 490 nm againsta blank containing 200 ml TBS and 60 ml L-DOPA [12].

2.8.4. Superoxide anion analysis (NBT assay)Briefly, 100 ml haemolymph in anticoagulant solution was

deposited in triplicate onmicroplates previously coated with 100 mlpoly L lysine solution (0.2%) to improve cell adhesion. Microplateswere centrifuged at 300� g for 20 min at 4 �C. Plasmawas removedand 100 ml zymosan (0.1% in Hank’s balanced salt solution) wasadded and allowed to react for 30 min at room temperature. 100 mlof NBT solution (0.3%) was added, stained for 30 min at roomtemperature and then 100 ml of methanol was added. The mixedsolution was discarded, and the microplates were washed 3 timeswith 100 ml of 70% methanol and air dried. The formazan was dis-solved by addition of 120 ml of 2 M KOH and 140 ml of DMSO(Dimethyl sulphoxide). The optical density at 630 nm for theshrimps superoxide anionwas measured using a microplate readerand expressed as NBT reduction in 10 ml of haemolymph [38].

2.8.5. Superoxide dismutase analysisSuperoxide dismutase (SOD) activity of haemolymph of shrimp

was measured by its ability to inhibit superoxide radical dependentreactions using the Ransod Kit (Randox, Crumlin, UK). The reactionmixture (1.7 ml) contained Xanthine (0.05 mM) and 2-(4-

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564554

iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride (INT,0.025 mM) were dissolved in CAPS 50 mM (pH 10.2) and EDTA(0.94 mM). In the presence of xanthine oxidase (80 U 1�1, 250 mL),superoxide and uric acid were produced from xanthine. Then, thesuperoxide radical reacted with INT to produce a red formazan dye.The optical density was measured at 505 nm at 37 �C and the rate ofreaction was estimated from the absorbance readings 30 s and3 min after adding xanthine oxidase. A reference standard SODwassupplied with the Ransod Kit. One unit of SOD was defined as theamount required to inhibit the rate of xanthine reduction by 50%.Specific activity was expressed as SOD Unit/ml [39].

2.8.6. Phagocytic activityThe haemolymph (0.5 ml) was mixed with 0.5 ml of KC-199

[40]. Hemocytes were separated by centrifugation at 6500 rpm at4 �C and washed with KC-199. Hemocytes were then diluted to107 cells and 0.2 ml was taken and mixed with 0.2 ml of latex beads(108/ml, particle diameter 1.094 mm) on a clean glass slide. Themixturewas incubated in a moisture chamber at room temperaturefor 30 min. The cells were fixed by 2.5% glutaraldehyde for 5 min,after which the non-adherent cells were removed with 0.85% NaCl,and then followed by air-drying, and staining with Wright stain.Numbers of ingested cells and ingesting cells were counted from200 cells and calculated for percentage of phagocytosis [36] asfollows:

Percentage phagocytosisð%Þ ¼ Number of cells ingesting beadNumber of cells observed

� 100

2.9. Real-time polymerase chain reaction (RT-PCR) analysis

After challenge experiment, the WSSV infection in P. monodonwas detected by RT-PCR analysis. The experimentally infectedshrimp were preserved in 70% ethanol and subsequently wererehydrated in distilled water for 1 h before the RT-PCR analysis.

2.9.1. DNA extractionTotal genomic DNA and associated WSSV viral DNA were

extracted by using a Qiagen DNeasy Kit. For the present study,20 mg of gill tissue was collected from control and experimentalshrimps separately and they were used for extraction of DNA. TheDNA concentration and quality were determined by measuringoptical density at 260 nm, by using a Genesys 5 spectrophotometer(Spectronic instrument, Inc., New York, USA).

2.9.2. Real-time PCR primer and probePrimer 3 software was used to design primers and the TaqMan

probe with the WSSV whole sequence (GenBank accession no.AF332093. Primers RT-WSSV-F154 (50-CCA GTT CAG AAT CGG ACGTT-30) and RT-WSSV-R154 (50-AAA GAC GCC TAC CCT GTT GA-30)produced a fragment of 154 bp after amplification. The TaqManprobe, RT-WSSV-TP154 (50-TCC ATA GTT CCT GGT TTG TAA TGT GCCG-30) was synthesized and labeled with the fluorescent dyes 5-carboxyfluorescein (FAM) on the 50 end and Black Hole Quencher(BHQ) on the 30 end.

2.9.3. Construction of positive control vectors and standards forquantification

A WSSV fragment containing a 154-bp target amplicon forreal-time PCR was ligated into the pGEM-T-Easy vector andcloned into Escherichia coli (DH5a). The target segment in therecombinant plasmid, pWSSV-154 was confirmed by using anautomated DNA sequencer (ABI 3730XL, Applied Biosystem Inc.,

USA). The copy number of the target amplicon in the plasmidwas estimated and 10-fold serial dilutions were made to use asabsolute standards for quantification. The instrument deter-mined fluorescence at the end of each annealing and extensioncycles.

2.9.4. Real-time PCR amplificationReal-time PCR was performed by using a Perfect Real-Time

premix (RR039A, Takara) containing a high-performance Taqantibody, Takara Ex Taq HS, for hot starts real-time PCR. A sample of40e200 ng total DNA was added to a PCR mixture containing0.25 mM of each primer and 0.125 mM TaqMan probe in a finalreaction volume of 20 mL. The amplification program consisted of30 s at 95 �C for initial denaturation, followed by 45 cycles of 5 sdenaturation and 20 s annealing and extension at 60 �C. Thermalcycling was performed on a Rotor Gene 6000 (Corbett ResearchInc., Australia).

2.9.5. Data analysisThe threshold was automatically detected and the preliminary

data were analyzed by using Rotor Gene Operating Softwareversion 1.7.61, resulting in a fractional cycle number (Ct value)assigned to each individual sample. A set of standard dilutions(from 5.9 � 106 to 5.9 � 100 viral copies mL/l) was created froma plasmid containing the target amplicon and run simultaneouslywith the specimen DNA. All the samples were run in duplicate.Regression between viral copy numbers and Ct values were used asa standard curve for determining viral load. For each new run, atleast 2 non-template controls (NTC) were performed as negativecontrols.

2.10. Statistical analysis

The data obtained in the present study were expressed asMean� SD and were analyzed using students ‘t’ test with a post hocmultiple comparison of SNK test at a significant level of 5% usinga computer software STATISTICA 06 (Statosoft, Bedford, UK).

3. Results

3.1. FT-IR analysis

The FT-IR analysis of fucoidan from S. wightii is given in the Fig.1.In the region of 3600e1600 cm�1, three bands appeared witha broad band centered at 3450.48 cm�1 assigned to hydrogenbonded OeH stretching vibrations, the weak signal at wavelength2146.37 cm�1 indicated the presence of C]C]O and the asym-metric stretching of carboxylate OeCeO vibration at 1604.82 cm�1.The band at 1422.70 cm�1 may be due to CeOH deformationvibration with contribution of OeCeO symmetric stretchingvibration of carboxylate group. The weak band at 1253.00 cm�1

indicated the presence of S]O stretching vibration of sulfate group.The band at 1037.21 cm�1 may also be due to CeO stretchingvibrations. The spectrum showed a band at 891.62 cm�1 assigned tothe C1eH deformation vibration of b-mannuronic acid residues.The band at 818.88 cm�1 seems to be characteristic of CeOeSstretching of sulfate group. The band at 618.68 cm�1 may be dueto C^CeH stretching vibration.

3.1.1. NMR analysisThe 13C NMR spectrum showed sharp absorptions correspond-

ing to a (1-6)-b-D-linked galacton at ppm 101.6 (C-1), 75.62 (C-5),72.8 (C-3), 70.77 (C-2), 69.82 (C-6), 69.17 (C-4). Similarly, the 1HNMR spectrum showed the correlation of these signals with theppm of 4.464 (H-1), 3.901 (H-5), 3.682 (H-3), 3.556 (H-2), 4.075/

3450.4

8

2146.3

7

1604.8

2

1422.7

0 1253.0

0

1037.2

1

891.6

2

818.8

8

618.6

8

500100015002000250030003500

500100015002000250030003500

Wavenumber cm-1

02

04

06

08

01

00

02

04

06

08

01

00

Tra

nsm

itta

nce

[%

]

Fig. 1. FT-IR analysis of fucoidan extracted from brown seaweed S. wightii.

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 555

3.959 (H-6/H-60) and 4.075 (H-4), respectively. The 13C absorptionat ppm 101.6, 99.94, 96.12 correlated with 1H absorption at ppm5.684, 5.107 and 5.097 are corresponding to a terminal a-L fucose, 3-linked a-L fucose and 3,4 distribution of a-L-fucose, which sug-gested the presence of 3 sulfated 4 linked and 4 sulfated 3-linked a-L-fucose. The 13C NMR spectrum showed absorptions correspond-ing to a b-D mannuronic acid at ppm 101.6 (C-1), 71.78 (C-2), 72.83(C-3), 77.69 (C-4), 75.62 (C-5) and 175.26 (C-6). Similarly, the 1HNMR spectrum showed the correlation of these signals with theppm of 5.09 (H-1), 3.901 (H-2), 3.74 (H-3), 3.95 (H-4), 3.79 (H-5)and 1.11 (H-6), respectively (Figs. 2 and 3).

Fig. 2. 1H NMR analysis of fucoidan extra

3.2. WSSV challenge experiment

3.2.1. Cumulative mortality percentageThe cumulative mortality percentage of shrimp P. monodon after

WSSV challenge experiment is given in Fig. 4. The shrimp weresuccumbed to death started from 3rd day of challenge test. Themortality of control group was 4% on 3rd day. At the same time, inthe experimental groups no mortality was recorded. In lowestconcentration of (0.1%) fucoidan supplemented diets fed shrimps,the mortality recorded was 2% during 5th day of challenge test. Butin the highest concentrations (0.2 and 0.3%) of fucoidan

cted from brown seaweed S. wightii.

Fig. 3. 13C NMR analysis of fucoidan extracted from brown seaweed S. wightii.

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564556

supplemented diets fed groups, the mortality observed was 2 and1% during 6th and 7th days of challenge test, respectively.When theduration of the challenge experiment increased, the cumulativemortality was increased gradually. Finally, within 10 days, 100%mortality was observed in control group, whereas in the experi-mental groups, the survival of P. monodon was prolonged up to 21days of challenge experiment. Within 21 days, 72, 62 and 51%mortality was recorded respectively in 0.1, 0.2 and 0.3% concen-trations of fucoidan supplemented diets fed shrimps.

3.2.2. Cumulative mortality index (CMI) and reduction in mortalityThe cumulative mortality index and reduction in mortality

percentage of control and experimental groups of P. monodon afterchallenged with WSSV is given in Table 2. The CMI of control group

Fig. 4. Cumulative mortality percentage (%) of shrimp P. monodon fed on differentconcentrations of fucoidan supplemented diets after challenged with WSSV in 21 daysinterval.

was 21,462 and it was significantly (P < 0.05) reduced in experi-mental groups. The reduction in mortality over control group was50.81, 59.80 and 68.06%, respectively in 0.1, 0.2 and 0.3% of fucoidansupplemented diets fed groups of shrimps.

3.3. Immunological analysis

3.3.1. Total hemocyte count (THC)At the beginning of the challenge experiment, THC in the hae-

molymph sample of control groupwas 52.4�105 cells ml�1. But theTHC was significantly (P < 0.05) increased with increasingconcentrations of fucoidan. At the lowest concentration (0.1%) offucoidan, the THC was 72.3 � 105 cells ml�1, whereas it was 79.6and 88.9 � 105 cells ml�1, respectively in 0.2 and 0.3% fucoidansupplemented diets fed shrimp. With respect to the WSSV chal-lenge duration, THC in all the groups of shrimp decreased consid-erably during 10th day. For instance, in control group, it was23.8 � 105 cells ml�1, whereas 56.4, 68.9 and 80.4 � 105 cells ml�1,respectively in 0.1, 0.2 and 0.3% fucoidan supplemented diets fed

Table 2Cumulative mortality index (CMI) and percentage reduction in mortality of shrimpP. monodon fed on different concentrations of fucoidan supplemented diets afterchallenged with WSSV against control.

Concentration offucoidan (%)

CMI Reduction in mortality (%)

Control 21,462 � 227.22a 0 � 00.1 10,556 � 183.71b 50.81 � 0.140.2 8626 � 163.29c 59.80 � 0.160.3 6854 � 126.15d 68.06 � 0.13

Each value is a Mean � SD of three replicates; within each column, means with thedifferent superscript letters are statistically significant (t-test; P < 0.05 and subse-quent post hoc multiple comparison with SNK test).

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 557

shrimp. However, at the end of the challenge experiment (21stday), the THC was recovered and increased with increasingconcentrations of fucoidan, the THC recorded was 75.5, 81.6 and90.3 � 105 cells ml�1 respectively in 0.1, 0.2 and 0.3% fucoidansupplemented diets fed shrimp (Fig. 5).

3.3.2. Prophenoloxidase (PO) activityDuring the start of the challenge experiment (0 day), the PO

activity of control group was 0.1418 (OD), whereas it was signifi-cantly (P< 0.05) increased (0.1725e0.1820 OD) in the experimentalgroups fed with different concentrations (0.1e0.3%) of fucoidansupplemented diets. When the duration of WSSV challengeexperiment increased, the PO activity decreased in both control andexperimental groups. For instance on 10th day, the PO activitydecreased between 0.1005 and 0.1189 OD in experimental groups,whereas it was 0.0594 OD in control group. Invariably at the end ofthe challenge study, the PO activity gradually increased to 0.1676,0.1707 and 0.1765 OD in 0.1, 0.2 and 0.3% fucoidan supplementeddiets fed shrimp, respectively (Fig. 6).

3.3.3. Respiratory burst activity (NBT assay)The respiratory burst activity of control group was 0.0375 (OD)

on 0 day, but it was significantly (P < 0.05) increased to 0.0482,0.0598 and 0.0686 OD in 0.1, 0.2 and 0.3% of fucoidan supplementeddiets fed shrimp, respectively (Fig. 7). When the duration of WSSVchallenge experiment increased to 10th day, the respiratory burstactivity was significantly (P < 0.05) increased in experimentalgroups (0.0506e0.0698 OD), whereas in control group, it wasdecreased (0.0078 OD). Invariably at the end of the challengeexperiment, the respiratory burst activity was graduallydecreased to 0.0467, 0.0573 and 0.0643 OD in 0.1, 0.2 and 0.3% offucoidan supplemented diets fed shrimp, respectively.

3.3.4. Superoxide dismutase activity (SOD)In control group, 36.85 Unit/ml of SOD was recorded at the

beginning of the challenge experiment, whereas in experimentalgroups, the SOD was increased with increasing concentrations offucoidan (56.13e62.14 Unit/ml in 0.1e0.3%). On 10th day of WSSVchallenge experiment, the SOD activity decreased in all the groups.For instance, it was only 9.30 Unit/ml in the control group,whereas it was 50.20e55.96 Unit/ml in the respectiveexperimental groups. Finally within 21 days, the shrimp recovered

Fig. 5. Total hemocyte count (THC) of shrimp P. monodon fed on different concentrations ofinterval. Each value is the mean � SD of three replicates; bars with different letters are scomparison with SNK test) (NC: Negative control; PC: Positive control).

SOD and it was recorded as 53.14, 57.20 and 59.06 Unit/ml,respectively in 0.1, 0.2 and 0.3% fucoidan supplemented diets fedshrimp (Fig. 8).

3.3.5. Phagocytic activityThe phagocytic activity of control and experimental groups of

shrimp was significantly (P < 0.05) varied at the beginning of thechallenge study. For instance, the phagocytic activity of controlgroup was 5.71%, whereas it was increased from 6.85 to 7.74% in therespective (0.1e0.3%) concentrations of fucoidan supplementeddiets fed shrimps. When the WSSV challenge duration prolongedto 10 days, the phagocytic activity decreased. It was only 3.20% inthe control group, whereas it was 5.59, 5.98 and 6.62% in theexperimental groups. Finally at the end of the challenge study,the phagocytic activity again increased from 6.03 to 7.15% in0.1e0.3% fucoidan supplemented diets fed shrimp, respectively(Fig. 9).

3.4. WSSV quantification through RT-PCR

WSSV-infected P. monodon of both control and experimentalgroups were screened by RT-PCR analysis for the quantification ofWSSV DNA (Table 3 and Fig. 10). The WSSV infection in positivecontrol group showed 1.42 � 106 WSSV DNA copies within 16.96threshold cycles (Ct FAM). But in the experimental groups, the copynumber of WSSV DNA was decreased with increasing concentra-tions of fucoidan. The experimental group with low concentration(0.1%) of fucoidan supplemented diet fed shrimp displayed 756WSSV DNA copies within 27.23 threshold cycles. But in 0.2 and 0.3%concentrations of fucoidan supplemented diet fed groups, 52.6 and11.0 WSSV DNA copies were recorded, respectively within 30.86and 36.26 threshold cycles. The negative controls did not displayany amplification. Strong linear correlation R2 ¼ 0.9920 was ob-tained between the threshold cycles (Ct) and the amount of WSSVDNA copies in RT-PCR with reaction efficiency E ¼ 1.08 and properslope M ¼ �3.139 indicating that the assay had a large dynamicrange (Fig. 11).

4. Discussion

S. wightii is one of the major diversified brown seaweed speciesalong the south west coast of India. From this, the polysaccharide-

fucoidan supplemented diets during challenge experiment with WSSV in different daystatistically significant from each other (t-test; P < 0.05 subsequent post hoc multiple

Fig. 6. Prophenoloxidase activity (OD) of shrimp P. monodon fed on different concentrations of fucoidan supplemented diets during challenge experiment with WSSV in differentdays interval. Each value is the mean � SD of three replicates; bars with different letters are statistically significant from each other (t-test; P < 0.05 subsequent post hoc multiplecomparison with SNK test) (NC: Negative control; PC: Positive control).

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564558

fucoidan was extracted and investigated for its biological activitiesagainst WSSV in shrimp P. monodon. The fucose and sulfatecontents of extracted fucoidanwere 70.61 and 45.06%, respectively.Similarly, Yang et al. [29] have reported that the fucoidan ofU. pinnatifida contain 78.8% fucose and 41.5% sulphates. Thebiochemical component of fucoidan has been reported that thecomparable amount (42e66%) of carbohydrates with smalleramount (11.5e34.2%) of sulphates [24,41e45]. The biochemicalcomposition of fucoidan is mainly depending on the species ofseaweed, anatomical regions, growing conditions, extractionprocedures and analytical methods [24,41,42,44,46e49].

The FT-IR analysis revealed that the presence of sulfate group(S]O stretching) at 1253.00 cm�1 in the fucoidan fraction. Thespectrum shows a band at 891.62 cm�1 assigned to the C1eHdeformation vibration of mannuronic acid residues. The band at

Fig. 7. NBT assay (OD) of shrimp P. monodon fed on different concentrations of fucoidan suppvalue is the mean � SD of three replicates; bars with different letters are statistically significatest) (NC: Negative control; PC: Positive control).

818.88 cm�1 seems to be characteristic of CeOeS stretching ofsulfate group. 1H NMR and 13C spectroscopy is a reliable method forthe determination of the composition and structure of fucoidanmolecules [50]. In the present study, the C & H NMR analyses wereperformed after hydrolysis of fucoidan fraction. The result of C & HNMR indicated the presence of carbon (69.17e101.6; 101.6e96.12;71.78e175.26 ppm) and anomeric proton (3.556e4.464;5.097e5.684; 1.11e5.09 ppm) of (1e6)-b-D-linked galacton, a-L-fucose and b-D mannuronic acid in purified fucoidan. According tothe molecular characterization, the sulfate group, fucose, b-D-gal-actan and b-D-mannuronic acid present in the S. wightii fucoidan.The sulfate group of fucoidan may act against WSSV infection andthe fucose, galactan and mannuronic acid stimulate immunesystem of shrimps against diseases. Similarly, Chandıa and Matsu-hiro [51] suggested that the IR-spectrum of fucoidan of Lessonia

lemented diets during challenge experiment with WSSV in different days interval. Eachnt from each other (t-test; P < 0.05 subsequent post hocmultiple comparison with SNK

Fig. 8. SOD activity (Unit/ml) of shrimp P. monodon fed on different concentrations of fucoidan supplemented diets during challenge experiment with WSSV in different daysinterval. Each value is the mean � SD of three replicates; bars with different letters are statistically significant from each other (t-test; P < 0.05 subsequent post hoc multiplecomparison with SNK test) (NC: Negative control; PC: Positive control).

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 559

vadosa showed the characteristic band of SeO stretching vibrationat 1259 cm�1 and a band at 849.5 cm�1 due to CeOeS vibrationwhich was assigned to sulfate group linked to axial secondaryalcoholic group. The asymmetric deformation of OeSeO groupabsorption at 582.1 cm�1 confirmed the presence of significantamount of sulfate groups. Bilan et al. [44] studied the IR-spectrumof fucoidan from Fucus serratus, has an intense absorption band at1240 cm�1 (S]O) common to all the sulfate esters. An additionalsulfate absorption band at 824 cm�1 (CeOeS, secondary equatorialsulfate) and a shoulder at 844 cm�1 (CeOeS, secondary axialsulfate) indicated the majority of sulfate groups occupy positions 2and/or 3, and only a minor part of sulfate was located at position 4of fucopyranose residues. They also observed the NMR spectra offucoidan of F. serratus contained correlation peaks at 5.08/3.94 and5.08/4.00 ppm corresponding to H-10/H-3 and H-10/H-4 interac-tions usual for (1-3)-linked fucobioside fragments as well as peak at4.98/3.87 and 4.98/1.30 ppm corresponding to H-10/H-4 and H-10/

Fig. 9. Phagocytic activity (%) of shrimp P. monodon fed on different concentrations of fucinterval. Each value is the mean � SD of three replicates; bars with different letters are scomparison with SNK test) (NC: Negative control; PC: Positive control).

H-6 interactions for (1-4)-linked fucobioside fragments. Chizhovet al. [46] reported that the NMR analysis of fucoidan from thebrown seaweed Chorda filum, it attached with O2 in two different a-(1,3)-linked fucose residues (C-1: 94.7 and 93.4 ppm, H-1: 5.25 and5.32 ppm, respectively). a-(1,3 and 1,2)-fucose residues give signalsof anomeric carbons at 93.4 and 92.9 ppm; this splitting possiblyarises due to the effect of the acetate attached to a neighboringfucose residue.

The fucoidan was supplemented with pellet diets at threedifferent concentrations (0.1, 0.2 and 0.3%) and fed to shrimpP. monodon for 45 days. After feeding experiment, the WSSV chal-lenge test was performed and the mortality percentage of experi-mental groups of shrimp reduced from 50.81 to 68.06% over controlgroup. The reduction in mortality of all the tested groups increasedwith increasing the concentrations of fucoidan. In accordance withthese, Chotigeat et al. [14] have reported that the oral administra-tion of fucoidan from brown algae S. polycystum reduced the impact

oidan supplemented diets during challenge experiment with WSSV in different daystatistically significant from each other (t-test; P < 0.05 subsequent post hoc multiple

Table 3Quantification of WSSV DNA copies by RT-PCR analysis of shrimp P. monodon fed ondifferent concentrations of fucoidan supplemented diets after challenged withWSSV.

Concentration of fucoidan (%) Ct FAM (Cycles) No of DNA copies

Negative control e e

Positive Control 16.96 1.42 � 106

0.1 27.23 7560.2 30.86 52.60.3 36.26 11.0

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564560

of the WSSV infection in P. monodon. They suggested that, 4.4, 14and 44% of survival rate respectively in juvenile shrimps fed on thecrude fucoidan of 100, 200 and 400 mg/kg of body weight/day afterchallenged with WSSV. Oral administration of partially purifiedfucoidan (PPF) from Cladosiphon okamuranus was also reported tocontrol WSSV in Penaeus japonicus [27]. They conducted two sets ofexperiment depending upon the size of shrimp (8.2 g in firstexperiment and 12.3 g in second experiment). They suggested thesurvival rate in the first experimentwas 75.0 and 70.0% respectivelyin 60 and 100 mg PPF-fed groups of shrimps after 10 days of WSSVchallenge experiment. These values were significantly (P < 0.01)higher than the value of control group. In the second experiment,the survival rate was 78.9 and 82.4% respectively in 60 and 100 mgPPF-fed group after 10 days of WSSV challenge experiment, thesevalues were also significantly (P < 0.01) higher than the value ofcontrol group. There were no significant differences between the60 mg and 100 mg PPF-fed groups in either experiment. The b-1,3-glucan have been reported to improve the immunity effectively andincreased the resistance to WSSV in post larvae and juveniles ofP. monodon [12,52]. In their study, all shrimp in the WSSV chal-lenged, glucan free (control) group died within 5 days. But themean survival in the WSSV challenged glucan fed group was 12.2%on 6th day. Similarly in the present study, the mortality of controlgroup showed 100% within 10th day of WSSV challenge experi-ment. But the fucoidan supplemented diets fed shrimp displayed51e72% mortality within 21 days of WSSV challenge experiment.

Themechanism of inhibition of fucoidan against the virus is thatthe negative charges of the sulfate group of the polysaccharide bindwith positive charges of amino acids at V3 loop of viral envelopeglycoprotein (gp 120). The V3 loop is essential for virus attachmentto cell surface heparan sulfate, a primary binding, before morespecific binding occurs to the CD4 receptor of CD4þ cell. Thisprocess prevents the entry of virus into the host cell [53,54]. The

Fig. 10. Amplification curve showing 10-fold serial dilutions of standards, control and differsamples.

sulfate content was reported to affect the cell proliferation of CCL39fibroblast whereby the content of sulfate higher than 20% reducedproliferation of the cells in a dose dependent manner [55].

In the present study, the immunological parameters wereanalyzed during WSSV challenge experiment. The hemocytes playan important role in the cellular immune response including clot-ting, non-self recognition, phagocytosis, melanization, encapsula-tion, cytotoxicity and cell-to-cell communication [56]. In thepresent study, at the beginning of the challenge experiment, theTHC was significantly (P < 0.05) increased with increasingconcentrations of fucoidan (72.3 � 105e88.9 � 105 cells ml�1 in0.1e0.3%) than the control group (52.4 � 105 cells ml�1). When theWSSV challenge duration prolonged, the THC in all the groups ofshrimp decreased considerably during 10th day. At the end of thechallenge experiment (21st day), the THC was recovered andincreased with increasing concentrations of fucoidan(75.5e90.3 � 105 cells ml�1 in 0.1e0.3%). Similarly, Chang et al.[12] have reported the dietary effect of b-1,3-glucan (BG) on totalhemocyte count of P. monodon challenged with WSSV. Theyobserved that the shrimp THC was significantly (P < 0.0001)reduced after WSSV challenge experiment. All infected shrimp hada THC of 17e21 � 106 cells ml�1, which was less than that of thepositive control group (27� 106 cells ml�1) at day 1 after challenge.By day 3e9, the BG free and BG 0.1% groups showed a THC less than20% that of the positive control group and died subsequently. Butthe shrimp fed on diet supplemented with 0.2, 1, and 2% BGgroups recovered THC at days 12e24. Pholdaeng and Pongsamart[9] have studied the effect of polysaccharide gel (PG) extractedfrom Durio zibethinus on THC in shrimp P. monodon against WSSV.They prepared 1, 2 and 3% PG-supplemented diets and a basal dietwithout PG was used as control and fed to shrimp daily approxi-mately 8% of body weight for 12 weeks. After feeding experiment,the THC level of 1e3% PG-supplemented diets fed shrimp showedhigher values (1.48e1.74 � 107 cells ml�1) than that of the controlgroup (1.33 � 107). Then the immunostimulated shrimp (after 12weeks of feeding experiment) were challenged with WSSV andthe shrimp showed higher relative percentage survival (RPS) thanthe control group after 14 days of challenge experiment.Balasubramanian et al. [19] have studied the effect of C. dactylonextract on THC of P. monodon challenged with WSSV. They sug-gested that the shrimp were treated by both in vitro (intramuscularinjection) and in vivo (orally with feed) methods at the concen-tration of 2 mg per animal and 2% of the C. dactylon extract incor-porated with commercially available artificial pellet feed,

ent concentrations (0.1e0.3%) of fucoidan supplemented diets fed experimental shrimp

Fig. 11. Standard curve of WSSV TaqMan real-time PCR.

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 561

respectively. The control shrimp hemocyte count was significantly(P < 0.05) reduced by WSSV infection in both in vivo and in vitromethods, but shrimp administeredwith plant extract to treatWSSVinfection showed a significant decrease at the early stage of WSSVchallenge and recovered to normal level. The antiviral plant extractalone-administered shrimp showed no significant changes in totalhemocyte count.

The proPO system is acknowledged to be the most importantimmune system in crustaceans [57,58]. The activated proPO systemis involved with some important molecules that are released toperform crucial immune responses, including non-self recognition,melanin formation, adhesion, and cell-to-cell communication[59,60]. In the present study, the prophenoloxidase activity (PO) ofexperimental shrimp was significantly (P < 0.05) increased thancontrol shrimp during WSSV challenge test. At the beginning (0day) the PO activity of control group was 0.1418 (OD), but it was0.1725e0.1820 (OD) in the experimental groups. When theduration of challenge experiment increased (10th day), the POactivity decreased, but at the end of the experiment, the shrimprecovered normal PO activity in fucoidan supplemented diets fedshrimp. Similarly, Chang et al. [12] have reported that the dietaryeffect of b-1,3-glucan on PO activity of P. monodon challenged withWSSV. They suggested that the shrimp fed on glucan diets showedhigher PO activity than BG free group before challenge. One dayafter challenge, the PO activity of the challenged groups becamesignificantly lower than the positive control group. But the shrimpsfed with 0.2, 1.0 or 2% BG diets, the PO activity significantly(P < 0.001) rebounded following an initial drop, and by day 9, itattained the highest level. Pholdaeng and Pongsamart [9] havereported that the shrimp P. monodon fed with 1e3% polysaccharidegel (PG) of D. zibethinus e supplemented diets for 12 weeksdemonstrated significantly (P < 0.05) higher PO activity(721e534 Units/min/mg protein) than the control group(405 Units/min/mg protein). The immunostimulated shrimp werethen challenged with WSSV and the shrimp showed higherrelative percentage survival (RPS) against WSSV than controlgroup after 14 days of challenge experiment. Balasubramanianet al. [19] have studied the effect of C. dactylon extract on proPOactivity of P. monodon challenged with WSSV. They observed thatthe proPO activity among the different experimental groups ofshrimp had a significant (P < 0.05) increase when compared withthe control group. Shrimp treated with plant extract by in vitro orin vivo showed a significant (P < 0.05) increase in proPO activitythan the control group. In shrimp treated with antiviral plantextract, proPO activity attained the highest level at day 2 and day 5of in vitro and in vivo experiments respectively, but followingrecovery, it started decreasing and reached normal level. In WSSV-infected shrimp, the proPO level was significantly (P < 0.05)

increased and reached double the normal value. Plant extractalone-administered shrimp showed a significant enhancement ofproPO activity and attained the highest level at day 2 and day 5 ofin vitro and in vivo experiments respectively, then decreasedgradually with days post administration.

Reactive oxygen intermediates (ROIs) including superoxideanion are released during respiratory bursts of phagocytosis, whichrepresent a defense mechanism against microbial infection [61].However, the excessive accumulation of ROIs is extremely toxic tohost cells. The harmful effects of ROIs are effectively neutralized bythe antioxidant defense system of the host organisms [62]. In thisstudy, the respiratory burst activity (superoxide anion activity) ofexperimental groups of shrimp was significantly (P < 0.05) higherthan control group after WSSV challenge experiment. At thebeginning, the respiratory burst activity of control groupwas 0.0375(OD), whereas it was in-between 0.0482 and 0.0686 OD in 0.1e0.3%fucoidan supplemented diets fed shrimp. When the duration of thechallenge experiment increased, the respiratory burst activity wasincreased in experimental groups, whereas in control group, it wasdecreased. Similarly, Chang et al. [12] have reported that the die-taryeffect ofb-1,3-glucanon superoxide anionactivityof P.monodonchallenged with WSSV. They observed that the oral BG administra-tion significantly enhanced O2 concentration before WSSV chal-lenge. After the shrimp were infected with WSSV, O2 concentrationdecreased drastically and further recovered gradually. In 0.2, 1 and2% BG groups, O2 concentration attained the highest level on day 12.Huynh et al. [63] have reported that the white shrimp Litopenaeusvannamei immersed in seawater containing fucoidan rich Sargassumhemiphyllum var. chinense powder and its extract showed increasedimmunity and resistance against WSSV. They suggested that theshrimp were immersed in 10 L of seawater containing 0, 100, 300,and 500mg L�1 powder and extract, separately. After 0, 1, 3, and 5 hof the immersion test, the respiratoryburst activity (RB)was directlyincreased with the concentration of powder and extract. The RB ofL. vannamei immersed in seawater containing the powder at500mg L�1 was significantly (P< 0.05) higher than those of controlshrimp after 3 h of immersion, but the RB was slightly decreasedafter 5 h of immersion. The RB activity of L. vannamei immersed inseawater containing the extract at 100, 300, and 500 mg L�1 weresignificantly higher than those of control shrimp after 3 h ofimmersion, but the RB activity was slightly decreased after 5 h ofimmersion. After 3 h of immersion test, the shrimpwere challengedwith WSSV and the survival rate of shrimp immersed in 300 and500mg L�1 powder and extractswere significantly (P< 0.05) higherthan those of shrimp immersed in 100mg L�1 powder or extract andthe control shrimp after 72e120 h. Balasubramanian et al. [19] havereported that the effect of C. dactylon extract on RB activity ofP. monodon challenged with WSSV. They suggested that the shrimp

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564562

infected with WSSV by intramuscular injection and oral adminis-tration showed a significant (P < 0.05) increase of RB activity thanthe control group. In WSSV treated groups both in vivo and in vitro,there was a significant (P < 0.05) increase in RB activity up to day 5post infection and following recovery and reached normal level.Plant extract alone-administered shrimp showed a significant(P < 0.05) increase in superoxide anion level and attained thehighest level at day 2 and day 5 of in vitro and in vivo experimentsrespectively, then decreased gradually with days postadministration.

Superoxide dismutase (SOD) is one of the main antioxidantdefense enzymes generated in response to oxidative stress [64]. Inthe present study, the level of SOD in experimental groups ofshrimp was significantly (P < 0.05) increased when compared tocontrol group after WSSV challenge experiment. In control group,36.85 Unit/ml of SOD was observed at the beginning of the chal-lenge experiment, at the same time, in the experimental groups,SOD was increased with increasing concentrations of fucoidan.When the duration of the challenge experiment increased to 10days, the SOD activity decreased. Finally at the end of the challengestudy, the shrimp recovered SOD activity. Similarly, Chang et al. [12]have reported that the dietary effect of b-1,3-glucan (BG) on SODactivity of P. monodon challenged with WSSV. They suggested thatthe SOD activity of the shrimp fed on 0.2,1 and 2% BG dietary sourcewas significantly (P < 0.001) higher than that of the BG free groupbefore challenge. Subsequently, the SOD activity decreased signif-icantly when the shrimp were infected with WSSV. The reductionwas especially obvious in the BG free group. Following the initialdecrease, the SOD activity of the BG fed groups recovered andattained the highest levels at day 12. Balasubramanian et al. [19]have studied the effect of C. dactylon extract on SOD activity ofP. monodon challenged with WSSV. They pointed out that theshrimp infected with WSSV by in vivo and in vitro showeda significant decrease in SOD activity than the normal shrimp. InWSSV and plant extract treated groups both in vivo and in vitro,there was a significant decrease in SOD activity at the early stageand following recovery, the SOD activity increased and reachednormal level. Plant extract alone-administered shrimp showed nosignificant difference in SOD activity, when compared with thecontrol shrimp.

Phagocytosis is an important cellular defense mechanism per-formed by hemocytes, the lymphoid organs, and the hepatopan-creas in crustaceans [65,66]. This is widely used to evaluatea decapod crustacean’s health status under different treatments ofprobiotics, immunostimulants and environmental stress. Moststudies showed that animals with better phagocytic activity havebetter disease resistance [67,68]. In the present study, the phago-cytic activity of experimental groups of shrimp was significantly(P< 0.05) increased than control group after challenge experiment.At the beginning of the challenge experiment, the phagocyticactivity of experimental groups was 6.85e7.74%, whereas in thecontrol group it was only 5.71%. When the challenge durationprolonged to 10th day, the phagocytic activity decreased in bothcontrol and experimental groups. However, at the end of thechallenge study (21st day), the phagocytic activity significantlyincreased from 6.03 to 7.15 % in 0.1e0.3% fucoidan supplementeddiets fed shrimp. In accordance with these, Chotigeat et al. [14]have pointed out that the fucoidan of S. polycystum enhanced thephagocytic activity of P. monodon challenged with WSSV. Theyobserved a higher (9.1%) level of phagocytosis in the experimentalgroup than the control group (3.72%). Chang et al. [12] have alsosuggested that the dietary effect of b-1,3-glucan on phagocyticactivity of P. monodon challenged withWSSV. They pointed out thatthe hemocyte phagocytic capability, as depicted by phagocyticindex (PI) was significantly higher in the BG fed groups than the BG

free group before challenge. Followed by the initial depression afterchallenge, PI of the 0.2, 1 and 2% BG groups returned to the level ofthe control group by day 12.

The immunostimulants such as b-glucan, laminarin, Lipopoly-saccharides, peptidoglycan and zymosan could activate theprophenoloxidase system and stimulate the superoxide anionproduction in shrimp [59,69,70]. In the present study, the fucoidancould activate proPO-activating system and increases the POactivity as well as RB activity, which indicated that fucoidan couldtrigger innate immunity of shrimp. Similarly, Huynh et al. [62] re-ported that b-glucan, the powder and the extract of S. hemiphyllumvar. chinense could increase PO activity by triggering proPO acti-vation system and increase RB in vitro indicated that both thepowder and the extract could trigger innate immunity of shrimp.

The Sargassum sp. polysaccharides contain large proportion offucose and sulfate, therefore known as sulfated polysaccharide [71].In penaeid shrimp, lipopolysaccharide and b-glucan bindingprotein (LGBP) and b-glucan binding protein (bGBP) contain a glu-canase motif, two polysaccharide recognition motifs (poly-saccharide- binding motif and b-glucan recognition motif), and twointegrin-binding motifs, RGD and RGD [72]. In tiger shrimpP. monodon, bGBP has the ability to bind curdlan, zymosan, but notLPS [73]. The binding mixture of bGBP with curdlan, laminarin, andLPS activated the proPO-activating system [74].

In the present study, the WSSV infection and DNA copies ofWSSV in all the tested groups was confirmed and quantified by RT-PCR analysis. The WSSV infection in positive control group showed1.42 � 106 WSSV DNA copies within 16.96 threshold cycles (CtFAM). But in the experimental groups, the copy numbers of WSSVDNA was positively decreased (756e11 within 27.23e36.26threshold cycles). The result was statistically proved andaccordingly a strong linear correlation (r2 ¼ 0.999) was obtainedbetween threshold cycles (ct FAM) and WSSV DNA copies.Similarly, Zhu and Zhang [75] have reported that the quantifica-tion of WSSV DNA in shrimp Marsupenaeus japonicus after treatedwith antiviral vp28-siRNA expressed in bacteria and challengedwith WSSV. They suggested that the WSSV DNA copies in shrimptreated with vp28-siRNAwere significantly decreased (P < 0.01) bycomparisonwith that of the positive control (WSSV only). Jang et al.[76] have quantified the rate of WSSV infection in hatchery-rearedpost larvae of fleshy shrimp, Fenneropenaeus chinensis throughTaqMan real-time PCR assay method. They observed that the postlarvae collected from 7 hatcheries showed a range of 2.6e713.6WSSV DNA copies ng�1. Balasubramanian et al. [19] have reportedfor the conformation ofWSSV infection in antiviral plant extracts ofC. dactylon treated P. monodon by RT-PCR analysis. They suggestedthat the RT-PCR and western blot analysis were carried out innormal shrimp, positive control shrimp and 1 or 2% of plant extractincorporated pellet diet fed shrimp challenged with WSSV and theWSSV VP28-specific transcript was detected by RT-PCR. The resultsindicated that VP28 specific transcript was not detected in hae-molymph of the shrimp fed on 2% plant extract supplemented dietas in negative control group. But the VP28 transcript was observedin all the organs of shrimp fed on 1% plant extract supplementeddiet as like positive control group. Dechamag et al. [26] have re-ported the expression of Phagocytosis Activating Protein (PAP) genein fucoidan treated WSSV challenged experimental groups ofshrimps by RT-PCR analysis. The results showed that the expressionof PAP gene activated by the intramuscular injection of fucoidanhad increased 6 h of post injection and its highest expression hadoccurred by week 2 post injections (4.8 fold, P < 0.05) and haddisappeared after week 5.

The results of the present study suggested that the brownseaweed S. wightii is major source of fucose and sulfate containingbiologically active polysaccharide-fucoidan, it may be considered as

G. Immanuel et al. / Fish & Shellfish Immunology 32 (2012) 551e564 563

good immunostimulant in shrimp diseases additionally or insteadof commercial antibiotics.

Acknowledgments

The first author wishes to thank University Grants Commission(UGC), New Delhi, India, for its financial support in the form ofresearch grant (F. No: 36-172/2008/SR).

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