Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 4 (5) 1011-1020 January 2011. ISSN 0973-8916in few drops <strong>of</strong> sterilized brackish water prior tonegative staining. For negative staining, one drop<strong>of</strong> suspension was mixed with 4 drops <strong>of</strong> themixture <strong>of</strong> 0.1% bovine serum albumin <strong>and</strong> 2%phosphotungstic acid (1:2, pH 7.0). The mixturewas placed on a grid for 30 to 60 s <strong>and</strong> excesssuspension was removed with filter paper. Thepreparation was allowed to dry before beingexamined. The results were observed under TEM(Technai, 20; Philips series) (15).4. Histopathology : The histopathology <strong>of</strong> gills,hepatopancreas, muscle <strong>and</strong> gut were performedfrom WSSV infected <strong>and</strong> healthy shrimps. T<strong>issue</strong>swere blotted free <strong>of</strong> heymolymph, fixed in Bouin’sfluid, sections were cut at 5-8µm <strong>and</strong> stained withH & E. The photographs were taken withAxioplan image analyzer at 40X <strong>and</strong> 100X.5. SDS PAGE Analysis : Muscles <strong>and</strong>hepatopancreas, 0.5gm each, from infected <strong>and</strong>healthy shrimps were homogenized in 10ml TNbuffer (20 mM Tris-HCl, 400 mM NaCl, pH 7.4),centrifuged at 8510×g for 10min at -4ºC <strong>and</strong>supernatant was subjected to polyacrylamide gelelectrophoresis using 10% polyacrylamideresolving gel <strong>and</strong> 5% stacking gel (16). Theresolving gel (30ml) was polymerized by 300µl <strong>of</strong>10% ammonium peroxodisulphate <strong>and</strong> 70µl <strong>of</strong> N,N, N, N-Tetramethylenediamine (TEMED).Stacking gel (10ml) was polymerized by adding50µl APS <strong>and</strong> 20µl TEMED. The samples weremixed with sample buffer, boiled for 5 min <strong>and</strong> allgel were run using a dual midi vertical gelelectrophoresis system at constant 100V in 1Xbuffer (25mM Tris, 25mM glycine, pH 8.3, 0.1%SDS). Proteins were visualized by silver stainingkit (Genie, Bangalore).Results1. Morphological symptoms : The infectedshrimps exhibited lethargy, anoxia, opaquemusculature <strong>and</strong> were detected near pond edges.They were detected with white spots on the1013carapace, on appendages <strong>and</strong> on other body partsas well as with pink to red body colouration (Fig.1). The other symptoms <strong>of</strong> infected P. monodonwere loose binding <strong>of</strong> the epidermis with cuticle<strong>and</strong> enlarged hepatopancreas with yellowishwhite colouration.Fig. 1 White spots <strong>of</strong> P. monodon. Arrow indicates the whitespots on the cuticle <strong>and</strong> other body parts.2. Scanning electron microscopy : The scanningelectron micrographs show white spots on bothupper <strong>and</strong> lower surfaces <strong>of</strong> carapace <strong>of</strong> infectedshrimps. On the inner surface <strong>of</strong> the carapace,spots were larger in size, usually rounded in shapemeasuring 25µm to 135µm in diameter. On theouter surface <strong>of</strong> carapace, white spots were verysmall in size <strong>and</strong> measuring between 25 to 32µmin size (Fig. 2a <strong>and</strong> 2b).Figure 2a-2b Scanning electron micrograph <strong>of</strong> whitespots <strong>of</strong> WSSV infected shrimps. Flattened White spotcovering the cuticle <strong>of</strong> carapace (a, b).EDX quantification <strong>of</strong> elements like C, O,Ca <strong>and</strong> P in carapace from both inner <strong>and</strong> outersurface with SEM in infected <strong>and</strong> control shrimpindicated that composition <strong>of</strong> inner <strong>and</strong> outersurface <strong>of</strong> white spots have not been changed<strong>and</strong> were measured as 38%; 32 to 33 %; 0.8 toProtein expression in WSSV
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)10140.7% <strong>and</strong> 29% respectively. At the same time, ascompared to control in white spot region, Ca <strong>and</strong>O were increased; whereas C has decreased (Fig3a-3d; Table 1).Figure 3a-3d shows the EDX graphs <strong>of</strong> different levels <strong>of</strong> carbon (C), oxygen (O), calcium (Ca) <strong>and</strong> phosphorus(P) <strong>of</strong> carapace in infected as well as healthy shrimps (Infected lower surface-3a, Control lower surface-3b,Infected upper surface-3c <strong>and</strong> Control upper surface-3d).Table 1 shows the values <strong>of</strong> elements for both upper <strong>and</strong> lower surfaces <strong>of</strong> WSSS infected <strong>and</strong> uninfectedshrimps by EDX analysis (Mean, n=4).Bhatt et al
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