MUD CRAB SCYLLA SERRATA (FORSKAL) AS CARRIER OF WHITE SPOT SYNDROME VIRUS (WSSV) IN CORINGA MANGROVES, ANDHRA PRADESH

Vijaya Bharathi. T 1 , Myla S. Chakravarty 2 and P. R. C. Ganesh 2 . 1. State Institute of Fisheries Technology, Jagannaickpur, Kakinda 2. Andhra University, Department of Marine Living Resources, College of Science and Technology, Visakhapatnam. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Extraction of DNA:-
Crab samples were brought to the laboratory in live condition. In the laboratory, the gills were dissected out and collected in sterile plastic sachets. Digestion Buffer (Guanidium Chloride) was added (800µl) to the plastic sachet containing the gills of the live crabs and the material was macerated in the sachet and incubated at room temperature for about 10-15 minutes. The homogenate was transferred to 1.5 ml micro centrifuge tube and centrifuged (Remi, C-24) at 4000 rpm for 5 minutes. 150 µl of supernatant was transferred to a fresh micro centrifuge tube and to it DNA precipitation Buffer (Absolute Ethyl Alcohol) was added (450 µl). The tube was centrifuged at 10,000-12,000 rpm for 10 minutes at 4 0 C. Then the supernatant was discarded. DNA washing Buffer (70% Ethyl Alcohol) (450 µl) was added and centrifuged at 10,000-12,000 rpm for 5 minutes at 4 0 C. The supernatant was then discarded and the pellet was dried at room temperature. After drying the pellet, it was dissolved in 100 µl of double distilled water, which will contain the DNA of White Spot Syndrome Virus, if present, in the sample.
DNA Amplification:-2 µl of template DNA was taken into the master mix tubes (reagent mixture tubes that contain amplification buffer, dNTPs, amplification grade water, Mg ++ and Taq polymerase). Nested PCR amplification was carried out in a Thermocycler (Eppendorf) for WSSV following a temperature profile, with an initial denaturation for 5 min. at 94 0 C followed by 30 cycles at 94 0 C for 30 seconds; 55º C for 30 seconds; 72 0 C for 30 seconds and a final extension at 72 0 C for 5 min. PCR was carried out using species-specific primers namely 486 bp fragment in first step and 310 bp in the second step.
Termocycler was programmed as: I step-initial denaturation at 94 0 C for 5 min followed by each cycle of denaturation at 94 0 C for 30 sec, annealing at 42 0 C for 30 sec, extension at 72 0 C for 40 sec and a final delay at 72 0 C for 7 min, the same conditions were followed in the second step also. In every batch of reaction, a positive control and negative control was included.
DNA Detection:-PCR products, after DNA amplification in the thermocycler were separated by agarose gel (1%) containing 0.5 µg per ml ethidium bromide and observed in a DNA transilluminator (Genei). Depending on the size of the electrophoresis tank, the quantity of agarose gel (1%) required was prepared. The agarose in 1x TAE (Tris Acetate EDTA) buffer was heated till the agarose was completely melted and cooled to room temperature. Before pouring the gel on to the already set gel platform, 5-µl ethidium bromide per 100 ml was added.
After the gel was set, it was placed in the electrophoresis chamber. 20 µl of product was taken after DNA amplification and it was mixed with 4-µl gel loading buffer (Bromophenol Blue) and loaded the gel. 8-µl molecular weight marker (100 bp DNA size marker) was loaded in a separate lane of the gel. The gel was run at 80 volts for 10 2701 minutes and then at 120 volt till the tracking dye moves about 2 cm from the edge of gel. Finally the gel was viewed under the UV transilluminator and the results were documented with a UV documentation unit (Uvidoc).

Results:-
A total of 357 crab samples were tested for WSSV with PCR in the study area. Out of these, 58 are positive (16%) and 299 are negative in the first step PCR whereas 132 are positive (37%) and 225 are negative in the nested PCR (Fig.2).

WSSV Prevalence:-
In the 1 st half of the year (2007) at Kakinada bay, the incidence of WSSV was 10.71% in the first step and 32.14% in the second step. In the 2 nd half 22.22% were positive and 44.44% in the second step. In Coringa creek, the incidence was 8% in the 1 st step and 32% in the 2 nd step in the 1 st half. In the 2 nd half 16.22% were positive in first step and 37.84% in second step. In the 1st half of the year in Gaderu creek, 18.52% in the 1 st step and 40.74% in the 2 nd step. In the 2 nd half 20% were positive in first step and 37.14% in second steps (Fig. 3).
In the 1st half of the year (2008) at Kakinada Bay, the incidence was 20% in the 1 st step and 44% in the 2 nd step. In 2 nd half 12.12% were positive in first step and 24.24% were positive in second step. In Coringa creek in the 1 st half, 19.23% were positive in first step and 50% in second step. In 2 nd half 13.33% were positive in first step and 30% were positive in second step. At Gaderu creek in the 1 st half 17.86% were positive in first step and 42.86% were positive in second step. In the 2 nd half 14.81% were positive in first step and 29.63% were in second step (Fig. 4).
Out of the crab samples tested 16% were positive in the first step whereas 37% positive in the nested PCR. At Kakinada Bay, 16.4% in first step and 36% were positive in the second step. In Coringa Creek, 14.4% and 37.3% in first step and second step respectively were positive to the disease. In Gaderu, the percentage incidence was 17.1 in first step and 37.6 in the second step (Fig.5).

Discussion:-
WSSV has become a major threat in shrimp culture causing overnight mass mortalities and there are no reports of incidence of WSSV in India before 1994. Karunasagar et al (1997) have reported severe mortalities of cultured shrimp Penaeus monodon due to WSSV. The white spot virus is believed to have been transmitted through seed and brooders brought to India from Southeast Asian countries, where the virus has been very much prevalent (Shankar and Mohan, 1998). A list of susceptible species of WSSV has been presented by Lo et al (1996). Crustaceans other than shrimps may also serve as carriers of the disease though they do not exhibit any clinical signs of WSSV externally and are also active and healthy. The mud crab S. serrata has also been found to be the same.
Mud crabs are known as carriers and vectors of WSSV . Natural WSSV infections have been found in captured and cultured specimens of the mud crab S. serrata in many countries of Asia Kanchanaphumet al., 1998). WSSV has been detected in around 60% of the benthic larvae of mud crab, S. Serrata both under natural and experimental conditions (Chen et al., 2000). Although mud crab is known to be a carrier of WSSV in India, studies are limited. In the present study, nested PCR has been used for better sensitivity. It has also been suggested by Lo et al (1996) that the two-step PCR 10 3 to 10 4 times more sensitive than 1-step PCR. Kou et al (1998) have opined that the sensitivity of the 2-step amplification is 10 3 to 10 4 times greater than that of the 1-step amplification.
Otta et al (1999) have carried out a study to evaluate the crustaceans as potential carriers of WSSV using PCR. After the WSSV outbreaks in India during late 1994, in the shrimp culture ponds, several instances of White spots on the exoskeleton of mud crab, S. serrata have been observed. Mud crab has been shown to be a carrier of WSSV in Taiwan  and Thailand , Supamattaya et al., 1998. Otta et al (1999) have tested one mud crab with external symptoms and 20 healthy samples without white spots and reported that the one with external symptoms is positive in the 1 st step PCR whereas 3 out of 20 healthy specimens are positive in the 2 nd step PCR indicating that S. serrata is also a carrier of WSSV. In India, it has been noticed that the natural prevalence of WSSV in crab is about 5.06% while in shrimp culture ponds it is about 30% (CIBA, 2009  The wild populations of mud crabs harbouring WSSV as carriers may adversely affect the shrimp production in the culture ponds, since shrimp culture ponds has been located in the vicinity of crab fishery. According to Rajendran et al (1999) S. serrata which is orally infected with WSSV has shown 10-20% mortality in 30 days. The infected crabs have shown marked histopathological changes similar to those reported earlier for WSSV in naturally infected shrimp (Wongteerasupaya et al., 1995). Similar results have been observed by Hossain et al (2001). Chakraborty et al (2002) have reported that all the specimens of S. serrata collected from different localities are positive to WSSV.
The prevalence of the disease is more during wet season (June-Sep) than the dry season (Jan-June) in both years. Of the three regions, the prevalence is 36% at Kakinada Bay, 37.3% in Coringa and 37.6 % in Gaderu. It may be due to the discharge of waters from shrimp ponds, containing pathogen into the creeks and drainage canals, since the shrimp culture is very high in the region (about 675 ha near Coringa and 1050 ha near Gaderu). Much difference in the percentage of positivity has also been observed in samples of different size classes. It is evident that among the three size classes 0-4 cm, 5-8 cm, 9-13 cm, the smallest size class showed higher percentage of positivity i.e. 52.38% in 2007 and 52.63% in 2008.The same is noticed in all three areas of study. The lowest of positivity was noticed in the size class 9-13 cm confirming that the smaller ones are more susceptible to this virus.