Songklanakarin Journal of Science and Technology SJST R1 Zainathan

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1 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan FIRST DETECTION OF WHITE SPOT SYNDROME VIRUS (WSSV) IN WILD MUDCRAB Scylla spp. (de Haan, ) FROM SETIU WETLANDS, MALAYSIA. Journal: Songklanakarin Journal of Science and Technology Manuscript ID SJST-0-00.R Manuscript Type: Original Article Date Submitted by the Author: -Jul-0 Complete List of Authors: Norizan, Nurshafiqah; University Malaysia Terengganu, School of Fisheries and Aquaculture Sciences Sharoum, Faizah; University Malaysia Terengganu, School of Fisheries and Aquaculture Sciences Hassan, Marina; institute of Tropical Aquaculture, Musa, Najiah; Universiti Malaysia Terengganu, Musa, Nadirah; Universiti Malaysia Terengganu, Dept Fisheries Science Abdul Wahid, Mohd Effendy; University Malaysia Terengganu, School of Fisheries and Aquaculture Sciences Zainathan, Sandra Catherine; University Malaysia Terengganu, School of Fisheries and Aquaculture Sciences; Universiti Malaysia Terengganu, Institute of Marine Biotechnology Keyword: mudcrab, Scylla spp., White Spot Syndrome Virus, PCR, Setiu Wetlands

2 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of FIRST DETECTION OF WHITE SPOT SYNDROME VIRUS (WSSV) IN WILD MUDCRAB Scylla spp. (de Haan, ) FROM SETIU WETLANDS, MALAYSIA. NURSHAFIQAH NORIZAN,,FAIZAH SHAHAROM-HARRISON, MARINA HASSAN, NAJIAH MUSA, NADIRAH MUSA, MUHD EFFENDY ABD WAHID,, SANDRA CATHERINE ZAINATHAN,* School of Fisheries and Aquaculture Sciences, 00 Universiti Malaysia Terengganu, Malaysia Institute of Marine Biotechnology, 00 Universiti Malaysia Terengganu, Malaysia Institute of Tropical Aquaculture, 00 Universiti Malaysia Terengganu, Malaysia *Corresponding author: sandra@umt.edu.my / Office: +00/ Mobile: +0/ Fax: +000 Abstract In this study, tissue samples from 0 wild mudcrabs (Scylla spp. including S. olivacea, S. tranquebarica and S. paramamosain) collected during pre-monsoon, monsoon and postmonsoon in Setiu Wetlands, Terengganu, Malaysia were screened for the presence of white spot syndrome virus (WSSV) by PCR. This study was conducted to detect the presence or absence of WSSV in wild Scylla spp. from Setiu Wetlands at different time of sampling. WSSV DNA was detected in % of the mudcrabs. The DNA sequence of a bp genome region amplified from a crab by PCR was identified to be most similar (% nucleotide sequence, % amino acid sequence) to a WSSV strain detected in Mexico (KU.) and Taiwan WSSV strain (AY00.). The data indicate that mudcrabs in the Setiu Wetland might act as a WSSV reservoir of risk to shrimp aquaculture. Our findings are the first detection of WSSV form wild mudcrabs, Scylla spp. from Setiu Wetlands, Terengganu, Malaysia. Keywords: Mudcrab, Scylla spp., White Spot Syndrome Virus, PCR, Setiu Wetlands

3 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Introduction The,000ha Setiu Wetland resides between the Setiu River Basin and larger Setiu-Chalok- Bari-Merang Basin Wetland in Terengganu on the east coast of the Malaysian Peninsular. The wetland possess high biodiversity and unique ecosystems including mangrove swamps and supports small-scale but economically-important oyster farming and brackish water cage, pond and pen culture of Scylla spp. mudcrabs (Azwad, 0). Scylla spp. present in intertidal zones and estuaries in the Setiu Wetland are dominated by Scylla olivacea (S. olivacea)(herbst, ), followed by Scylla tranquebarica (S. tranquebarica) (Fabricius, ) and Scylla paramamosain (S. paramamosain) (Estampador, ) (Keenan, Davie, & Mann, ; Zaidi, Hilmi, Ikhwanuddin, & Bachok, 0). Mudcrab culture is widely extensive with extremely low input and it has been accompanied with the development of many infectious disease, especially due to viruses, which cause mass mortalities during disease outbreak resulting in vast economic loss. Many new diseases have been reported with significant pathogenicity and high impact to the production of mudcrab across the world including White Spot Baculovirus (WSBV), White Spot Syndrome Virus (WSSV), Mud Crab Reovirus (MCRV) and Muscle Necrosis Virus (MNV) (Anderson & Prior, ; Chen, Lo, Chiu, Chang, & Kou, 000; Song et al., 00; Weng, Guo, Sun, Chan, & He, 00; Jithendran, Poornima, Balasubramanian, & Kulasekarapandian, 00; Somboonna et al., 00; Owen, Liessmann, La Fauce, Nyugen, & Zeng, 00; Bonami, & Zhang, 0; Liu, Qian, & Yan, 0; Deng et al., 0). One of the most severe viral infections in Scylla sp. is the WSSV infection. WSSV, which was discovered in in Southeast Asia, has become one of the most serious viral pathogen (Flegel, 00). To date, no decapod crustacean from marine and brackish or freshwater sources has been

4 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of reported to be resistant (Flegel, ; Lightner, ; Lo & Kou, ; Maeda et al., 000; Stentiford, Bonami, & Alday-Sanz, 00). WSSV has a wide host range and it has been observed not only in shrimp but also in crabs and other arthropods (Lo et al., a; Lo et al., b). Among these carriers, mudcrabs have been considered to be a threat to the shrimp farms because they are generally believed [based on a report for Scylla serrata (Forskål, )] to be highly tolerant to WSSV and remain infected for a long period of time without signs of disease (Flegel, 00; Somboonna et al., 00). WSSV can be reisolated from previously infected mudcrabs (Villareal, 00; Flegel, 00; Somboonna et al., 00). S. olivacea and S. tranquebarica have been reported to be susceptible to WSSV infection (Somboonna et al., 00). S. olivacea and S. paramamosain showed wide variation in response to challenge with WSSV as S. olivacea was shown to be more susceptible than S. paramamosain (Somboonna et al., 00). The East Coast fisherman in Setiu Wetlands depend on wild species such as Portunus pelagicus, Scylla spp., Squilla mantis, Macrobrachium lancesteri and cultured species, Litopenaeus vannamei for their living stipend. An outbreak of WSSV infection has affected one of the shrimp farm in Setiu Wetlands in March 0 (Unpublished). This incident has raised questions whether: the WSSV infections could have been transmitted horizontally via water to the surrounding rivers and wetlands area or WSSV infected wild crustaceans had transmitted the disease to the cultured farm. Wild organisms in natural reservoirs might represent a potential source of viral disease, thus it is necessary to understand the dynamics of viruses in the environment, in terms of the mode of infection, transmission and natural reservoir, which is dependent on its prevalence (Macias- Rodriguez et al., 0). Thus, this paper describes the first detection of WSSV in wild Scylla spp. from Setiu Wetlands, Terengganu, Malaysia.

5 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Materials and Method Sample collection A total of 0 samples of wild Scylla spp. including Scylla olivacea (S. olivacea) (n = ), Scylla tranquebarica (S. tranquebarica) (n = ) and Scylla paramamosain (S. paramamosain) (n = ) were collected from Setiu Wetlands, Terengganu (N 0 0.0, E 0.). The sampling trips were conducted during the post-monsoon (March 0), pre-monsoon (August 0) and monsoon seasons (December 0). The water quality parameters (ph, temperature, salinity and dissolved oxygen) were measured using YSI Multiparameter Water Quality Meter and API Freshwater Aquarium Master Test Kit (Mars Fishcare North America, Inc). The samples were kept in polystyrene box and brought back alive to laboratory of Aquatic Organisms Health in Universiti Malaysia Terengganu. The length and weight of the samples were measured and any external and internal clinical signs were observed. The gills and internal organs (hepatopancreas, stomach and heart) were collected for sample processing and further analysis by PCR was conducted based on Lo et al. (a, b) and Nunan and Lightner (0). DNA extraction The DNA extraction of the samples was conducted using NucleoSpin Tissue Extraction Kit (Macherey-Nagel) according to the protocol provided by the manufacturers. About mg of the pooled organs (hepatopancreas, heart and stomach) and gills were cut up to small pieces and placed in a. ml microcentrifuge tube. The extracted DNA was stored at -0 C prior to PCR analysis.

6 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of PCR amplification The conventional nested PCR was carried out according to the method by Lo et al. () a and b as described in OIE (0). The primers were based on the sequence published by Lo et al. (a, b): Forward primer F- ( -ACT-ACT-AAC-TTC-AGC-CTA-TCTAG- ) and reverse primer R- ( -TAA-TGC-GGG-TGT-AAT-GTT-CTT-ACG-A- ) for primary reaction with the expected size of bp. Followed by the second primer set F - ( -GTA- ACT-GCC-CCT-TCC-ATC-TCC-A- ) and R - ( -TAC-GGC-AGC-TGC-TGC-ACC- TTG-T- ) in the nested reaction which were expected to yield an amplicon of bp. A total of µl PCR mixture containing:. µl X MyTaqMix (Bioline), 0. µl Rnase-free water, 0. µl F (0 µm) and 0. µl R (0 µm) were added to µl extracted DNA. For the primary reaction, the PCR thermal profile was programmed as followed: a cycle of C for minutes, followed by cycles of C for minute, C for minute, and C for minutes and a final -minutes extension at C. The nested reaction was carried out with a total of µl PCR mixture containing:. µl X MyTaqMix (Bioline), 0. µl Rnase-free water, 0. µl F (00 µm) and 0. µl R (00 µm) were added to µl PCR product. The amplification was conducted with the following programme: a cycle of C for minutes, followed by cycles of C for minute, C for minute, and C for minutes and a final -minutes extension at C. The amplified PCR products from both reactions were visualized by gel electrophoresis through % (w/v) agarose gel in TAE buffer and stained with SYBR Safe DNA gel stain (Invitrogen) for minutes at 0 volt.

7 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Gel extraction and DNA sequencing analysis Each sample representative from PCR positive samples from each host species was chosen for sequencing analysis to examine the sequence difference of WSSV between the different host species.the expected bands from sample from site (S-, host species: S. paramamosain), sample from site (S-, host species: S. olivacea) and sample from site (S-, host species: S. tranquebarica) were excised and purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) based on the standard protocols. The purified DNA was diluted into 0 ngµl - as required by the protocol before it was sent for sequencing to First Base Laboratory (Selangor, Malaysia). Phylogenetic analysis The DNA sequencing results were used for the phylogenetic analysis. The sequences were used to interrogate the NCBI BLAST database to confirm its likely identity. Then the multiple alignment were aligned using Clustal X.0. with other WSSV- related sequences. Finally, the phylogenetic tree was inferred among the sequences of WSSV obtained in this study with known sequences contained in GenBank using Molecular Evolutionary Genetics Analysis (MEGA). Statistical analysis The effects of time of sampling on the prevalence of virus was determined using one-way ANOVA analysis. ANOVA analyses were performed using SPSS statistic software version.0. Values were identified as significantly different if p < 0.0. The Tukey HSD post hoc test

8 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of was applied at a significance level of α 0.0, to determine differences between the explanatory variable. Results No clinical signs were observed on all of the samples; externally and internally. A total of samples were detected with the presence of Octolasmis sp. on their gills. The average weight and carapace width of the samples were tabulated according to the different seasons (Table ). Water parameter at sampling location A significant difference was observed between the water parameter and the three seasons (Table ). These results indicated the occurrence of fluctuations in water parameters during the three seasons. Conventional nested PCR for the detection of WSSV inscylla spp. A total of samples (%) of Scylla olivacea (S. olivacea), Scylla tranquebarica (S. tranquebarica) and Scylla paramamosain (S. paramamosain) sampled during post-monsoon, pre-monsoon and monsoon season were found to be positive for the presence of WSSV. Higher number of positives were detected during the pre-monsoon and monsoon season in Setiu Wetlands; samples than post-monsoon season, samples. Based on the results, samples of S. olivacea showed the highest presence (.%) of WSSV compared to S. paramamosain (.%) and S. tranquebarica (.%) (Table ).The primary reaction of conventional nested PCR analysis of pooled gills and tissues (hepatopancreas, heart and stomach) demonstrated negative results for the presence of WSSV (Figure ). The secondary reaction of conventional nested PCR

9 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan analysis of pooled samples of gills and tissues (hepatopancreas, heart and stomach) demonstrated positive band at expected size of bp for the presence of WSSV (Figure ). A significant difference was observed between the season and the prevalence of WSSV as determined by one-way ANOVA (p< 0.0; p = 0.0). Tukey post-hoc test revealed that the prevalence of WSSV infection demonstrated a significant difference between the pre-monsoon and monsoon season (α < 0.0; α = 0.00). The total prevalence of WSSV infection was not influenced by the different number of mud crabs sampled during post-monsoon season (n=) pre-monsoon season (n=) and monsoon season (n=0). Comparison of total positives for WSSV infection based on the season showed highest prevalence during monsoon season (Figure ). Sequencing and phylogenetic analysis. A multiple alignment of nucleotide sequences of the amplified PCR products confirmed that sample S- ( S. paramamosain), S- (S. olivacea) and S-(S. tranquebarica) were from the same member of WSSV. These results confirmed that all the positive samples was from the same member of WSSV. S- (S. olivacea) and S-(S. tranquebarica) showed % while S- (S. paramamosain) showed % sequence similarity to the whole genome of WSSV Mexican strain WSSV-MX0 (GenBank accession no. KU.). Multiple alignment of nucleotide sequences of the amplified PCR product confirmed that samples S- (S. paramamosain) were from the same lineage with WSSV Mexican strain (WSSV-MX0) (Rodriguez-Anaya et al., 0), samples S- (S. olivacea) with Taiwan WSSV strain (Reville et al., 00) and samples S- (S. tranquebarica) with WSSV complete genome strain (van Hulten et al., 00) with minor variations (Figure ).

10 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page 0 of Discussion The water quality parameters which was taken at each of the sampling location include temperature, salinity, dissolve oxygen (DO) and ph. The temperature range ( C - C) was normal for tropical coastal waters (Alongi et al., 00). The lowest salinity was recorded at site, in the downstream of Setiu Wetlands (combination of freshwater and seawater) during the monsoon season. The highest salinity was recorded at sampling site, which was located in the inshore area, during the pre-monsoon. Scylla spp. are not known to be affected by elevated salinity due to their high tolerancy to wide range of salinities (FAO, 0). The dissolve oxygen (DO) was in range of. mgl - to. mgl - with the lowest DO recorded due to aquaculture activities at the site. Aquaculture discharges contain high levels of organic matter such as feed and faeces. The degradation of organic matter by microorganism will consume more oxygen, hence, lowering the DO content in water (Hargrave, Duplisea, Pfeiffer, & Wildish, ) and Scylla spp. can tolerate low oxygen level (FAO, 0). The results for the water parameters were consistent with previous study regarding water quality at Setiu Wetlands where aquaculture activities can affect water quality parameter (Suratman, Hussein, Latif, & Weston, 0). A total of samples of Scylla spp. (%) including S. olivacea, S. tranquebarica and S. paramamosain sampled from March to December 0 demonstrated positive result for the presence of WSSV. The result from this study is consistent with several papers which showed positive result for WSSV infection in Scylla spp. using nested PCR (Lo et al., ; Otta et al., ; Chen et al., 000; Vaseeharan, Jayakumar, & Ramasamy, 00; Somboonna et al., 00;

11 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Liu et al., 0; Joseph, Roswin, Anbu Rajan, Surendran, & Lalitha, 0). Natural WSSV infections have been found in wild-caught and farmed mud crabs in various stages in many countries of Asiatic region (Lo et al., a; Flegel, ; Flegel, 00; Kou, Peng, Chiu, & Lo, ; Otta et al., ; Lavilla-Pitogo, Marcial, Pedrajas, Quinitio, & Millamena, 00). Natural benthic larvae of wild Scylla serrata were found to be positive for WSSV infection at 0% prevalence (Lo et al., ). WSSV infection (0% prevalence) was detected in benthic larvae of wild mud crab, Scylla serrata captured from Taiwan s coastal water (Chen et al., 000). WSSV was detected on diseased cultured mud crabs collected from Zhejiang Province of China during which showed a prevalence of.% (Liu et al., 0). The number of WSSV positive Scylla spp. were detected throughout the three seasons; monsoon season in December (%), pre-monsoon season in August (%) and monsoon season in March (%). Consistent prevalence for WSSV infections in mud crab population captured in August and September were shown by Chen et al. (000). The results of one-way ANOVA showed that there was a significant difference between the three seasons and the prevalence of WSSV infection in wild mud crab, Scylla spp. This result is consistent with Chen et al. (000) that stated the infection rate of WSSV might be persistent throughout the year (Chen et al., 000). The high prevalence of WSSV infection during monsoon season demonstrated that WSSV outbreak is affected by the change of environmental factors; which will depress the immune system of crustacean (Gunalan, Soundarapandian, & Dinakaran, 00). Temperature is the most important environmental factor as it has a profound effect on disease expression. The average water temperatures between C to 0 C can play an important role in inducing outbreaks of WSSV (Vidal, Granja, Aranguren, Brock, & Salazar, 00). This study showed that the water temperature during monsoon season is lower compared to post-monsoon and pre-monsoon 0

12 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of season due to the incoming freshwater during the rainy season (Sankar, Ramamoorthy, Sakkaravarthi, & Vanitha, 0). Low water temperature has been known to influence the dispersal of WSSV (Jiravanichpaisal, Soderhall, & Soderhall, 00; Gunalan et al., 00). A BLAST search of the WSSV_S- (S. paramamosain) segments showed % sequence identity with MX0 (KU.), CN0 (KT.), CN0 (KT0.), CN0 (KT.), EG (KR0.), K-LV (JX.), Taiwan WSSV (AY00.), CM-VN (JX.), White Spot Syndrome Virus with partial sequence (AF.) and WSSV complete genome (AF0.). The published sequences compared to S- (S. paramamosain) were mostly sequences from cultured white shrimp, Litopenaeus vannamei (L. vannamei). WSSV_S- (S. olivacea) and WSSV_S- (S. tranquebarica) were found to be % similar with the above published sequences. S- (S. paramamosain) showed % similarity with the whole genome of a WSSV Mexican strain (WSSV-MX0), which was the first WSSV isolated from cultured L. vannamei from Mexican farms (Rodriguez-Anaya et al., 0). White spot syndrome virus (WSSV) was first discovered in from Taiwanese cultured Penaeus japonicus (Flegel, 00). Since then, WSSV has spread across Asian countries including Vietnam, Thailand, Korea, India, China, and Malaysia (Nakano et al., ; Flegel, ; Mohan, Shankar, Kulkarni, & Sudha, ; Zhan et al., ; Wang, Nunan, & Lightner, 000). The sequence similarity of S- (S. paramamosain) to L. vannamei indicated that the WSSV infections could have been transmitted to the wild environment from farm or vice versa. WSSV can be transmitted horizontally and vertically (Lo & Kou, ; Chou, Huang, Wang, Lo, & Kou, ; Otta et al., ). The horizontal transmission of WSSV was shown to be via ingestion and waterborne routes (Chou et al., ). The water disposal from shrimp farm was shown to transmit WSSV infectious agent into the natural enzootic areas (Lo & Kou, ;

13 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Esparza-Leal et al., 00). A cannibalistic behavior of mud crab also contributes to the transmission of WSSV (Lo et al., ). Consequently, the virus can be transmitted to whole population of the cultured or wild crab and shrimp (OIE, 0). However, the phylogenetic tree analysis showed that sample WSSV_S- (S. olivacea) shared a common ancestry relationships with the Taiwan WSSV strain (GenBank accession no. AY00.) while sample WSSV_S- (S. tranquebarica) with the WSSV complete genome strain (GenBank accession no. AF0.). Based on the published sequences; WSSV Mexican strain (WSSV-MX0) (Rodriguez-Anaya et al., 0), Taiwan WSSV strain (Reville et al., 00) and WSSV complete genome (van Hulten et al., 00), it is inheritable that WSSV was originally found in L.vannamei and Penaeus monodon. Both published sequences; Taiwan WSSV strain (Reville et al., 00) and WSSV complete genome (van Hulten et al., 00) originated from Thailand. Since Thailand is one of Malaysia s neighboring country, WSSV could be transmitted to the crustacean population via the horizontal route of infection through release of ship ballast water and illegal importation of live post-larvae (Lo et al., ; Sanchez- Martinez, Aguirre-Guzman, & Mejia-Ruiz, 00; Muller, Andrade, Tang-Nelson, Marques, & Lightner, 00). Genetic variation have been observed among the WSSV detected from the different host species of Scylla spp.. The host species play a role in the selection of a mutant within a viral population which led to a genetic variation of WSSV (Wang et al., 000). The genetic variation of WSSV in different host species could be explained where the passage of the virus through different hosts can induce a genomic alteration and alter the pathogenicity of the virus (Waikhom, John, George, & Jeyaseelan, 00). The non-static properties of WSSV genome enable itself to adapt to any environmental condition (Sablok et al., 00). This could further be

14 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of strengthen by previous study that the occurrence of genomic mutations resulted from the adaptation of the virus to different environmental conditions (Waikhom et al., 00) and due to the mutations in viral genome of WSSV after its introduction into the different countries (Muller et al., 00). Conclusions A total of 0 samples of wild Scylla spp. including Scylla olivacea (S. olivacea), Scylla tranquebarica (S. tranquebarica) and Scylla paramamosain (S. paramamosain) were sampled from Setiu Wetlands, Terengganu during post-monsoon, pre-monsoon and monsoon season. The water parameter taken from the sampling sites showed a normal range for the Scylla spp. habitat. A total of samples sampled during post-monsoon, pre-monsoon and monsoon season were found to be positive for the presence of WSSV. The sequenced sample of S- (S. olivacea) and S- (S. tranquebarica) showed high similarity (%) while S- (S. paramamosain) showed % to the WSSV Mexican Strain (WSSV-MX0). In conclusion, our findings are the first detection of WSSV form wild mudcrabs, Scylla spp. from Setiu Wetlands, Terengganu, Malaysia (a new location). Acknowledgements This work was funded by the projects under the Niche-area Research Grant Scheme (NRGS Vot. No. ).

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19 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan implications of inclusion of these diseases in European legislation. Aquaculture,, -. Suratman, S., Hussein, A. N. A. R., Latif, M. T., & Weston, K. (0). Reassessment of Physico- Chemical Water Quality in Setiu Wetlands, Malaysia. Sains Malaysiana,, -. Van Hulten, M. C. W., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M., Sandbrink, H., Lankhorst, R.K., & Vlak, J. M. (00). The white spot syndrome virus DNA genome sequence. Virology,,. Vaseeharan, B., Jayakumar, R., & Ramasamy, P. (00). PCR-based Detection of White Spot Syndrome Virus (WSSV) in Cultured and Captured Crustaceans in India. Letters in Applied Microbiology,, -. Vidal, O. M., Granja, C. B., Aranguren, F., Brock, J. A., & Salazar, M. (00). A Profound Effect of Hyperthermia on Survival of Litopenaeus vannamei Juveniles Infected with White Spot Syndrome Virus. World Aquaculture Society,, -. Villarreal, L. P. (00). Viruses and the evolution of life. Pp Washington (DC): ASM Press. Waikhom, G., John, K. R., George, M. R., & Jeyaseelan, M. J. P. (00). Differential host passaging alters pathogenicity and induces genomic variation in white spot syndrome virus. Aquaculture,,. Wang, Q., Nunan, L. M., & Lightner, D. V. (000). Identification of genomic variation among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization. Diseases of Aquatic Organisms,, -. Weng, S. P., Guo, Z. X., Sun, J. J., Chan, S. M., & He, J. G. (00). A reovirus disease in cultured mud crab, Scylla serrata in southern China. Journal of Fish Diseases, 0, -. Zaidi, Z. M., Hilmi, M. G., Ikhwanuddin, M., & Bachok, Z. (0).Population Dynamics of Mud Crab, Scylla olivacea from Setiu Wetland Areas, Terengganu, Malaysia. Proceedings of Universiti Malaysia Terengganu Annual Symposium 0. Zhan, W. B., Wang, Y. H., Fryer, J. L., Yu, K. K., Fukuda, H., & Meng, Q. X. (). White spot syndrome virus infection of cultured shrimp in China. Journal of Aquatic Animal Health, 0, 0 0.

20 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page 0 of FIGURE (Pg) FIGURES Figure : A) Primary PCR amplification of tissues from Scylla spp. from Setiu Wetlands. ) Lane (from left) 00 bp DNA ladder, ) Lane -: samples, ) Lane 0: Negative control, ) Lane and : Synthetic positive control of WSSV, bp. B) Nested PCR amplification of tissues from Scylla spp. from Setiu Wetland ) Lane (from left): 00bp DNA ladder, ) Lane - : Tissue samples, ) Lane 0: Negative control, ) Lane : Synthetic positive control of WSSV, bp. ) Lane and - Positive results for the presence of WSSV bp, ) Lane until - Negative results for the presence of WSSV.

21 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan FIGURE (Pg ) Figure : Mean score of the prevalence of WSSV infection in wild mud crab depend on the season. p < 0.0 (p = 0.0). A different lower case letters denote a significant differences for a Tukey post-hoc test, α < 0.0. Letter a and aa : no significant differences, α > 0.0; a = 0.0, aa = 0.0 and Letter b: significant differences, α < 0.0; ab = 0.00.

22 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of FIGURE (Pg ) Figure : Phylogenetic neighbour-joining tree deduced from analysis nucleotide sequences of WSSV strain. A BLAST search of the WSSV_S-, WSSV_S- and WSSV_S- segments showed similarities sequence identity with MX0 (KU.), CN0 (KT.), CN0 (KT0.), CN0 (KT.), EG (KR0.), K-LV (JX.), Taiwan WSSV (AY00.), CM-VN (JX.), White Spot Syndrome Virus with partial sequence (AF.) and WSSV complete genome (AF0.).

23 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan TABLE (Pg ) Average weight (g) Average carapace length (cm) TABLE Season Post-monsoon Pre-monsoon Monsoon Table : Average weight and carapace length of mud crab samples during post-monsoon, premonsoon and monsoon season.

24 Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan Page of TABLE (Pg ) Water parameter Temperature ( C) Salinity (ppt) Dissolve oxygen (mg/l) Season Post-monsoon Pre-monsoon Monsoon p value ph Table : Average water parameter for each season; post-monsoon, pre-monsoon and monsoon season at the sampling location. p value were calculated using one-way ANOVA to determine the differences for the water parameter according to season. p value significant at 0.0 level (p < 0.0).

25 Page of Songklanakarin Journal of Science and Technology SJST-0-00.R Zainathan TABLE (Pg ) Time/ Season March 0 (Postmonsoon) August 0 (Premonsoon) December 0 (Monsoon) Species Scylla olivacea Scylla tranquebarica Scylla Paramamosain Number tested Positive for WSSV infection Scylla olivacea Scylla tranquebarica Scylla paramamosain Scylla olivacea Scylla tranquebarica Scylla Paramamosain Total positives Total 0 Table : Summary of PCR analyses for the detection of WSSV in Scylla spp. from Setiu Wetlands during post-monsoon, pre-monsoon and monsoon season.