Long‐lasting Effect Against White Spot Syndrome Virus in Shrimp Broodstock,Litopenaeus vannamei,by LvRab7 Silencing

The white spot syndrome virus (WSSV) remains the most devastating viral pathogen of shrimp culture worldwide. Gene silencing by RNA interference (RNAi) using double stranded RNA (dsRNA) has been considered a powerful tool for conferring protection against WSSV when viral genes are silenced, as documented in several shrimp species. However, this effect is not long lasting. Our results provide the first evidence that long‐term silencing of the LvRab7 endogen produced antiviral effect against WSSV, which endured at least 21 d after dsRNA treatment (dat). Until now, the most efficient way to implement RNAi with dsRNA into the shrimp is by injection. Consequently, its application to broodstock in hatcheries is possible, minimizing the risk of vertical transmission of the virus. We show that the expression of Rab7 in hemocytes is lowest at 2 dat and finally recovers to basal status. In contrast, in gills and pleopods, gene expression silencing continued for at least 21 d. We challenged Litopenaeus vannamei broodstock with WSSV at 7, 14, or 21 dat reaching mortality rates of 0, 40, and 27%, respectively. In conclusion, the LvRab7 gene silencing is progressive and effective against WSSV. However, further studies are necessary to elucidate the functions of Rab7 in shrimp cells before applying this methodology at a commercial level.     

Production of specific dsRNA against white spot syndrome virus in the yeast Yarrowia lipolytica

White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double‐stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA‐specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei. From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA‐ORF89‐producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA‐ORF89. Yeast colonies positive to the WSSV‐ORF89 gene were selected. The expression of dsRNA‐ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA‐ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA‐ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01‐AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA‐ORF89 able to protect WSSV‐challenged shrimp.     

Effect of hot water extracts of brown seaweeds Sargassum spp. on growth and resistance to white spot syndrome virus in shrimp Penaeus monodon postlarvae

An experiment was conducted to evaluate the effect of a hot water extract of brown seaweeds Sargassum duplicatum and Sargassum wightii on the growth and white spot syndrome virus (WSSV) resistance in shrimp Penaeus monodon postlarvae (PL). Artemia nauplii (instar II) were enriched with both seaweed extracts at various concentrations (250, 500 and 750 mg L^?1) and fed to the respective P. monodon (PL15–35) group for 20 days. A control group was also maintained without seaweed extract supplementation. The weight gain of the experimental groups was significantly higher (0.274–0.323 g) than the control group (0.261 g). Similarly, the specific growth rate was also significantly higher (16.27–17.06%) in the experimental groups than in the control group (16.03%). After 20 days of the feeding experiment, the shrimp PL were challenged with WSSV for 21 days. During the challenge test, the control shrimp displayed 100% mortality within 8 days. In contrast, the mortality percentage of the highest concentration (750 mg L^?1) of seaweed extract enriched Artemia nauplii fed shrimp was 54–79%. Comparatively, low mortality was observed in S. wightii extract‐enriched Artemia nauplii fed shrimp. The polymerase chain reaction analysis indicated the concentration‐dependent infection of WSSV in P. monodon PL.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.     

Prevalence of white spot syndrome virus infection detected by one-step and nested PCR in selected tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) hatcheries

White spot syndrome (WSS) is considered as a great threat to commercial farming of the tiger shrimp (Penaeus monodon). The causal agent of WSS is a DNA virus called white spot syndrome virus (WSSV). The prevalence of this dreadful virus infection has been studied in five randomly selected hatcheries located in the Cox’s Bazar district of Bangladesh. Both one-step and nested polymerase chain reaction (PCR) involving two pairs of primers, namely, 146F1/146R1 and 146F2/146R2, amplifying the 1447 bp and 941 bp fragments, respectively, were conducted to detect the WSSV. Out of 60 randomly collected shrimps, 12 (20%) were found to be positive by one-step PCR, while 18 (30%) were found to be positive by nested PCR. The nested PCR was found to be much more sensitive than the one-step PCR. The shrimp specimens showing clinical signs of WSS were positive for WSSV by both one-step and nested PCR. Some of the apparently healthy samples were also found to be positive for WSSV by nested PCR. Among the two primer-pairs, the inner pair amplifying the 941 bp fragment was more sensitive than the outer primer pair amplifying the 1447 bp fragment when used in one-step PCR.     

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Limited evidence for genetic variation for resistance to the white spot syndrome virus in Indian populations of Penaeus monodon

There has been a highly detrimental impact of the white spot syndrome virus (WSSV) on black tiger shrimp (Penaeus monodon) aquaculture in India. Currently, no cost‐effective measures are available for controlling the disease. One alternative is to improve WSSV resistance through a selective breeding programme for disease‐resistant shrimp, provided that genetic variation exists for this trait. The aim of this study was to evaluate the evidence for genetic variation in resistance to WSSV in P. monodon sourced from Indian populations. Post‐larval shrimp (n=1950) from 54 full‐sibling families were challenged with WSSV using WSSV‐infected mince meat. The heritability was estimated using four different statistical models fitted to the resulting time to death data, including two linear models and two Weibull proportional hazard frailty models. None of the estimated heritabilities were significantly different from zero. We suggest three possible explanations for these results: there actually is very little variation between P. monodon in WSSV resistance and all individuals are highly susceptible to the disease; there is genetic variation in resistance to WSSV in P. monodon but we did not find it in our experiment because the level of challenge in the experiment was too high to allow genetic differences to be expressed; the variation is due to mutations conferring resistance, which are at a low frequency in the population, and we did not sample a broad enough genetic base to capture these mutations.     

Passive immunity in tiger shrimp (Penaeus monodon) fed with monoclonal antibody to white spot syndrome virus

A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p < 0.05), respectively, during 15 days post-challenge. MAb was stable in shrimp for passive immunization against WSSV and could be a potential tool for prophylaxis against the virus.     

Antiviral Effect of Fucoidan Extracted from the Brown Seaweed,Sargassum wightii,on Shrimp Penaeus monodon Postlarvae against White Spot Syndrome Virus

This study was carried out to determine the effect of seaweed polysaccharide, fucoidan from brown seaweed, Sargassum wightii, on Penaeus monodon postlarvae (PL) against white spot syndrome virus (WSSV). The fucoidan was extracted from S. wightii, and the yield was observed as 2.832 ± 0.204%. Artemia franciscana nauplii were enriched with extracted fucoidan at four different concentrations (100, 200, 300, and 400 mg/L) and fed to shrimp P. monodon PL for 20 d. After feeding experiment, the P. monodon PL were challenged with WSSV, and the mortality percentage was recorded daily up to 21 d. During the challenge test, the control PL showed 100% cumulative mortality within 9 d, but the fucoidan‐enriched Artemia nauplii fed groups of PL exhibited 60–94% cumulative mortality within 21 d. The reduction in mortality percentage of experimental groups of PL over control PL was ranged between 33.71 and 61.65%. The polymerase chain reaction analysis confirmed the concentration‐dependent variation in WSSV infection.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

A Microsatellite DNA Marker Developed for Identifying Disease‐resistant Population of Giant Black Tiger Shrimp,Penaeus monodon

White spot disease caused by white spot syndrome virus (WSSV) poses major problems that result in huge economic losses each year in shrimp aquaculture throughout the world. In the present study, microsatellite‐based DNA fingerprints have been compared between naturally occurring WSSV disease‐resistant and susceptible populations of giant black tiger shrimp, Penaeus monodon, to find DNA markers. For the first time, we report here a microsatellite locus, which, after amplification by polymerase chain reaction, provides a highly statistically significant DNA fingerprint of 71 bp, only in disease susceptible populations but not in disease‐resistant shrimp populations, whereas a 317 bp band is common in both. The absence of the former DNA marker will be very useful to identify disease‐resistant broodstock of P. monodon for marker‐assisted selection in breeding programs to generate disease‐free shrimps (P. monodon) in the aquaculture industry.     

The incidence of suspected white spot syndrome virus in semi‐intensive and extensive shrimp farms in Bangladesh: implications for management

The study was conducted to assess key factors influencing suspected white spot syndrome virus (WSSV) disease and associated shrimp production and economic performance in three contrasting black tiger shrimp (Penaeus monodon) culture technologies promoted by the United States Agency for International Development funded Shrimp Quality Support Project (SQSP) in Bangladesh. A total of 350 traditional, 315 Modified Traditional Technology1 (MTT1), 36 MTT2 and 88 Closed System Technology (CST) farmers from 10 sub‐districts in three districts of Khulna division were surveyed following random sampling at the end of the project. Binomial probit regression analysis revealed that smaller newly constructed ponds (known locally as gher) were less susceptible to WSSV, provided aquatic weeds were controlled using chemicals. Removal of sludge from ghers also had a positive effect, irrespective of technology and location. It was also shown that stocking of screened shrimp postlarvae (PL) does not guarantee protection against WSSV (t = 1.39, P > 0.05). Higher shrimp production was obtained by farmers practicing CST, followed by those operating MTTs and traditional technology respectively. Farmers who adopted CST also gained higher profitability followed by those operating MTT1, MTT2 and traditional technology.     

Assessment of transmission risk in WSSV-infected shrimp Litopenaeus vannamei upon cooking

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.     

White spot syndrome virus (WSSV) prevalence associated with disease resistance among wild populations of black tiger shrimp,Penaeus monodon (Fabricius)

White spot disease caused by white spot syndrome virus (WSSV) is the major issue of huge economic destruction globally in the shrimp aquaculture industry. In the present investigation, WSSV prevalence associated with disease resistance was studied among wild black tiger shrimp, Penaeus monodon (Fabricius) from four distant geographic locations along the East coast of India during 2009–2010. Results suggested that the WSSV prevalence in wild P. monodon was the highest (56.2%) in Chennai, Tamil Nadu followed by Digha, West Bengal (10.9%), Visakhapatnam, Andhra Pradesh (0.6%) and Chilika, Orissa (0%). Quantitative data suggested that the mean copy number of WSSV among these four places was 1.4 × 10⁶, 4.6 × 10⁴, 1.6 × 10² and 2.3 × 10² copies μg^?1 shrimp genomic DNA respectively. The disease resistant prevalence using the 71 bp microsatellite DNA marker was the highest among Chilika, Orissa (63.6%) and Visakhapatnam, Andhra Pradesh (63.5%). Higher WSSV prevalence in Chennai, Tamil Nadu and Digha, West Bengal corresponded to lower disease resistant prevalence (24% and 40.2%). Conclusively, probably collection of broodstock of P. monodon from places like Chilika and Visakhapatnam would be a much safer approach for the development of specific pathogen‐resistant shrimp aquaculture.     

Effect of high water temperature (33 °C) on the clinical and virological outcome of experimental infections with white spot syndrome virus (WSSV) in specific pathogen-free (SPF) Litopenaeus vannamei

White spot syndrome virus (WSSV) is the most lethal pathogen of cultured shrimp. Previous studies done with undefined WSSV titers showed that high water temperature (32-33 °C) reduced/delayed mortality of WSSV-infected shrimp. This study evaluated the effect of high water temperature on the clinical and virological outcome of a WSSV infection under standardized conditions. Groups of specific pathogen-free Litopenaeus vannamei were challenged either by intramuscular or oral routes with a low (30 SID₅₀) or a high (10,000 SID₅₀) virus titer. Shrimp were kept (i) continuously at 27 °C, (ii) 30 °C or (iii) 33 °C; (iv) maintained at 33 °C before challenge and 27 °C afterwards, or (v) kept at 27 °C before challenge and 33 °C afterwards. Shrimp were maintained at the respective temperatures for 120 h before challenge and 120-144 h post challenge (hpc). Gross signs and mortality were monitored every 12 h until the end of the experiment. Dead and surviving shrimp were screened for WSSV infection (VP28-positive cells) by indirect immunofluorescence (IIF). Shrimp kept continuously at 27 °C or 30 °C, or switched to 27 °C post challenge developed gross signs within 24 hpc, first mortalities at 36-60 hpc and 100% cumulative mortality between 60 and 144 hpc depending on the virus titer. All dead shrimp were WSSV-positive. In contrast, shrimp kept at 33 °C continuously or after WSSV challenge showed no signs of disease and low mortalities (0-30%) regardless of the virus titer. Dead and surviving shrimp were WSSV-negative. Further, early virus replication was studied in two groups of shrimp: one maintained at 27 °C before and after challenge and one switched from 27 °C to 33 °C after challenge with 10,000 SID₅₀. Immunohistochemistry (IHC) analysis showed that WSSV-positive cells were first displayed at 12 hpc in shrimp kept at 27 °C and by 24 hpc the infection became systemic. In contrast, shrimp kept at 33 °C did not display WSSV-positive cells at 12 or 24 hpc. This work confirms previous reports that high water temperature prevents the onset of disease and significantly reduces mortality of WSSV-inoculated shrimp regardless of the route of inoculation or virus titer used. This strategy may have practical applications to control WSSV in tropical shrimp farming countries.     

凡纳滨对虾LvRab5B蛋白与IHHNV病毒蛋白的互作

为了探究LvRab5B蛋白在凡纳滨对虾抗病毒感染中的作用,实验分别构建了LvRab5B蛋白在昆虫和酵母细胞中的融合表达载体,将不同的载体导入不同的细胞中,利用免疫荧光和酵母双杂交的方法研究了Lv Rab5B蛋白在昆虫细胞中的表达以及Lv Rab5B蛋白与病毒IHHNV之间的互作关系;通过qRT-PCR方法研究了该蛋白在健康对虾不同组织中的表达情况以及凡纳滨对虾分别感染IHHNV和WSSV后不同时间点的相对表达量。结果显示,Lv Rab5B基因融合蛋白能够在昆虫细胞中表达;Lv Rab5B蛋白与IHHNV病毒衣壳蛋白CP无相互作用,而与非结构蛋白NS1相互作用明显,与非结构蛋白NS2作用较弱。qRT-PCR结果显示,LvRab5B基因在凡纳滨对虾心脏、鳃腺、肠道、胃、肝胰脏和肌肉中都表达,在肠道中表达量最高,肝胰脏次之;Lv Rab5B蛋白在凡纳滨对虾机体感染病毒前后的表达情况不同,感染初期表达降低,随后迅速上升,末期下降。研究表明,LvRab5B基因参与凡纳滨对虾抵抗IHHNV和WSSV病毒的先天免疫过程,为进一步研究Lv Rab5B蛋白在对虾机体中的免疫功能及作用机制奠定了基础。