White Spot Syndrome Virus Load Research Articles

Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene PvProsaposin during white spot syndrome virus infection.

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in Penaues vannamei hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene, PvProsaposin, was evident and their interaction was confirmed by a reduction in luciferase activity in vitro. Suppression of PvProsaposin in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when PvProsaposin was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of PvProsaposin transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the PvProsaposin, an anti-apoptotic gene. PvProsaposin inhibition disrupts hemocyte homeostasis, rendering the shrimp's inability to withstand WSSV invasion.IMPORTANCEGene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp's survival during infection. The upregulated miRNA pva-miR-166 could repress PvProsaposin expression in shrimp hemocytes infected with WSSV. The significance of PvProsaposin in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in PvProsaposin-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-PvProsaposin on the expression of PvProsaposin also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene PvProsaposin by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.

Comparative transcriptomic analysis of Chinese shrimp Fenneropenaeus chinensis infected with white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most lethal viruses in shrimp aquaculture. Compared with other shrimp species, the Chinese shrimp (Fenneropenaeus chinensis) is more susceptible to WSSV. The mortality of WSSV-infected F. chinensis can reach 100% within a week. No effective solution currently exists to solve the WSSV problem in F. chinensis aquaculture. After years of research, we found that individual F. chinensis from the same family can carry different viral load at the same time post-WSSV infection. To better understand the anti-WSSV mechanism of F. chinensis, we analysed the transcriptomic data from F. chinensis with high and low WSSV load at the same time post-WSSV infection and from healthy F. chinensis, in a WSSV-resistant group and a WSSV-susceptible group, respectively. We identified more differentially expressed genes in F. chinensis with high WSSV load than in those with low WSSV load at the same time post-WSSV infection. Many differentially expressed genes were enriched in Gene Ontology terms related to binding function and the extracellular matrix-receptor interaction pathway, which might play important roles in the anti-WSSV processes among F. chinensis.

Luteolin in Lonicera japonica inhibits the proliferation of white spot syndrome virus in the crayfish Procambarus clarkii

White spot disease caused by the white spot syndrome virus (WSSV) is a highly lethal infectious disease of crustacean aquaculture, with no effective means of control. Active compounds from herbal medicines are promising candidates for the development of antiviral agents for WSSV. We evaluated the efficacy of 12 herbal medicines in inhibiting WSSV proliferation in a WSSV infection model (Procambarus clarkii), Lonicera japonica extract had the strongest inhibitory effect on WSSV replication. Luteolin (LUT), the major bioactive compound of L. japonica extract, was further investigated and revealed to have a strong inhibitory effect on WSSV (96.04%, 50 mg/kg). LUT treatment decreased the WSSV load and the expression of important viral genes (immediate-early gene ie1, DNA polymerase DNApol and envelope protein Vp28) in a dose-dependent manner and increased the survival of WSSV-infected crayfish by 60.00% (50 mg/kg). Co-incubation and time-of-addition assays revealed that LUT did not affect the infectivity of viral particles or viral absorption and entry. Expression of immune-related genes such as barrier-to-autointegration (BAF), heat shock protein 70 (Hsp70) and signal transducer and activator of transcription (STAT), which may be beneficial for viral replication, were significantly decreased (P < 0.01) in LUT-treated crayfish. LUT also modified the expression of antioxidants (SOD, CAT and GST), pro-inflammatory factor (COX-1 and COX-2), and apoptosis-related (BAX and BI-1) genes (P < 0.01) to enhance antioxidant defenses, mitigate inflammation and induce apoptosis, respectively. Overall, LUT could be used as a prophylactic or therapeutic agent against WSSV infection in crustacean aquaculture.

Intestinal microbiota in white spot syndrome virus infected red swamp crayfish (Procambarus clarkii) at different health statuses

Intestinal microbiota play an important role in animal health and disease. Until recently, the intestinal microbiota in white spot syndrome virus (WSSV)-infected red swamp crayfish (Procambarus clarkii) at different health statuses have not been studied. This study investigated the intestinal microbiota of healthy crayfish (HC), WSSV-infected active crayfish (IAC) and WSSV-infected diseased crayfish (IDC) by high throughput sequencing technology. The relative abundances of certain phyla changed significantly in WSSV-infected crayfish, shown as decreased Tenericutes and increased Proteobacteria in the IAC and IDC groups and increased Bacteroidetes in the IAC group. More core genera and the significantly increased relative abundance of Anaerorhabdus furcosa resulted in increased intestinal species diversity in the IAC group. The relative abundances of intestinal opportunistic Aeromonas, Citrobacter and WSSV loads significantly increased in the IDC group. The IDC group possessed three significantly increased level-2 KEGG pathways with relative abundances over 5%, including membrane transport, amino acids and carbohydrates metabolism, and the largest increased pathway was glycan biosynthesis and metabolism. The findings provide a basis for developing microbial strategies for the prevention and therapy of WSS disease.

Molecular identification of white spot syndrome virus (WSSV) and associated risk factors for white spot disease (WSD) prevalence in shrimp (Penaeus monodon) aquaculture in Bangladesh

White spot disease has caused significant economic losses in the shrimp farming industry of Bangladesh over the last two decades. The responsible virus, WSSV, may show severe disease with significant mortality depending on farm management and environmental and seasonal changes. Data on farm management and environmental parameters were collected from the southwest region of Bangladesh in 2018, and WSSV infection was confirmed by the species-specific gene VP28 using conventional PCR, real-time PCR and sequencing. Through bivariate analysis, nine significant risk factors for WSD were identified, viz. farm age, presence of nursery pond, reservoir of PL, weed in farm area, control of weed, stocking density, stocking frequency, ammonia and oxygen concentration. This study detected 46 WSSV-infected shrimp farms by conventional PCR, whereas real-time PCR identified 47 WSSV-positive out of 49 farms. WSSV prevalence was highest in the Khulna region, with 100% positivity in all seasons. WSSV loads ranged from 5.62 × 109 to 2.01 × 1015 copies/g of shrimp tissue. The VP28 gene sequence confirmed that 15 representative samples were 100% identical to the 2018 WSSV strain of India. The relationships among risk factors, prevalence and severity of disease, and origin of WSSV strains could be impactful for WSD management.

Survival and immune response of white shrimp Litopenaeus vannamei following single and concurrent infections with WSSV and Vibrio parahaemolyticus

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

Circulatory white spot syndrome virus in South-West region of Bangladesh from 2014 to 2017: molecular characterization and genetic variation

White Spot Syndrome Virus (WSSV), the etiological agent of White Spot Disease (WSD) is a major impediment for shrimp aquaculture in the worldwide. A critical threshold level of WSSV load in infected shrimp is an important trait for disease manifestation and WSSV transmission in cultured shrimp and subsequently make outbreaks. The present study investigated 120 naturally infected cultured shrimp samples by SYBR Green based qPCR assay for WSD diagnosis and quantification of WSSV load. Among them, 94 samples resulted a variable count of WSSV load ranging from 2.1 × 108 to 2.64 × 1014 copies/g of shrimp tissue. The severity of WSSV infection was assessed based on the established critical threshold load of WSSV in shrimp tissue. Compared to the established critical threshold value of WSSV load in shrimp tissue, our findings showed the horrifying scenario of the severity of WSSV infection in cultured shrimps of Bangladesh that was found to be above the critical limit to initiate an outbreak in the Bangladeshi shrimp aquaculture industry. The latest phylogenetic pattern was altered from the former monophyletic history among WSSVs of Bangladesh due to a variation at 500th nucleotide of VP28 coding gene. Viruses characterized from recent outbreaks in 2015 and 2017 displayed amino acid substitution at position 167 (G→E) on the surface of VP28 protein which has demonstrated the probable replacement of indigenous virus pool. Therefore, it is imperative to take initiative for the management and prevention of WSSV outbreak to sustain shrimp aquaculture in South-West region of Bangladesh.

Relationship between white spot syndrome virus (WSSV) loads and characterizations of water quality in Litopenaeus vannamei culture ponds during the tropical storm.

An in-situ experiment was conducted to investigate the effect of tropical storm on the white spot syndrome virus (WSSV) loads in Litopenaeus vannamei rearing ponds. White spot syndrome virus loads, heterotrophic bacteria, Vibrio and water quality (including temperature, dissolved oxygen (DO), salinity, pH, NH4-N, and NO2-N) were continually monitored through one tropical storm. The WSSV loads decreased when tropical storm made landfall, and substantially increased when typhoon passed. The variation of WSSV loads was correlated with DO, temperature, heterotrophic bacteria count, and ammonia-N concentrations. These results suggested that maintaining high level DO and promoting heterotrophic bacteria growth in the shrimp ponds might prevent the diseases' outbreak after the landfall of tropical storm.

Effect of multiple infections with white spot syndrome virus and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.): mortality and viral replication

Multiple infections are commonly found in practical shrimp culture and may cause more serious consequences than infections by one pathogen only. Therefore, this study was conducted to evaluate the effect of multiple infections with white spot syndrome virus (WSSV) and Vibrio anguillarum on Pacific white shrimp Litopenaeus vannamei (L.), mortality, WSSV replication in vivo and host immune response. In the WSSV single-infection group (WSSV load, 2 × 10(2) copies μL(-1)), mean cumulative mortality was 29.2%. In the V. anguillarum single-infection group, cumulative mortality was 12.5% when shrimp were challenged by 10(5) CFU mL(-1) of bacteria. In the co- and super-infection groups, 37.5% and 50% cumulative mortalities, respectively, were observed at a lower bacterial concentration of 10(3) CFU mL(-1), suggesting that shrimp with multiple infections died earlier and more frequently than singly infected shrimp. WSSV load after injection was tracked over time by TaqMan quantitative PCR. WSSV load increased more rapidly in the multiple-infection groups than in the single-infection group. Additionally, mRNA expression of the genes encoding prophenoloxidase 1 and 2, which are closely involved in innate immunity in shrimp, was down-regulated more extensively in multiple-infection groups than in single-infection groups, as indicated by quantitative reverse-transcription PCR.

TCTP Is a Critical Factor in Shrimp Immune Response to Virus Infection

The translationally controlled tumor protein (TCTP) is an abundant, ubiquitous, and conserved protein which plays important roles in a number of biological processes. In the present study, the TCTP in shrimp Litopenaeus vannamei was analyzed. The TCTP of L.vannamei, a 168-amino-acid polypeptide, shares a high degree of similarity with TCTPs from other species, having two TCTP protein signatures at the 45–55 aa and 123–145 aa motif. The mRNA and protein levels from different tissues were detected with the highest in muscle and the lowest in heart among all examined tissues. In addition, temporal TCTP expression was significantly up-regulated at 16 h and 48 h following infection with white spot syndrome virus (WSSV). Lastly, silencing of TCTP with dsRNA led to a significant increase of WSSV loads. These results provide new insights into the importance of TCTP as an evolutionarily conserved molecule for shrimp innate immunity against virus infection.

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 × 10(3) 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.

Screening and characterization of antimicrobial secondary metabolites from Halomonas salifodinae MPM-TC and its in vivo antiviral influence on Indian white shrimp Fenneropenaeus indicus against WSSV challenge

Antimicrobial secondary metabolites from extremophiles play a significant role in the pharmacological industry due to their stable and strong activity and it is used in the treatment of microbial infections. In the present work, Halomonas salifodinae MPM-TC (M. Peter Marian-T. Citarasu) was isolated from the solar salt works in India and identified by 16S rRNA sequencing. The secondary metabolites were extracted from H. salifodinae MPM-TC and tested for antibacterial activity against aquatic bacterial pathogens such as Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Aeromonas hydrophila isolated from infected fish/shrimp, and it effectively controlled them with more than 10mm of zone of inhibition. The metabolites were purified through silica column chromatography and in vitro antiviral activity was performed against White Spot Syndrome Virus (WSSV) using different fractions. Among the different tested fractions, fraction-III (F-III) was able to suppress WSSV replication. Shrimps challenged with a WSSV inoculum incubated with F-III and treated Fenneropenaeus indicus survived around twice as many as the controls. Gas chromatography–mass spectroscopic (GC–MS) analysis revealed that the antiviral active fraction contains around eight compounds including Perfluorotributylamine, Cyclopentane, 1-butyl-2-ethyl and 1,1′-Biphenyl]-3-amine. Further the active fraction F-III was incorporated in the artificial diets at the concentration of 200 (HS1), 400 (HS2) and 800 (HS3) μgkg−1 and fed to F. indicus for 30days. After 30days of culture, shrimps were challenged with virulent WSSV and studied for WSSV VP 28 gene expression, biochemical, haematological and immunological changes. Surprisingly, groups treated with lower concentrations of fraction F-III (HS1 or HS2) significantly (P<0.05) suppressed the viral replication. Different levels of protein and glucose, improved total haemocyte count (THC), coagulase activity and oxyhaemocyanin level all were comparable to controls. Also, immunological parameters such as prophenol oxidase and intracellular superoxide anion production were significantly increased (F=97.18; P⩽0.001 and F=5.70; P⩽0.05) in the groups treated with the three test concentrations. The presence of antiviral and immunostimulant active principles in the F-III fraction effectively suppressed the WSSV load and boosted F. indicus’s immune system. This research will help to develop novel antiviral drugs from plants against aquatic important pathogens.

Contribution of the Argonaute-1 Isoforms to Invertebrate Antiviral Defense

Argonaute (Ago) protein, the central component of the RNA interference (RNAi) pathway, plays important roles in host innate antiviral immunity. Most organisms harbor a large number of different Ago proteins and isoforms; however, the roles of Ago isoforms in immune defense against pathogens remain unclear. In the present study, three Argonaute-1 (Ago1) isoforms, termed Ago1A, Ago1B, and Ago1C, were found in Marsupenaeus japonicus shrimp. Quantitative real-time PCR (polymerase chain reaction) revealed that isoforms Ago1A and Ago1B containing an insertion sequence in the PIWI domain, were significantly up-regulated in lymphoid organ and hemolymph, and also upon white spot syndrome virus (WSSV) challenge, indicating the involvement of Ago1A and Ago1B in antiviral immunity. The results showed that silencing of Ago1A with a sequence-specific siRNA led to a significant increase of WSSV loads. It was revealed that knockdown of Ago1B mRNA by 37–70% resulted in higher virus loads in shrimp. However, upon silencing Ago1B by more than 85%, a two-fold increase in Ago1A mRNA was observed but viral load was the same as untreated controls challenged with WSSV, suggesting that the simultaneous up-regulation of Ago1A might compensate for the loss of Ago1B. These data indicated that Ago1A played more important roles in the antiviral immune response than Ago1B. The simultaneous inhibition of Ago1A and Ago1B resulted in a greater increase in viral loads than Ago1A or Ago1B alone, indicating that Ago1A and Ago1B isoforms were involved in shrimp antiviral immunity. It was revealed that Ago1C had no effect on virus infection. Therefore, the current study presented the first report on the contribution of Ago isoforms in the invertebrate defense against virus infection.

Tolerance ofFenneropenaeus chinensis“Huanghai No. 2” To While Spot Syndrome Virus

In this study, postlarvae of the fleshy shrimp Fenneropenaeus chinensis “Huanghai No. 2” (a new shrimp variety bred in China) were fed with white spot syndrome virus (WSSV)-toxic bait with amount of 25% of the total shrimp body weight and were evaluated WSSV loads at different postinfection periods (i.e. early, peak and late periods). A total of 83 of 4,424 shrimp (1.88%) survived at the end of the experiment, which lasted 358 h, 662 h, and 232 h in 3 ponds, respectively. The WSSV loads were 8.8 × 104–1.5×105,4.4×105–1.2×106, and 8.5×105–4.6×106 copies/ng DNA, respectively, averaging 9.5×105 copies per anograms DNA of all sampled shrimp (n = 229). The viral loads in the shrimp that died during the late postinfection period were significantly higher than in those shrimp that died during the early and peak periods (P < 0.01). The viral load in surviving shrimp was 2.2×105 (n = 44) copies/ng DNA. Among the surviving shrimp, 18.2% (n = 8) had a viral load greater than 1.0×104 copies/ng DNA and 52.3% (n = 23) had less than 100 copies/ng DNA. Based on these result, the range of 7.0×104–107 copies/ng DNA is suggested to be the lethal amount for shrimp death caused by WSSV. These results could be an important reference in an advance warning of a WSSV outbreak in the culture process of these shrimp, and could provide valuable information in further selective breeding of WSSV-tolerant or -resistant varieties of F. chinensis.

White Spot Syndrome Virus (WSSV) Concentrations in Crustacean Tissues - A Review of Data Relevant to Assess the Risk Associated with Commodity Trade

We have reviewed the available peer reviewed literature on pathogen load for white spot syndrome virus (WSSV) in species susceptible to infection. Data on pathogen load in traded commodities are relevant for undertaking import risk assessments for a specific pathogen. Data were available for several of the major penaeid shrimp species farmed for aquaculture and for one crab and crayfish species. Most data are based on experimental infection, but some data were available for farmed or wild shrimp. Owing to the unavailability of immortal cell lines to determine viral load of viable virus, quantitative PCR was the main method used for quantification. The viral loads measured in shrimp at the onset of mortality events were extremely high (in the order of 10(9) -10(10) copy numbers gram(-1) of tissue). In a farm setting, the onset of increased mortalities will often trigger emergency harvests. Therefore, shrimp obtained from emergency harvests are likely to carry substantial concentrations of viral particles. Viral load did not vary greatly with tissue type. The WSSV load in wild crustaceans, farmed crustaceans not undergoing a mortality event or survivors of a mortality event was significantly lower (usually by multiple logs). Studies have also been undertaken in 'vaccinated' shrimp. One of the 'vaccines' led to a significant reduction of viral load in WSSV-exposed animals. The data obtained from the literature review are put into context with published information on minimal infectious dose and WSSV survival in frozen commodity shrimp.

TaqMan real‐time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan-based real-time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L⁻¹) for 10 days and challenged with WSSV by feeding WSSV-positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post-infection. The TaqMan real-time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33-1.42) and small correlation of variability (CV) (1.89-3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L⁻¹ of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L⁻¹ of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L⁻¹ (TAN) had increased susceptibility to WSSV.

White spot syndrome virus quantification in blue crab Portunus trituberculatus hatchery-produced larvae and wild populations by TaqMan real-time PCR, with an emphasis on the relationship between viral infection and crab health

The blue crab, Portunus trituberculatus, is one of the most important fishery resources in the Yellow Sea of Korea, but the wild stock of this species has been reduced due to over-fishing and the destruction of the natural habitat in this area. Hatchery-produced seeds of blue crab have been released into the sea to enhance stock, but information on viral infection in the larvae as well as wild crabs of this species is very limited. In the present study, TaqMan real-time PCR was applied to quantify white spot syndrome virus (WSSV) in hatchery-produced larvae and wild populations of P. trituberculatus in South Korea. Out of 140 P. trituberculatus zoea from seven commercial hatcheries, 96.4% were WSSV-positive. The mean WSSV copies were 6.0 per ng DNA, or 3216.0 per larva. In 222 adult crabs from four wild populations captured in different seasons, the WSSV-positive rate was 79.3%, and the WSSV load was 5.2 copies per ng DNA, or 2116.5 copies per mg tissue. Both the WSSV-positive rate and the load of the winter population were significantly lower than those of the other three populations. Statistical analysis showed no significant correlations between WSSV infection loads and growth (CL, CW, and BW). The results suggest that low viral load of WSSV may not affect the growth of P. trituberculatus in a wild environment.

Real-time PCR Quantification of White Spot Syndrome Virus (WSSV) and Hepatopancreatic Parvovirus (HPV) Loads in Shrimp and Seawaters of Shrimp Ponds on the West Coast of South Korea

Viral diseases are major emerging problems of shrimp that have affected the production, and even complete losses for shrimp farms. In this study, we developed a sensitive TaqMan real-time PCR method to quantify white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) in the shrimp and pond water in which fleshy shrimp, Fenneropenaeus chinensis, and Pacific white shrimp, Litopenaeus vannamei, are reared. WSSV and HPV in pond seawaters ranged from <TEX>$1.65{\times}10^3$</TEX> to <TEX>$2.43{\times}10^9$</TEX> and from 0 to <TEX>$4.43{\times}10^5$</TEX> copies/L of seawater, respectively. Of 20 ponds analyzed, all pond water and shrimp were positive for WSSv. L. vannamei showed higher susceptibility to WSSV than F chinensis. HPV was detected only in the pond water for F chinensis. In shrimp tissue, however, HPV was found in both species, with 23-times higher infection rate in F chinensis than L. vannamei. The total bacterial counts in the pond water ranged from <TEX>$2.23{\times}l0^3$</TEX> to <TEX>$1.98{\times}l0^5\;CFU/mL$</TEX>. The variations in total bacterial count for each pond appeared to correlate to the variations of the WSSV load. Statistical analysis indicated that there was no significant difference (P>0.05) between the WSSV load in pond water and shrimp, and there was no relationship between total bacterial load and viral load in the pond water. However, a significant difference (P<0.01) was found between HPV load and L. vannamei and F chinensis pond water.

Enhancement of white spot syndrome virus load in hatchery-reared mud crab Scylla serrata (Forsskål, 1775) juveniles at a low temperature

This study was funded by the European Union’s International Cooperation project on Culture and Management of the crab Scylla (INCO-CAMS) headed by Dr. Lewis Le Vay. We thank our co-workers in the hatchery, nursery and grow-out for providing the crab samples — E. T. Quinitio, F. D. Parado-Estepa, E. M. Rodriguez, J. H. Primavera, J. Bin as and J. de Pedro. We also thank the staff of Fish Health Section for their assistance with the diagnosis — M. G. Paner, C. D. Sombito and R. D. Travina.

The effect of acute salinity change on white spot syndrome (WSS) outbreaks in Fenneropenaeus chinensis

In order to observe the effect of salinity on disease resistance and white spot syndrome virus (WSSV) proliferation in Fenneropenaeus chinensis, shrimps with latent WSSV were subjected to two acute salinity changes from the original salinity of 22 ppt to 18 and 14 ppt in an hour, respectively. The total haemocyte count (THC) of the challenged group showed no evident change under salinity adjustments, but the phenoloxidase (PO) index declined significantly ( P < 0.05) corresponding to continuing acute salinity changes from the 24th to the 72nd hour. According to the WSSV load detected by quantitative real-time PCR method, it was found that WSSV carried by the challenged group and control group were significantly different ( P < 0.05); acute salinity change from 22 to 14 ppt led to the WSSV carried in the challenged group being significantly higher ( P < 0.05) than that of those surviving in 22 ppt, but salinity change from 22 to 18 ppt had no such effect. At the end of the 72-h experiment, the challenged group subjected to salinity change from 22 to 14 ppt had nearly 3 times the WSSV load as the control group with no salinity change. Therefore, salinity changes over a particular range could result in a decrease of immunocompetence and obvious WSSV proliferation in the shrimps, leading to white spot syndrome developing from a latent infection to an acute outbreak.