Silencing Pacific white shrimp Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus

White spot syndrome virus (WSSV) is a major threat for farmed shrimp worldwide. RNA interference (RNAi) is the most recent tool against viral diseases. Rab7 silencing effectively inhibited virus infections in juvenile shrimp, but the antiviral effect in brooders remains unknown. This study found a homologue Penaeus monodon Rab7 gene in Litopenaeus vannamei brooders from Mexico. Sequence identity was >99% to a Thai LvRab7 sequence and >94% to Rab7 sequences from P. monodon or Marsupenaeus japonicus. Animals treated with a partial (494 bp) or a complete (618 bp) LvRab7 dsRNA sequences and challenged 48 h post treatment (hpt) with a high WSSV dose showed 80–88% mortality respectively. Shrimp treated with 4 or 20 μg LvRab7 dsRNA and challenged with a WSSV high dose had 80% mortality each, but it was reduced to 33% and 40%, respectively, with a low dose. Efficacy of dsRNA to reduce shrimp mortality was dependent on virus dose used regardless of dsRNA concentration. A significant reduction in LvRab7 mRNA levels was observed at 120 hpt. In conclusion, silencing LvRab7 in brooders showed a mild antiviral effect against a WSSV challenge at 48 hpt.     

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.     

White spot syndrome virus (WSSV) infection and immunity responses in white shrimp (Litopenaeus vannamei) exposed to sublethal levels of metals

To determine if exposure to a sublethal mixture of metals (Cd, Cu, Fe, Mn, Pb and Zn) increases susceptibility to White spot syndrome virus (WSSV) infection, Litopenaeus vannamei juveniles were fed WSSV‐infected shrimp tissues after 21 days of exposure to the metal mixture (WS‐MM treatment). Other treatments consisted of shrimp not exposed to metals and fed infected tissues (WS), and shrimp fed healthy tissues and exposed (MM) or not exposed to metals (C). The presence of viral DNA and inclusion bodies was detected at 32 hr postinfection (hpi) in the stomach epithelium of shrimp from the WS treatment, and eight hours later in shrimp from the WS‐MM treatment, possibly because of an initial negative effect of metals in viral replication. At 40 hpi, the severity of infection represented by the histopathological index increased in both WS and WS‐MM treatments, and values were higher in WS‐MM than in WS shrimp at the end of the experiment. From 56 hpi to the end of experiment, total hemocyte counts were lower in both WS‐MM and WS treatments, and concentrations were particularly low in WS‐MM shrimp. Conversely, phenoloxidase activity was higher in the WS‐MM treatment from 32 to 56 hpi, suggesting a possible role of the prophenoloxidase (proPO) system in the antiviral defense against WSSV. The presence of heavy metals at sublethal concentrations may increase shrimp susceptibility to WSSV; this is supported by a decrease in circulating hemocytes, an increase in the humoral response, and the development of a higher number of WSSV inclusion bodies.     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north‐western Mexico

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10‐fold serial dilution of tissue infected with strain WSSV Mx‐H, and the virulence of four WSSV strains from north‐western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD₅₀ of the Mx‐H strain was a dilution dose of 10^?7.5; the mortality titre was 10^9.2 LD₅₀ per gram. In shrimp injected with 10^2.5 to 10^6.5 LD₅₀, no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx‐F strain (intermediate virulence) and the Mx‐C strain (high virulence) showed more genetic differences than those observed between the Mx‐G (low‐virulence) and Mx‐H (high‐virulence) strains, in ORF94 and ORF125. The application of high‐viral‐load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

Potential role of LvDscam in specific immune response of Litopenaeus vannamei against white spot syndrome virus by oral delivery of VP28 using Bacillus subtilis

Envelope protein VP28 has been suggested as a candidate vaccine component to evoke a better protection against white spot syndrome virus (WSSV). We have reported that Bacillus subtilis spores harbouring VP28 (rVP28‐bs) can specifically protect shrimp against WSSV. However, the mechanism that supports the production of unique molecules induced by rVP28‐bs to trigger specific immunity is originally unknown. It has recently been suggested that Dscam (Down syndrome cell adhesion molecule) plays an essential role in the alternative adaptive immunity of invertebrates. In this study, we compared the diversity of Litopenaeus vannamei Dscam (LvDscam) variable regions by different antigens immunization. A total of 13, 15 and 11 expressed alternative sequences were identified for N‐terminal Ig2, N‐terminal Ig3 and the entire Ig7 domain, respectively. More than half of the unique variants (16 out of 22) were found in the Ig2/Ig3 domains. Further analysis of the interaction between VP28 and unique Ig2/Ig3 variants was confirmed by both yeast two‐hybrid and GST pull‐down approach. We also found that the percentage of haemocytes phagocytosing WSSV was significantly higher (P < 0.001) in the shrimp injected with control‐siRNA (43.8 ± 2.2) than those with Dscam‐siRNA (11.3 ± 5.4) in the rVP28‐bs groups. With Dscam‐siRNA injection, survivorship significantly decreased (P < 0.001) in the rVP28‐bs group after WSSV challenge. Our data suggested that LvDscam‐mediated pathway may be involved in the specific immune response of shrimp against WSSV induced by rVP28‐bs.     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Immune responses of whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.     

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.     

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan.     

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.     

Validation of a feeding strategy to deliver bacterially expressed dsRNA to marine amoeba from the genus Neoparamoeba

RNA interference (RNAi) mediated by double stranded RNA (dsRNA) has emerged as one of the most promising techniques to study gene function of non‐model protozoan parasites. We have previously demonstrated that bacterially expressed dsRNA delivered by immersion elicited successful knockdown in Neoparamoeba pemaquidensis, the non‐infective species closely related to the causative agent of salmonid amoebic gill disease (AGD). However, considering that amoebae naturally feeds on microorganisms, direct ingestion of bacteria designed to express dsRNA could allow rapid and low‐cost analysis of gene function on a large‐scale. Therefore, the main objective of this study was to investigate whether ingestion of bacteria‐expressing dsRNAs would also induce suppression of N. pemaquidensis β‐actin and EF1α. Despite effective bacterial uptake, no significant variation in EF1α relative copy number was triggered by dsRNA ingestion. β‐actin, on the other hand, presented similar silencing efficiency to what was observed in our previous soaking study. However, the observed RNAi response was delayed by at least 72 h. The present work provides evidence that delivery of bacterially expressed dsRNA through feeding can be successfully achieved in N. pemaquidensis, albeit not as efficiently as by soaking. Therefore, further investigation is required to develop more efficient and specific RNAi delivery systems in Neoparamoeba species. To our knowledge, this is the first time that RNAi‐mediated knockdown through ingestion was attempted to manipulate gene function of a marine amoeba.     

Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks

White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major ‘indel regions’ found at ORF14/15 and ORF23/24 (WSSV‐Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV‐infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non‐outbreak ponds. Although a higher mean RU number was observed in the improved‐extensive system than in the rice‐shrimp or semi‐intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.     

Highly efficient double‐stranded RNA transfection of penaeid shrimp using cationic liposomes

Yellow head virus (YHV) is a viral pathogen of shrimp that has caused high economic loss in the Thai shrimp farming industry, and the RNA interference mechanism has potential as a virus control strategy, as previous studies have shown that injection of virus specific double‐stranded RNA (dsRNA) can inhibit viral replication in shrimp. However, to date, complete inhibition viral replication has not been achieved. This study sought to determine whether cationic liposomes mixed with an antiviral dsRNA (YHV‐Pro‐dsRNA) were able to enhance protection against YHV. The results showed that injection of a YHV‐Pro‐dsRNA/cationic liposome complex markedly increased protection against YHV as compared with naked YHV‐Pro‐dsRNA, suggesting that cationic liposomes enhance the uptake of protective dsRNA into shrimp cells.     

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms

White spot syndrome virus (WSSV)‐infected shrimp samples collected from grow‐out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next‐generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV‐NS (new strain) relative to WSSV‐TH and WSSV‐LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV‐NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV‐LS and 402 bp for WSSV‐NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV‐LS by WSSV‐NS in Indian shrimp culture system.