Comparison of five commercial kits for isolation of total RNA in samples of WSSV-infected shrimp.

Viral diseases are the most serious threat to the expansion and development of shrimp aquaculture. Rapid diagnosis of the white spot syndrome virus (WSSV), a lethal shrimp pathogen, is essential to restrict its spread and reduce the mortality of infected shrimp. This virus has globally affected the shrimp farming industry, with a devastating economic impact. Several studies have focused on the expression of WSSV transcripts to understand the molecular mechanisms governing the pathological development of the disease. Since gene expression studies and molecular diagnostics at the early stages of infection depend on the efficient isolation of high-quality RNA, the extraction methods should be carefully selected. However, previous comparisons of the performance of RNA isolation kits have yet to be systematically investigated. In this study, 5 commercial RNA extraction methods were compared in WSSV-infected shrimp. The highest total RNA yield (ng mg-1 tissue) was obtained using TRIzol. Even though the 260/280 nm absorption ratios showed significant differences, the methods showed good purity values (>2.0). RNA integrity was evaluated in a denaturing agarose gel electrophoresis, and degradation was observed after the total RNA samples were treated with DNase I. Finally, the method that allowed the earlier detection of WSSV transcripts by qRT-PCR was the Zymo Direct-zol RNA MiniPrep kit. This study shows that the amount of observed (or estimated) WSSV transcripts might be affected because of the RNA isolation method. In addition, these results may contribute to improve the accuracy of the results obtained in gene expression studies, for more sensitive and robust detection of WSSV.

Effect of functional diets on intestinal microbiota and resistance to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) of Pacific white shrimp (Penaeus vannamei).

AIMS: The present study evaluated the effect of four functional diets and a reference diet on the survival and intestinal bacterial community of shrimp Penaeus vannamei infected with acute hepatopancreatic necrosis disease (AHPND). METHODS AND RESULTS: After 42 days of feeding trail, shrimp were inoculated with a Vibrio parahaemolyticus (CIB-0018-3) carrying the plasmid encoding for the PirAB toxins responsible for AHPND. After 120 h postinfection (hpi), shrimp fed with a diet containing 2% of a mix with Curcuma longa and Lepidium meyenii (TuMa) and a diet containing 0.2% of vitamin C (VitC) showed a significantly higher survival (85%) compared to the remaining treatments (50%-55%) (p < 0.05). Infected shrimp fed with TuMa diet, showed a significant reduction of Vibrionales, and VitC diet promoted an increase of Alteromonadales. CONCLUSIONS: Our findings suggest that the TuMa diet conferred protection against AHPND and could be attributed to a combined effect of antibacterial properties against Vibrionales, and promoting a desirable bacterial community in the shrimp intestine, while the VitC diet protection could be attributed to their antioxidant capacity and in a lower proportion to a bacterial modulation in shrimp gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Acute hepatopancreatic necrosis disease is a devastating disease that significantly affects aquaculture production of shrimps. Therefore, the use of functional diets that promote resistance to AHPND represents a valuable tool to reduce the mortality of farmed shrimp.

Adaptive mitochondrial response of the whiteleg shrimp Litopenaeus vannamei to environmental challenges and pathogens.

In most eukaryotic organisms, mitochondrial uncoupling mechanisms control ATP synthesis and reactive oxygen species production. One such mechanism is the permeability transition of the mitochondrial inner membrane. In mammals, ischemia-reperfusion events or viral diseases may induce ionic disturbances, such as calcium overload; this cation enters the mitochondria, thereby triggering the permeability transition. This phenomenon increases inner membrane permeability, affects transmembrane potential, promotes mitochondrial swelling, and induces apoptosis. Previous studies have found that the mitochondria of some crustaceans do not exhibit a calcium-regulated permeability transition. However, in the whiteleg shrimp Litopenaeus vannamei, contradictory evidence has prevented this phenomenon from being confirmed or rejected. Both the ability of L. vannamei mitochondria to take up large quantities of calcium through a putative mitochondrial calcium uniporter with conserved characteristics and permeability transition were investigated in this study by determining mitochondrial responses to cations overload. By measuring mitochondrial swelling and transmembrane potential, we investigated whether shrimp exposure to hypoxia-reoxygenation events or viral diseases may induce mitochondrial permeability transition. The results of this study demonstrate that shrimp mitochondria take up large quantities of calcium through a canonical mitochondrial calcium uniporter. Neither calcium nor other ions were observed to promote permeability transition. This phenomenon does not depend on the life cycle stage of shrimp, and it is not induced during hypoxia/reoxygenation events or in the presence of viral diseases. The absence of the permeability transition phenomenon and its adaptive meaning are discussed as a loss with biological advantages, possibly enabling organisms to survive under harsh environmental conditions.

White spot syndrome virus down-regulates expression of histones H2A and H4 of Penaeus vannamei to promote viral replication.

The white spot syndrome virus (WSSV) is a highly lethal and contagious pathogen to most cultivated shrimp species. The WSSV genome contains the ICP11 gene and its encoded protein acts as a multifunctinal DNA mimic protein that disrupts the nucleosome assembly by binding to the histone proteins H2A and H3. In addition, WSSV provokes severe nuclear hypertrophy and DNA damage. However, little is known about the influence of WSSV on the expression of the host's genes encoding for histones. Therefore, we investigated the effect of WSSV infection on the expression of the genes encoding histones in shrimp Penaeus vannamei. An RT-qPCR assay was performed to evaluate the temporal expression of H2A and H4 transcripts in the shrimp. Significant changes were observed in the expression of these genes, which coincided with the dynamics of replication of the virus. H2A reached its maximum expression levels at 12 hpi. Thus, it may be suggested that this is a viral strategy to evade the host's immune response in order to promote viral replication.

The white spot syndrome virus hijacks the expression of the Penaeus vannamei Toll signaling pathway to evade host immunity and facilitate its replication.

The white spot syndrome virus (WSSV), the most lethal pathogen of shrimp, is a dsDNA virus with approximately a 300,000 base pairs and contains approximately 180-500 predicted open reading frames (ORFs), of which only 6% show homology to any known protein from other viruses or organisms. Although most of its ORFs encode enzymes for nucleotide metabolism, DNA replication, and protein modification, the WSSV uses some of its encoded proteins successfully to take control of the metabolism of the host and avoid immune responses. The contribution of the shrimp innate immune response to prevent viral invasions is recognized but yet not fully understood. Thus, the role of several components of Toll pathway of the shrimp Penaeus vannamei against WSSV has been previously described, and the consequential effects occurring through the cascade remain unknown. In the current study the effects of WSSV over various components of the shrimp Toll pathway were studied. The gene expression of Spätzle, Toll, Tube, Cactus and Dorsal was altered after 6-12 h post inoculation. The expression of LvToll3, LvCactus, LvDorsal, decreased ~4.4-, ~3.7- and ~7.3-fold at 48, 24 and 48 hpi, respectively. Furthermore, a remarkable reduction (~18-fold) in the expression of the gene encoding LvCactus in WSSV infected specimens was observed at 6 hpi. This may be a sophisticated strategy exploited by WSSV to evade the Toll-mediated immune action, and to promote its replication, thereby contributing to viral fitness.

Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

Selection and validation of candidate reference genes for quantitative real-time PCR studies in the shrimp Penaeus vannamei under viral infection.

The decapod Penstyldensovirus 1 (PstDV-1) represents one of the most serious threats for penaeid shrimp farming. Studies aimed at defining relevant molecular effects of this virus over its host are imperative in the attempt to increase our understanding of its pathogenesis. Unfortunately, few studies have focused on the definition of the expression profile of reference genes in shrimp challenged with a pathogen. As a result, there are no studies on the selection of reference genes for the normalization of target gene expression changes yielding reliable data of the effects following PstDV-1 infection in shrimp. Therefore, the aim of the present study was to evaluate and validate the appropriateness of four candidate reference genes (ef1-α, gapdh, rpl8 and ß-tubulin) for their use as reference genes to normalize qPCR data in gene expression studies of PstDV-1-shrimp interactions. By analyzing the expression profile of those genes, gapdh was validated as a suitable reference gene to normalize expression data gathered from a PstDV1-challenge, while ef1-α, ß-tubulin, and rpl8 were identified as unstably expressed during the infectious process. The suitability of gapdh as a common reference gene in studies of host gene response to viral infections is underlined.

High Occurrence of the Decapod Penstyldensovirus (PstDV1) Detected in Postlarvae of Penaeus vannamei Produced in Commercial Hatcheries of Mexico.

The decapod penstyldensovirus (PstDV1) is a widely spread shrimp pathogen that causes high mortalities in the shrimp Penaeus stylirostris, while in P. vannamei, it has been associated with induction of the runt deformity syndrome. Using shrimp post-larvae (PL, stages PL13-PL21) collected from 16 commercial hatcheries from Mexico, and a sensitive PCR protocol for its detection, a survey of the PstDV1 prevalence in larvae was undertaken. A high overall prevalence of PstDV1 (49.5 %) in shrimp PL from the studied hatcheries was found. This study reveals that PstDV1 occurs persistently in PL populations, which may have significant implications for its dispersal.

An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp.

The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico.

The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock.

Marine viruses: the beneficial side of a threat.

Marine viruses are ubiquitous, extremely diverse, and outnumber any form of life in the sea. Despite their ecological importance, viruses in marine environments have been largely ignored by the academic community, and only those that have caused substantial economic losses have received more attention. Fortunately, our current understanding on marine viruses has advanced considerably during the last decades. These advances have opened new and exciting research opportunities as several unique structural and genetic characteristics of marine viruses have shown to possess an immense potential for various biotechnological applications. Here, a condensed overview of the possibilities of using the enormous potential offered by marine viruses to develop innovative products in industries as pharmaceuticals, environmental remediation, cosmetics, material sciences, and several others, is presented. The importance of marine viruses to biotechnology should not be underestimated.

The endemic copepod Calanus pacificus californicus as a potential vector of white spot syndrome virus.

The susceptibility of the endemic copepod Calanus pacificus californicus to white spot syndrome virus (WSSV) was established by the temporal analysis of WSSV VP28 transcripts by quantitative real-time PCR (qRT-PCR). The copepods were collected from a shrimp pond located in Bahia de Kino Sonora, Mexico, and challenged per os with WSSV by a virus-phytoplankton adhesion route. Samples were collected at 0, 24, 48 and 84 h postinoculation (hpi). The VP28 transcripts were not detected at early stages (0 and 24 hpi); however, some transcript accumulation was observed at 48 hpi and gradually increased until 84 hpi. Thus, these results clearly show that the copepod C. pacificus californicus is susceptible to WSSV infection and that it may be a potential vector for the dispersal of WSSV. However, further studies are still needed to correlate the epidemiological outbreaks of WSSV with the presence of copepods in shrimp ponds.

Evaluation of the productive and physiological responses of Litopenaeus vannamei infected with WSSV and fed diets enriched with Dunaliella sp.

An experimental investigation was performed to evaluate the productive and physiological responses of Litopenaeus vannamei that were infected with WSSV and fed diets enriched with Dunaliella sp., which had a high ß-carotene content induced by nitrogen reduction in the culture medium. A basal diet containing 35% crude protein and experimental diets T1 and T2, which included 1% and 2% microalgae meal, respectively, were evaluated. Positive (infected juveniles) and negative (non-infected) controls were also evaluated. Survival was significantly higher (80%) in the two treatment groups compared to the positive control group (56%). In the negative control group, survival was 100%. Some variation was recorded for hemolymph metabolites among treatments and at distinct times post-infection; although the tendencies were not clear, some metabolites (glucose and triglycerides) appeared to decrease on the last days of the trial, probably due to their use as energy for the shrimp to thrive despite the infection. The results of the study suggest a positive effect of the dietary inclusion of Dunaliella meal on shrimp survival and an unclear effect on hemolymph metabolites.

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans.

BACKGROUND: The White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry. METHODS: In this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations. RESULTS: A universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method. CONCLUSION: The designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a single-stranded DNA virus that infects several penaeid shrimp species, provoking economic losses in farmed shrimp populations estimated at several million of dollars. Furthermore, IHHNV has historically been considered an important threat for wild shrimp populations, but its real measurable impact remains unknown. Currently no treatments are available against IHHNV, and research to develop potential antiviral strategies depends on a detailed understanding of the viral life cycle. However, the exact pathophysiological events underlying the development of metabolic changes in IHHNV-infected shrimp are still unknown. Thus, the biochemical changes caused by the IHHNV infection in plasma and hepatopancreas of the economically important shrimp species Penaeus vannamei were evaluated. Glucose, lactate, total protein, glycogen, triacylglycerides, cholesterol, and total lipids were measured in healthy and IHHNV-infected shrimp. Significant changes were observed in energy substrates (glucose, lactate, triacylglycerides and cholesterol), in plasma and hepatopancreas. These changes may indicate a temporal sequestration of the host-cell metabolic pathways by the virus to maximize its replication and propagation.

Experimental infection and detection of necrotizing hepatopancreatitis bacterium in the American lobster Homarus americanus.

Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.