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.

Differential expression of heat shock protein genes in Rhipicephalus sanguineus (Acari: Ixodidae) exposed to temperature and humidity variations.

The brown dog tick (Rhipicephalus sanguineus), a well-recognized parasite that is an important vector of many pathogens, widely distributed in the world, can be found in dogs and many other host animals, including humans. However, the response of this species to climate changes is not fully understood yet. Therefore, this study aimed to analyze the expression of the genes encoding for hsp20 and hsp70 of nymphs and eggs of R. sanguineus in response to different relative humidity and temperature. While no significant differences were observed in hsp70 levels in nymphs, striking variations were found for hsp20. On the contrary, significant differences were found on the expression levels of both HSPs when exposed at different treatments. Expression of hsp20 was more affected by relative humidity than by temperature, which could imply that hsp20 may have a more critical protective role during vulnerable developmental stages of the tick than hsp70.

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.

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.

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.

The Pacific oyster (Crassostrea gigas) Hsp70 modulates the Ostreid herpes virus 1 infectivity.

The Ostreid herpes virus type 1 (OsHV-1) is one of the most devastating pathogen in oyster cultures. Among several factors, as food limitation, oxygen depletion, salinity and temperature variations, episodes of "summer mortality" of the Pacific oyster Crassostrea gigas have also been associated with OsHV-1 infection. Mortalities of C. gigas spat and juveniles have increased significantly in Europe, and contemporary mortality records of this mollusk in México have been associated with the occurrence of OsHV-1. In the present study, the expression of the heat shock protein 70 gene from the Pacific oyster correlates with the abundance of DNA polymerase transcripts from the OsHV-1. This may suggest that the induction on the expression of the Pacific oyster hsp70 may potentially participate in the immune response against the virus. Furthermore, this study reports for the first time a TEM representative image of the OsHV-1 in aqueous solution, which possesses an icosahedral shape with a diameter of 70 nm × 100 nm. Finally, the examined sequence encoding the ORF4 of the OsHV-1 isolate from northwest Mexico showed specific sequence variations when compared with OsHV-1 isolates from distant geographical areas.

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.

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV.

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.

Genome dynamics in three different geographical isolates of white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV), the sole member of the monotypic family Nimaviridae, is considered an extremely lethal shrimp pathogen. Despite its impact, some essential biological characteristics related to WSSV genome dynamics, such as the synonymous codon usage pattern and selection pressure in genes, remain to be elucidated. The results show that compositional limitations and mutational pressure determine the codon usage bias and base composition in WSSV. Furthermore, different forces of selective pressure are acting across various regions of the WSSV genome. Finally, this study points out the possible occurrence of two major recombination events.

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.

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.

A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites.

Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA, which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the Km for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The Km for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase.

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response.

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda).

Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsin-like (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Cas-like to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L-1. The K M values ranged from 0.072 mmol L-1 for the Try-like activity of P. vannamei to 0.309 mmol L-1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing ~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

Usage of energy reserves in crustaceans during starvation: status and future directions.

In this paper, we review the current knowledge about the usage of carbohydrates, lipids and proteins as energy source by marine crustaceans during starvation. Crustaceans are a large and diverse group including some economically important species. The efforts to culture them for human consumption has prompted the interest to understand the preferences of energy sources to be applied for feed formulation and cost reduction. Important differences have been found among species and appear to be related not only to the biochemistry and physiology of nutrition, but also to the living environment of the crustaceans. Furthermore, crustaceans undergo morphological, physiological and behavioral changes due to their natural growing process that affect their feeding habits, an aspect that should be carefully considered. We discuss the current information on marine crustaceans about energy usage and describe areas of future research, where starvation studies render important insights.

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.

Starvation and diet composition affect mRNA levels of the high density-lipoprotein-beta glucan binding protein in the shrimp Litopenaeus vannamei.

A high density lipoprotein-beta glucan binding protein (HDL-BGBP) is synthesized in the hepatopancreas of the white shrimp Litopenaeus vannamei and secreted to the hemolymph. Recently, we reported the HDL-BGBP full length cDNA sequence and found that the predicted polypeptide is larger than the mature protein and also, that it contains a long 5'- and 3'-UTRs that may be involved in transcript level regulation. To test whether starvation and feeding may play a role in regulating HDL-BGBP mRNA levels, two different stimuli were evaluated: starvation and composition of diets. After 24 h, the steady state HDL-BGBP mRNA levels of starved shrimp decreased, suggesting that synthesis of the lipoprotein is less required in the absence of food. When shrimp were fed with diets containing different concentrations of protein and lipids, changes in HDL-BGBP mRNA levels were also detected. Shrimp fed the lower concentration of protein and lipid feed accumulated higher levels of HDL-BGBP mRNA. These results indicate that feeding influences HDL-BGBP transcript levels in the hepatopancreas.

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.

Seasonal changes in gene expression and polymorphism of hsp70 in cultivated oysters (Crassostrea gigas) at extreme temperatures.

The HSP70 proteins are an important element of the response against thermal stress and infectious diseases, and they are highly conserved and ubiquitous. In some species, variations on the hsp70 encoding sequence resulted in intraspecific differential expression, which leads to variations on thermo-tolerance among individuals. This phenomenon has not been described in the Pacific oyster Crassostrea gigas, which is cultivated in Mexico under temperature conditions highly above the optimal for this species. The present study was aimed to identify associations between hsp70 genotypes and their expression levels in C. gigas. By analyzing a 603 bp fragment from the 3' end of the hsp70 gene, 21 different genotypes with 60 nucleotide polymorphic sites were detected, of which 34 sites were found in heterozygous condition. Although no correlation was found between genotype-expression-season, a minimum expression threshold that should be taken into account as an important feature for a future breeding program is proposed.