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.

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.

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.

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.