Biofiltering capacity of Chambardia rubens (Bivalvia: Unionidae) may ameliorate stress and growth inhibition induced by the neonicotinoid insecticide acetamiprid in zebrafish Danio rerio.

Neonicotinoid insecticides specifically target insect subtypes of nicotinic acetylcholine receptors. Acetamiprid (ACE: C10H11ClN4), the neonicotinoid insecticide, is used to control crop insect pests worldwide. It is a nitrile, monochloropyridine, and carboxamidine that is highly soluble and accessible to waterways. There, it causes neurotoxic and oxidative perturbance to non-target organisms. The unionid mussel Chamabradia rubens is a common Northern River Nile suspension feeder. The current study aimed to assess ACE filtration from waters by C. rubens, and whether this biological power can reduce ACE effects on fish. Removal of ACE by C. rubens was assessed using LC-MS/MS. Zebrafish Danio rerio adults were exposed to different sublethal doses of ACE in the presence or absence of C. rubens in their aquaria. The results showed that mussels could remove significant ACE amounts from water, where it accumulated mostly in the digestive gland. The presence of C.rubens in zebrafish aquaria having ACE was accompanied by significant upregulation of antioxidant enzyme gene transcripts and total H2O2 scavenging, in contrast to mussel-free ACE-exposed groups. Meanwhile, liver triglycerides rose 5-6-fold in response to ACE in the "Fish-Only" groups, indicating an ACE-induced hepatotoxicity. Also, Insulin-like growth factor 1 (igf1) and fish body mass increased more in "Fish+Mussel" groups than in the "Fish-Only" ones. In aggregate, these findings suggest that the Nile mussel could reduce the oxidative stress and metabolic changes induced in fish by ACE. This can contribute valuable environmental and economic benefits upon the use of this mussel as a biofilter.

Virus discovery in cultured portunid crabs: Genomic, phylogenetic, histopathological and microscopic characterization of a reovirus and a new bunyavirus.

Portunid crabs are distributed worldwide and highly valued in aquaculture. Viral infections are the main limiting factor for the survival of these animals and, consequently, for the success of commercial-scale cultivation. However, there is still a lack of knowledge about the viruses that infect cultured portunid crabs worldwide. Herein, the genome sequence and phylogeny of Callinectes sapidus reovirus 2 (CsRV2) are described, and the discovery of a new bunyavirus in Callinectes danae cultured in southern Brazil is reported. The CsRV2 genome sequence consists of 12 dsRNA segments (20,909 nt) encode 13 proteins. The predicted RNA-dependent RNA polymerase (RdRp) shows a high level of similarity with that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 belongs to the genus Cardoreovirus. The CsRV2 particles are icosahedral, measuring approximately 65 nm in diameter, and exhibit typical non-turreted reovirus morphology. High throughput sequencing data revealed the presence of an additional putative virus genome similar to bunyavirus, called Callinectes danae Portunibunyavirus 1 (CdPBV1). The CdPBV1 genome is tripartite, consisting of 6,654 nt, 3,120 nt and 1,656 nt single-stranded RNA segments that each encode a single protein. Each segment has a high identity with European shore crab virus 1, suggesting that CdPBV1 is a new representative of the family Cruliviridae. The putative spherical particles of CdPBV1 measure ∼120 nm in diameter and present a typical bunyavirus morphology. The results of the histopathological analysis suggest that these new viruses can affect the health and, consequently, the survival of C. danae in captivity. Therefore, the findings reported here should be used to improve prophylactic and pathogen control practices and contribute to the development and optimization of the production of soft-shell crabs on a commercial scale in Brazil.

Investigating conspecific CsRV1 transmission in Callinectes sapidus.

A reo-like virus, CsRV1, is found in blue crabs, Callinectes sapidus, from every North American location assessed, including Chesapeake Bay and the Atlantic and Gulf coasts, USA and associated with blue crabs in softshell production. CsRV1-associated crab mortality is prevalent in captive crabs, but it is still unknown how CsRV1 is transmitted. The purpose of this study was to examine the role that conspecific predation or scavenging may play in per os transmission in single exposure and repeated exposure experiments. For viruses without cell culture propagation, repeated exposure experiments have the challenge of presenting the virus consistently during the experiment and across time replicates. In a single-exposure experiment, none of the crabs fed muscle tissue of crabs carrying intense infections of CsRV1 developed CsRV1 infections. In a repeated-exposure trial, using infected muscle tissue prepared in alginate blocks, CsRV1 was detected in 11% of the crabs fed infected tissue but was not significantly different from the control group fed alginate lacking CsRV1. For repeated per os exposure experiments, the study demonstrated the utility of using alginate to present the same homogenous sample of virus, both injected and per os, over time for oral challenge experiments. Conspecific predation and scavenging could be a transmission route, but future work into this and other possible routes of transmission for CsRV1 is important to better understand the role this virus plays in wild crab populations and the soft-shell crab industry.

Cross-Hemispheric Genetic Diversity and Spatial Genetic Structure of Callinectes sapidus Reovirus 1 (CsRV1).

The movement of viruses in aquatic systems is rarely studied over large geographic scales. Oceanic currents, host migration, latitude-based variation in climate, and resulting changes in host life history are all potential drivers of virus connectivity, adaptation, and genetic structure. To expand our understanding of the genetic diversity of Callinectes sapidus reovirus 1 (CsRV1) across a broad spatial and host life history range of its blue crab host (Callinectes sapidus), we obtained 22 complete and 96 partial genomic sequences for CsRV1 strains from the US Atlantic coast, Gulf of Mexico, Caribbean Sea, and the Atlantic coast of South America. Phylogenetic analyses of CsRV1 genomes revealed that virus genotypes were divided into four major genogroups consistent with their host geographic origins. However, some CsRV1 sequences from the US mid-Atlantic shared high genetic similarity with the Gulf of Mexico genotypes, suggesting potential human-mediated movement of CsRV1 between the US mid-Atlantic and Gulf coasts. This study advances our understanding of how climate, coastal geography, host life history, and human activity drive patterns of genetic structure and diversity of viruses in marine animals and contributes to the capacity to infer broadscale host population connectivity in marine ecosystems from virus population genetic data.

Characterization of Two Novel Toti-Like Viruses Co-infecting the Atlantic Blue Crab, Callinectes sapidus, in Its Northern Range of the United States.

The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab (Callinectes sapidus) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for -1 ribosomal frameshifting, which may induce the expression of an encoded ORF1-ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.

Diversity and classification of reoviruses in crustaceans: A proposal.

A variety of reoviruses have been described in crustacean hosts, including shrimp, crayfish, prawn, and especially in crabs. However, only one genus of crustacean reovirus - Cardoreovirus - has been formally recognized by ICTV (International Committee on Taxonomy of Viruses) and most crustacean reoviruses remain unclassified. This arises in part from ambiguous or incomplete information on which to categorize them. In recent years, increased availability of crustacean reovirus genomic sequences is making the discovery and classification of crustacean reoviruses faster and more certain. This minireview describes the properties of the reoviruses infecting crustaceans and suggests an overall classification of brachyuran crustacean reoviruses based on a combination of morphology, host, genome organization pattern and phylogenetic sequence analysis.

Near-Complete Sequence of a Highly Divergent Reovirus Genome Recovered from Callinectes sapidus.

This report describes the sequence of a reovirus genome, discovered in Callinectes sapidus in Brazil. The genome sequence of Callinectes sapidus reovirus 2 (CsRV2) consists of 12 segments that encode 13 putative proteins. The predicted RNA-dependent RNA polymerase is highly similar to that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 also belongs to the genus Cardoreovirus.

Effects of road salt on microbial communities: Halophiles as biomarkers of road salt pollution.

Increased use of salting to de-ice roadways, especially in urban areas, is leading to elevated salinity levels in soil as well as surface- and ground water. This salt pollution may cause long-term ecological changes to soil and aquatic microbial communities. In this study, we examined the impact on microbial communities in soils exposed to urban road salt runoff using both culturing and 16S amplicon sequencing. Both methods showed an increase in halophilic Bacteria and Archaea in samples from road salt-exposed areas and suggested that halophiles are becoming persistent members of microbial communities in urban, road salt-impacted soils. Since salt is a pollutant that can accumulate in soils over time, it is critical to begin assessing its impact on the environment immediately. Toward this goal, we have developed a facile semi-quantitative assay utilizing halophilic microbes as biomarkers to evaluate on-going salt pollution of soils.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase.

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Prevalence of the pathogenic crustacean virus Callinectes sapidus reovirus 1 near flow-through blue crab aquaculture in Chesapeake Bay, USA.

Understanding the ecology of diseases is important to understanding variability in abundance, and therefore management, of marine animals exploited commercially. The blue crab Callinectes sapidus fills a crucial benthic-pelagic niche in Atlantic estuarine ecosystems and supports large commercial fisheries in both North and South America. In the USA, pre-molt blue crabs are typically held in short-term shedding (ecdysis) facilities to produce soft-shell crabs of increased value. However, mortality rates in these facilities are high and commonly associated with the pathogenic C. sapidus reovirus 1 (CsRV1). To assess whether crab mortalities in these facilities might increase CsRV1 prevalence in wild crab populations, tissue sampled from crabs collected over 2 summers either near to or far from shedding facilities using flow-through water systems were tested by reverse transcription quantitative PCR (RT-qPCR) for the presence of CsRV1 RNA. In support of our hypothesis, PCR data identified the probability of detecting CsRV1 in wild crabs sampled close to shedding facilities to be 78% higher than in crabs sampled from far sites. PCR detections were also 61-72% more probable in male crabs and 21% more likely in male and female crabs over the minimum landing size. As the prevalence at which CsRV1 was detected varied within seasons, among locations and between years, blue crab migration and/or population fluctuations appear to also be involved.

Does a blue crab putative insulin-like peptide binding protein (ILPBP) play a role in a virus infection?

Insulin-like peptides (ILPs) have regulatory roles in reproduction, development and metabolism in invertebrates. The mode of ILP actions has not been well studied in invertebrates in regard to the role of binding partners, i.e., ILP binding protein (ILPBP). In this study, the full-length cDNA of Callinectes sapidus ILPBP (Cas-ILPBP, 960 bp) has been isolated using RACE cloning, having short 5' and 3' UTRs of 30 and 162 bp, respectively. The predicted precursor of Cas-ILPBP (255 aa) contains, in order a signal peptide (23 aa), an insulin-like growth factor (IGF) binding (IB) domain (79 aa), a kazal-type serine protease inhibitor (KI) domain (36 aa) and an immunoglobulin (Ig) domain (101 aa). Phylogenetic analysis shows that Cas-ILPBP is grouped with the ILPBPs of other crustacean species, and it shares the closest relationship with the ILPBP from another crab species, Scylla paramamosain. Transcripts of Cas-ILPBP are found in all examined tissues, with the highest levels in the nervous tissues (eyestalk ganglia, brain and thoracic ganglia complex) and followed by midgut, the pericardial organ, abdominal muscle and the heart. As Cas-ILPBP contains a putative Ig domain, it is hypothesized that this protein may be involved in immunity, particularly in the adult females infected with a reo-like virus (CsRV1). The expression levels of Cas-ILPBP are examined in several tissues (hemocytes, midgut, eyestalk ganglia) from the animals carrying varying levels of CsRV1 at 17 and 23 °C water temperatures. Cas-ILPBP levels in the midgut are most significantly affected by high levels of CsRV1 infection. Reduction in Cas-ILPBP levels in the midguts is noted from the animals infected with high levels of CsRV1 that show reduced or stop feeding activity, indicating that it may play an important role in midgut functions such as digestion and nutrient absorption.

Genome Sequence Analysis of CsRV1: A Pathogenic Reovirus that Infects the Blue Crab Callinectes sapidus Across Its Trans-Hemispheric Range.

The blue crab, Callinectes sapidus Rathbun, 1896, which is a commercially important trophic link in coastal ecosystems of the western Atlantic, is infected in both North and South America by C. sapidus Reovirus 1 (CsRV1), a double stranded RNA virus. The 12 genome segments of a North American strain of CsRV1 were sequenced using Ion Torrent technology. Putative functions could be assigned for 3 of the 13 proteins encoded in the genome, based on their similarity to proteins encoded in other reovirus genomes. Comparison of the CsRV1 RNA-dependent RNA polymerase (RdRP) sequence to genomes of other crab-infecting reoviruses shows that it is similar to the mud crab reovirus found in Scylla serrata and WX-2012 in Eriocheir sinensis, Chinese mitten crab, and supports the idea that there is a distinct "Crabreo" genus, different from Seadornavirus and Cardoreovirus, the two closest genera in the Reoviridae. A region of 98% nucleotide sequence identity between CsRV1 and the only available sequence of the P virus of Macropipus depurator suggests that these two viruses may be closely related. An 860 nucleotide region of the CsRV1 RdRP gene was amplified and sequenced from 15 infected crabs collected from across the geographic range of C. sapidus. Pairwise analysis of predicted protein sequences shows that CsRV1 strains in Brazil can be distinguished from those in North America based on conserved residues in this gene. The sequencing, annotation, and preliminary population metrics of the genome of CsRV1 should facilitate additional studies in diverse disciplines, including structure-function relationships of reovirus proteins, investigations into the evolution of the Reoviridae, and biogeographic research on the connectivity of C. sapidus populations across the Northern and Southern hemispheres.

Investigating physiological, cellular and molecular effects in juvenile blue crab, Callinectus sapidus, exposed to field-collected sediments contaminated by oil from the Deepwater Horizon Incident.

Juvenile blue crabs, Callinectus sapidus, were exposed for 31 days to six different sediments collected within the Pass a Loutre State Wildlife Management Area approximately 6 months or 1.5 years post-capping of the Macondo-252 well-head following the Deepwater Horizon (DWH) Incident. Based on forensic analysis to fingerprint for DWH oil, these sediments differed in their levels of DWH oil contamination, and included one reference sediment collected from a location with no detectable DWH oil present. The concentration of 50 individual parent and alkylation group polycyclic aromatic hydrocarbons (PAHs), saturated hydrocarbons (37 total), and total extractable hydrocarbons were determined in each sediment, as were biologically relevant metals, grain size distribution, percent total organic carbon, and percent total solids. Total concentrations of 50 PAHs (TPAH50) of initial treatment sediments ranged from 187 µg kg(-1) (reference site) to 2,086,458 µg kg(-1) (the highest DWH oil contaminated site). Multiple biological endpoints were measured including mortality, growth, and ecdysis. Additionally, early biomarkers of biological stress were examined in the hemolymph and hepatopancreas of crabs, including DNA damage (Comet assay) and expression of genes encoding Cu-metallothionein (CuMT), glutathione-S-transferase (GST), and manganese superoxide dismutase (MnSOD). Over the 31 day exposure, there were no treatment related mortalities in juvenile blue crabs. The overall growth and molting of the crabs were not substantially different between the various sediment exposures over the exposure period. Additionally, none of the early biomarkers of biological stress were correlated with PAH concentrations. Overall, juvenile blue crabs did not appear to be negatively impacted during the 31 day exposure by DWH oil contaminated sediments collected at least 6 months post-capping of the Macondo-252 well-head.

Temperature correlates with annual changes in Hematodinium perezi prevalence in blue crab Callinectes sapidus in Florida, USA.

Blue crabs Callinectes sapidus were monitored biannually throughout Florida, USA, for 2 yr using a highly sensitive, quantitative polymerase chain reaction (qPCR) to determine the spatial and temporal changes in prevalence and intensity of Hematodinium perezi infections during drought years. Despite persistent drought conditions, H. perezi infections were not universally found. Overall prevalence was 25.3% (95% CI: 22.8-28.1%) in 1066 crabs sampled from 6 locations (Jacksonville, Ormond Beach, Everglades City, Tampa Bay, Steinhatchee, and Panama City) from 2011 to 2012. Presence of H. perezi was consistently highest in winter season samples, ranging from 4.2-51.1% (3 locations) in 2011, to 32-83% (5 locations) in 2012. The highest prevalence and intensities were observed in the winter samples from Everglades City. Previous studies have found that the prevalence of H. perezi in C. sapidus in temperate regions of the US East Coast shows seasonal peaks in early winter in Maryland and South Carolina and in fall and spring in Georgia. The seasonality of infections in the subtropical waters of Florida reinforces the concept that temperature is a strong factor that may override other drivers, such as drought. Seasonal H. perezi infections in Florida appear to be triggered by the parasite responding to an optimal temperature during the annual rise from the low temperature of winter when salinity is elevated. However, salinity alone is not sufficient to trigger an increase in prevalence of H. perezi in Florida.

Disease, parasite, and commensal prevalences for blue crab Callinectes sapidus at shedding facilities in Louisiana, USA.

Blue crab diseases, parasites, and commensals are not well studied in the Gulf of Mexico, and their prevalence rates have only been sporadically determined. Commercial soft shell shedding facilities in Louisiana experience high mortality rates of pre-molt crabs, and some of these deaths may be attributable to diseases or parasites. During the active shedding season in 2013, we determined the prevalence of shell disease, Vibrio spp., Lagenophrys callinectes, and Hematodinium perezi at 4 commercial shedding facilities along the Louisiana coast. We also detected Ameson michaelis and reo-like virus infections. Shell disease was moderately prevalent at rates above 50% and varied by shedding facility, collection month, and crab size. Vibrio spp. bacteria were prevalent in the hemolymph of 37% of the pre-molt crabs. Lagenophrys callinectes was highly prevalent in the pre-molt crabs, but because it is a commensal species, it may not cause high mortality rates. Hematodinium perezi was absent in all pre-molt crabs.

Draft Genome Sequence of the Oyster Larval Probiotic Bacterium Vibrio sp. Strain OY15.

We report the draft genome sequence of Vibrio sp. strain OY15, a Gram-negative marine bacterium isolated from an oyster (Crassostrea virginica) digestive tract and shown to possess probiotic activity. The availability of this genome sequence will facilitate the study of the mechanisms of probiotic activity as well as virulence capacity.

Draft Genome Sequence of the Shellfish Bacterial Pathogen Vibrio sp. Strain B183.

We report the draft genome sequence of Vibrio sp. strain B183, a Gram-negative marine bacterium isolated from shellfish that causes mortality in larval mariculture. The availability of this genome sequence will facilitate the study of its virulence mechanisms and add to our knowledge of Vibrio sp. diversity and evolution.

Variation in spatial and temporal incidence of the crustacean pathogen Hematodinium perezi in environmental samples from Atlantic Coastal Bays.

BACKGROUND: Hematodinium perezi, a parasitic dinoflagellate, infects and kills blue crabs, Callinectes sapidus, along the Atlantic and Gulf coasts of the United States. The parasite proliferates within host hemolymph and tissues, and also produces free-swimming biflagellated dinospores that emerge from infected crabs. Infections in C. sapidus recur annually, and it is not known if biotic or environmental reservoirs contribute to reinfection and outbreaks. To address this data gap, a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed to asses the temporal and spatial incidence of the parasite in Delaware and Maryland coastal bays. RESULTS: A previously-used PCR assay for H. perezi, based on the small subunit rRNA gene sequence, was found to lack adequate species specificity to discriminate non-Hematodinium sp. dinoflagellate species in environmental samples. A new ITS2-targeted assay was developed and validated to detect H. perezi DNA in sediment and water samples using E. coli carrying the H. perezi rDNA genes. Application of the method to environmental samples identified potential hotspots in sediment in Indian River Inlet, DE and Chincoteague Bay, MD and VA. H. perezi DNA was not detected in co-occurring shrimp or snails, even during an outbreak of the parasite in C. sapidus. CONCLUSIONS: H. perezi is present in water and sediment samples in Maryland and Delaware coastal bays from April through November with a wide spatial and temporal variability in incidence. Sampling sites with high levels of H. perezi DNA in both bays share characteristics of silty, organic sediments and low tidal currents. The environmental detection of H. perezi in spring, ahead of peak prevalence in crabs, points to gaps in our understanding of the parasite's life history prior to infection in crabs as well as the mode of environmental transmission. To better understand the H. perezi life cycle will require further monitoring of the parasite in habitats as well as hosts. Improved understanding of potential environmental transmission to crabs will facilitate the development of disease forecasting.

The Alveolate Perkinsus marinus: biological insights from EST gene discovery.

BACKGROUND: Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date. RESULTS: To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated>31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value

Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus.

Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of double-stranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in >50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.