Salinity and temperature affect the symbiont profile and host condition of Florida USA blue crabs Callinectes sapidus.

Subtropical Florida blue crabs, Callinectes sapidus, exhibit differing life history traits compared to their temperate counterparts, likely influencing symbiont infection dynamics. Little information exists for Florida C. sapidus symbiont profiles, their distribution among various habitats, and influence on crab condition. Using histopathology, genomics, and transmission electron microscopy, we describe the first symbiont profiles for Florida C. sapidus occupying freshwater to marine habitats. Twelve symbiont groups were identified from 409 crabs including ciliophorans, digenean, microsporidian, Haplosporidia, Hematodinium sp., Nematoda, filamentous bacteria, gregarine, Callinectes sapidus nudivirus, Octolasmis sp., Cambarincola sp., and putative microcell. Overall, 78% of C. sapidus were documented with one or more symbiont groups demonstrating high infection rates in wild populations. Environmental variables water temperature and salinity explained 48% of the variation in symbiont groups among Florida habitats, and salinity was positively correlated with C. sapidus symbiont diversity. This suggests freshwater C. sapidus possess fewer symbionts and represent healthier individuals compared to saltwater populations. Crab condition was examined using the reflex action mortality predictor (RAMP) to determine if reflex impairment could be linked to symbiont prevalence. Symbionts were found positively correlated with crab condition, and impaired crabs were more likely to host symbionts, demonstrating symbiont inclusion may boost predictive ability of the RAMP application. The microsporidian symbiont group had a particularly strong effect on C. sapidus reflex response, and impairment was on average 1.57 times higher compared to all other symbiont groups. Our findings demonstrate the importance of considering full symbiont profiles and their associations with a spatially and temporally variable environment to fully assess C. sapidus population health.

Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida): A microsporidian pathogen of the Jonah crab (Cancer borealis).

The microsporidian diversity catalogued so far has resulted in the development of several taxonomic groups, one of which is the Enterocytozoonida - a group of generalist 'ultimate opportunists', which infect many fished and aquacultured animals, as well as a broad suite of host taxa, including humans. In this study, we provide phylogenetic, ultrastructural, developmental, and pathological evidence for the creation of a new genus and species to hold a microsporidian parasite of the Jonah crab, Cancer borealis. Cancer borealis represents a species of commercial interest and has become the target of a recently developed fishery on the USA and Canadian Atlantic coast. This species was found to harbour a microsporidian parasite that develops in the cytoplasm of alpha and beta cells of the hepatopancreas. We retrieved a 937 bp fragment of the parasite SSU region, alongside developmental and ultrastructural data that suggests this species is âˆ¼ 87 % similar to Parahepatospora carcini and develops in a similar manner in direct association with the host cell cytoplasm. The mature spores are ovoid in shape and measure 1.48 ± 0.15 µm (SD) in length and 1.00 ± 0.11 µm (SD) in width. Phylogenetically, the new parasite clades in the Enterocytozoonida on the same branch as P. carcini. We provide a new genus and species to hold the parasite: Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida) and explore the likelihood that this species may fit into the Hepatoporidae family.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

High prevalence of Paramarteilia canceri infecting velvet swimming crabs Necora puber in Ireland.

The velvet swimming crab Necora puber has been fished in Ireland since the early 1980s and contributes significant income to smaller fishing vessels. From 2016 onwards, reduced landings have been reported. We undertook a full pathological investigation of crabs from fishing grounds at 3 sites on the west (Galway), southwest (Castletownbere) and east (Howth) coasts of Ireland. Histopathology, transmission electron microscopy and molecular taxonomic and phylogenetic analyses showed high prevalence and infection level of Paramarteilia canceri, previously only reported from the edible crab Cancer pagurus. This study provides the first molecular data for P. canceri, and shows its phylogenetic position in the order Paramyxida (Rhizaria). Other parasites and symbionts detected in the crabs were also noted, including widespread but low co-infection with Hematodinium sp. and a microsporidian consistent with the Ameson and Nadelspora genera. This is the first histological record of Hematodinium sp. in velvet crabs from Ireland. Four N. puber individuals across 2 sites were co-infected by P. canceri and Hematodinium sp. At one site, 3 velvet crabs infected with P. canceri were co-infected with the first microsporidian recorded from this host; the microsporidian 18S sequence was almost identical to Ameson pulvis, known to infect European shore crabs Carcinus maenas. The study provides a comprehensive phylogenetic analysis of this and all other available Ameson and Nadelspora 18S sequences. Together, these findings provide a baseline for further investigations of N. puber populations along the coast of Ireland.

Resistance to white spot syndrome virus in the European shore crab is associated with suppressed virion trafficking and heightened immune responses.

Introduction: All decapod crustaceans are considered potentially susceptible to White Spot Syndrome Virus (WSSV) infection, but the degree of White Spot Disease (WSD) susceptibility varies widely between species. The European shore crab Carcinus maenas can be infected with the virus for long periods of time without signs of disease. Given the high mortality rate of susceptible species, the differential susceptibility of these resistant hosts offers an opportunity to investigate mechanisms of disease resistance. Methods: Here, the temporal transcriptional responses (mRNA and miRNA) of C. maenas following WSSV injection were analysed and compared to a previously published dataset for the highly WSSV susceptible Penaeus vannamei to identify key genes, processes and pathways contributing to increased WSD resistance. Results: We show that, in contrast to P. vannamei, the transcriptional response during the first 2 days following WSSV injection in C. maenas is limited. During the later time points (7 days onwards), two groups of crabs were identified, a recalcitrant group where no replication of the virus occurred, and a group where significant viral replication occurred, with the transcriptional profiles of the latter group resembling those of WSSV-susceptible species. We identify key differences in the molecular responses of these groups to WSSV injection. Discussion: We propose that increased WSD resistance in C. maenas may result from impaired WSSV endocytosis due to the inhibition of internal vesicle budding by dynamin-1, and a delay in movement to the nucleus caused by the downregulation of cytoskeletal transcripts required for WSSV cytoskeleton docking, during early stages of the infection. This response allows resistant hosts greater time to fine-tune immune responses associated with miRNA expression, apoptosis and the melanisation cascade to defend against, and clear, invading WSSV. These findings suggest that the initial stages of infection are key to resistance to WSSV in the crab and highlight possible pathways that could be targeted in farmed crustacean to enhance resistance to WSD.

Identification and Full Characterisation of Two Novel Crustacean Infecting Members of the Family Nudiviridae Provides Support for Two Subfamilies.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

Global mRNA and miRNA Analysis Reveal Key Processes in the Initial Response to Infection with WSSV in the Pacific Whiteleg Shrimp.

White Spot Disease (WSD) presents a major barrier to penaeid shrimp production. Mechanisms underlying White Spot Syndrome Virus (WSSV) susceptibility in penaeids are poorly understood due to limited information related to early infection. We investigated mRNA and miRNA transcription in Penaeus vannamei over 36 h following infection. Over this time course, 6192 transcripts and 27 miRNAs were differentially expressed-with limited differential expression from 3-12 h post injection (hpi) and a more significant transcriptional response associated with the onset of disease symptoms (24 hpi). During early infection, regulated processes included cytoskeletal remodelling and alterations in phagocytic activity that may assist WSSV entry and translocation, novel miRNA-induced metabolic shifts, and the downregulation of ATP-dependent proton transporter subunits that may impair cellular recycling. During later infection, uncoupling of the electron transport chain may drive cellular dysfunction and lead to high mortalities in infected penaeids. We propose that post-transcriptional silencing of the immune priming gene Dscam (downregulated following infections) by a novel shrimp miRNA (Pva-pmiR-78; upregulated) as a potential mechanism preventing future recognition of WSSV that may be suppressed in surviving shrimp. Our findings improve our understanding of WSD pathogenesis in P. vannamei and provide potential avenues for future development of prophylactics and treatments.

Patterns of infection in a native and an invasive crayfish across the UK.

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson's Index) in P. leniusculus ('Pacifastacus leniusculus Bacilliform Virus' - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, 'Austropotamobius pallipes bacilliform virus' (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats.

How do abiotic environmental conditions influence shrimp susceptibility to disease? A critical analysis focussed on White Spot Disease.

White Spot Syndrome Virus (WSSV) causes White Spot Disease (WSD) and is historically the most devastating disease in the shrimp industry. Global losses from this disease have previously exceeded $3 bn annually, having a major impact on a global industry worth US$19 bn per annum. Shrimp are cultured predominantly in enclosed ponds that are subject to considerable fluctuations in abiotic conditions and WSD outbreaks are increasingly linked to periods of extreme weather, which may cause major fluctuations in pond culture conditions. Combined with the intensity of production in these systems, the resulting suboptimal physicochemical conditions have a major bearing on the susceptibility of shrimp to infection and disease. Current knowledge indicates that pond temperature and salinity are major factors determining outbreak severity. WSSV appears to be most virulent in water temperatures between 25 and 28 °C and salinities far removed from the isoosmotic point of shrimp. Elevated temperatures (>30 °C) may protect against WSD, depending on the stage of infection, however the mechanisms mediating this effect have not been well established. Other factors relating to water quality that may play key roles in determining outbreak severity include dissolved oxygen concentration, nitrogenous compound concentration, partial pressure of carbon dioxide and pH, but data on their impacts on WSSV susceptibility in cultured shrimps is scarce. This illustrates a major research gap in our understanding of the influence of environmental conditions on disease. For example, it is not clear whether temperature manipulations can be used effectively to prevent or mitigate WSD in cultured shrimp. Therefore, developing our understanding of the impact of environmental conditions on shrimp susceptibility to WSSV may provide insight for WSD mitigation when, even after decades of research, there is no effective practical prophylaxis or treatment.

A Novel RNA Virus, Macrobrachium rosenbergii Golda Virus (MrGV), Linked to Mass Mortalities of the Larval Giant Freshwater Prawn in Bangladesh.

Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single­stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.

Diagnosis and prevalence of two new species of haplosporidians infecting shore crabs Carcinus maenas: Haplosporidium carcini n. sp., and H. cranc n. sp.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

Isolation of a Chinook Salmon Bafinivirus (CSBV) in Imported Goldfish Carassius auratus L. in the United Kingdom and Evaluation of Its Virulence in Resident Fish Species.

This is the first record of a fish nidovirus isolated from a consignment of goldfish at the United Kingdom (UK) border. The full-length viral genome was 25,985 nt, sharing a 97.9% nucleotide identity with the Chinook salmon bafinivirus (CSBV) NIDO with two deletions of 537 and 480 nt on the ORF Ia protein. To assess the potential impact on UK fish species, Atlantic salmon, common carp and goldfish were exposed to the virus via an intraperitoneal (IP) injection and bath challenge. Moribundity was recorded in only 8% of IP-injected goldfish. A high viral load, ≈107 of the CSBV PpIa gene, was measured in the kidney of moribund goldfish. Mild histopathological changes were observed in the kidneys of challenged carps. Ultrastructural observations in renal tubule epithelial cells of goldfish showed cylindrical tubes (≈15 nm in diameter) and tubular structures budding spherical virions (≈200 nm in diameter) with external spike-like structures. Negative staining showed both circular and bacilliform virions. Seroconversion was measured in common carp and goldfish but not in Atlantic salmon. This study reinforces the potential risk of novel and emerging pathogens being introduced to recipient countries via the international ornamental fish trade and the importance of regular full health screens at the border inspection posts to reduce this risk.

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries.

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6-77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 µg ml-1), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.

A New Family of DNA Viruses Causing Disease in Crustaceans from Diverse Aquatic Biomes.

Panulirus argus virus 1 (PaV1) is the only known virus infecting the Caribbean spiny lobster (Panulirus argus) from the Caribbean Sea. Recently, related viruses, Dikerogammarus haemobaphes virus 1 (DhV1) and Carcinus maenas virus 1 (CmV1), have been detected in the demon shrimp (Dikerogammarus haemobaphes) and the European shore crab (Carcinus maenas), respectively, from sites in the United Kingdom. The virion morphology of these crustacean viruses is similar to that of iridoviruses. However, unlike iridoviruses and other nucleocytoplasmic large DNA viruses (NCLDVs), these viruses complete their morphogenesis in the host cell nucleus rather than in the cytoplasm. To date, these crustacean viruses have remained unclassified due to a lack of genomic data. Using an Illumina MiSeq sequencer, we sequenced the complete genomes of PaV1, CmV1, and DhV1. Comparative genome analysis shows that these crustacean virus genomes encode the 10 hallmark proteins previously described for the NCLDVs of eukaryotes, strongly suggesting that they are members of this group. With a size range of 70 to 74 kb, these are the smallest NCLDV genomes identified to date. Extensive gene loss, divergence of gene sequences, and the accumulation of low-complexity sequences reflect the extreme degradation of the genomes of these "minimal" NCLDVs rather than any direct relationship with the NCLDV ancestor. Phylogenomic analysis supports the classification of these crustacean viruses as a distinct family, "Mininucleoviridae," within the pitho-irido-Marseille branch of the NCLDVs.IMPORTANCE Recent genomic and metagenomic studies have led to a dramatic expansion of the known diversity of nucleocytoplasmic large DNA viruses (NCLDVs) of eukaryotes, which include giant viruses of protists and important pathogens of vertebrates, such as poxviruses. However, the characterization of viruses from nonmodel hosts still lags behind. We sequenced the complete genomes of three viruses infecting crustaceans, the Caribbean spiny lobster, demon shrimp, and European shore crab. These viruses have the smallest genomes among the known NCLDVs, with losses of many core genes, some of which are shared with iridoviruses. The deterioration of the transcription apparatus is compatible with microscopic and ultrastructural observations indicating that these viruses replicate in the nucleus of infected cells rather than in the cytoplasm. Phylogenomic analysis indicates that these viruses are sufficiently distinct from all other NCLDVs to justify the creation of a separate family, for which we propose the name "Mininucleoviridae" (i.e., small viruses reproducing in the cell nucleus).

The first clawed lobster virus Homarus gammarus nudivirus (HgNV n. sp.) expands the diversity of the Nudiviridae.

Viral diseases of crustaceans are increasingly recognised as challenges to shellfish farms and fisheries. Here we describe the first naturally-occurring virus reported in any clawed lobster species. Hypertrophied nuclei with emarginated chromatin, characteristic histopathological lesions of DNA virus infection, were observed within the hepatopancreatic epithelial cells of juvenile European lobsters (Homarus gammarus). Transmission electron microscopy revealed infection with a bacilliform virus containing a rod shaped nucleocapsid enveloped in an elliptical membrane. Assembly of PCR-free shotgun metagenomic sequencing produced a circular genome of 107,063 bp containing 97 open reading frames, the majority of which share sequence similarity with a virus infecting the black tiger shrimp: Penaeus monodon nudivirus (PmNV). Multiple phylogenetic analyses confirm the new virus to be a novel member of the Nudiviridae: Homarus gammarus nudivirus (HgNV). Evidence of occlusion body formation, characteristic of PmNV and its closest relatives, was not observed, questioning the horizontal transmission strategy of HgNV outside of the host. We discuss the potential impacts of HgNV on juvenile lobster growth and mortality and present HgNV-specific primers to serve as a diagnostic tool for monitoring the virus in wild and farmed lobster stocks.

In vitro gill cell monolayer successfully reproduces in vivo Atlantic salmon host responses to Neoparamoeba perurans infection.

An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (106 copies at 1 h to 103 at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1ß, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1ß, MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.

Green crab Carcinus maenas symbiont profiles along a North Atlantic invasion route.

The green crab Carcinus maenas is an invader on the Atlantic coast of Canada and the USA. In these locations, crab populations have facilitated the development of a legal fishery in which C. maenas is caught and sold, mainly for use as bait to capture economically important crustaceans such as American lobster Homarus americanus. The paucity of knowledge on the symbionts of invasive C. maenas in Canada and their potential for transfer to lobsters poses a potential risk of unintended transmission. We carried out a histological survey for symbionts of C. maenas from their native range in Northern Europe (in the UK and Faroe Islands), and invasive range in Atlantic Canada. In total, 19 separate symbiotic associations were identified from C. maenas collected from 27 sites. These included metazoan parasites (nematodes, Profilicollis botulus, Sacculina carcini, Microphallidae, ectoparasitic crustaceans), microbial eukaryotes (ciliates, Hematodinium sp., Haplosporidium littoralis, Ameson pulvis, Parahepatospora carcini, gregarines, amoebae), bacteria (Rickettsia-like organism, milky disease), and viral pathogens (parvo-like virus, herpes-like virus, iridovirus, Carcinus maenas bacilliform virus and a haemocyte-infecting rod-shaped virus). Hematodinium sp. were not observed in the Canadian population; however, parasites such as Trematoda and Acanthocephala were present in all countries despite their complex, multi-species lifecycles. Some pathogens may pose a risk of transmission to other decapods and native fauna via the use of this host in the bait industry, such as the discovery of a virus resembling the previously described white spot syndrome virus (WSSV), B-virus and 'rod-shaped virus' (RV-CM) and amoebae, which have previously been found to cause disease in aquaculture (e.g. Salmo salar) and fisheries species (e.g. H. americanus).

Development and application of a duplex PCR assay for detection of Crangon crangon bacilliform virus in populations of European brown shrimp (Crangon crangon).

Crangon crangon bacilliform virus (CcBV) was first discovered in 2004 in European brown shrimp (Crangon crangon) caught along the English coast. This study describes a duplex PCR assay developed for the detection of CcBV, based on amplification of the lef-8 gene (211 bp) of CcBV and the E75 gene (105 bp) of C. crangon as an internal amplification control. The lef-8 and E75 primer pairs were designed based on preliminary genome sequencing information of the virus and transcriptomic data available for C. crangon, respectively. Sequencing of the resulting amplicons confirmed the specificity of this PCR assay and sequence analysis of the lef-8 fragment revealed amino acid identity percentages ranging between 31 and 42% with members of the Nudiviridae, proposing that CcBV may reside within this family. Finally, the duplex PCR assay was applied to samples of C. crangon hepatopancreas tissue collected along the Belgian coast to screen for the presence of CcBV. The prevalence of CcBV averaged 87%, which is comparable to previous reports of high prevalence, based upon histological analysis, in shrimp collected along the English coast. Development of a specific and sensitive PCR assay to detect CcBV will provide a useful tool for future aquaculture and research programs involving C. crangon.

Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia.

Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first sequenced genome from the Enterocytozoonidae - a major family of human and animal-infecting microsporidians - appeared to have lost genes for glycolysis. Here, we sequence and analyse genomes from additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei, a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri, a pathogen that lives exclusively inside epithelial cell nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri's adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host-pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.