Amoebic crab disease (ACD) in edible crab Cancer pagurus from the English Channel, UK.

The genera Paramoeba and Neoparamoeba (Amoebozoa, Dactylopodida, Paramoebidae) include well-known opportunistic pathogens associated with fish (N. peruans; amoebic gill disease), lobsters, molluscs and sea urchins, but only rarely with crabs (grey crab disease of blue crabs). Following reports of elevated post-capture mortality in edible crabs Cancer pagurus captured from a site within the English Channel fishery in the UK, a novel disease (amoebic crab disease, ACD) was detected in significant proportions of the catch. We present histopathological, transmission electron microscopy and molecular phylogenetic data, showing that this disease is defined by colonization of haemolymph, connective tissues and fixed phagocytes by amoeboid cells, leading to tissue destruction and presumably death in severely diseased hosts. The pathology was strongly associated with a novel amoeba with a phylogenetic position on 18S rRNA gene trees robustly sister to Janickina pigmentifera (which groups within the current circumscription of Paramoeba/Neoparamoeba), herein described as Janickina feisti n. sp. We provide evidence that J. feisti is associated with ACD in 50% of C. pagurus sampled from the mortality event. A diversity of other paramoebid sequence types, clustering with known radiations of N. pemaquidensis and N. aestuarina and a novel N. aestuarina sequence type, was detected by PCR in most of the crabs investigated, but their detection was much less strongly associated with clinical signs of disease. The discovery of ACD in edible crabs from the UK is discussed relative to published historical health surveys for this species.

Haplosporidium acetes n. sp. infecting the hepatopancreas of jelly prawns Acetes sibogae australis from Moreton Bay, Australia.

Wild Acetes sibogae australis from northern Moreton Bay, Australia displaying opacity of the hepatopancreas were sampled and examined histologically, revealing infection by multinucleate plasmodia of a haplosporidian-like parasite in the epithelial cells of the hepatopancreas. A morphological and phylogenetic investigation identified the parasite as a novel species of the order Haplosporida, and the parasite is described as Haplosporidium acetes n. sp. This is the first report of disease caused by a haplosporidian in wild Australian decapod crustaceans, and the first record of haplosporidiosis in sergestid shrimp. Infections of H. acetes were observed in all cell types (R, B, F and E) within the hepatopancreas. Infected epithelial cells became hypertrophied as they filled with haplosporidian parasites and, in heavy infections, caused almost complete displacement of normal hepatopancreas tissue. Although sporulation was not observed, infected jelly prawns appeared terminally diseased. Infections became grossly evident in around 5% of wild prawns during early autumn at a time of year when jelly prawn populations decline rapidly with decreasing water temperatures, however histopathology indicated at least 13% of apparently normal jelly prawns were also infected. Further studies are required in order to determine if this parasite influences jelly prawn population dynamics. In addition, we report co-infection of a novel microsporidian parasite in the Enterocytozoon Group Microsporidia (EGM) infecting nuclei of hepatopancreatic epithelial cells. The microsporidian was phylogenetically distinct from Enterocytozoon hepatopenaei (EHP) known to infect penaeid shrimp in Asia.

A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Sustainable aquaculture through the One Health lens.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics - underpinned by evidence, policy and legislation - that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production - and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design.

Pseudokabatana alburnus n. gen. n. sp., (Microsporidia) from the liver of topmouth culter Culter alburnus (Actinopterygii, Cyprinidae) from China.

We describe the type species of a novel genus of microsporidian parasite, Pseudokabatana alburnus n. gen. n. sp., infecting the liver of topmouth culter, Culter alburnus Basilewsky, 1855, from Lake Poyang off Xingzi county, Jiangxi Province, China. The parasite elicits formation of spherical xenomas of up to 1.2 mm in diameter containing all observed life stages from early merogonal plasmodia to mature spores contained within the cytoplasm of host hepatocytes. Merogonal plasmodia existed in direct contact with the host cytoplasm and contained up to 20 visible nuclei. Plasmotomy of the multinucleate plasmodium led to formation of uninucleate cells in which the nucleus underwent further division to form bi-nucleate presporonts, sporonts (defined by cells with a thickened endospore) and eventually sporoblasts (containing pre-cursors of the spore extrusion apparatus). Mature spores were pyriform and monokaryotic, measuring 2.3 ± 0.19 µm long and 1.3 ± 0.10 µm wide. Spores possessed a bipartite polaroplast and 5-6 coils of a polar filament, in a single rank. The obtained partial SSU rRNA gene sequence, 1383 bp in length, did not match any of microsporidia available in GenBank. SSU rDNA-based phylogenetic analysis indicated a new taxon branching with Kabatana rondoni, a parasite infecting the skeletal muscle of Gymnorhamphichthys rondoni from the Amazon River. Due to different host and tissue tropism, the novel taxon did not fit the diagnostic criteria for the genus Kabatana. Further, based on SSU rDNA-inferred phylogenetic analyses, different ultrastructural features of developmental stages, and ecological considerations, a new genus Pseudokabatana and type species Pseudokabatana alburnus n. sp. was erected for the parasite in topmouth culter.

Characterization of microsporidian Ameson herrnkindi sp. nov. infecting Caribbean spiny lobsters Panulirus argus.

The Caribbean spiny lobster Panulirus argus supports a large and valuable fishery in the Caribbean Sea. In 2007-2008, a rare microsporidian parasite with spore characteristics typical of the Ameson genus was detected in 2 spiny lobsters from southeast Florida (FL). However, the parasite species was not confirmed by molecular analyses. To address this deficiency, reported here are structural and molecular data on single lobsters displaying comparable 'cotton-like' abdominal muscle containing ovoid microsporidian spores found at different locations in FL in 2014 and 2018 and in Saint Kitts and Nevis Islands in 2017. In the lobster from 2014, multiple life stages consistent with an Ameson-like monokaryotic microsporidian were detected by transmission electron microscopy. A partial (1228 bp) small subunit (SSU) rRNA gene sequence showed each microsporidia to be identical and positioned it closest phylogenetically to Ameson pulvis in a highly supported clade also containing A. michaelis, A. metacarcini, A. portunus, and Nadelspora canceri. Using ecological, pathological, ultrastructural, and molecular data, the P. argus microsporidian has been assigned to a distinct species: Ameson herrnkindi.

Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens 'O-type' and 'M-type'. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within 'M. refringens' are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3' end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

Evidence for trophic transfer of Inodosporus octospora and Ovipleistophora arlo n. sp. (Microsporidia) between crustacean and fish hosts.

Within aquatic habitats, the hyper-abundant Order Crustacea appear to be the predominant host group for members of the Phylum Microsporidia. The musculature, a common site of infection, provides access to biochemical (carbohydrate-rich) and physiological (mitochondria-rich) conditions conducive to prolific parasite replication and maturation. The significant proportion of body plan devoted to skeletal musculature in Crustacea provides the location for a highly efficient intracellular parasite factory. In this study, we utilize histological, ultrastructural and phylogenetic evidence to describe a previously known (Inodosporus octospora) and novel (Ovipleistophora arlo n. sp.) microsporidian parasites infecting the musculature of the common prawn (Palaemon serratus) from the same site, at the same time of year. Despite similar clinical signs of infection, both parasites are otherwise distinct in terms of pathogenesis, morphology and phylogeny. Based upon partial subunit ribosomal RNA (SSU rDNA) sequence, we show that that I. octospora may be identical to a Kabatana sp. previously described infecting two-spot goby (Gobiusculus flavescens) in Europe, or at least that Inodosporus and Kabatana genera are synonyms. In addition, SSU rDNA sequence for O. arlo places it within a distinct clade containing Ovipleistophora mirandellae and Ovipleistophora ovariae, both infecting the oocytes of freshwater fish in Europe. Taken together, our data provide strong evidence for trophic-transfer between crustacean and fish hosts for two different microsporidians within clade 5 of the phylum. Furthermore, it demonstrates that morphologically and phylogenetically distinct microsporidians can infect the same tissues of the same host species to impart clinical signs which mimic infection with the other.

A taxonomic review of viruses infecting crustaceans with an emphasis on wild hosts.

Numerous infections by viral pathogens have been described from wild and cultured crustacean hosts, yet relatively few of these pathogens have been formally characterised and classified. To date viruses have generally been tentatively assigned to families based upon morphological and developmental characteristics and their location of infection within the host cell. Often nucleotide sequence information is unavailable. Some of these viral infections have caused well-documented devastating consequences on the global crustacean farming industry whilst their effects on wild populations remain largely unstudied. This paper provides an up to date review of all known viruses described infecting crustacean hosts. Full characterisation and harmonisation of these descriptions utilising specifications proposed by the International Committee on Taxonomy of Viruses (ICTV) is required to synonymise numerous examples of differential naming or abbreviation of naming, of the same virus in some cases. Development and application of techniques such as viral purification and high throughput sequencing of viral genomes will assist with these full descriptions and, provide appropriate diagnostic targets for surveillance of known and novel relatives. This review also highlights the importance of comparative study with viruses infecting insects and other arthropods to assist this process.

Determining Good Environmental Status under the Marine Strategy Framework Directive: Case study for descriptor 8 (chemical contaminants).

The European Union Marine Strategy Framework Directive (MSFD) requires individual member states to develop a robust set of tools for defining eleven qualitative descriptors of Good Environmental Status (GES), such as demonstrating that "Concentrations of contaminants are at levels not giving rise to pollution effects" (GES descriptor 8). Adopting the recommendations of the ICES/OSPAR Study Group for the Integrated Monitoring of Contaminants and Biological Effects (SGIMC), we present a case study demonstrating how the proposed approach, using chemical contaminant (metals and polycyclic aromatic hydrocarbons and polychlorinated biphenyls) and biological effects (EROD, bile metabolites and pathology) data in different matrices (sediment and biota), could be used to contribute to the determination of GES in a region of the North Sea region off the east coast of the UK.

Hyperspora aquatica n.gn., n.sp. (Microsporidia), hyperparasitic in Marteilia cochillia (Paramyxida), is closely related to crustacean-infecting microspordian taxa.

The Paramyxida, closely related to haplosporidians, paradinids, and mikrocytids, is an obscure order of parasitic protists within the class Ascetosporea. All characterized ascetosporeans are parasites of invertebrate hosts, including molluscs, crustaceans and polychaetes. Representatives of the genus Marteilia are the best studied paramyxids, largely due to their impact on cultured oyster stocks, and their listing in international legislative frameworks. Although several examples of microsporidian hyperparasitism of paramyxids have been reported, phylogenetic data for these taxa are lacking. Recently, a microsporidian parasite was described infecting the paramyxid Marteilia cochillia, a serious pathogen of European cockles. In the current study, we investigated the phylogeny of the microsporidian hyperparasite infecting M. cochillia in cockles and, a further hyperparasite, Unikaryon legeri infecting the digenean Meiogymnophallus minutus, also in cockles. We show that rather than representing basally branching taxa in the increasingly replete Cryptomycota/Rozellomycota outgroup (containing taxa such as Mitosporidium and Paramicrosoridium), these hyperparasites instead group with other known microsporidian parasites infecting aquatic crustaceans. In doing so, we erect a new genus and species (Hyperspora aquatica n. gn., n.sp.) to contain the hyperparasite of M. cochillia and clarify the phylogenetic position of U. legeri. We propose that in both cases, hyperparasitism may provide a strategy for the vectoring of microsporidians between hosts of different trophic status (e.g. molluscs to crustaceans) within aquatic systems. In particular, we propose that the paramyxid hyperparasite H. aquatica may eventually be detected as a parasite of marine crustaceans. The potential route of transmission of the microsporidian between the paramyxid (in its host cockle) to crustaceans, and, the 'hitch-hiking' strategy employed by H. aquatica is discussed.

Single and multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of farmed and wild crustacean hosts.

Almost half of all known microsporidian taxa infect aquatic animals. Of these, many cause disease in arthropods. Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolated Hepatospora spp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship between Hepatospora isolated from E. sinensis and C. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyse Hepatospora isolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminate Hepatospora isolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description of H. eriocheir in Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential for Hepatospora to exist as a cline across wide geographies and habitats.

Microsporidia - Emergent Pathogens in the Global Food Chain.

Intensification of food production has the potential to drive increased disease prevalence in food plants and animals. Microsporidia are diversely distributed, opportunistic, and density-dependent parasites infecting hosts from almost all known animal taxa. They are frequent in highly managed aquatic and terrestrial hosts, many of which are vulnerable to epizootics, and all of which are crucial for the stability of the animal-human food chain. Mass rearing and changes in global climate may exacerbate disease and more efficient transmission of parasites in stressed or immune-deficient hosts. Further, human microsporidiosis appears to be adventitious and primarily associated with an increasing community of immune-deficient individuals. Taken together, strong evidence exists for an increasing prevalence of microsporidiosis in animals and humans, and for sharing of pathogens across hosts and biomes.

Changes in the water quality conditions of Kuwait's marine waters: Long term impacts of nutrient enrichment.

This work analyses a 30 year water quality data set collated from chemical analyses of Kuwait's marine waters. Spatial patterns across six sites in Kuwait Bay and seven sites located in the Arabian Gulf are explored and discussed in terms of the changing influences associated with point and diffuse sources. Statistical modelling demonstrated significant increases for dissolved nutrients over the time period. Kuwait marine waters have been subject to inputs from urban development, untreated sewage discharges and decreasing river flow from the Shatt al-Arab River. Chlorophyll biomass showed a small but significant reduction; the high sewage content of the coastal waters from sewage discharges likely favouring the presence of smaller phytoplankton taxa. This detailed assessment of temporal data of the impacts of sewage inputs into Kuwait's coastal waters establishes an important baseline permitting future assessments to be made as sewage is upgraded, and the river continues to be extracted upstream.

Paradoxium irvingi n.gen. n.sp. (Microsporidia) infecting the musculature of European pink shrimp Pandalus montagui.

This paper utilises histological, ultrastructure and molecular phylogenetic data to describe a novel genus and species (Paradoxium irvingi n.gen., n.sp.) within clade 5 of the phylum Microsporidia. The parasite infects the musculature of the pink shrimp Pandalus montagui captured from United Kingdom waters. The novel microsporidium is morphologically and phylogenetically dissimilar to its nearest phylogenetic branch relative Thelohania butleri infecting the sister shrimp taxon Pandalus jordani. Furthermore, it is morphologically distinct from the type species of the genus Thelohania, Thelohania giardi infecting European brown shrimp Crangon crangon. Since phylogenetic data pertaining to type T. giardi is not currently available, our discovery places some doubt on the likelihood that T. butleri represents the proposed surrogate for the type taxon. Further it demonstrates potential for significant morphological plasticity in this clade of muscle-infecting microsporidians of crustaceans which contains the genera Myospora, Cucumispora, Thelohania, and now Paradoxium. Since it cannot be stated with certainty that T. butleri (or other taxa within the clade) represent true close relatives of T. giardi, clarity on this issue will only occur with re-discovery and genotyping of type T. giardi infecting C. crangon from European waters.

Histopathological assessment of liver and gonad pathology in continental slope fish from the northeast Atlantic Ocean.

The deep-sea environment is a sink for a wide variety of contaminants including heavy metals and organic compounds of anthropogenic origin. Life history traits of many deep-water fish species including longevity and high trophic position may predispose them to contaminant exposure and subsequent induction of pathological changes, including tumour formation. The lack of evidence for this hypothesis prompted this investigation in order to provide data on the presence of pathological changes in the liver and gonads of several deep-water fish species. Fish were obtained from the north east region of the Bay of Biscay (north east Atlantic Ocean) by trawling at depths between 700 and 1400 m. Liver and gonad samples were collected on board ship and fixed for histological processing and subsequent examination by light microscopy. Hepatocellular and nuclear pleomorphism and individual cases of ovotestis and foci of cellular alteration (FCA) were detected in black scabbardfish (Aphanopus carbo). Six cases of FCA were observed in orange roughy (Hoplostethus atlanticus) (n = 50) together with a single case of hepatocellular adenoma. A wide variety of inflammatory and degenerative lesions were found in all species examined. Deep-water fish display a range of pathologies similar to those seen in shelf-sea species used for international monitoring programmes including biological effects of contaminants. This study has confirmed the utility of health screening in deep-water fish for detecting evidence of prior exposure to contaminants and has also gained evidence of pathology potentially associated with exposure to algal toxins.

Histopathological survey of potential biomarkers for the assessment of contaminant related biological effects in species of fish and shellfish collected from Kuwait Bay, Arabian Gulf.

The marine environment in Kuwait is dominated by Kuwait Bay, a shallow, depositional habitat vital for the breeding and propagation of marine organisms. The bay receives effluent inputs from industrial centres, ports, sewage outflows along with discharges from power and desalination plants. The major classes of pollutant discharged into the bay include petroleum hydrocarbons, metals, nutrients, cooling water and hyper-saline water. Further, the bay has been historically impacted by a deliberate release of oil and contamination with ordnance and shipwrecks during the 1991 Gulf war. With an aim to establish an integrated pollution effects monitoring programme in Kuwait, this paper describes the application of a quality assured approach to conduct a histopathology baseline survey in oriental sole (Synaptura orientalis) and the large-toothed flounder (Pseudorhombus arsius), which are two potential sentinel flatfish species present in the Arabian Gulf. Liver and gonadal histopathology revealed a range of pathologies similar to those previously observed in European and American pollution effects surveys that utilise flatfish (including pathology markers indicative of possible carcinogenesis and endocrine disruption). Further, we extended these studies to invertebrates (Jinga prawn, Metapenaeus affinis and the grooved tiger prawn, Penaeus semisulcatus) found within the Arabian Gulf. Such baseline data is essential before attempts are made to develop integrated monitoring programmes that aim to assess the health of fish and shellfish in relation to chemical contamination.

Areospora rohanae n.gen. n.sp. (Microsporidia; Areosporiidae n. fam.) elicits multi-nucleate giant-cell formation in southern king crab (Lithodes santolla).

This paper utilises histological, ultrastructure and molecular phylogenetic data to describe a novel genus and species (Areospora rohanae n.gen., n.sp.) within the phylum Microsporidia. Phylogenetic and morphological distinction from other known lineages within the phylum also provide strong support for erection of a new family (Areosporiidae n. fam) to contain the parasite. Recognised via lesions observed by workers in king crab processing facilities in southern Chile, the parasite elicits giant cell formation in infected crabs. Merogony within haemocytes and fixed phagocytes proceeds apparent fusion of infected cells to produce multinucleate syncitia in which further development of the parasite occurs. Subsequent recruitment of adjacent cells within the haemal spaces of the hepatopancreas, the podocytes of the gill, and particularly in the subcuticular connective tissues, characterises the pathogenesis of A. rohanae. In late stages of infection, significant remodelling of the subcuticular tissues corresponds to the clinical lesions observed within processing plants. Sporogony of A. rohanae also occurs within the syncitial cytoplasm and culminates in production of bizarre spores, ornamented with distinctive tubular bristles. Spores occur in sets of 8 within a sporophorous vesicle. The description of A. rohanae offers considerable insight into the pathogenesis of giant-cell forming Microsporidia, signifies a new lineage of giant-cell forming Microsporidia in marine hosts, and may reflect emergence of a commercially-significant pathogen in the southern ocean Lithodes santolla fishery.

Baseline histopathological survey of a recently invading island population of 'killer shrimp', Dikerogammarus villosus.

Dikerogammarus villosus, an invasive amphipod, has recently been detected in UK freshwaters. To assess the potential for pathogen introduction with the invader, a year-long histopathology survey of the D. villosus population inhabiting the initial site of detection (Grafham Water, Cambridgeshire, UK) was conducted. Additional samples were collected from 2 other subsequently identified populations within the UK (Cardiff Bay and Norfolk Broads), and from established populations in France (River Rhine) and Poland (River Vistula). The data revealed a range of pathogens and commensals. Several pathogens occurring within continental populations were not present within the UK populations. Microsporidian parasites and a novel viral pathogen were amongst those not observed in the UK. The absence of these pathogens at UK sites may therefore impart significant survival advantages to D. villosus over native fauna, thereby increasing its success as an invader. The contrast in pathogen profile between UK and continental-invasive populations of D. villosus provides preliminary evidence for so-called 'enemy release' in UK populations of D. villosus and is suggestive of single-point introductions, rather than continual incursion events as previously observed throughout its continental invasive range. This baseline survey provides important data on the pathogen and commensal profile of a high-impact, invasive species early in its invasion history of the UK. It can be utilised to assess potential for temporal pathogen acquisition by non-native invasive aquatic species and to investigate competitive advantages placed upon this invader due to absence of important pathogens experienced within its native range.