In the last decade, declines in the population of wild blue mussels Mytilus edulis in the Tamar estuary (United Kingdom) have been noted. In archived samples collected from 2013 to 2019, between 7% (in 2013) and 18% (in 2019) showed large granulocytoma and haemocytic infiltration in the interstitial tissue of the digestive gland. Four samples were selected for 16S rRNA gene Nanopore sequencing. A consensus sequence of 1449 bp showed nucleotide similarities between 99.93–100% with published sequences of Francisella halioticida. In situ hybridisation (ISH) confirmed the presence of F. halioticida DNA within individual granulocytes of granulocytomas and also in prokaryotic-like inclusion bodies within the digestive epithelial cells. The design of diagnostic tests for surveillance of F. halioticida, including more specific ISH probes and sequencing the genome of the isolates infecting mussels, will shed more light on the pathogenicity and spread of this pathogen.

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.

Agapollen is a traditional heliothermic marine oyster lagoon in western Norway, representing the northernmost site of any Marteilia sp. protists detected in Europe. The semi-closed lagoon is a unique site to study the life cycle and development of M. pararefringens in naïve mussels. Two baskets with uninfected mussels were deployed in the lagoon outlet in May and October 2018, respectively, and sampled every 6 wk. The parasite was first detected in the mussels by PCR in early July and by histology in late August. By then, M. pararefringens had developed into mature stages, indicating a rapid development during mid-summer. Sporulation occurred during autumn. Mussels deployed in October never became infected, indicating that transmission was restricted to the warmest period of the year. Pronounced pathology was observed in infected mussels, including degenerated digestive tubules and infiltration of haemocytes. Mussel mortality was observed in the baskets, but whether this was due to infections of M. pararefringens or other environmental factors could not be determined. Plankton samples from the lagoon were also collected for PCR analysis. These samples, dominated by copepods, were positive for M. pararefringens in summer. In sorted samples, M. pararefringens was detected in the Acartia spp. and Paracartia grani fractions between July and October. These plankton copepods are therefore potentially involved in the life cycle of M. pararefringens.

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.

An unusual condition affecting market size rainbow trout was investigated. This condition was prevalent for several years at low levels but affected a large proportion of stock during 2018 and 2019. Chronic fibrosis affecting cranial tissues and the jaw was observed in samples collected in 2018. A larger sampling was then conducted in 2019 to investigate the presence of an infectious agent(s). An extensive inflammatory response in the mandibular region was the main finding, however infectious agents in the lesions were not identified through classical virology and bacteriology analysis. Tetracapsuloides bryosalmonae infection, calcinosis, and a Gram-positive bacterial infection of a single fish cardiac tissue was observed, however, a correlation of these pathologies and the cranial mandibular fibrosis (CMF) syndrome was not established. The gene expression of a panel of 16 immune-related genes was studied. Among these, tgf-b, sIgM, il11, hspa, and the antimicrobial peptides lys and cath1 were up-regulated in jaw sections of CMF-affected fish, showing a strong positive correlation with the severity of the lesions. Idiopathic chronic fibrosis with the activation of the Tfg-B pathway and local hyper-immunoglobulaemia was therefore diagnosed. Initiating factors and causative agent(s) (biotic or abiotic) of CMF remain, at present, unclear.

ABSTRACTThis study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting generaEchinogammarusandOrchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% inEchinogammarussp. and 15% inOrchestiasp., but was seldom detected during the rest of the year. The parasite infected predominantly haemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host haemocytes, resulting in haemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite asTxikispora philomaiosn. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detectedT. philomaiosin the platyhelminthProcerodessp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

This chapter provides an introduction to important marine pathogens, providing an overview of the diversity of pathogen types and how they affect different hosts in the marine environment. The chapter focuses on wild and cultured species and highlights that single infections are relatively rare, with co- and secondary infections being commonplace. The authors highlight the importance of understanding “normal” host tissue structure prior to interpreting pathological changes and outline the role of histology to assess pathogenicity of emerging diseases, linking presence of individual pathogens and co-infections with degree of host response. Fact sheets focus on the pathology (i.e., interaction of a specific pathogen group with the host cell/system) with high-quality histology and TEM images, emphasizing tissue changes caused by pathogens, and point the reader to presumptive diagnosis via histology while highlighting the need for confirmatory testing via other means. The pathobiome concept is introduced and explained, and the utility for predicting outcomes at the individual and population levels discussed.

Red mark syndrome (RMS) is a skin condition in Rainbow trout Oncorhynchus mykiss that has been reported worldwide but was first seen in the United Kingdom (UK) in 2003. The current study was conducted to examine if there was an association between a Midichloria-like organism (MLO) and RMS using a statistically appropriate sample set, whilst determining if there is a lack of association with Flavobacterium psychrophilum implicated in disease in previous studies. Fish in this study were obtained from three sites positive for RMS in the UK and United States (US), and three sites in the UK and the Netherlands that had no previous history of this condition. Samples taken from RMS-affected sites were found to show typical RMS pathology. Analysis of the major organs of affected fish by quantitative polymerase chain reaction (qPCR) demonstrated a significantly higher presence of the MLO in the RMS-affected tissues. Although most of the tissues were positive for the MLO, the highest correlation was seen in the skin, whilst the tissues from the unaffected fish were all negative. Thus, a strong positive correlation was found between the MLO and RMS-affected fish, whilst no association was found between the RMS-affected fish and F. psychrophilum other than superficial presence in the skin. The use of immunohistochemistry showed positive staining of what was considered to be MLO-related antigens in the internal organs of most RMS-affected fish. Attempts were made to culture the MLO, but no MLO was isolated.

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 Sigma PBDEs and Sigma 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 mu g 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. Crown Copyright (C) 2020 Published by Elsevier Ltd. All rights reserved.

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.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

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.

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.

In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalization of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical iridovirus morphology, that is enveloped, with icosahedral and/or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as infectious spleen and kidney necrosis virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow-up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR-positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.

The sea bottom of the Skagerrak Strait (North Sea) contains munitions loaded with chemical warfare agents (CWA), mostly stored in shipwrecks scuttled intentionally after the end of the World War II. The munition shells inside the wrecks are in different states of deterioration and corrosion and their environmental risk potential is unknown. The Atlantic hagfish (Myxine glutinosa), a sediment-dwelling chordate, was used as a model organism to study the potential impact of dumped CWA on the local ecosystem by using biochemical biomarkers. The hagfish were collected in 2017 and 2018 at three sampling sites: in the immediate vicinity of a wreck with CWA in the Skagerrak, a few kilometres from the wreck, and a reference site 21 km from the wreck, considered to be free of CWA. Significant differences were observed between the wreck site and the reference sites in the activities of glutathione reductase, superoxide dismutase and glutathione S-transferase, while the activity levels of catalase and acetylcholinesterase were identical at all sites. The recorded differences demonstrated negative biological effects in the hagfish sampled close to the dumped chemical munitions. Due to the limited knowledge of hagfish biology and of the extent of CWA contamination in Skagerrak, the results presented here warrant more research to further elucidate the potential environmental risks of the scuttled wrecks. The usefulness of the species as a bioindicator organism is further discussed.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Within the framework of the international project DAIMON (Decision Aid for Marine Munitions), the impact of dumped chemical munitions on fish health was investigated. The Skagerrak Straight (North Sea, at 600 m depth) contains munitions with chemical warfare agents (CWA), scuttled after the end of World War II. Studies of liver histopathology in Atlantic hagfish (Myxine glutinosa) were carried out at three sampling sites: at a wreck with CWA in the Skagerrak (n = 82), a Skagerrak reference site considered to be free of CWA (n = 14) and at a reference site in the northern North Sea outside the Skagerrak (n = 17). Liver lesions were diagnosed and categorized according to standardized ICES and BEQUALM protocols and OSPAR guidelines. Non-specific liver lesions were found in 87.6% of 113 hagfish examined. The prevalence of pre-neoplastic lesions was 7.1% and of neoplastic lesions 6.2%. There was no statistically significant difference in prevalence between hagfish samples from the wreck site and from the reference site near the wrecks. However, at the reference site in the northern North Sea, the prevalence of non-specific lesions was low and neither pre-neoplastic nor neoplastic lesions were observed.

The growth of aquaculture over the past 50 years has been accompanied by the emergence of aquatic animal diseases, many of which have spread to become pandemic in countries or continents. An analysis of 400 emerging disease events in aquatic animals that were logged by the Centre for Environment, Fisheries and Aquaculture Science between 2002 and 2017 revealed that more than half were caused by viruses. However, in molluscs, most events were parasitic. Categorising these events indicated that the key processes underpinning emergence were the movement of live animals and host switching. Profiles of key pathogens further illustrate the importance of wild aquatic animals as the source of new infections in farmed animals. It is also clear that the spread of new diseases through the largescale movement of aquatic animals for farming, for food and for the ornamental trade has allowed many to achieve pandemic status. Many viral pathogens of fish (e.g. infectious salmon anaemia, viral haemorrhagic septicaemia) and shrimp (e.g. white spot syndrome virus) affect a large proportion of the global production of key susceptible species. Wild aquatic animal populations have also been severely affected by pandemic diseases, best exemplified by Batrachochytrium dendrobatidis, a fungal infection of amphibians, whose emergence and spread were driven by the movement of animals for the ornamental trade. Batrachochytrium dendrobatidis is now widespread in the tropics and subtropics and has caused local extinctions of susceptible amphibian hosts. Given the rising demand for seafood, aquacultural production will continue to grow and diseases will continue to emerge. Some will inevitably achieve pandemic status, having significant impacts on production and trade, unless there are considerable changes in global monitoring and the response to aquatic animal diseases.

Simultaneous and sequential infections often occur in wild and farming environments. Despite growing awareness, co-infection studies are still very limited, mainly to a few well-established human models. European salmonids are susceptible to both Proliferative Kidney Disease (PKD), an endemic emergent disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae, and Viral Haemorrhagic Septicaemia (VHS), an OIE notifiable listed disease caused by the Piscine Novirhabdovirus. No information is available as to how their immune system reacts when interacting with heterogeneous infections. A chronic (PKD) + acute (VHS) sequential co-infection model was established to assess if the responses elicited in co-infected fish are modulated, when compared to fish with single infections. Macro- and microscopic lesions were assessed after the challenge, and infection status confirmed by RT-qPCR analysis, enabling the identification of singly-infected and co-infected fish. A typical histophlogosis associated with histozoic extrasporogonic T. bryosalmonae was detected together with acute inflammation, haemorrhaging and necrosis due to the viral infection. The host immune response was measured in terms of key marker genes expression in kidney tissues. During T. bryosalmonae/VHSV-Ia co-infection, modulation of pro-inflammatory and antimicrobial peptide genes was strongly influenced by the viral infection, with a protracted inflammatory status, perhaps representing a negative side effect in these fish. Earlier activation of the cellular and humoral responses was detected in co-infected fish, with a more pronounced upregulation of Th1 and antiviral marker genes. These results reveal that some brown trout immune responses are enhanced or prolonged during PKD/VHS co-infection, relative to single infection.

The Lyme Bay marine protected area (MPA) hosts a valuable population of king scallop Pecten maximus L. Recently, an Endozoicomonas-like organism (ELO), infecting host gill epithelial tissue, was associated with king scallop mass mortality events within the Lyme Bay MPA. Currently, very little is known about its transmission and survival outside the host. In this investigation, animals collected outside of reported mortality events showed high levels of ELO infection. Gill tissue disruption and the release of bacteria into the interlamellar space was seen histologically, suggesting shedding of ELO from host animals. To investigate pathogen survival outside the host, infected scallops were maintained in static water for a 24 h period, and then removed. Over the subsequent 8 d, water samples were collected and the quantity of ELO 16S rRNA transcript was measured by TaqManTM quantitative PCR (qPCR). The 16S rRNA transcript quantity was stable outside the host for 6 d before bacteria survival declined 2 logs (7.9 × 108 16S rRNA to 2.3 × 106 transcripts), suggesting that ELO can survive independently outside the host organism. The ELO-specific qPCR probe can therefore be used in future field studies of ELO prevalence within the environment and fauna of the Lyme Bay MPA.