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 health survey of the reef forming scleractinian cold-water corals Lophelia pertusa and Madrepora oculata in a remote submarine canyon on the European continental margin, NE Atlantic.

Monitoring of cold-water corals (CWCs) for pathogens and diseases is limited due to the environment, protected nature of the corals and their habitat and as well as the challenging and sampling effort required. It is recognised that environmental factors such as temperature and pH can expedite the ability of pathogens to cause diseases in cold-water corals therefore the characterisation of pathogen diversity, prevalence and associated pathologies is essential. The present study combined histology and polymerase chain reaction (PCR) diagnostic techniques to screen for two significant pathogen groups (bacteria of the genus Vibrio and the protozoan Haplosporidia) in the dominant NE Atlantic deep-water framework corals Lophelia pertusa (13 colonies) and Madrepora oculata (2 colonies) at three sampling locations (canyon head, south branch and the flank) in the Porcupine Bank Canyon (PBC), NE Atlantic. One M. oculata colony and four L. pertusa colonies were collected from both the canyon flank and the south branch whilst five L. pertusa colonies were collected from the canyon head. No pathogens were detected in the M. oculata samples. Neither histology nor PCR detected Vibrio spp. in L. pertusa, although Illumina technology used in this study to profile the CWCs microbiome, detected V. shilonii (0.03%) in a single L. pertusa individual, from the canyon head, that had also been screened in this study. A macroborer was observed at a prevalence of 0.07% at the canyon head only. Rickettsiales-like organisms (RLOs) were visualised with an overall prevalence of 40% and with a low intensity of 1 to 4 (RLO) colonies per individual polyp by histology. L. pertusa from the PBC canyon head had an RLO prevalence of 13.3% with the highest detection of 26.7% recorded in the south branch corals. Similarly, unidentified cells observed in L. pertusa from the south branch (20%) were more common than those observed in L. pertusa from the canyon head (6.7%). No RLOs or unidentified cells were observed in corals from the flank. Mean particulate organic matter concentration is highest in the south branch (2,612 µg l-1) followed by the canyon head (1,065 µg l-1) and lowest at the canyon flank (494 µg l-1). Although the route of pathogen entry and the impact of RLO infection on L. pertusa is unclear, particulate availability and the feeding strategies employed by the scleractinian corals may be influencing their exposure to pathogens. The absence of a pathogen in M. oculata may be attributed to the smaller number of colonies screened or the narrower diet in M. oculata compared to the unrestricted diet exhibited in L. pertusa, if ingestion is a route of entry for pathogen groups. The findings of this study also shed some light on how environmental conditions experienced by deep sea organisms and their life strategies may be limiting pathogen diversity and prevalence.

Histopathological survey for parasite groups in Gammarus varsoviensis (Amphipoda).

Invasive non-native amphipods (Crustacea) are becoming a model system in which to explore the impact and diversity of invasive parasites-parasites that are carried along an invasion route with their hosts. Gammarus varsoviensis is a freshwater amphipod species that has a recently explored invasion history. We provide a histopathological survey for a putatively invasive non-native population of this amphipod, identifying 8 symbiotic groups: Acanthocephala, Rotifera, Digenea, ciliated protozoa, Haplosporidia, Microsporidia, 'Candidatus Aquirickettsiella', and a putative nudivirus, at various prevalence. Our survey indicates that the parasites have no sex bias and that each has the potential to be carried in either sex along an invasion route. We discuss the pathology and prevalence of the above symbiotic groups and whether those that are parasitic may pose a risk if G. varsoviensis were to carry them to novel locations.

Co-occurrence of pathogen assemblages in a keystone species the common cockle Cerastoderma edule on the Irish coast.

Despite coinfections being recognized as the rule in animal populations, most studies focus on single pathogen systems. Pathogen interaction networks and the drivers of such associations are lacking in disease ecology studies. Common cockle Cerastoderma edule populations are exposed to a great diversity of pathogens, thus making them a good model system to investigate. This study examined the diversity and prevalence of pathogens from different taxonomic levels in wild and fished C. edule on the Irish coast. Potential interactions were tested focussing on abiotic (seawater temperature and salinity) and biotic (cockle size and age, and epiflora on shells) factors. No Microsporidia nor OsHV-1µVar were detected. Single infections with Haplosporidia (37.7%) or Vibrio (25.3%) were more common than two-pathogen coinfected individuals (9.5%), which may more easily succumb to infection. Fished C. edule populations with high cockle densities were more exposed to infections. Higher temperature and presence of epiflora on cockle shells promoted coinfection in warmer months. Low seawater salinity, host condition and proximity to other infected host species influenced coinfection distribution. A positive association between two Minchinia spp. was observed, most likely due to their different pathogenic effect. Findings highlight the major influence that ecological factors have on pathogen interactions and host­pathogen interplay.

Detection of haplosporidian protistan parasites supports an increase to their known diversity, geographic range and bivalve host specificity.

Haplosporidian protist parasites are a major concern for aquatic animal health, as they have been responsible for some of the most significant marine epizootics on record. Despite their impact on food security, aquaculture and ecosystem health, characterizing haplosporidian diversity, distributions and host range remains challenging. In this study, water filtering bivalve species, cockles Cerastoderma edule, mussels Mytilus spp. and Pacific oysters Crassostrea gigas, were screened using molecular genetic assays using deoxyribonucleic acid (DNA) markers for the Haplosporidia small subunit ribosomal deoxyribonucleic acid region. Two Haplosporidia species, both belonging to the Minchinia clade, were detected in C. edule and in the blue mussel Mytilus edulis in a new geographic range for the first time. No haplosporidians were detected in the C. gigas, Mediterranean mussel Mytilus galloprovincialis or Mytilus hybrids. These findings indicate that host selection and partitioning are occurring amongst cohabiting bivalve species. The detection of these Haplosporidia spp. raises questions as to whether they were always present, were introduced unintentionally via aquaculture and or shipping or were naturally introduced via water currents. These findings support an increase in the known diversity of a significant parasite group and highlight that parasite species may be present in marine environments but remain undetected, even in well-studied host species.

Biotic and abiotic factors influencing haplosporidian species distribution in the cockle Cerastoderma edule in Ireland.

The Phylum Haplosporidia consists of four genera (Minchinia, Haplosporidium, Urosporidium and Bonamia) that are endoparasitic protists of a wide range of marine invertebrates including commercial bivalve species. Characterization of haplosporidian species remains a challenge due to their patchy spatial and temporal distributions, host-restricted occurrence, and poorly known life cycles. However, they are commonly associated with significant mortality events in bivalves. Due to the recent sporadic mortality events that have occurred in cockles in Europe, the objectives of this study were to determine the diversity, distribution and seasonality of haplosporidian species in Cerastoderma edule populations at several Irish sites. The role of abiotic (temperature, salinity and dissolved oxygen in water) and biotic (cockle size and age) factors as drivers or inhibitors of haplosporidian infection were also assessed. Cockles (n = 998) from the intertidal were sampled from April/July 2018 to April 2019 at three sites with no commercial fishing activity on the south coast (Celtic Sea) and one site on the northeast coast (Irish Sea) with an active commercial fishery. Screening of the cockles by molecular techniques (PCR, Sanger sequencing) and by histopathology was carried out. Two species were identified and confirmed in Irish C. edule for the first time, Minchinia mercenariae -like (14.8%) and Minchinia tapetis (29.6%). Similar to other haplosporidian parasites, the Minchinia spp. detected in our study were present year-round at all sites, except for M. tapetis in Youghal Bay (Celtic Sea). Coinfection of both Minchinia species was only observed in Cork Harbour (Celtic Sea) and Dundalk Bay (Irish Sea), where Minchinia spp. showed a higher presence compared to Youghal Bay and Dungarvan Harbour (Celtic Sea). Moreover, haplosporidians detected with generic primers, were present at all of the sample sites throughout the year but had a higher occurrence during the winter months and were positively correlated with dissolved oxygen. Likewise, smaller and older C.edule seemed to be more vulnerable to the haplosporidian infection. Furthermore, haplosporidian distribution displayed spatial variability between and within sample sites, with the highest presence being observed in cockles at one of the commercially fished Dundalk beds, while the lowest presence was observed in cockles at the second Dundalk bed that was more influenced by freshwater runoff when the tide was out. Findings from this study provide additional information on the distribution and seasonal presence of novel haplosporidian species and their potential abiotic and biotic drivers/inhibitors of infection.

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa.

Bonamia exitiosa is an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of the Bonamia genus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

Loop-Mediated Isothermal Amplification for the Fast Detection of Bonamia ostreae and Bonamia exitiosa in Flat Oysters.

The haplosporidian parasites Bonamia ostreae (BO) and B. exitiosa (BE) are serious oyster pathogens. Two independent laboratories evaluated fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for rapidly detecting these parasites. Specific LAMP assays were designed on the BO actin-1 and BE actin genes. A further generic assay was conceived on a conserved region of the 18S gene to detect both Bonamia species. The optimal reaction temperature varied from 65 to 67 °C depending on the test and instrument. Melting temperatures were 89.8-90.2 °C, 87.0-87.6 °C, and 86.2-86.6 °C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/µL in a 30 min run. The BO and BE test sensitivity was ~1 log lower than a real-time PCR, while the generic test sensitivity was similar to the real-time PCR. Both the BO and BE assays were shown to be specific; however, the generic assay potentially cross-reacts with Haplosporidium costale. The performance of the LAMP assays evaluated on samples of known status detected positives within 7-20 min with a test accuracy of 100% for the BO and generic tests and a 95.8% accuracy for BE. The ease of use, rapidity and affordability of these tests allow for field deployment.

Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite.

This study aimed to examine the pathobiology of a haplosporidian-like infection in juvenile (pre-recruit) edible crabs (Cancer pagurus) from two locations in South West Wales, UK. Infected crabs showed no external symptoms of the disease but dissection revealed an infected and hypertrophic antennal gland. Histological examination showed extensive parasitisation of the antennal gland overlying the hepatopancreas. Heavily infected crabs also showed the presence of parasites with morphological similarities to Haplosporidia in the labyrinth of the antennal gland and in the gills. The spread of the infection from the antennal gland to the gills suggests that these parasites are released into the haemolymph. Attempts to characterise the haplosporidian-like organism using several primers previously shown to amplify members of the phylum Haplosporidia failed. The prevalence of infection in juvenile edible crabs varied throughout the sampling period of November 2011 to July 2012 with the lowest level of ca. 15% in November peaking at 70% in March. This parasite may represent a threat to the sustainability of edible crab fisheries in this region if the damage observed in the antennal gland and gills results in host mortality. The identification of these parasites as members of the phylum Haplosporidia based on morphology alone must be seen as tentative in the absence of sequence data.