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.

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.

Role of the intertidal predatory shore crab Carcinus maenas in transmission dynamics of ostreid herpesvirus-1 microvariant.

Ostreid herpesvirus-1 microVar (OsHV-1 µVar) has been responsible for significant mortalities globally in the Pacific oyster Crassostrea gigas. While the impact of this virus on the Pacific oyster has been significant, this pathogen may have wider ecosystem consequences. It has not been definitively determined how the virus is sustaining itself in the marine environment and whether other species are susceptible. The shore crab Carcinus maenas is a mobile predator and scavenger of C. gigas, commonly found at Pacific oyster culture sites. The aim of this study was to investigate the role of the crab in viral maintenance and transmission to the Pacific oyster. A field trial took place over 1 summer at different shore heights at 2 Irish Pacific oyster culture sites that are endemic for OsHV-1 µVar. Infection of OsHV-1 µVar in tissues of C. maenas at both shore heights of both sites was detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), in situ hybridization and direct Sanger sequencing. In addition, a laboratory trial demonstrated that transmission of the virus could occur to naïve C. gigas within 4 d, from C. maenas previously exposed to the virus in the wild. These findings provide some insight into the possibility that the virus can be transmitted through marine food webs. The results also suggest viral plasticity in the hosts required by the virus and potential impacts on a range of crustacean species with wider ecosystem impacts if transmission to other species occurs.

The role of the mussel Mytilus spp. in the transmission of ostreid herpesvirus-1 microVar.

The Pacific oyster Crassostrea gigas contributes significantly to global aquaculture; however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can harbour the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400-500 m higher up the shore from the trestles and up to 26 km at non-culture sites were screened for OsHV-1 and variants by all the World Organization for Animal Health (OIE) recommended diagnostic methods including polymerase chain reaction (PCR), quantitative PCR (qPCR), histology, in situ hybridization and confirmation using direct sequencing. The particular primers that target OsHV-1 and variants, including OsHV-1 microVar (µVar), were used in the PCR and qPCR. OsHV-1 µVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels can harbour OsHV-1 µVar; however, the impact of OsHV-1 µVar on Mytilus spp. requires further investigation.

Oral immunostimulation of the oyster Ostrea edulis: Impacts on the parasite Bonamia ostreae.

Bioactive compounds were orally administered to the native European oyster Ostrea edulis to evaluate the immune response and the progression of infection of the protozoan parasite Bonamia ostreae. The immunostimulants lipopolysaccharide and zymosan directly administrated to the water column induced an increase in lysozyme activity and the percentage of granulocytes in naïve oysters over a period of 7 days. In another set of experiments, zymosan and curdlan were microencapsulated in alginate and also administered to the water column to naïve and B. ostreae infected O. edulis. Oyster mortality, prevalence and intensity of infection and several immune parameters were evaluated up to 28 days post-administration. Lysozyme activity, nitric oxide production and the expression of galectin, lysozyme and superoxide dismutase increased after 24 h in both infected and uninfected oysters. Zymosan immunostimulated oysters displayed a decrease in the prevalence of B. ostreae infection not attributed to mortalities but which could be associated to the enhanced ability of immunostimulants to evoke an enhanced immune response in the oysters and reduce infection.

A health status survey of clams, Mya arenaria and Ensis siliqua, in the Irish Sea.

The soft shell clam, Mya arenaria, and the razor clam, Ensis siliqua, are widely distributed in Irish waters. Though the reproductive biology and other aspects of the physiology of these species has been previously investigated, little or no data are currently available on their health status. As this knowledge is essential for correct management of a species, M. arenaria and E. siliqua were examined to assess their current health status using histological and molecular methods, over a period of sixteen months. No pathogens or disease were observed in M. arenaria, and low incidences of Prokaryote inclusions, trematode parasites, Nematopsis spp. and eosinophilic bodies were recorded in razor clams for the first time in Northern European waters.

The health status of mussels, Mytilus spp., in Ireland and Wales with the molecular identification of a previously undescribed haplosporidian.

Both wild and cultured mussels (Mytilus edulis, Mytilus galloprovincialis and hybrids), are found along most of the Irish coastline. M. edulis is widespread along all Irish coasts and is the only mussel species present on both the east coast of Ireland and the Welsh coast in the Irish Sea. M. galloprovincialis and hybrids are found along the Irish coastline except for the east coast. Samples of Mytilus spp. were collected from twenty-four sites, encompassing all coasts of Ireland and the Welsh coast, at different times of the year and over several years (2008-2011). In total, 841 mussels were examined histologically to assess their health status and the presence of any parasites or commensals. Mussels from 14 of the 24 sites were screened using polymerase chain reaction (PCR) to determine which mytilid species were present. A range of parasites were observed, generally at low levels. The most diverse community of parasites was observed at a sheltered site with poor water quality. Of significance, a previously undescribed haplosporidian was detected in a single mussel sample in the Menai Strait, Wales, by PCR and was confirmed by direct sequencing and is most closely related to Minchina chitonis and a haplosporidian of the Florida marsh clam Cyrenoida floridana. While M. edulis were infected by a variety of micro- and macro-parasites, only trematodes were observed in M. galloprovincialis and hybrids. Habitat description and the environmental factors influencing the study sites, including water quality and exposure, were recorded.

The occurrence of haplosporidian parasites, Haplosporidium nelsoni and Haplosporidium sp., in oysters in Ireland.

The phylum Haplosporidia is a group of obligate protozoan parasites that infect a number of freshwater and marine invertebrates. Haplosporidian parasites have caused significant mortalities in commercially important shellfish species worldwide. In this study, haplosporidia were detected in Pacific oysters Crassostrea gigas originating in Ireland and were subsequently identified independently in laboratories both in Ireland and in Spain as Haplosporidium nelsoni. In Ireland, H. nelsoni plasmodia were also observed in the heart tissue of a single Ostrea edulis. A range of techniques including heart smear screening, histology, standard polymerase chain reaction (PCR), direct sequencing and in situ hybridisation with an H. nelsoni specific DNA probe were carried out to confirm diagnosis. This is the first reporting of H. nelsoni in oysters in Ireland and this is the first reporting of the detection of this haplosporidian in O. edulis. In Ireland, another haplosporidian was also observed in a single O. edulis during heart smear screening. PCR and DNA sequencing were carried out and indicated the presence of a Haplosporidium sp., most likely Haplosporidium armoricanum. The low prevalence and intensity of infection of both haplosporidian species in Irish C. gigas and in particular O. edulis may indicate that their presence is inconsequential.

A previously undescribed ostreid herpes virus 1 (OsHV-1) genotype detected in the pacific oyster, Crassostrea gigas, in Ireland.

Significant mortalities of the Pacific oyster, Crassostrea gigas, have been reported worldwide since the 1950s. The impact these re-occurring mortality events have had on the C. gigas industry has highlighted the necessity to determine the factors that may be causing these mortalities. This study investigated the possible role of ostreid herpes virus (OsHV-1) in C. gigas mortalities over 2 successive summers at 2 study areas in Ireland. A single sample of adult C. gigas, which had been experiencing mortalities at one of the sites was screened. Successive cohorts of C. gigas spat obtained from a hatchery outside Ireland was relayed to both sites in 2003 and in 2004. Spat were screened each year prior to relaying. Samples were collected every 2 weeks and mortality counts were recorded and observed at both sites. Polymerase chain reaction (PCR) analysis and subsequent sequencing indicated that a previously undocumented variant genotype of OsHV-1 was present in the single cohort of adult C. gigas and in seed and juveniles at both sites, in both years. Analysis suggests that the Irish OsHV-1 µvar variant genotype is closely related to OsHV-1 µvar, first described in France in 2008.

Influence of disseminated neoplasia, trematode infections and gametogenesis on surfacing and mortality in the cockle Cerastoderma edule.

Cerastoderma edule is a widely distributed bivalve mollusc, commercially exploited throughout Europe and is also an important food source for birds and crustaceans. Recently, mass surfacing and mortalities of cockles have been observed and reported at sites in Ireland and elsewhere, particularly in the summer months. One such site is Flaxfort Strand, Courtmacsherry Bay, County Cork, Ireland, an important feeding area used by many seabirds during the summer months. For the past few years large numbers of surfaced cockles have been observed at the site in a moribund condition. Samples of cockles from this area were collected over the summer months and their health status assessed. Cockles that had surfaced (moribund) and those still buried in the sediment were quantified and screened: sex, gonadal maturity and size class of cockles were also determined. Disseminated neoplasia and trematodes were observed in screened cockles. The most significant finding during the study was that mortalities and surfacing of cockles was related to a greater incidence of disseminated neoplasia. No neoplasia was observed in the smallest and largest size classes. There was a significantly higher prevalence of neoplasia in moribund cockles than in buried cockles, whereas in both groups a similar concentration of trematode metacercariae was observed in the screened tissues. Also, most of the cockles that had surfaced were either in the process of spawning or were spent. Overall a much larger percentage of moribund cockles exhibited both trematode infections plus neoplasia compared with buried cockles. A combination of the presence of neoplasia and trematodes, along with stress related to spawning, may immunocompromise the cockless, causing the animals to surface and become moribund.

Observations raise the question if the Pacific oyster, Crassostrea gigas, can act as either a carrier or a reservoir for Bonamia ostreae or Bonamia exitiosa.

This study investigated the ability of the Pacific oyster, Crassostrea gigas, to act as a carrier or reservoir of the protistan Bonamia ostreae. Studies were carried out independently in Ireland and in Spain. Naïve C. gigas were exposed to B. ostreae both in the field and in the laboratory via natural exposure or experimental injection. Naïve flat oysters, Ostrea edulis, were placed in tanks with previously exposed C. gigas. Oysters were screened for B. ostreae by examination of ventricular heart smears and by polymerase chain reaction (PCR) screening of tissue samples (gill and/or heart) and shell cavity fluid. PCR-positive oysters were further screened using histology and in situ hybridization (ISH). B. ostreae DNA was detected in the tissues and/or shell cavity fluid of a small number of C. gigas in the field and in the laboratory. B. ostreae-like cells were visualized in the haemocytes of 1 C. gigas and B. ostreae-like cells were observed extracellularly in the connective tissues of 1 other C. gigas. When C. gigas naturally exposed to B. ostreae were held with naïve O. edulis, B. ostreae DNA was detected in O. edulis; however, B. ostreae cells were not visualized. In Spain, B. exitiosa DNA was also detected in Pacific oyster tissues. The results of this study have important implications for C. gigas transfers from B. ostreae-endemic areas to uninfected areas and highlight B. ostreae and B. exitiosa's ability to survive extracellularly and in other non-typical hosts.

Temporal variation of Meiogymnophallus minutus infections in the first and second intermediate host.

In order to study seasonal patterns of Meiogymnophallus minutus infections in its intermediate hosts, bivalve samples were collected monthly between April 2008 and March 2009 from a high intertidal flat at Courtmacsherry Bay, Ireland. Infection rates in the first intermediate host Scrobicularia plana did not fluctuate significantly with season. Completely developed M. minutus cercariae appeared in daughter sporocysts from June and prevailed from July to October, indicating that transmission of M. minutus from its first to its second intermediate host is confined to this period of the year. All analysed individuals of the second intermediate host Cerastoderma edule were found to be infected with metacercariae. Infection levels significantly increased in September, suggesting recent cercarial invasions. Throughout the year, the majority of metacercariae were hyperinfected by the pathogenic microsporidian Unikaryon legeri. Spreading of hyperinfections was confined to spring and summer. Newly settled metacercariae were not affected by hyperparasitism and presumably retained their infectivity for half a year. Our findings suggest that the spreading of hyperinfections is correlated with higher water temperatures and that the entire metacercarial population has to rebuild every year as a consequence of hyperparasite-induced mortality.

Infection of Mytilus edulis by the trematode Echinostephilla patellae (Digenea: Philophthalmidae).

The blue mussel Mytilus edulis is described as second intermediate host for Echinostephilla patellae from the common limpet Patella vulgata. Mussels were infected with metacercariae of E. patellae under laboratory conditions. Average infection rates increased with increasing temperature, whereas numbers of cercariae, to which individual mussels were exposed, had no effect on relative infection success. The round to slightly oval metacercariae with an average cyst diameter of 208 microm (range 186-243 microm) encysted exclusively in the foot tissue of M. edulis. Morphologically similar metacercariae were found in naturally infected mussels at sites where parasitized P. vulgata and M. edulis are sympatric. This is the first report of E. patellae in blue mussels. The detection of M. edulis being a second intermediate host is of particular interest with regard to the abundance of the parasite and host organisms in intertidal rocky shore ecosystems. The potential role of the common limpet P. vulgata as an alternative secondary host is discussed.

Investigating the possible role of benthic macroinvertebrates and zooplankton in the life cycle of the haplosporidian Bonamia ostreae.

Bonamia ostreae is a protistan parasite of the European flat oyster, Ostrea edulis. Though direct transmission of the parasite can occur between oysters, it is unclear if this represents the complete life cycle of the parasite, and the role of a secondary or intermediate host or carrier species cannot be ruled out. In this preliminary study, benthic macroinvertebrates and zooplankton from a B. ostreae-endemic area were screened for the presence of parasite DNA, using polymerase chain reaction (PCR). Eight benthic macroinvertebrates and nineteen grouped zooplankton samples gave positive results. Certain species, found positive for the parasite DNA, were then used in laboratory transmission trials, to investigate if they could infect naïve oysters. Transmission of B. ostreae was effected to two naïve oysters cohabiting with the brittle star, Ophiothrix fragilis.

Impacts of microcystins on the feeding behaviour and energy balance of zebra mussels, Dreissena polymorpha: a bioenergetics approach.

Microcystins are produced by bloom-forming cyanobacteria and pose significant health and ecological problems. To investigate the impacts of these biotoxins on the physiology of the zebra mussels, Dreissena polymorpha, a series of short-term feeding experiments were conducted in the laboratory. We used five microalgal diets consisting of single-cell suspensions of the green algae, Chlorella vulgaris, the diatom, Asterionella formosa, the cryptophyte, Cryptomonas sp. and two strains of the toxic cyanobacterium, Microcystis aeruginosa (strains CCAP 1450/06 and CCAP 1450/10). A sixth diet was a mixture of the diatom and the CCAP 1450/10 cyanobacterial strain. The low-toxicity strain CCAP 1450/06 contained 7.4 microg l(-1) of the MC-LR variant while the very toxic strain CCAP 1450/10 contained 23.8 microg l(-1) of MC-LR and 82.9 microg l(-1) of MC-LF. A flow-through system was designed to measure the following feeding parameters: clearance, filtration, ingestion and absorption rates. Ultimately the scope for growth (SFG) was determined as a net energy balance. We observed that mussels cleared the cyanobacterial species containing MC-LF (mean+/-95% confidence interval) at a significant lower rate (498+/-82 ml h(-1) g(-1) for the single cell suspension and 663+/-100 ml h(-1) g(-1) for the mixture diet) than all of the non-toxic species and the cyanobacterium containing MC-LR (all above 1l h(-1) g(-1)). The same pattern was observed with all the feeding parameters, particularly absorption rates. Furthermore, MC-LF caused an acute irritant response manifested by the production of 'pseudodiarrhoea', unusually fluid pseudofaeces, rich in mucus and MC-LF-producing Microcystis cells, ejected through the pedal gape of the mussels. This overall response therefore demonstrates selective rejection of MC-LF-producing cyanobacteria by zebra mussels, enhancing the presence of the very toxic MC-LF-producing M. aeruginosa in mixed cyanobacterial blooms and in the benthos. Finally, we observed that the SFG (mean+/-95% confidence interval) of mussels feeding on M. aeruginosa containing MC-LF was significantly lower (34.0+/-18.8 J h(-1) g(-1) for the single cell suspension and 83.1+/-53.0 J h(-1) g(-1) for the mixture diet) than for mussels ingesting non-toxic diets, except for C. vulgaris (all above 200 J h(-1)g(-1)). This reveals a sublethal, stressful effect of microcystins (particularly MC-LF) on the feeding behaviour and energy balance of the zebra mussel.

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis (L.).

Lysozyme activity and protein concentration in the haemolymph of the flat oyster Ostrea edulis were investigated. These biochemical constituents of the haemolymph could be an indication of the physiological condition and vitality of the defence system of an animal. Haemolymph protein and lysozyme in oysters were examined over an 18 month period to determine their relationship with the strain of oyster, the season, the site, and parasitism by Bonamia ostreae. Haemolymph protein concentration exhibited seasonal fluctuations and varied between strains. Levels of protein in oysters highly infected with B. ostreae were slightly depressed but not significantly so. Haemolymph lysozyme varied greatly between individuals but no correlation was found between lysozyme levels and infection of oysters by B. ostreae.

Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia.

The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7 % of the 'lightly' infected oysters, and 66.7 % of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia.