Sharon A. Lynch

Toxicity of copper oxide nanoparticles in the blue mussel, Mytilus edulis: a redox proteomic investigation

Relatively little is known about the fate and effects of nanomaterials even in relatively simple organisms such as Mytilus edulis. Here, copper oxide nanoparticles (CuO NP) are shown to induce dose-dependent toxic effects at the biochemical, physiological and tissue levels in the blue mussel. Stable CuO NP suspensions were sized by differential light scattering and nanoparticle tracking analysis to yield average particle diameters of approximately 100 nm. These were administered to M. edulis, at doses of 400, 700 and 1000 ppb. Ingested copper was predominantly located in the gill tissue with small amounts in digestive gland. Fifteen coomassie-stained spots were excised from two dimensional gel electrophoresis separations of gill tissue extacts and identified by peptide mass fingerprinting. These contained six unique proteins (alpha- and beta-tubulin, actin, tropomyosin, triosephosphate isomerase and Cu-Zn superoxide dismutase). Of these, two spots (actin and triosephosphate isomerase) showed decreased protein thiols while three (alpha-tubulin, tropomyosin and Cu-Zn superoxide dismutase) showed increased carbonylation which is indicative of protein oxidation of cytoskeleton and enzymes in response to CuO NP. The neutral red retention time (NRRT) assay revealed toxicity due to the CuO NPs which was comparable with toxic metal oxide nanoparticles such as chromium and cobalt. In contrast, non-toxic titanium and gold metal oxide nanoparticles gave no NRRT effects at similar NP concentrations. Histology revealed deposition of pigmented brown cells in response to CuO NP, located predominantly along the mantle and gill margin but also lining digestive tubules and some of the sinuses and distributed throughout the connective tissue and in the adductor muscle.

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.

Future Oceanic Warming and Acidification Alter Immune Response and Disease Status in a Commercial Shellfish Species, Mytilus edulis L.

Increases in atmospheric carbon dioxide are leading to physical changes in marine environments including parallel decreases in ocean pH and increases in seawater temperature. This study examined the impacts of a six month exposure to combined decreased pH and increased temperature on the immune response and disease status in the blue mussel, Mytilus edulis L. Results provide the first confirmation that exposure to future acidification and warming conditions via aquarium-based simulation may have parallel implications for bivalve health. Collectively, the data suggests that temperature more than pH may be the key driver affecting immune response in M. edulis. Data also suggests that both increases in temperature and/or lowered pH conditions may lead to changes in parasite abundance and diversity, pathological conditions, and bacterial incidence in M. edulis. These results have implications for future management of shellfish under a predicted climate change scenario and future sustainability of shellfisheries. Examination of the combined effects of two stressors over an extended exposure period provides key preliminary data and thus, this work represents a unique and vital contribution to current research efforts towards a collective understanding of expected near-future impacts of climate change on marine environments.

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. exitiosas ability to survive extracellularly and in other non-typical hosts.

THE SUSCEPTIBILITY OF YOUNG PRESPAWNING OYSTERS, OSTREA EDULIS, TO BONAMIA OSTREAE

Abstract Young prespawning oysters, Ostrea edulis, were held over 6 mo at two different Bonamia ostreae-endemic sites in Ireland, to determine to what extent they could become infected with this protozoan parasite. Prevalence and intensity of infection were monitored, using the traditional method of ventricular heart smears and polymerase chain reaction (PCR). Results showed that 0+ and 1+ oysters were susceptible to infection. Infection was observed in the naïve and previously exposed oysters 2 months post relaying. Of ventricular heart smears and PCR, PCR was the more sensitive diagnostic technique in detecting B. ostreae in most of the oysters. Current methods recommended by the Office International Epizooties (OIE) and the European Union (EU), histology and screening of heart smears for B. ostreae, may be inadequate because certain low levels of infection may go undetected.

Bonamia parasites: a rapidly changing perspective on a genus of important mollusc pathogens

Organisms of the genus Bonamia are intracellular protistan parasites of oysters. To date, 4 species have been described (B. ostreae, B. exitiosa, B. perspora and B. roughleyi), although the status of B. roughleyi is controversial. Introduction especially of B. ostreae and B. exitiosa to naïve host populations has been shown to cause mass mortalities in the past and has had a dramatic impact on oyster production. Both B. ostreae and B. exitiosa are pathogens notifiable to the World Organisation for Animal Health (OIE) and the European Union. Effective management of the disease caused by these pathogens is complicated by the extensive nature of the oyster production process and limited options for disease control of the cultured stocks in open water. This review focuses on the recent advances in research on genetic relationships between Bonamia isolates, geographical distribution, susceptible host species, diagnostics, epizootiology, host-parasite interactions, and disease resistance and control of this globally important genus of oyster pathogens.

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.

Thirty-year history of Irish (Rossmore) Ostrea edulis selectively bred for disease resistance to Bonamia ostreae.

The protistan pathogen Bonamia ostreae was first detected in Ostrea edulis at Rossmore, Cork Harbour, on the south coast of Ireland in 1987. A selective breeding programme commenced in 1988 by Atlantic Shellfish Ltd. to produce B. ostreae-resistant oysters using 3 to 4 yr old survivors as broodstock for controlled spawning in land-based spatting ponds. On-growing of oyster spat settled on mussel cultch was carried out on designated beds within Cork Harbour. Oyster production subsequently increased successfully, resulting in 3 yr old Rossmore O. edulis being marketed from 1993 onwards and a record tonnage of 4 yr old oysters being produced in 1995 and 1996. O. edulis production, B. ostreae prevalence and oyster mortalities have been monitored and recorded at Rossmore for over 30 yr. The collation and analysis of this data from 52 samples and 3190 oysters demonstrate the introduction and progression of bonamiosis and subsequent interventions to ameliorate disease effects during this period at Rossmore. Results suggest that O. edulis mortalities are now negligible during the first 4 yr of growth, prevalence of B. ostreae infection is low, and no correlation exists between prevalence of infection and oyster mortalities. This study, when compared to other studies of bonamiosis-infected oyster populations, suggests that an intervention in the form of a selective breeding programme is required to reduce the impact of the disease.

The Reproductive Biology of the Softshell Clam, Mya arenaria, in Ireland, and the Possible Impacts of Climate Variability

Little is known about the biology of the softshell clam in Europe, despite it being identified as a potential species to culture for food in the future. Monthly samples of the softshell clam, Mya arenaria, were collected intertidally from Co. Wexford, Ireland, over a period of sixteen months. The mean weight of sampled individuals was 74±4.9 g and mean length was 8.2±0.2 cm. Histological examination revealed a female-to-male ratio of 1 : 1.15. In 2010, M. arenaria at this site matured over the summer months, with both sexes either ripe or spawning by August. A single spawning event was recorded in 2010, completed by November. Two unusually cold winters, followed by a warmer-than-average spring, appear to have affected M. arenaria gametogenesis in this area, potentially affecting the time of spawning, fertilisation success, and recruitment of this species. No hermaphrodites were observed in the samples collected, nor were any pathogens observed. Timing of development and spawning is compared with the coasts of eastern North America and with other European coasts.

Development and Assessment of a Sensitive and Cost-Effective Polymerase Chain Reaction to Detect Ostreid Herpesvirus 1 and Variants

ABSTRACT Ostreid herpesvirus 1 (OsHV-1) and variants (OsHV-1 var, OsHV-1 µvar, Irish genotype) have had a significant impact on the Pacific oyster, Crassostrea gigas, industry worldwide and on the survival of larvae and juveniles of several other bivalve species in Europe. Diagnostic techniques used to screen for this pathogen include histology, polymerase chain reaction (PCR), and quantitative polymerase chain reaction (qPCR), which allows for quantification of the virus. Techniques used to confirm infection include PCR, in situ hybridization, and transmission electron microscopy, which have variable sensitivity and specificity. In this study, several new primers were designed to amplify the C region in the ORF4 gene of the virus. This region is variable and diagnostic among OsHV-1 and variants. To date, the routinely used C2/C6 primers, which are used to screen for OsHV-1 and variants, amplify a product of 709 bp whereas the new primers described in this study amplify products of 296 bp, 385 bp, and 400 bp. Several C. gigas samples, which had been exposed to herpes virus and variants were screened, and a sample of wild mussels (Mytilus spp.) was used as a negative control. The performance of the new PCR and primers was compared with the performance of the C2/C6 primers and qPCR. The effect of tissue storage and DNA extraction on PCR performance was also examined. The results of this study indicate that the new PCR and primers are more rapid, sensitive, and cost-effective compared with the C2/C6 PCR, and are as sensitive as qPCR. The results also highlight the impact that sample storage, tissue selection, DNA extraction method used, and subsequent storage of DNA have on PCR success or failure.