Haplosporidium Nelsoni Research Articles

Molecular and ultrastructural characterization of Haplosporidium diporeiae n. sp., a parasite of Diporeia sp. (Amphipoda, Gammaridea) in the Laurentian Great Lakes (USA).

BackgroundThe phylum Haplosporidia contains coelozoic and histozoic, spore-forming, obligate protozoan endoparasites that infect a number of freshwater and marine invertebrates including bivalves, crustaceans, and polychaetes. In amphipods, haplosporidians cause systemic infection resulting in a range of pathologies. While amphipods belonging to the genus Diporeia (Gammarideae) have been shown to host haplosporidians, the taxonomic relationship of the Diporeia haplosporidian(s) is largely unknown due to the lack of phylogenetic and detailed ultrastructural studies.MethodsThe infection characteristics and taxonomic identity of a haplosporidian infecting Diporeia spp. (Diporeia) were based on microscopical investigation, electron microscopy, and Bayesian phylogenetic inference using haplosporidian 16S rRNA gene sequences.ResultsIn stained sections, the haplosporidian was observed to cause systemic infections in Diporeia that were often accompanied with host tissue degeneration. The haplosporidian appeared as binucleate plasmodia and sporocysts containing different spore maturation stages in the coelom, connective tissue, digestive tissue, and muscle. All of the observed systemic infections progressed to sporogenesis. Transmission electron microscopy revealed that fixed mature spores were slightly ellipsoidal and had a mean spore length X width of 5.34 ± 0.17 × 4.09 ± 0.15 μm. A hinged opercular lid with a length of 3.1 ± 0.17 μm was observed for a number of developing spores. The average thickness of the cell wall was 90.0 ± 8.33 nm. Thin filaments (70 nm) composed of spore wall material were observed projecting from an abopercular thickening of the spore wall. Phylogenetic analysis showed that the haplosporidian is novel bearing some similarities with the oyster pathogen Haplosporidium nelsoni, yet distinctly different.ConclusionsBased on its morphology, genetic sequence, and host, it became evident that the Diporeia haplosporidian is taxonomically novel and we propose its nomenclature as Haplosporidium diporeiae. This is the first report of a haplosporidian infecting Diporeia in Lake Superior.

A Comparison of Two Methods for Colorimetric in situ Hybridization Using Paraffin‐Embedded Tissue Sections and Digoxigenin‐Labeled Hybridization Probes

Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals.

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

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.

Triploid Oysters in the Chesapeake Bay: Comparison of Diploid and TriploidCrassostrea virginica

Diploid and triploid Eastern oysters, Crassostrea virginica, were tested at 3 sites characterized by low or moderate salinity regimes in the Virginia part of the Chesapeake Bay from November 2005 through October 2007. Both diploid and triploid cultures were replicated 3 times by producing separate spawns from different broodstock. Ploidy had a generally consistent effect on the performance of C. virginica at the 3 test sites. At the end of the study, in October 2007, and across all sites, triploid oysters had lower cumulative mortality than diploids (-34%), and greater shell height ( 25%), whole weight ( 88%), and yield ( 152%). as well as a higher proportion of market-size oysters ( 114%) than diploids. Both diploids and triploids were similarly infected by Perkinsus marinus and, to a lesser extent, by Haplosporidium nelsoni. In a closer look, growth parameters (shell height growth, whole weight, yield, and percentage of marketable oysters) were always higher in triploids than in diploids regardless of the parental source, strongly supporting the superior advantage of triploids. Similar results were obtained for cumulative mortality, but to a lesser extent as a result of the large variation in mortality for both diploid and triploid cohorts among sites, as well as a significant site-by-cohort interaction. Our report is the first clear illustration of variation for the cumulative mortality exhibited among different spawns in triploids, and comes with the lesson that care must be taken in experiments in which the goal is to test the effect of ploidy on this trait. Our results support the notion that selective breeding programs to reduce mortality, coupled with triploid production to increase growth, can further optimize yield. The best-performing replicate spawn had 80% survival after 2.5 y, and reached an average shell height of 92 mm, weighing 142 g.

Development of resistance to an introduced marine pathogen by a native host

In 1957–1959, the introduced protistan parasite, Haplosporidium nelsoni, killed 90–95% of the oysters (Crassostrea virginica) in lower Delaware Bay and about half of those in the upper bay. Shortly thereafter, H. nelsoni-caused mortality in the wild population of the lower bay declined, approximating that of first-generation selectively bred oysters. For nearly three decades thereafter no further change in survival of the wild population was evident, although steady improvement was achieved by continued selective breeding. Survival of the wild population is thought to have plateaued because the great majority of oysters inhabited the upper bay where they were protected from H. nelsoni infection and selective mortality by low salinity. Consequently, they contributed most of the offspring to the bay population. From 1957 through 1987, H. nelsoni prevalence was cyclic, but overall high (annual maxima of 60 to 85%) in the lower bay. Since 1988, however, prevalence in wild oysters has rarely exceeded 30% anywhere in the bay, even though unselected oysters continue to become heavily infected when exposed, and molecular evidence indicates that the parasite remains present throughout the bay. This apparent “second step” in the development of resistance in the wild oysters occurred after a drought-associated incursion of H. nelsoni into the upper bay in the mid-1980s. Mortalities were widespread, heavy and more extreme than during the 1957–59 epizootic. Resistant survivors of the second epizootic have apparently repopulated the bay. When compared to unselected stocks, common-garden exposure to H. nelsoni of oysters from both upbay and downbay sites indicates that a high degree of resistance to the development of MSX disease has become widespread in the wild oyster population of Delaware Bay after two major selection events separated by nearly 30 years.

Haplosporidium nelsoni (MSX) in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China

The protozoan parasite Haplosporidium nelsoni (MSX) was identified in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China by histopathologic examination, polymerase chain reaction (PCR) amplification, and in situ hybridization (ISH) assay. H. nelsoni plasmodia-like structures were identified in the digestive glands of scallops by histologic examination, but no parasite spores were observed. PCR using the Hap-F2, R2 primer pair produced a sequence with 100% homology with the corresponding small subunit rDNA region of H. nelsoni. An ISH assay using the oligonucleotide probe MSX1347 produced a positive reaction with the Japanese scallop parasite. This is the first report of H. nelsoni in P. yessoensis in China.

Declining impact of an introduced pathogen: ­Haplosporidium nelsoni in the oyster Crassostrea virginica in Chesapeake Bay

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 432:1-15 (2011) - DOI: https://doi.org/10.3354/meps09221 FEATURE ARTICLE Declining impact of an introduced pathogen: ­Haplosporidium nelsoni in the oyster Crassostrea virginica in Chesapeake Bay Ryan B. Carnegie*, Eugene M. Burreson Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, Virginia 23062, USA *Email: carnegie@vims.edu ABSTRACT: Disease caused by the parasite Haplosporidium nelsoni has devastated Crassostrea virginica in Chesapeake Bay, exacerbating effects of overharvesting and adversely impacting the ecology of the bay. H. nelsoni is thought to persist as an impediment to oyster restoration because strong reproductive contributions from oysters in low-salinity refugia from parasitism have prevented ­development of disease resistance. On the contrary, long-term data indicate that while infection pressure on naïve sentinels has grown, H. nelsoni levels in wild oysters have fallen, with prevalence typically below 20% and advanced infections uncommon. A transplant experiment comparing naïve sentinels with oysters from ­disease-enzootic populations indicated that these observations ­represent true disease resistance, and its ­geo­graphical ­distribution was revealed by annual fall surveys, and by intensive sampling in 2007 and 2008. Re­sistance is best developed in the small, polyhaline Lynnhaven River, where salinities are never low enough to suppress H. nelsoni. Resistance gradually decreases up the large tributaries and the bay, as revealed by infections reaching higher levels further upstream when drought allows H. nelsoni to colonize unselected oyster populations. Elevated disease in small oysters from the Rappahannock River suggests that a non-resistant component of the population persists due to reproductive contributions from unselected oysters in upriver sanctuaries from disease selection, and from ­susceptible but pre-disease oysters in the lower river. Contributions of these unselected oysters notwithstanding, our results point to substantial reproduction by resistant oysters in disease-enzootic waters, and suggest that reefs in mesohaline-polyhaline salinities, and not only those in disease-free lower mesohaline areas, should be the focus of conservation and restoration efforts. KEY WORDS: Haplosporidium nelsoni · Multinucleated sphere X disease · MSX · Crassostrea virginica · Disease ­resistance · Oyster restoration Full text in pdf format Information about this Feature Article NextCite this article as: Carnegie RB, Burreson EM (2011) Declining impact of an introduced pathogen: ­Haplosporidium nelsoni in the oyster Crassostrea virginica in Chesapeake Bay. Mar Ecol Prog Ser 432:1-15. https://doi.org/10.3354/meps09221 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 432. Online publication date: June 27, 2011 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2011 Inter-Research.

Widespread survey finds no evidence of Haplosporidium nelsoni (MSX) in Gulf of Mexico oysters

The advent of molecular detection assays has provided a set of very sensitive tools for the detection of pathogens in marine organisms, but it has also raised problems of how to interpret positive signals that are not accompanied by visual confirmation. PCR-positive results have recently been reported for Haplosporidium nelsoni (MSX), a pathogen of the oyster Crassostrea virginica in 31 of 40 oysters from 6 sites in the Gulf of Mexico and the Caribbean Sea. Histological confirmation of the PCR results was not undertaken, and no haplosporidian has been reported from the numerous histological studies and surveys of oysters in the region. To further investigate the possibility that H. nelsoni is present in this region, we sampled 210 oysters from 40 sites around the Gulf of Mexico and Puerto Rico using PCR and 180 of these using tissue-section histology also. None of the oysters showed evidence of H. nelsoni by PCR or of any haplosporidian by histology. We cannot, therefore, confirm that H. nelsoni is present and widespread in the Gulf of Mexico and the Caribbean Sea. Our results do not prove that H. nelsoni is absent from the region, but taken together with results from previous histological surveys, they suggest that for the purposes of controlling oyster importation, the region should continue to be considered free of the parasite.

Haplosporidium nelsoni and H. costale in the Pacific oyster Crassostrea gigas from China’s coasts

We examined 220 Pacific oysters Crassostrea gigas obtained from 11 locations along China's coasts for the presence of the 2 protistan parasites Haplosporidium nelsoni (MSX; multinucleated sphere X) and H. costale (SSO; seaside organism). Haplosporidium-like plasmodia were histologically observed in 9 oysters (4.09%) from 7 locations. Five oysters had mixed infections, and 4 oysters were infected only with H. nelsoni as determined by in situ hybridization (ISH) and polymerase chain reaction (PCR). This is the first report of H. nelsoni and H. costale infection in bivalves in Chinese waters.

A 1-year investigation of the parasite Haplosporidium nelsoni (MSX) in the Pacific oyster Crassostrea gigas from Dayaowan Bay, China

The infection prevalence of the protozoan parasite Haplosporidium nelsoni (MSX) in Pacific oysters (Crassostrea gigas), collected from Dayaowan Bay on the north coast of the Yellow Sea, China, was investigated in 2007. The traditional histological method of diagnosing H. nelsoni infection in oysters was compared to that of polymerase chain reaction (PCR). Histology and the first PCR analysis detected infection in 21 (a total of 240 oysters) (8.75%) oysters, and the second PCR revealed infection in 26 oysters (10.83%). Only local or epithelial infections were found; no systemic infections were detected. Infection by H. nelsoni mostly occurred from April through October, and the monthly prevalence ranged from 5% to 25%, with a peak in August. These results suggest that the prevalence of the parasite is low in Dayaowan Bay. The prevalence of H. nelsoni is thought to be controlled in some way by temperature and salinity.

Survival and Growth of TriploidCrassostrea virginica(Gmelin, 1791) andC. ariakensis(Fujita, 1913) in Bottom Environments of Chesapeake Bay: Implications for an Introduction

Survival and growth of triploid Crassostrea virginica and triploid C. ariakensis were investigated at four sites surrounding Chesapeake Bay, United States, that varied in salinity, tidal regime, water depth, predation intensity and disease pressure. Four experimental treatments were established at each site: C. virginica; C. ariakensis; 50:50 of C. virginica: C. ariakensis; and shell only. Oysters were deployed at mean shell heights of 12.80 mm and 13.85 mm (C. virginica and C. ariakensis, respectively), at an overall density of 347.5 oysters m-2. Oyster survival and growth varied significantly with site and species. Survival was significantly higher in C. virginica than C. ariakensis at the intertidal site, and significantly higher in C. ariakensis than C. virginica at the highest salinity, subtidal site. Survival did not differ significantly between species at the mid and low salinity, subtidal sites. For both species, survival differed significantly between sites, with lowest survival in both species occurring at the intertidal site. Among the subtidal sites, C. virginica survival varied inversely with salinity, whereas C. ariakensis had the lowest survival at the mid salinity site. Eight months after deployment C. ariakensis were significantly larger than C. virginica at all sites. This difference generally persisted throughout the experiment, though the size differences between oyster species at the lowest salinity site were small (< 10%). Shell heights within single-species treatments differed significantly between sites; highest growth rates were observed at the high salinity, subtidal site, whereas lowest growth rates were observed at the high salinity, intertidal site. At low and mid salinity subtidal sites, C. ariakensis shell heights were significantly greater in the single-species treatment compared with the mixed-species treatment. Perkinsus marinus infections occurred in both species at all sites, with prevalences varying between sites. In C. virginica, moderate and high intensity infections were only common at the two higher salinity sites, whereas infections in C. ariakensis were generally low to rare. Haplosporidium nelsoni infections in C. virginica were only observed at the two higher salinity sites and prevalences were generally low. Two out of 53 C. ariakensis tested at the high salinity, subtidal site had rare H. nelsoni infections. Bonamia spp. infections were never observed. Our study supports previous laboratory findings and observations from its native range that C. ariakensis survives poorly in intertidal habitats. In subtidal habitats, however, C. ariakensis displayed broad environmental tolerances, often exceeding native oyster survival and growth rates. Post-introduction C. ariakensis populations would be shaped by the survival and growth patterns described here, but also by their reproductive success, larval survival, predator-prey interactions and prevailing disease dynamics.

Using bivalves as particle collectors with PCR detection to investigate the environmental distribution of Haplosporidium nelsoni

Recently, PCR technology has been applied to search for marine microorganisms in environmental samples. Such sampling, however, has drawbacks, including the need to collect and filter large volumes of water, and the presence of substances in environmental samples that may destroy DNA or interfere with DNA isolation and amplification. We explored the possibility of using suspension-feeding bivalves in conjunction with PCR to investigate the environmental distribution of microparasites using the oyster pathogen Haplosporidium nelsoni (the MSX disease agent) with oysters and mussels in Delaware Bay, USA, as a model system. Delaware Bay oysters Crassostrea virginica have become very resistant to H. nelsoni infection development and rarely exhibit histologically detectable lesions. The ribbed mussel Geukensia demissa is also resistant. Infections were detected in only 6% of the histologically examined oysters in the present study, although PCR-positive signals for H. nelsoni were found in feces, gill or heart samples of up to 100% of oysters. Positive signals were found in the feces and gill (but not heart) samples of up to 50% of mussels. The temporal evolution of PCR signals suggested a wave-like pattern of putative infective particles, which mimicked a pattern documented in early mortality studies. The present study demonstrated that H. nelsoni persists throughout Delaware Bay, although it is rarely detected using standard histology. We propose that the use of suspension-feeding bivalves as particle collectors in combination with PCR could be a method for detecting the presence of marine microparasites and might help answer questions about factors that prevent outbreaks of epizootics in certain regions.

Differential modulation of eastern oyster (Crassostrea virginica) disease parasites by the El-Niño-Southern Oscillation and the North Atlantic Oscillation

The eastern oyster (Crassostrea virginica) is affected by two protozoan parasites, Perkinsus marinus which causes Dermo disease and Haplosporidium nelsoni which causes MSX (Multinucleated Sphere Unknown) disease. Both diseases are largely controlled by water temperature and salinity and thus are potentially sensitive to climate variations resulting from the El Nino-Southern Oscillation (ENSO), which influences climate along the Gulf of Mexico coast, and the North Atlantic Oscillation (NAO), which influences climate along the Atlantic coast of the United States. In this study, a 10-year time series of temperature and salinity and P. marinus infection intensity for a site in Louisiana on the Gulf of Mexico coast and a 52-year time series of air temperature and freshwater inflow and oyster mortality from Delaware Bay on the Atlantic coast of the United States were analyzed to determine patterns in disease and disease-induced mortality in C. virginica populations that resulted from ENSO and NAO climate variations. Wavelet analysis was used to decompose the environmental, disease infection intensity and oyster mortality time series into a time–frequency space to determine the dominant modes of variability and the time variability of the modes. For the Louisiana site, salinity and Dermo disease infection intensity are correlated at a periodicity of 4 years, which corresponds to ENSO. The influence of ENSO on Dermo disease along the Gulf of Mexico is through its effect on salinity, with high salinity, which occurs during the La Nina phase of ENSO at this location, favoring parasite proliferation. For the Delaware Bay site, the primary correlation was between temperature and oyster mortality, with a periodicity of 8 years, which corresponds to the NAO. Warmer temperatures, which occur during the positive phase of the NAO, favor the parasites causing increased oyster mortality. Thus, disease prevalence and intensity in C. virginica populations along the Gulf of Mexico coast is primarily regulated by salinity, whereas temperature regulates the disease process along the United States east coast. These results show that the response of an organism to climate variability in a region is not indicative of the response that will occur over the entire range of a particular species. This has important implications for management of marine resources, especially those that are commercially harvested.

Prospect of Oyster Culture in Pakistan: Pathology Assessment of Two Commercially Important Oyster Species

Nine different species of oysters belonging to the genera Crassostrea, Saccostrea, and Ostrea occur naturally along Pakistan's coastline in the northern Arabian Sea. At the present time, no commercial harvesting or hatchery culture of oysters exists in Pakistan. The world's oyster aquaculture industry is seriously affected by diseases caused by parasites, such as Bonamia ostrea in the European flat oyster Ostrea edulis, and Haplosporidium nelsoni or Perkinsus marinus in the Eastern oyster Crassostrea virginica. Pathogens may be endemic or recently introduced and may cause epizootic mortalities and catastrophic economic losses. To evaluate if there are parasites or diseases in Pakistan's oysters that could limit prospective aquaculture development, histopathological analyses were performed on two oyster species, Ostrea nomades and Crassostrea belcheri. Oysters were sampled year-around, and fixed and processed for histology. Slides (217 cases for O. nomades and 76 cases for C. belcheri) were examined for the presence of parasites or pathological changes. Ostrea nomades was infected by a protozoan parasite, Nematopsis sp. Sneider, 1892 (Apicomplexa, Porosporidae), at 67% prevalence, and C. belcheri at 1.3% prevalence. Nematopsis spp. did not induce pathological changes in the oysters. Stegotricha-like ciliates (Thigmotrichida, Ancistrocomidae) were present in the stomachs of O. nomades with a prevalence of 1%, and 25% of C. belcheri presented ceroid deposits in their tissues. Historically, Ostrea and Crassostrea genera have been associated with epizootic diseases, but in the present samples, no known, economically important parasites or diseases were found. Absence of any pathology in these samples projects a positive future for oyster aquaculture in Pakistan. These oyster populations should be protected from nonendemic bivalve importations, which could transmit new parasites to these potentially susceptible, previously unexposed, native oysters.

Characteristics of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin 1791) in the Discharge Area of a Nuclear Power Plant in Central Chesapeake Bay

Since 1978, the Maryland Department of Natural Resources has investigated the uptake and depuration by C. virginica of radionuclides released by the Calvert Cliffs Nuclear Power Plant (CCNPP), which is located on the western shore of Chesapeake Bay in southern Maryland. This ongoing program presented a unique opportunity to compare radionuclide transfer dynamics and other parameters such as growth, mortality, disease status, and meat condition for Crassostrea virginica and C. ariakensis in a side-by-side, in-situ environmental setting. Introducing the Asian oyster, C. ariakensis, into Chesapeake Bay has been proposed to help restore an oyster population to historical levels, to enhance the Bay ecosystem, and to contribute to the revival of the commercial oyster fishery. The population of the indigenous C. virginica has declined dramatically throughout the bay in part as a result of infection by two non-native protozoan parasites that cause MSX (Haplosporidium nelsoni) and Dermo (Perkinsus marinus) diseases. Other factors including habitat loss, reduced water quality, and overfishing have also reduced population size. Crassostrea ariakensis is known to be more tolerant to these diseases and thus may have greater potential for survival and growth in Chesapeake Bay than the native oyster. Triploid C. ariakensis and diploid C. virginica were immersed in cages in the vicinity of the CCNPP discharge for two exposure periods between July and December 2004. After retrieval, oysters were analyzed for radionuclide concentrations, growth rate, mortality, disease status, and meat condition. Fouling of oysters and cages was also observed. Quantities of radionuclides released by the CCNPP for both exposure periods were insufficient to produce detectable concentrations in either C. virginica or C. ariakensis, precluding a comparison of uptake of Co58, Co60, or Ag-110 m, radionuclides that have been detectable historically. Shell lengths were not significantly different between the two species for the two sampling periods, despite the fact that there were considerable age differences between them, but C. ariakensis did show a greater rate of growth. Meat weights and condition indices for C. ariakensis were significantly greater than for C. virginica (P < 0.01). Mortality and Dermo prevalence and intensity were also significantly greater for C. virginica than for C. ariakensis (P < 0.01). Although quantities of radionuclides were insufficient to produce detectable concentrations in either species, this study provided a unique opportunity to examine C. ariakensis in an area of the Maryland Chesapeake Bay where C. ariakensis has not previously been observed.

Prevalence of Perkinsus marinus (Dermo), Haplosporidium nelsoni (MSX), and QPX in Bivalves of Delaware's Inland Bays and Quantitative, High‐Throughput Diagnosis of Dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters ( Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166 bp polynucleotide probe while the second used a 30 bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested ( Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian ( Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220 bp region and detected Minchinia sp. DNA from 50 ng of genomic DNA extracted from the tissues of infected oysters and 10 fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.

HAPLOSPORIDIUM NELSONI (MSX) rDNA DETECTED IN OYSTERS FROM THE GULF OF MEXICO AND THE CARIBBEAN SEA

The known range of the oyster pathogen Haplosporidium nelsoni Haskin, Stauber, and Mackin (MSX) extends along the North American Atlantic coast from Nova Scotia to Florida. Our study demonstrates that H. nelsoni is also present throughout the Gulf of Mexico. Thirty of 41 oysters (73%) sampled from sites ranging from Florida to as far south as Venezuela were positive for MSX by PCR amplification of the ribosomal rRNA gene complex. DNA sequences cloned from oysters positive for H. nelsoni were $99% identical to H. nelsoni and clearly divergent (<89% identical) from known haplosporidian congeners, including Haplosporidium costale Wood and Andrews (SSO). The absence of MSX epizootics in the Gulf of Mexico, despite the wide distribution of H. nelsoni infections, raises questions about pathogenicity and the host-parasite relationship in subtropical latitudes.

Flow cytometry as a tool to quantify oyster defence mechanisms

The fast growing oyster aquaculture industry is greatly hindered by Perkinsus marinus and Haplosporidium nelsoni which can kill up to 80% of the production. The relationship between parasites and oyster defence mechanisms is unclear. Two defence mechanisms of the Eastern Oyster ( Crassostrea virginica) were quantified at the single cell level utilising flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the H 2O 2-specific increase in dichlorofluorescein-associated fluorescence upon stimulation. These two assays distinguished three populations of haemocytes (granulocytes, hyalinocytes and intermediate cells) with unique functional characteristics. Granulocytes were most active at phagocytosis and H 2O 2 production while hyalinocytes were relatively inactive. The intermediate cells had moderate phagocytic and respiratory burst activity. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system.