Search

Aims To investigate the relationships between individual health status of oysters, particularly with regard to parasitic infection, and variability in abundance of human-pathogenic Vibrio species. Methods and Results Aquacultured eastern oysters, Crassostrea virginica, were analysed individually for infection by the protozoan parasite Perkinsus marinus through quantitative PCR, and total Vibrio vulnificus and total and pathogenic Vibrio parahaemolyticus abundance was assessed using a most probable number (MPN)-qPCR approach. Additionally, perspective on general oyster health and other parasitic infections was obtained through histopathology. Perkinsus marinus infection and human-pathogenic Vibrio species levels were not correlated, but through histology, analyses revealed that oysters infected by Haplosporidium nelsoni harboured more V. vulnificus. Conclusions The highly prevalent parasite P. marinus had little influence on human-pathogenic Vibrio species levels in eastern oysters, but the less prevalent parasite, H. nelsoni, may influence V. vulnificus levels, highlighting the potential nuances of within-oyster dynamics of Vibrio species. Significance and Impact of the Study Human-pathogenic bacteria continue to be a concern to the oyster industry and causes for individual oyster variation in bacterial levels remain unknown. The major oyster pathogen P. marinus does not appear to affect levels of these bacteria within oysters, suggesting that other factors may influence Vibrio spp. levels in oysters.

Goniodomin A (GDA, 1) is a phycotoxin produced by at least four species of Alexandrium dinoflagellates that are found globally in brackish estuaries and lagoons. It is a linear polyketide with six oxygen heterocyclic rings that is cyclized into a macrocyclic structure via lactone formation. Two of the oxygen heterocycles in 1 comprise a spiro-bis-pyran, whereas goniodomin B (GDB) contains a 2,7-dioxabicyclo[3.3.1]nonane ring system fused to a pyran. When H2O is present, 1 undergoes facile conversion to isomer GDB and to an α,β-unsaturated ketone, goniodomin C (GDC, 7). GDB and GDC can be formed from GDA by cleavage of the spiro-bis-pyran ring system. GDA, but not GDB or GDC, forms a crown ether-type complex with K+. Equilibration of GDA with GDB and GDC is observed in the presence of H+ and of Na+, but the equilibrated mixtures revert to GDA upon addition of K+. Structural differences have been found between the K+ and Na+ complexes. The association of GDA with K+ is strong, while that with Na+ is weak. The K+ complex has a compact, well-defined structure, whereas Na+ complexes are an ill-defined mixture of species. Analyses of in vitro A. monilatum and A. hiranoi cultures indicate that only GDA is present in the cells; GDB and GDC appear to be postharvest transformation products.

Contamination of oysters with a variety of viruses is one key pathway to trigger outbreaks of massive oyster mortality as well as human illnesses, including gastroenteritis and hepatitis. Much effort has gone into examining the fate of viruses in contaminated oysters, yet the current state of knowledge of nonlinear virus-oyster interactions is not comprehensive because most studies have focused on a limited number of processes under a narrow range of experimental conditions. A framework is needed for describing the complex nonlinear virus-oyster interactions. Here, we introduce a mathematical model that includes key processes for viral dynamics in oysters, such as oyster filtration, viral replication, the antiviral immune response, apoptosis, autophagy, and selective accumulation. We evaluate the model performance for two groups of viruses, those that replicate in oysters (e.g., ostreid herpesvirus) and those that do not (e.g., norovirus), and show that this model simulates well the viral dynamics in oysters for both groups. The model analytically explains experimental findings and predicts how changes in different physiological processes and environmental conditions nonlinearly affect in-host viral dynamics, for example, that oysters at higher temperatures may be more resistant to infection by ostreid herpesvirus. It also provides new insight into food treatment for controlling outbreaks, for example, that depuration for reducing norovirus levels is more effective in environments where oyster filtration rates are higher. This study provides the foundation of a modeling framework to guide future experiments and numerical modeling for better prediction and management of outbreaks.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

Human-pathogenic Vibrio bacteria are acquired by oysters through filtering seawater, however, the relationships between levels of these bacteria in measured in oysters and overlying waters are inconsistent across regions. The reasons for these discrepancies are unclear hindering our ability to assess if -or when- seawater samples can be used as a proxy for oysters to assess risk. We investigated whether concentrations of total and human pathogenic Vibrio vulnificus (vvhA and pilF genes) and Vibrio parahaemolyticus (tlh, tdh and trh genes) measured in seawater reflect concentrations of these bacteria in oysters (Crassostrea virginica) cultured within the US lower Chesapeake Bay region. We measured Vibrio spp. concentrations using an MPN-qPCR approach and analyzed the data using structural equation modeling (SEM). We found seawater concentrations of these bacteria to predictably respond to temperature and salinity over chlorophyll a, pheophytin or turbidity. We also inferred from the SEM results that Vibrio concentrations in seawater strongly predict their respective concentrations in oysters. We hypothesize that such seawater-oyster coupling can be observed in regions of low tidal range. Due to the ease of sampling and processing of seawater samples compared to oyster samples, we suggest that under low tidal range conditions, seawater samples can foster increased spatial and temporal coverage and complement data associated with oyster samples.

Harmful algal blooms (HABs) cause damage to fisheries, aquaculture, and human health around the globe. However, the impact of HABs on water column microbiomes and biogeochemistry is poorly understood. This study examined the impacts of consecutive blooms of the ichthyotoxic dinoflagellates Margalefidinium polykrikoides and Alexandrium monilatum on the water microbiome in the York River Estuary, Chesapeake Bay, USA. The samples dominated by single dinoflagellate species and by a mix of the two dinoflagellates had different microbiome compositions than the ones with low levels of both species. The M. polykrikoides bloom was co-dominated by Winogradskyella and had increased concentrations of dissolved organic carbon. The A. monilatum bloom had little impact on the prokaryotic portion of the whole community but was associated with a specific group of prokaryotes in the particle-attached (3 µm) fraction including Candidatus Nitrosopumilus, Candidatus Actinomarina, SAR11 Clade Ia, Candidatus Bealeia, and Rhodobacteraceae HIMB11. Thus, blooms of these two algal species impacted the estuarine microbiome in different ways, likely leading to shifts in estuarine carbon and nutrient cycling, with M. polykrikoides potentially having a greater impact on carbon cycling in the estuarine ecosystem than A. monilatum.

The Caribbean spiny lobster Panulirus argus supports a large and valuable fishery in the Caribbean Sea. In 2007-2008, a rare microsporidian parasite with spore characteristics typical of the Ameson genus was detected in 2 spiny lobsters from southeast Florida (FL). However, the parasite species was not confirmed by molecular analyses. To address this deficiency, reported here are structural and molecular data on single lobsters displaying comparable 'cotton-like' abdominal muscle containing ovoid microsporidian spores found at different locations in FL in 2014 and 2018 and in Saint Kitts and Nevis Islands in 2017. In the lobster from 2014, multiple life stages consistent with an Ameson-like monokaryotic microsporidian were detected by transmission electron microscopy. A partial (1228 bp) small subunit (SSU) rRNA gene sequence showed each microsporidia to be identical and positioned it closest phylogenetically to Ameson pulvis in a highly supported clade also containing A. michaelis, A. metacarcini, A. portunus, and Nadelspora canceri. Using ecological, pathological, ultrastructural, and molecular data, the P. argus microsporidian has been assigned to a distinct species: Ameson herrnkindi.

Despite global investment in shellfish restoration activities, relatively little attention has been given to predicting optimal restoration sites and testing these expectations. We used a coupled biological-physical connectivity model as a guide to plant two distinct hatchery-spawned strains of the eastern oyster, Crassostrea virginica, in the Lafayette River, Virginia during the summer of 2013 at two locations corresponding to virtual spawning locations within the connectivity model. We utilized single nucleotide polymorphism markers to test the model predictions by genotyping oysters recruited after planting two hatchery-spawned strains and examining interannual recruitment variability for two successive years. Two spat were identified as hybrids of one of the planted strains and resident oyster genotypes. We also observed a genetic influence from an oyster strain used previously for restoration. Differences in environmental conditions between the two years of monitored recruitment likely affected larval dispersal and survival, contributing to observed interannual differences in the newly recruiting cohorts. Oyster spat from 2013 were genetically more similar to resident adults sampled in the Lafayette River, while the 2014 spat exhibited genotypic frequencies more similar to adults from surrounding rivers. The winds during the spawning seasons differed between years providing conditions for retention in 2013 and mixing of water masses in 2014. We recommend that the monitoring of restoration activities should consider relevant environmental conditions and observe multiple years of recruitment to assess the genetic impacts of restoration plantings and variable reproductive success.

Blooms of Margalefidinium (previously Cochlodinium) polykrikoides occur almost annually in summer in the lower Chesapeake Bay and its tributaries (e.g., the James and York Rivers). The Lafayette River, a sub-tributary of the lower James River, has been recognized as an initiation location for blooms in this region. The timing of bloom initiation varies interannually, ranging from late June to early August. To fully understand critical environmental factors controlling bloom initiation and interactions between physical and biological processes, a numerical module simulating M. polykrikoides blooms was developed with a focus on the bloom initiation. The module also includes life cycle and behavioral strategies such as mixotrophy, vertical migration, cyst dynamics and grazing suppression. Parameterizations for these behaviors were assigned based on published laboratory culture experiments. The module was coupled with a 3D physical-biogeochemical model for the James River that examined the contribution of each environmental factor and behavioral strategy to bloom initiation and development. Model simulation results highlight the importance of mixotrophy in maintaining high growth rates for M. polykrikoides in this region. Model results suggest that while many factors contribute to the initiation process, temperature, physical transport processes, and cyst germination are the three dominant factors controlling the interannual variability in the timing of bloom initiation.

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.

During a survey of the production of goniodomin A (GDA) by Alexandrium pseudogonyaulax in Danish coastal waters, Krock et al. (2018) obtained mass spectral evidence for the presence of a truncated congener, herein termed GD754, having a molecular weight 14 Da lower than GDA and assigned it as goniodomin B (GDB). An erroneous structure of GDB involving deletion of a methylene group between rings B and D had previously been reported by Espina et al. (2016) but without experimental details. HPLC properties reported by Krock for GD754 point to it being a homolog of GDA. Comparison of mass spectral fragmentation data reported for GD754 with fragmentation data for GDA, show it to be a truncated form of GDA with the deletion involving a CH2 group from ring F or one of the two methyl substituents on ring F, not elsewhere on the molecule. On biosynthetic grounds, the GD754 congener is proposed to be 34-desmethyl-GDA. Further experimental work will be required to confirm this hypothesis.

Throughout human history there are few foods that have been at the center of as much conflict, intrigue, murders, passionate emotions and poetry as the humble oyster. The history of the oyster in the Chesapeake Bay region includes a boom and bust period and is peppered with many colorful and fascinating anecdotes. It was a primary food source for Native Americans and early European settlers, and the targeted species of a thriving fishing industry in the 1800s during which over exploitation and competition resulted in “oyster wars” fought on several fronts. Today the oyster is the focus of a rapidly growing aquaculture industry and is recognized as a keystone species in the Bay providing important ecological functions such as water filtration and habitat through reef formation for a diverse community of Bay organisms including juvenile fish and crabs. This has motivated oyster restoration efforts led by both governmental agencies and non-profit organizations. Future challenges for the Chesapeake Bay oyster populations and aquaculture industry include multiple stressors; disease, changing climate, hypoxia, and harmful algal blooms.

A monoclonal Perkinsus chesapeaki isolate was established from 1 of 10 infected Australian Anadara trapezia cockles. Morphological features were similar to those of described P. chesapeaki isolates, and also included a unique vermiform schizont cell-type. Perkinsus olseni-specific PCR primers amplified DNAs from all 10 cockles. Perkinsus chesapeaki-specific primers also amplified DNAs from 4/10 cockles, including DNA from the isolate source cockle. Three different sets of DNA sequences from the monoclonal isolate grouped with the homologous, previously deposited, P. chesapeaki sequences in phylogenetic analyses. In situ hybridization assays detected both P. chesapeaki and P. olseni cells in histological sections from the source cockle for monoclonal isolate ATCC PRA-425.

The parasitic dinoflagellate Hematodinium perezi infects the American blue crab Callinectes sapidus and other decapods along the Eastern seaboard and Gulf of Mexico coast of the USA. Large juvenile and adult blue crabs experience high mortality during seasonal outbreaks of H. perezi, but less is known about its presence in the early life history stages of this host. We determined the prevalence of H. perezi in megalopae and early benthic juvenile crabs from multiple locations along the Virginia portion of the Delmarva Peninsula. The DNA of H. perezi was not detected in any megalopae collected from several locations within the oceanic coastal bay complex in which H. perezi is found at high prevalence levels. However, prevalence levels were high in early benthic juveniles from 2 oceanic coastal embayments: South Bay and Cobb Bay. Prevalence levels were lower at locations within Chesapeake Bay, including Cherrystone Creek, Hungars Creek, and Pungoteague Creek. Sampling over different seasons and several consecutive years indicates that disease transmission occurs rapidly after megalopae settle in high-salinity bays along the Delmarva Peninsula during the late summer and fall. Infected juvenile crabs can overwinter with the parasite and, when subjected to increasing water temperatures in spring, infections progress rapidly, culminating in transmission to other crabs in late spring and early summer. In high-salinity embayments, H. perezi can reach high prevalence levels and may significantly affect recruitment of juvenile blue crabs into the adult fishery.

In 1968 Burkholder and associates (J. Antibiot. (Tokyo) 1968, 21, 659–664) isolated the antifungal toxin goniodomin from an unidentified Puerto Rican dinoflagellate and partially characterized its structure. Subsequently, a metabolite of Alexandrium hiranoi was isolated by Murakami et al. from a bloom in Japan and its structure was established (Tetrahedron Lett. 1988, 29, 1149–1152). The Japanese substance had strong similarities to Burkholder's but due to uncertainty as to whether it was identical or only similar, Murakami named his toxin goniodomin A. A detailed study of this question now provides compelling evidence that Burkholder's goniodomin is identical to goniodomin A. Morphological characterization of the dinoflagellate suggests that it was the genus Alexandrium but insufficient evidence is available to make a definite identification of the species. This is the only report of goniodomin in the Caribbean region.

Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia's coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods.

We describe the introduction, taxonomic identification and genetic parameters for farm traits of the Midori strain of the Pacific oyster Crassostrea gigas - a possible alternative to the widely farmed Miyagi strain on the US West Coast. Broodstock (G0) oysters collected from the mouth of the Midori river and near Iwajima Island, Ariake Bay, Japan were identified as C. gigas based on the nuclear internal transcribed spacer (ITS) region and 28S gene as well as a mitochondrial (COI) marker. G0 and G1 generations and G2 larvae and spat were maintained in quarantine conditions and subjected to numerous histological exams and PCR tests for detection of pathogens of concern. After the G1 generation and G2 early life stages were found to be free of detectable diseases, G2 spat were planted at farm test sites and adults were used to produce a G3 generation. This G3 generation was cultured under non-quarantine conditions and families were planted at sub-tidal and inter-tidal test sites, in Oregon and California, respectively. Field performance traits were measured to determine the potential for improvement through selection. Overall, Midori oysters showed significantly lower values for harvest yield, survival, individual average weight and dry meat weight compared with oysters from a selected stock of the Miyagi strain of Pacific oyster; however, shell shape index (a measure of shell depth) was significantly greater in Midori oysters. Heritability estimates for group traits at harvest (survival, yield and individual average harvest weight) were high, whereas heritability estimates for individual traits (individual weight, wet meat weight and dry meat weight) and for shell dimension measurements (shell length, shell width and shell depth) were moderate-to-high. Family-by-site (GxE) correlations were low-to-moderate for all measured traits. Genetic correlations were positive and moderate-to-high among group traits and shell dimensions. In contrast, correlations between shell shape and other traits had small-to-moderate negative values but these were not statistically significant. Estimated potential genetic gain for dry meat weight was high (46%) whereas estimated gains for yield, individual average weight, individual weight were high-to-moderate (approximately 10%). In contrast, shell shape showed very little potential for genetic gain through selection. Overall, the Midori strain of the Pacific oyster shows promise as a deep-cupped oyster for the half-shell trade. Statement of relevance We describe the introduction of a new strain of Pacific oyster (Midori) collected from southern Japan to the US West Coast. Rigorous quarantine protocols and disease tests were applied to ensure that the risks of introducing diseases were minimized. The Midori strain was found to have a deeper cup than the commonly farmed Miyagi strain but the potential estimated gain for improving this trait through selection was small.

Resource allocation to reproduction is a primary physiological concern for individuals, and can vary with age, environment, or a combination of both factors. In this study we quantified the impact of environment and individual age on the reproductive output of female oysters Crassostrea virginica. We determined the relative fecundity, egg total lipid content, and overall and omega-3/omega-6 (ω3/ω6) fatty acid signatures (FAS) of eggs spawned by female oysters over a 2-year period (n = 32 and n = 64). Variation was quantified spatially and ontogenetically by sampling young and old oyster populations from two rivers in Chesapeake Bay, totaling four collection sites. During Year 1, when oysters underwent oogenesis in different locations, overall and ω3/ω6 egg FAS varied significantly by river, with no significant differences observed in the FAS of oysters by age in Year 1. In Year 2, when oysters from different sites underwent oogenesis in a single location, no significant differences in the overall egg FAS or ω3/ω6 egg FAS by river or age were observed. These findings suggest that oysters integrate environment into their reproductive output, but that time spent growing at a specific location (in this case, represented by oyster age) plays a relatively minor role in the biochemical composition of oyster eggs. These results have consequences for our understanding of how resources are allocated from the female oyster to eggs and, more generally, the impact of environment and ontogeny on reproductive physiology.

The recent discovery of Perkinsus honshuensis, a new Perkinsus species infecting Manila clams Ruditapes philippinarum (Sowerby, 1852), in Japan, suggested that, based on proximity, P. honshuensis could also be in Korean waters, where to date, P. olseni was believed to be the only Perkinsus species present. Perkinsus sp. infections consistently occurred among Ruditapes variegatus clams on a pebble beach on Jeju Island, off the south coast of Korea. The typical 'signet ring' morphology of the parasite was observed in the connective tissue of the digestive gland, and infection intensity was comparatively low (3.3 × 103 ± 1.2 × 104 to 1.3 × 104 ± 6.1 × 104 cells g-1 gill weight). Further DNA analyses of internal transcribed spacer (ITS-1, 5.8S and ITS-2) and non-transcribed spacer (NTS) regions of the parasite showed 98.9-99.8 and 98.5-99.5% similarity to those of P. honshuensis from Japan, respectively. Phylogenetic analyses using ITS and NTS sequences indicated that Perkinsus sp. from Jeju formed a highly supported clade with P. honshuensis. This is the first report of P. honshuensis infections in clams in Korean waters and the first report of R. variegatus as a host for that parasite.

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

High salinity relay of Eastern oysters (Crassostrea virginica) was evaluated as a post-harvest processing (PHP) method for reducing Vibrio vulnificus. This approach relies on the exposure of oysters to natural high salinity waters and preserves a live product compared to previously approved PHPs. Although results of prior studies evaluating high salinity relay as a means to decrease V. vulnificus levels were promising, validation of this method as a PHP following approved guidelines is required. This study was designed to provide data for validation of this method following Food and Drug Administration (FDA) PHP validation guidelines. During each of 3 relay experiments, oysters cultured from 3 different Chesapeake Bay sites of contrasting salinities (10-21 psu) were relayed without acclimation to high salinity waters (31-33 psu) for up to 28 days. Densities of V. vulnificus and densities of total and pathogenic Vibrio parahaemolyticus (as tdh positive strains) were measured using an MPN-quantitative PCR approach. Overall, 9 lots of oysters were relayed with 6 exhibiting initial V. vulnificus10,000/g. As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the30 MPN/g densities requirements for V. vulnificus after 14 to 28 days of relay. Densities of total and pathogenic V. parahaemolyticus in relayed oysters were significantly lower than densities at the sites of origin suggesting an additional benefit associated with high salinity relay. While relay did not have a detrimental effect on oyster condition, oyster mortality levels ranged from 2 to 61% after 28 days of relay. Although the identification of the factors implicated in oyster mortality will require further examination, this study strongly supports the validation of high salinity relay as an effective PHP method to reduce levels of V. vulnificus in oysters to endpoint levels approved for human consumption.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l’Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l’Ebre.

Panama is a major hub for commercial shipping between two oceans, making it an ideal location to examine parasite biogeography, potential invasions, and the spread of infectious agents. Our goals were to (i) characterise the diversity and genetic connectivity of Perkinsus spp. haplotypes across the Panamanian Isthmus and (ii) combine these data with sequences from around the world to evaluate the current phylogeography and genetic connectivity of these widespread molluscan parasites. We collected 752 bivalves from 12 locations along the coast of Panama including locations around the Bocas del Toro archipelago and the Caribbean and Pacific entrances to the Panama Canal, from December 2012 to February 2013. We used molecular genetic methods to screen for Perkinsus spp. and obtained internal transcribed spacer region (ITS) ribosomal DNA (rDNA) sequences for all positive samples. Our sequence data were used to evaluate regional haplotype diversity and distribution across both coasts of Panama, and were then combined with publicly available sequences to create global haplotype networks. We found 26 ITS haplotypes from four Perkinsus spp. (1-12 haplotypes per species) in Panama. Perkinsus beihaiensis haplotypes had the highest genetic diversity, were the most regionally widespread, and were associated with the greatest number of hosts. On a global scale, network analyses demonstrated that some haplotypes found in Panama were cosmopolitan (Perkinsus chesapeaki, Perkinsus marinus), while others were more geographically restricted (Perkinsus olseni, P. beihaiensis), indicating different levels of genetic connectivity and dispersal. We found some Perkinsus haplotypes were shared across the Isthmus of Panama and several regions around the world, including across ocean basins. We also found that haplotype diversity is currently underestimated and directly related to the number of sequences. Nevertheless, our results demonstrate long-range dispersal and global connectivity for many haplotypes, suggesting that dispersal through shipping probably contributes to these biogeographical patterns.

The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

Since 2012, aquacultured eastern oysters Crassostrea virginica have been reported by oyster farmers to display mortality approaching 30%, and in some cases 85%, in areas of the lower Chesapeake Bay, VA. Based on accounts from industry, this mortality has typically affected 1-y-old oysters between May and early July, and has tended to occur in triploid oysters, which represent the vast bulk of production in the area. During this period, samples submitted for pathology have not revealed the presence of major pathogens as a cause. In 2015, to gain deeper insight into this mortality and determine whether specific sites, ploidy condition, or genetic lines were affected, oyster seed commercially produced in early 2014 were obtained from four lines, one diploid (2N DEBY) and three triploid (3N DEBY, 3N hANA, and 3N Northern). These lines were deployed in July 2014 at aquaculture farms at five Chesapeake Bay locations: Locklies Creek and Milford Haven on the western shore, and Pungoteague Creek, Nassawadox Creek, and Cherrystone Creek on the Eastern Shore. During this study, mortality was observed to peak in June at most sites, reaching a mean mortality across all tested lines of 17.0% and a cumulative mortality for the study period of 32.0% at Nassawadox Creek, the site most severely affected by mortality that followed the expected early summer mortality pattern. Interval mortality at all sites decreased to under 5% after June, but cumulative levels for the study period reached from 8.8% to 18.6% even at the sites least affected by mortality. This represents a high level of mortality given the documented absence of material involvement by major oyster pathogens such as Hapolosporidium nelsoni and Perkinsus marinus. Infiltration of gill tissues by hemocytes, observed in up to 33% of individuals at Nassawadox Creek coincident with the increase in mortality, was the only pathology observed. Harmful algal blooms were not associated with the mortality, nor were abnormal temperatures or salinities. There was no clear relationship of mortality to oyster genetic heritage, although there was variability in susceptibility among oyster lines and interactions between lines and specific sites. At some locations and in comparison with diploids, triploid oysters appeared to be more susceptible to mortality. Mortality in triploids was coincident with the timing of peak gametogenic development in diploids. Given the lack of involvement by major pathogens and the possible association of mortality with oyster gametogenesis, future work should seek to better understand the suite of environmental stressors potentially impacting cultured oysters in these systems and their interactions with the physiology and energetics of these animals.

Ostreobdella californiana n. sp. is described from rockfishes in the genus Sebastes in public display tanks in the Steinhart Aquarium, California Academy of Sciences, San Francisco, California, U.S.A., and in the Bodega Marine Laboratory, University of California, Davis, Bodega Bay, California. New species justification was based on external morphology, histology, and molecular sequence data. Leeches are small, up to 12 mm total length. Oral sucker is larger than caudal sucker, with 1 pair of eyes on the oral sucker and 10 ocelli on the caudal sucker. Segmental ocelli occur both dorsally and ventrally on the urosome. Small papillae are present on urosome segments, but pulsatile vesicles are absent. There are 6 pairs of testisacs. The male reproductive system has a muscular organ replacing the typical atrium and a complex system of conducting/vector tissue. Mycetomes are present, and postceca are fused with fenestrae at each ganglion.

Hematodiniumperezi (genotype III) is a parasitic dinoflagellate that infects blue crabs along the eastern seaboard and Gulf of Mexico, USA. In order to examine the intra-specific genetic variation of this parasite, eleven microsatellite markers from H. perezi (III) were amplified from 227 infected blue crabs collected during 2008–2009 from six sites in Virginia. Simultaneous infections with multiple genetic types in a single-host individual were common and observed in 42 % of the samples. The remaining 58 % of samples had a single allele per locus at all eight polymorphic loci suggesting that the life history stages of the parasite in the host hemolymph are likely haploid. The composition and distribution of multi-locus genotypes (MLG) from samples with infections of a single genetic type indicated high genotypic variation along the Delmarva Peninsula, Virginia, with no evidence of population structure. The lack of linkage disequilibrium combined with the large number of unique MLGs (84 %) is strong evidence for recombination in the life cycle, but the sexual stages remain undetermined. This is the first evidence of ploidy level, infections by multiple genetic types in an individual host animal, high levels of genotypic variation, and sexual reproduction for any species of Hematodinium.

Genetic population structure of anadromous striped bass along the US Atlantic coast was analyzed using 14 neutral nuclear DNA microsatellites. Young-of-the-year and adult striped bass (n = 1114) were sampled from Hudson River, Delaware River, Chesapeake Bay, North Carolina, and South Carolina. Analyses indicated clear population structure with significant genetic differentiation between all regions. Global multilocus FST was estimated at 0.028 (P < 0.001). Population structure followed an isolation-by-distance model and temporal sampling indicated a stable population structure more than 2 years at all locations. Significant structure was absent within Hudson River, whereas weak but significant genetic differences were observed between northern and southern samples in Chesapeake Bay. The largest and smallest effective striped bass population sizes were found in Chesapeake Bay and South Carolina, respectively. Coalescence analysis indicated that the highest historical gene flow has been between Chesapeake Bay and Hudson River populations, and that exchange has not been unidirectional. Bayesian analysis of contemporary migration indicated that Chesapeake Bay serves as a major source of migrants for Atlantic coastal regions from Albemarle Sound northward. In addition to examining population genetic structure, the data acquired during this project were capable of serving as a baseline for assigning fish with unknown origin to source region.

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid-Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR-RFLP analysis using cultures obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues' collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR-RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.

Hematodinium sp. infections are relatively common in some American blue crab (Callinectes sapidus) populations in estuaries of the western Atlantic Ocean. Outbreaks of disease caused by Hematodinium sp. can be extensive and can cause substantial mortalities in blue crab populations in high salinities. We examined several species of crustaceans to determine if the same species of Hematodinium that infects C. sapidus is found in other crustaceans from the same localities. Over a 2-yr period, 1,829 crustaceans were collected from the Delmarva Peninsula, Virginia, examined for the presence of infections. A portion of the first internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene complex from Hematodinium sp. was amplified and sequences were compared among 35 individual crustaceans putatively infected with the parasite, as determined by microscopic examination, and 4 crustaceans putatively infected based only on PCR analysis. Of the 18 crustacean species examined, 5 were infected with Hematodinium sp. after microscopic examination and PCR analysis, including 3 new host records, and an additional species was positive only via PCR analysis. The ITS1 rRNA sequences of Hematodinium sp. from the infected crustaceans were highly similar to each other and to that reported from C. sapidus (98%). The similarity among these ITS1 sequences and similarities in the histopathology of infected hosts is evidence that the same species of Hematodinium found in C. sapidus infects a broad range of crustaceans along the Delmarva Peninsula. Our data indicate that the species of Hematodinium found in blue crabs from estuaries along the east coast of North America is a host generalist, capable of infecting hosts in different families within the Order Decapoda. Additionally, evidence indicates that it may be capable of infecting crustaceans within the Order Amphipoda.

Although several different U.S. hatchery stocks of the Asian Suminoe oyster Crassostrea ariakensis were used in laboratory and field trials assessing performance, and in comparative studies with the native oyster Crassostrea virginica, the genetic composition of these hatchery stocks has not yet been examined comprehensively. Using eight microsatellite markers we investigated the genetic variability among five hatchery stocks and compared the genetic makeup of these stocks with 8 wild populations from Asia. Results showed significant genetic differentiation among the 5 hatchery stocks that was 6-fold larger than that observed among wild populations. A significant reduction in genetic diversity was observed in hatchery stocks compared with wild source populations, indicating a genetic bottleneck. Two long-established stocks showed significant decreases in both allelic diversity and heterozygosity compared with the wild Japanese source population, whereas three recently established stocks showed le...

In 2009 the U.S. Food and Drug Administration (FDA) announced its intention to implement postharvest processing (PHP) methods to eliminate Vibrio vulnificus from oysters intended for the raw, half-shell market that are harvested from the Gulf of Mexico during warmer months. FDA-approved PHP methods can be expensive and may be associated with unfavorable responses from some consumers. A relatively unexplored PHP method that uses relaying to high salinity waters could be an alternative strategy, considering that high salinities appear to negatively affect the survival of V. vulnificus. During relay, however, oysters may be exposed to rapid and large salinity increases that could cause increased mortality. In this study, the effectiveness of high salinity relay to reduce V. vulnificus to

In this study we describe, the first microsporidian parasite from nephropid lobsters. Metanephrops challengeri were captured from an important marine fishery situated off the south coast of New Zealand. Infected lobsters displayed an unusual external appearance and were lethargic. Histology was used to demonstrate replacement of skeletal and other muscles by merogonic and sporogonic stages of the parasite, while transmission electron microscopy revealed the presence of diplokaryotic meronts, sporonts, sporoblasts and spore stages, all in direct contact with the host sarcoplasm. Analysis of the ssrDNA gene sequence from the lobster microsporidian suggested a close affinity with Thelohania butleri, a morphologically dissimilar microsporidian from marine shrimps. Whilst morphological features of the lobster parasite are consistent with members of the family Nosematidae, molecular data place the parasite closer to members of the family Thelohanidae. Due to the contradiction between morphological and molecular taxonomic data, we propose the erection of a new genus in which the lobster parasite is the type species (Myospora metanephrops). Furthermore, we recommend the erection of a new family (Myosporidae) and a new order (Crustaceacida) to contain this genus. The taxonomic framework presented could be further applied to the re-classification of existing members of the Phylum Microsporidia.

Species of Hematodinium are highly pathogenic parasitic dinoflagellates and can cause substantial mortalities in many decapod crustaceans. Periodic outbreaks of Hematodinium disease have significantly impacted stocks of the blue crab Callinectes sapidus throughout the Delmarva Peninsula. Using a primer set targeting the internal transcribed spacer region 1 (ITS1) of the ribosomal RNA (rRNA) gene complex of Hematodinium , we confirmed the presence of the parasite's DNA in water and suspended sediment samples collected from Virginia estuaries of the Delmarva Peninsula by polymerase chain reaction (PCR) on DNA extracted from these environmental samples. Amplicon sequences were highly conserved and 99–100% similar to the ITS1 sequence of the endemic Hematodinium sp. reported from blue crabs in the region. A sensitive real-time PCR assay was developed and validated for the detection and quantification of the parasite DNA. The assay has a limit of detection of less than one parasite cell in 100 ml of environmental water or one gram of sediment; it will provide a valuable tool for our ongoing studies on the transmission dynamics of this parasite. The real-time PCR assay was further tested on a free-living stage of Hematodinium . Sporulation resulting in the release of millions of Hematodinium dinospores from heavily diseased crabs was observed in the laboratory. Some dinospores survived up to 7 days in aquaria outside of the crab host. We speculate that the Hematodinium sp. dinospores have a short free-living stage in the water column and that transmission to new hosts must occur relatively quickly in estuarine habitats.

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global θST (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise θST comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid-Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR-RFLP analysis using cultures obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues' collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR-RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.

A molecular genetic identification key for nine species of Crassostrea oysters was developed based on restriction fragment-length polymorphism (RFLP) analyses of nuclear and mitochondrial DNA markers. Seven of nine species were unambiguously differentiated based on digestion of the ITS-1 nuclear marker with Hae III and Hinf I. Individual species exhibited one or two RFLP patterns for each restriction endonuclease, and only C. madrasensis and C. iredalei were indistinguishable electrophoretically. All nine species were unambiguously differentiated based on digestion of the COI mitochondrial marker with Dde I and Hae III. Species exhibited one or two RFLP patterns for each restriction endonuclease, and species pairs unresolved by the first restriction enzyme were completely resolved with the second. The resulting key distinguishes among many Indo-Pacific Crassostrea oysters that overlap across some or all of their ranges, and establish as an expandable framework for future additions of other species to the key.

Severe over-fishing, habitat degradation, and recent disease impacts have devastated the eastern oyster (Crassostrea virginica) fishery in the Chesapeake Bay. Several restoration efforts are in progress, including the unconventional approach of seeding reefs with an aquaculture strain selected for disease resistance and fast growth in hopes of mitigating the negative effects of disease and low census numbers. Supplementation of four sites (The Great Wicomico, Lynnhaven, York, and Elizabeth Rivers) examined in this study totaled approximately 18,500,000 aquaculture oysters from 2002 to 2006. We collected locally recruited offspring (n = 6517) from 2002 to 2006 at these sites to determine if reproduction by the transplanted oysters produced detectable contributions to recruitment by examining the frequency of a composite mitochondrial haplotype that occurs at high frequencies in this aquaculture strain but is rare in wild Chesapeake Bay oysters. The estimated frequency of this haplotype in locally ...

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR-RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.

Oysters were collected from coastal locations in China from 1999-2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction-based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2-8 microm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species-specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.