Fine-scale population structure of the northern hard clam (Mercenaria mercenaria) revealed by genome-wide SNP markers.

Aquaculture is growing rapidly worldwide, and sustainability is dependent on an understanding of current genetic variation and levels of connectivity among populations. Genetic data are essential to mitigate the genetic and ecological impacts of aquaculture on wild populations and guard against unintended human-induced loss of intraspecific diversity in aquacultured lines. Impacts of disregarding genetics can include loss of diversity within and between populations and disruption of local adaptation patterns, which can lead to a decrease in fitness. The northern hard clam, Mercenaria mercenaria (Linnaeus, 1758), is an economically valuable aquaculture species along the North American Atlantic and Gulf coasts. Hard clams have a pelagic larval phase that allows for dispersal, but the level of genetic connectivity among geographic areas is not well understood. To better inform the establishment of site-appropriate aquaculture brood stocks, this study used DArTseq™ genotyping by sequencing to characterize the genetic stock structure of wild clams sampled along the east coast of North America and document genetic diversity within populations. Samples were collected from 15 locations from Prince Edward Island, Canada, to South Carolina, USA. Stringent data filtering resulted in 4960 single nucleotide polymorphisms from 448 individuals. Five genetic breaks separating six genetically distinct populations were identified: Canada, Maine, Massachusetts, Mid-Atlantic, Chesapeake Bay, and the Carolinas (F ST 0.003-0.046; p < 0.0001). This is the first study to assess population genetic structure of this economically important hard clam along a large portion of its native range with high-resolution genomic markers, enabling identification of previously unrecognized population structure. Results of this study not only broaden insight into the factors shaping the current distribution of M. mercenaria but also reveal the genetic population dynamics of a species with a long pelagic larval dispersal period along the North American Atlantic and Gulf coasts.

Co-occurrence of marine and freshwater phycotoxins in oysters, and analysis of possible predictors for management.

Oysters (Crassostrea virginica) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 µg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 µm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R2 = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R2 = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety.

Mass spectrometric characterization of the seco acid formed by cleavage of the macrolide ring of the algal metabolite goniodomin A.

Goniodomin A (GDA) is a polyketide macrolide produced by multiple species of the marine dinoflagellate genus Alexandrium. GDA is unusual in that it undergoes cleavage of the ester linkage under mild conditions to give mixtures of seco acids (GDA-sa). Ring-opening occurs even in pure water although the rate of cleavage accelerates with increasing pH. The seco acids exist as a dynamic mixture of structural and stereo isomers which is only partially separable by chromatography. Freshly prepared seco acids show only end absorption in the UV spectrum but a gradual bathochromic change occurs, which is consistent with formation of α,ß-unsaturated ketones. Use of NMR and crystallography is precluded for structure elucidation. Nevertheless, structural assignments can be made by mass spectrometric techniques. Retro-Diels-Alder fragmentation has been of value for independently characterizing the head and tail regions of the seco acids. The chemical transformations of GDA revealed in the current studies help clarify observations made on laboratory cultures and in the natural environment. GDA has been found to reside mainly within the algal cells while the seco acids are mainly external with the transformation of GDA to the seco acids occurring largely outside the cells. This relationship, plus the fact that GDA is short-lived in growth medium whereas GDA-sa is long-lived, suggests that the toxicological properties of GDA-sa in its natural environment are more important for the survival of the Alexandrium spp. than those of GDA. The structural similarity of GDA-sa to that of monensin is noted. Monensin has strong antimicrobial properties, attributed to its ability to transport sodium ions across cell membranes. We propose that toxic properties of GDA may primarily be due to the ability of GDA-sa to mediate metal ion transport across cell membranes of predator organisms.

Natural transmission of Hematodinium perezi in juvenile blue crabs (Callinectes sapidus) in the laboratory.

Hematodinium perezi is a dinoflagellate endoparasitic in marine crustaceans, primarily decapods. It occurs in juvenile blue crabs, Callinectes sapidus, at high prevalence levels and has severe pathogenic effects in this host. The life history outside the host has not been experimentally investigated and, until now, transmission using dinospores has not been successful. We investigated the natural transmission dynamics of H. perezi in the laboratory using small juvenile crabs, which are highly susceptible to infection in the field, and elevated temperatures, which are known to stimulate dinospore production. Natural water-borne transmission to naïve crabs varied between 7 and 100% and was not correlated with dinospore densities measured from their aquaria water. Infections appeared to develop quickly in naïve hosts at 25 °C, suggesting that elevated temperatures as seen in the late summer and early autumn have a strong influence on the transmission of H. perezi in natural systems.

Vibrio vulnificus and Vibrio parahaemolyticus in Oysters under Low Tidal Range Conditions: Is Seawater Analysis Useful for Risk Assessment?

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.

Blooms of the harmful algae Margalefidinium polykrikoides and Alexandrium monilatum alter the York River Estuary microbiome.

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.

A Deterministic Model for Understanding Nonlinear Viral Dynamics in Oysters.

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. IMPORTANCE The fate of viruses in contaminated oysters has received a significant amount of attention in the fields of oyster aquaculture, food quality control, and public health. However, intensive studies through laboratory experiments and in situ observations are often conducted under a narrow range of experimental conditions and for a specific purpose in their respective fields. Given the complex interactions of various processes and nonlinear viral responses to changes in physiological and environmental conditions, a theoretical framework fully describing the viral dynamics in oysters is warranted to guide future studies from a top-down design. Here, we developed a process-based, in-host modeling framework that builds a bridge for better communications between different disciplines studying virus-oyster interactions.

Impact of parasitism on levels of human-pathogenic Vibrio species in eastern oysters.

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.

Alkali Metal- and Acid-Catalyzed Interconversion of Goniodomin A with Congeners B and C.

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,ß-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.

Developing a 3D mechanistic model for examining factors contributing to harmful blooms of Margalefidinium polykrikoides in a temperate estuary.

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.

Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters.

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.

Oyster hatchery breakthrough of two HABs and potential effects on larval eastern oysters (Crassostrea virginica).

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.

Revisiting the toxin profile of Alexandrium pseudogonyaulax; Formation of a desmethyl congener of goniodomin A.

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 Espiña 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.

Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants.

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.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

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.

The toxin goniodomin, produced by Alexandrium spp., is identical to goniodomin A.

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.

Algal Toxin Goniodomin A Binds Potassium Ion Selectively to Yield a Conformationally Altered Complex with Potential Biological Consequences.

The marine toxin goniodomin A (GDA) is a polycyclic macrolide containing a spiroacetal and three cyclic ethers as part of the macrocycle backbone. GDA is produced by three species of the Alexandrium genus of dinoflagellates, blooms of which are associated with "red tides", which are widely dispersed and can cause significant harm to marine life. The toxicity of GDA has been attributed to stabilization of the filamentous form of the actin group of structural proteins, but the structural basis for its binding is not known. Japanese workers, capitalizing on the assumed rigidity of the heavily substituted macrolide ring, assigned the relative configuration and conformation by relying on NMR coupling constants and NOEs; the absolute configuration was assigned by degradation to a fragment that was compared with synthetic material. We have confirmed the absolute structure and broad features of the conformation by X-ray crystallography but have found GDA to complex with alkali metal ions in spite of two of the heterocyclic rings facing outward. Such an arrangement would have been expected to impair the ability of GDA to form a crown-ether-type multidentate complex. GDA shows preference for K+, Rb+, and Cs+ over Li+ and Na+ in determinations of relative affinities by TLC on metal-ion-impregnated silica gel plates and by electrospray mass spectrometry. NMR studies employing the K+ complex of GDA, formed from potassium tetrakis[pentafluorophenyl]borate (KBArF20), reveal a major alteration of the conformation of the macrolide ring. These observations argue against the prior assumption of rigidity of the ring. Alterations in chemical shifts, coupling constants, and NOEs indicate the involvement of most of the molecule other than ring F. Molecular mechanics simulations suggest K+ forms a heptacoordinate complex involving OA, OB, OC, OD, OE, and the C-26 and C-27 hydroxy groups. We speculate that complexation of K+ with GDA electrostatically stabilizes the complex of GDA with filamentous actin in marine animals due to the protein being negatively charged at physiological pH. GDA may also cause potassium leakage through cell membranes. This study provides insight into the structural features and chemistry of GDA that may be responsible for significant ecological damage associated with the GDA-producing algal blooms.

High salinity relay as a post-harvest processing method for reducing Vibrio vulnificus levels in oysters (Crassostrea virginica).

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. vulnificus >10,000/g. As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the <30 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.

Phylogeography and connectivity of molluscan parasites: Perkinsus spp. in Panama and beyond.

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.

A novel monoclonal Perkinsus chesapeaki in vitro isolate from an Australian cockle, Anadara trapezia.

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.