The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

The combined effect of salinity and temperature on the growth and toxin content of four Chilean strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated from an outbreak occurring in southern Chile in 2009

The toxic dinoflagellate Alexandrium catenella has been detected in the southern Chile since 1972, causing severe negative impacts on public health and aquaculture activities. Several environmental factors have been determined to affect growth and toxin production in Alexandrium strains. The aim of this study was to determine the effect of four combined conditions of two temperatures (10 and 15 °C) and two salinities (15 and 35 psu) on the growth and the Paralytic Shellfish Poisoning (PSP) toxin content and composition in four Chilean strains of A. catenella (PFB41, PFB42, PFB37 and PFB38), isolated during a summer outbreak occurred in southern Chile in 2009. The growth curves showed a higher effect of the salinity in strains PFB41 and PFB42 than in strains PFB37 and PFB38. The values of growth rates and maximum cell densities ranged from 0.25 to 0.73 div day⁻¹ and 1.1 × 10⁴ to 5.2 × 10⁴ cells mL⁻¹, respectively. All of the strains showed the highest values for both growth parameters at 15 °C and 35 psu. In general, growth parameters were higher at 35 psu independently of the temperature. On the other hand, the total PSP toxin content ranged widely from 3.99 to 239 fmol cell⁻¹. The highest values of PSP toxin content were attained at 10 °C and 35 psu for all of the strains, at both stages of growth. All of the strains displayed different toxin compositions, with neoSTX, GTX4-1, GTX3-2 and GTX5 being the main toxins detected. The results showed significant differences in the absolute values of growth and toxin production parameters among the strains grown under the same culture conditions, and for each strain grown under different combined conditions of temperature and salinity. These findings demonstrate that abiotic factors can differentially affect the population dynamics of the A. catenella toxic genotypes, thus making it extremely difficult to predict the ecological behavior of this species in the field in terms of the intensity of a potential outbreak.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Co-occurrence of Dinophysis tripos and pectenotoxins in Argentinean shelf waters

The species Dinophysis tripos is a widely distributed marine dinoflagellate associated with diarrheic shellfish poisoning (DSP) events, which has been recently identified as a pectenotoxin (PTX) producer. In two sampling expeditions carried out during austral autumns 2012 and 2013 along the Argentine Sea (≈38–56° S), lipophilic phycotoxins were measured by tandem mass spectrometry coupled to liquid chromatography (LC–MS/MS) in size-fractionated plankton samples together with microscopic analyses of potentially toxic phytoplankton. PTX-2, PTX-11 and PTX-2sa were recurrently detected in the 50–200 μm fractions, in association to D. tripos. PTX-2 was also widely distributed among the 20–50 μm fractions, mostly related to Dinophysis acuminata. Okadaic acid or its analogs were not detected in any sample. This is the first report of D. tripos related to PTX in the Argentine Sea and the first record of PTX-11 and PTX-2sa for this area. The morphological variability of D. tripos, including the presence of intermediate, small and dimorphic cells, is described. Also, the micro- and mesoplanktonic potential grazers of Dinophysis spp. were explored.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Estimating the contribution of N-sulfocarbamoyl paralytic shellfish toxin analogs GTX6 and C3 + 4 to the toxicity of mussels (Mytilus galloprovincialis) over a bloom of Gymnodinium catenatum

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3 + 4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3 + 4 and GTX6 and their contribution to shellfish toxicity. C3 + 4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63 × 10³ cells l⁻¹) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1 + 2 were clearly the dominant compounds, followed by C3 + 4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1 + 2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1 + 2, C3 + 4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3 + 4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3 + 4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.     

A shift in the dominant toxin-producing algal species in central California alters phycotoxins in food webs

In California, the toxic algal species of primary concern are the dinoflagellate Alexandrium catenella and members of the pennate diatom genus Pseudo-nitzschia, both producers of potent neurotoxins that are capable of sickening and killing marine life and humans. During the summer of 2004 in Monterey Bay, we observed a change in the taxonomic structure of the phytoplankton community—the typically diatom-dominated community shifted to a red tide, dinoflagellate-dominated community. Here we use a 6-year time series (2000–2006) to show how the abundance of the dominant harmful algal bloom (HAB) species in the Bay up to that point, Pseudo-nitzschia, significantly declined during the dinoflagellate-dominated interval, while two genera of toxic dinoflagellates, Alexandrium and Dinophysis, became the predominant toxin producers. This change represents a shift from a genus of toxin producers that typically dominates the community during a toxic bloom, to HAB taxa that are generally only minor components of the community in a toxic event. This change in the local HAB species was also reflected in the toxins present in higher trophic levels. Despite the small contribution of A. catenella to the overall phytoplankton community, the increase in the presence of this species in Monterey Bay was associated with an increase in the presence of paralytic shellfish poisoning (PSP) toxins in sentinel shellfish and clupeoid fish. This report provides the first evidence that PSP toxins are present in California's pelagic food web, as PSP toxins were detected in both northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax). Another interesting observation from our data is the co-occurrence of DA and PSP toxins in both planktivorous fish and sentinel shellfish. We also provide evidence, based on the statewide biotoxin monitoring program, that this increase in the frequency and abundance of PSP events related to A. catenella occurred not just in Monterey Bay, but also in other coastal regions of California. Our results demonstrate that changes in the taxonomic structure of the phytoplankton community influences the nature of the algal toxins that move through local food webs and also emphasizes the importance of monitoring for the full suite of toxic algae, rather than just one genus or species.     

Toxic mucus traps: A novel mechanism that mediates prey uptake in the mixotrophic dinoflagellate Alexandrium pseudogonyaulax

The functional role of harmful substances (i.e. toxins) produced by marine planktonic algae is still, in many cases, unknown. This study describes a novel mechanism by which the phototrophic dinoflagellate Alexandrium pseudogonyaulax secretes a toxic mucus trap where prey items are caught and immobilized prior to ingestion. Prey cells remain entrapped and immobile in the mucus trap, but most stay intact, readily available as whole-cell prey. It is shown that food uptake by A. pseudogonyaulax increases its growth rate considerably even in nutrient-replete, high-light conditions. The increase in growth rate was more enhanced in light-limited treatments and A. pseudogonyaulax grew significantly faster when fed Heterocapsa rotundata, than when fed Teleaulax acuta under both light conditions. For comparison, strains of Alexandrium catenella and Alexandrium minutum were studied for their mixotrophic capabilities. None of these strains were mixotrophic under the conditions provided. In addition, the toxic effects on various protistan targets of these Alexandrium strains as well as Alexandrium tamarense and Alexandrium ostenfeldii were compared to that of A. pseudogonyaulax. A. tamarense and A. catenella did immobilize and lyse target cells through substances leaked directly into the water, differing from all the strains of A. pseudogonyaulax studied. Results show that the toxic effect of A. pseudogonyaulax is non-specific causing nearly 100% immobilization of a variety of protistan targets at relatively low cell concentrations (500 cells ml⁻¹ of donor cell). A critical donor cell density was not required as only one A. pseudogonyaulax cell was able to cause immobilization of target cells. For the first time, the connection between excreted toxins and phagotrophy is evident in an Alexandrium species and this particular strategy has the potential to severely impact competing phytoplankton communities.     

Production of dinophysistoxin-1 and pectenotoxin-2 by a culture of Dinophysis acuminata (Dinophyceae)

The production of diarrhetic shellfish poisoning toxins (okadaic acid analogues and other lipophilic toxins) by a culture of Dinophysis acuminata, fed with the autotrophic ciliate Myrionecta rubra, was confirmed by LC–MS analysis, and the toxin profile compared with that in the field assemblage of the same species. The growth response of D. acuminata to the density of the food organism was also examined in laboratory experiments. In semi-continuous culture experiments, the growth rates of D. acuminata increased with increasing density of M. rubra and a maximum growth rate of 0.67 per day was calculated. In batch culture experiments; the cellular content of PTX2 and DTX1 were 14.7–14.8 and 2.5–4.8 pg cell^?1, respectively. Okadaic acid, dinophysistoxin-3, pectenotoxin-1, pectenotoxin-6, yessotoxin (YTX) and 45-OHYTX were not detected. PTX2 was detected (cellular toxin content: 22 pg cell^?1), but DTX1 was not detected, in an extract of D. acuminata collected from natural seawater at the same location where the cultured D. acuminata specimens were isolated. These results strongly suggest that D. acuminata produces these toxins during cell growth and that environmental factors influence variations in the toxin composition and specific cellular toxicity.     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon,southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

Which species,Alexandrium catenella (Group I) or A. pacificum (Group IV), is really responsible for past paralytic shellfish poisoning outbreaks in Jinhae-Masan Bay,Korea?

Paralytic shellfish poisoning (PSP) caused the deaths of four people in coastal area of Korea, mainly Jinhae-Masan Bay and adjacent areas, in April 1986 and in 1996. The PSP outbreaks were caused by the consumption of mussels, Mytilus edulis. The organism that caused PSP was identified, from morphological data only, as Alexandrium tamarense which is recently renamed as A. catenella, however recent studies have shown that the morphological diagnostic characteristics used to identify Alexandrium species have uncertainties and molecular tools and other criteria should be considered as well. The organism that caused past PSP outbreaks and incidents in Korea therefore need to be carefully reconsidered. The aim of this study was to re-evaluate the species really responsible for past outbreaks of PSP in Jinhae-Masan Bay, Korea. The temporal production and fluxes of the resting cysts of Alexandrium species were investigated for one year (from March 2011 to February 2012) using a sediment trap, and the morphology and phylogeny of vegetative cells germinated from the resting cysts were analysed. The production of Alexandrium species peaked in August and November, when temporal discrepancies were found in the water temperature (22.4 and 22.7 °C in August, 19.1 and 19.6 °C in November) and salinity (29.5 and 26.1 psu in August, 30.5 and 31.8 psu in November). The morphological data revealed that Alexandrium species germinated from resting cysts collected in August have a ventral pore on the 1′ plate, whereas the 1′ plate in Alexandrium species germinated from resting cysts collected in November lacks a ventral pore. Molecular phylogenetic data for the vegetative cells from the germination experiments allowed the August and November peaks to be assigned to Alexandrium catenella (Group I) and A. pacificum (Group IV), respectively. This indicates that the production of resting cysts of A. catenella can be enhanced by relatively high water temperature. This result is not consistent with those of previous studies that A. catenella responsible for PSP outbreaks was found at relatively low water temperature. In addition, large subunit ribosomal sequences data revealed that A. pacificum isolates from Korea were closely related to those from Australia, Japan and New Zealand where the PSP toxicity of shellfish and blooms occurred in the 1990s, indicating that the introduction of toxic dinoflagellates were related to ballast water from bulk-cargo shipping. Based on these results, we concluded that past PSP outbreaks in Jinhae-Masan Bay of Korea could have been caused by A. pacificum rather than by A. catenella.     

Spatial distribution of toxic Alexandrium tamiyavanichii (Dinophyceae) in the southeastern South China Sea-Sulu Sea: A molecular-based assessment using real-time quantitative PCR (qPCR) assay

In this study, a quantitative real-time PCR (qPCR) assay targeting the second internal transcribed spacer (ITS2) of the nuclear-encoded ribosomal RNA gene (rDNA) was developed for Alexandrium tamiyavanichii, a harmful tropical marine dinoflagellate. This species is of concern because it produces toxins that cause paralytic shellfish poisoning (PSP). The qPCR assay employed hydrolysis probe technology and showed high specificity, with a detection limit of 10² gene copies (less than one cell equivalent). Using this assay, the spatial distribution of A. tamiyavanichii was assessed, for the first time, in the southeastern South China Sea and the Sulu Sea. Plankton samples were collected from 71 stations during a scientific cruise from the Research Vessel Sonne as part of the joint EU project on Stratosphere ozone: Halogens in a Varying Atmosphere (SHIVA), conducted in November 2011. The highest cell densities were detected offshore of Kuching, southern Borneo (150 cells l⁻¹) and exceeded the threshold level of 20–40 cells l⁻¹ where the bioaccumulation of PSP toxins by shellfish is of concern. The distribution of A. tamiyavanichii was patchy horizontally with the highest cell concentrations found mainly offshore of southern Borneo, and a heterogeneous vertical distribution was observed above the pycnocline. The A. tamiyavanichii qPCR assay proved its applicability, specificity and sensitivity, and provides an alternative implementation tool for harmful microalgae monitoring programs.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Interaction between toxic dinoflagellate Alexandrium catenella exposure and disease associated with herpesvirus OsHV-1 μVar in Pacific oyster spat Crassostrea gigas

Blooms of toxic dinoflagellates can co-occur with mass mortality events associated with herpesvirus OsHV-1 μVar infection that have been decimating Pacific oyster Crassostrea gigas spat and juveniles every summer since 2008 in France. This study investigated the possible effect of a harmful dinoflagellate, Alexandrium catenella, a producer of Paralytic Shellfish Toxins (PSTs), upon the oyster spat–herpesvirus interaction. Oyster spat from a hatchery were challenged by cohabitation with oysters contaminated in the field with OsHV-1 μVar and possibly other pathogens. Simultaneously, the oysters were exposed to cultured A. catenella. Infection with OsHV-1 μVar and PST accumulation were measured after 4 days of experimental exposure.Exposure to Alexandrium catenella modified the host–pathogen interaction by reducing prevalence of OsHV-1 μVar infection. In addition, oysters challenged with OsHV-1 μVar and possibly other pathogens from the environment accumulated smaller amounts of PSTs than unchallenged oysters. Three possible mechanisms are suggested by these results: (i) possible direct interactions between A. catenella and herpesvirus (or associated pathogens) could reduce viral transmission and algal availability for oyster consumption; (ii) oyster feeding behavior or digestive functions may have been altered, thus decreasing both uptake of viral particles and consumption or digestion of toxic algae and consequent toxin accumulation; (iii) immuno-activation by A. catenella could enhance defense efficiency against OsHV-1 μVar infection. These findings suggest further research on relationships between OsHV-1 μVar and toxic dinoflagellates and their combined effects upon disease transmission and proliferation processes, as well as on oyster physiological and immunological involvement in this complex, tripartite interaction.     

Environmental control of harmful dinoflagellates and diatoms in a fjordic system

Fjordic coastlines provide an ideal protected environment for both finfish and shellfish aquaculture operations. This study reports the results of a cruise to the Scottish Clyde Sea, and associated fjordic sea lochs, that coincided with blooms of the diarrhetic shellfish toxin producing dinoflagellate Dinophysis acuta and the diatom genus Chaetoceros, that can generate finfish mortalities. Unusually, D. acuta reached one order of magnitude higher cell abundance in the water column (2840 cells L⁻¹) than the more common Dinophysis acuminata (200 cells L⁻¹) and was linked with elevated shellfish toxicity (maximum 601 ± 237 μg OA eq/kg shellfish flesh) which caused shellfish harvesting closures in the region. Significant correlations between D. acuta abundance and that of Mesodinium rubrum were also observed across the cruise transect potentially supporting bloom formation of the mixotrophic D. acuta. Significant spatial variability in phytoplankton that was related to physical characteristics of the water column was observed, with a temperature-driven frontal region at the mouth of Loch Fyne being important in the development of the D. acuta, but not the Chaetoceros bloom. The front also provided important protection to the aquaculture located within the loch, with neither of the blooms encroaching within it. Analysis based on a particle-tracking model confirms the importance of the front to cell transport and shows significant inter-annual differences in advection within the region, that are important to the harmful algal bloom risk therein.     

Toxin production,retention, and extracellular release by Dinophysis acuminata during extended stationary phase and culture decline

Dinophysis spp. produce diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins. The extent to which the dinoflagellate cells retain their toxicity in stationary phase, a period when cells are most toxic, and their transition into cell death is not known. Here we present results on the production, recycling, retention, and release of toxins from a monoculture of Dinophysis acuminata during these two important stages. Once stationary phase was reached, cultures were divided between light and dark treatments to identify if light influenced toxin dynamics. Light was required for long-term cell maintenance (>2 months) of D. acuminata in the absence of prey, however, in the dark, cells in stationary phase survived on reserves alone for four weeks before beginning to decline. Cells maintained relatively constant levels of intracellular OA (0.39 ± 0.03 pg/cell, 0.44 ± 0.05 pg/cell), DTX1 (0.45 ± 0.09 pg/cell, 0.64 ± 0.10 pg/cell) and PTX2 (10.4 ± 1.4 pg/cell, 11.0 ± 1.9 pg/cell) in the dark and light treatments, respectively, throughout stationary phase and into culture decline. Toxin production was only apparent during late exponential and early stationary growth when cells were actively dividing. In general, the concentration of dissolved (extracellular) toxin in the medium significantly increased upon culture aging and decline; cells did not appear to be actively or passively releasing toxin during stationary phase, but rather extracellular release was likely a result of cell death. Light availability did not have an apparent effect on toxin production, quotas, or intracellular vs. extracellular distribution. Together these results suggest that a bloom of D. acuminata would retain its cellular toxicity or potency as long as the population is viable, and that cells under conditions of low light (e.g., at the boundary or below euphotic zone) and/or minimal prey could maintain toxicity for extended periods.     

DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species

Alexandrium species can be very difficult to identify, with A. catenella, A. tamarense, and A. fundyense that compose “Alexandrium tamarense species complex” (Atama complex) as a distinct example. DNA barcoding is promising to offer a solution but remains to be established. In this study, we examined the utility of ITS in resolving the Atama species complex, by analyzing previously studied strains plus unstudied Chinese strains within the LSU- and SSU-rDNA based group/clade frameworks recently established. We further investigated the presence of intragenomic polymorphism and its implications in species delimitation. Similar to the previous SSU and LSU results, our ITS-based phylogenies divided the complex to five clusters, but with longer and evener branch lengths between the clusters. Based on the ITS region, the inter-cluster genetic distances (p = 0.134–0.216) were consistently and substantially greater than intra-cluster genetic distances (p = 0.000–0.066), with an average inter-cluster (species) distance (p = 0.167) 7.6-fold of the average intraspecific difference (p = 0.022), qualifying the approximately 510–520 bp ITS as a DNA barcode for Atama complex. We detected varying levels of intragenomic polymorphism in ITS but found that this did not impact the taxon-resolving power of this gene. With this DNA barcode, the new East and South China Sea strains and one Antarctic strain were placed in Clade IIC/Group IV, even though there were 7–10 polymorphic sites in their ITS, in contrast to none in SSU. Furthermore, our results suggest that the five clusters are recognizable as distinct species according to the phylogenetic species concept. Based on the phylogenetic placements of the type-locality strains of the existing three morphospecies and the dominant localities of other strains, we propose that Group I/Clade I be designated as A. fundyense, Group III/Clade IIB as A. tamarense, Group IV/Clade IIC as A. catenella, Group II/Clade IIA as A. mediterranis, and Group V/Clade IID as A. australis.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.