A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.     

Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.     

Okadaic acid accumulation in macrofilter feeders subjected to natural blooms of Dinophysis acuminata

Thermaikos Gulf is a eutrophic area located in the Northwestern part of the Aegean Sea in the Eastern Mediterranean. Interspecific differences among various filter feeders in their ability to accumulate okadaic acid, were observed during natural blooms of Dinophysis acuminata in the gulf. Okadaic acid analyses by high performance liquid chromatography (HPLC) were performed on benthic specimens and D. acuminata cell densities and cell toxin content were estimated in water samples. Seven filter feeding species were collected in the gulf during two DSP outbreaks in May 2003 and March 2004. The various species showed a different potential to accumulate okadaic acid in their tissues. The highest concentrations were found in the mussel populations (Mytilus galloprovincialis and Modiolus barbatus), while among the non-bivalve filter feeders, ascidians were the main accumulators of okadaic acid. The rest of shellfish populations (Flexopecten proteus, Chlamys varia and Venus verrucosa) were found to contain toxins only during 2004, when D. acuminata densities were found above 10000 cells l⁻¹. M. galloprovincialis was proved to be the most appropriate indicator for a safe warning of okadaic acid contamination in Thermaikos Gulf.     

Contribution to toxicity assessment of <Emphasis Type="BoldItalic">Dinophysis acuminata</Emphasis> (Dinophyceae)

Blooms of Dinophysis in French coastal waters are implicated in most bans on marketing commercial bivalves. However, the relation between Dinophysis cell density and shellfish toxicity is not always consistent. Discrepancies may be due to the simple fact that it is nearly impossible to compare an integral over a few days (shellfish toxin content) and water samples. Furthermore, it seems that cells may have a variable specific toxicity. This work focuses on the variability in cell toxicity taking into account recent findings and using liquid chromatography coupled to mass spectrometry with an ion trap and electrospray interface. Esterified analogues of okadaic acid (DTX-4 and diol-esters) have been identified in cultures of Prorocentrum lima, another okadaic acid producer. These analogues are inactive on some protein phosphatases, contrary to okadaic acid, and seem to protect the cell from harmful effects by the toxin and to be enzymatically hydrolyzed during cell lysis. In order to document specific toxicity and to validate the presence of these analogues, D. acuminata concentrates were subjected to two separate heating and freeze/thaw procedures, respectively inhibiting or promoting hydrolysis. This paper reports on the high variability of D. acuminata specific toxicity and the presence of esters found in half of the samples only.     

Occurrence of okadaic acid,a major diarrheic shellfish toxin,in natural populations ofDinophysis spp. from the eastern coast of North America

Okadaic acid, one of the principal toxin components implicated in cases of diarrheic shellfish poisoning (DSP), was identified for the first time in natural phytoplankton assemblages from North American waters. During periods in late summer when significant quantities of okadaic acid were detected in net haul samples in the lower estuary and Gulf of St Lawrence in eastern Canada, the phytoplankton community consistently contained species of the dinoflagellate genusDinophysis Ehrenberg. The presence of okadaic acid was detected by screening dinoflagellate extracts with an enzyme-linked immunological assay (ELISA); positive results were confirmed by reverse-phase high-performance liquid chromatography (HPLC) separation, followed by fluorescence detection. Okadaic acid was only found in phytoplankton samples in which the photosynthetic dinophysoid speciesD. norvegica andD. acuminata were prominent; blooms of the related heterotrophic speciesD. rotundata exhibited no trace of okadaic acid, nor other suspected DSP components.     

First report of a new rapid assay for diarrhetic shellfish poisoning toxins

Jellett Rapid Testing Ltd. has developed a rapid field test kit to screen for diarrhetic shellfish poisoning (DSP) toxins. The new test provides a qualitative (positive/negative) indication of the presence of okadaic acid (OA) and some of its analogues in about 30 min. It is designed as a screening method for regulatory labs to eliminate negative samples, thereby leaving a smaller number of positive samples to be tested with more sophisticated and time-consuming quantitative methods. Due to its simplicity and speed, the Rapid Test for DSP may eventually be used in other applications such as shellfish harvest management and toxin research. The test is based on easy-to-use lateral flow immunochromatographic (LFI) test strips, which operate the same way as Jellett Rapid Testing's Rapid Tests for paralytic shellfish poisoning (PSP) toxins and amnesic shellfish poisoning (ASP) toxins. The sensitivity of the antibodies to some of the analogues of the DSP family of toxins was investigated using pure compounds from the National Research Council of Canada. In the Rapid Test format, okadaic acid, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2) were detected similarly with 50% reduction in test line color intensity at 5 nM for the solutions applied to test strips. One of the DTX-3 esters eliminated the test line at 500 nM, indicating low cross-reactivity, whereas no effect was observed with one of the brevetoxins (PbTx-3), yessotoxin, gymnodimine, spirolide and pectenotoxins PTX2, PTX11, at concentrations up to 1000 nM. In the ELISA format, the distinction between analogues was more apparent than on test strips. Mid-points were at 8 nM for okadaic acid, and 40 nM and 25 nM for DTX1 and DTX2, respectively.     

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.     

DIFFERENCES IN THE PRODUCTION AND EXCRETION KINETICS OF OKADAIC ACID,DINOPHYSISTOXIN‐1, AND PECTENOTOXIN‐2 BETWEEN CULTURES OF DINOPHYSIS ACUMINATA AND DINOPHYSIS FORTII ISOLATED FROM WESTERN JAPAN1

We established clonal cultures of Dinophysis acuminata Clap. et Lachm. and D. fortii Pavill. isolated from western Japan and examined toxin production in them, focusing on intracellular production and extracellular excretion. At the end of incubations, the total amounts of pectenotoxin‐2 (PTX‐2), dinophysistoxin‐1 (DTX‐1), and okadaic acid (OA) in the D. acuminata cultures reached up to 672.7 ± 14.7 (mean ± SD), 88.1 ± 2.8, and 539.3 ± 39.7 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 5.1, 79.5, and 79.5% of the total amounts, respectively. Similarly, at the end of incubations, the total amounts of PTX‐2, DTX‐1, and OA in the D. fortii cultures reached up to 526.6 ± 52.6 (mean ±SD), 4.4 ± 0.4, and 135.9 ± 3.9 ng · mL^?1, respectively, and the excreted extracellular amounts were equivalent to 1.8, 80.1, and 86.6% of the total amounts, respectively. Further, we tested the availability of cell debris and dissolved organic substances that originated from the ciliate prey Myrionecta rubra for growth and toxin production in D. acuminata. Although no significant growth was observed in D. acuminata in the medium containing the cell debris and organic substances originated from M. rubra, the toxicity was significantly greater than that in the control (P < 0.05–0.001); this finding suggested the availability of organic substances for toxin production. However, toxin productivity was remarkably lower than that of Dinophysis species feeding on living M. rubra.     

Artificial neural network approaches to one-step weekly prediction of Dinophysis acuminata blooms in Huelva (Western Andalucía, Spain)

On the Atlantic coasts of Andalucía, chronic spring–summer (March–June) diarrhetic shellfish poisoning (DSP) outbreaks are associated with blooms of Dinophysis acuminata, Claparède and Lachmann. Artificial neural networks (ANNs) have been successfully used to model primary production and have recently been tested for the prediction of harmful algae blooms. In this study, we evaluated the performance of feed forward ANN models trained to predict D. acuminata blooms. ANN models were trained and tested using weekly data (5 previous weeks) of D. acuminata cell counts from eight stations of the Andalucía HAB monitoring programme in the coasts of Huelva between 1998 and 2004. Principal component analysis (PCA) were previously carried out to find out possible similarities within time series from each zone with the aim of reducing the number of areas to model. Our results show that ANN models with a low number of input variables are able to reproduce trends in D. acuminata population dynamics.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Phytoplankton composition of the Kandalaksha Gulf, Russian White Sea: Dinophysis and lipophilic toxins in the blue mussel (Mytilus edulis)

Dinophysis acuminata and D. norvegica were observed in plankton net samples during the summer of 2002 from the Kandalaksha Gulf in the White Sea (North European Russia). Prorocentrum lima was found as an epiphyte on subtidal macroalgae in August, but not observed in plankton net samples. Protein phosphatase 2A (PP2A) inhibition measured 127.8 ng OA-equivalent/g of mussel (Mytilus edulis) hepatopancreas from samples collected a few days after when Dinophysis was recorded at a density of 1550 cells L⁻¹. Liquid chromatography–mass spectrometry confirmed presence of several classes of lipophilic shellfish toxins associated with Dinophysis spp. in the mussels including okadaic acid, dinophysistoxin-1, pectenotoxins and yessotoxins. No azaspiracid was detected. This represents the first identification of phycotoxicity in the White Sea.     

Occurrence of Prorocentrum lima,a DSP toxin-producing species from the Atlantic coast of Canada

A species of Prorocentrum (Dinophyta, Prorocentrales), isolated from a phytoplankton net sample from the Atlantic coast of Nova Scotia, has been brought into unialgal culture. The sample was collected at an aquaculture site immediately following an incident of diarrhetic shellfish poisoning (DSP) due to the consumption of contaminated mussels. This clonal isolate has been identified as P. lima, based on its morphological characteristics. Analysis of the culture extract, using high performance liquid chromatography (HPLC) with fluorescence detection, indicated the presence of the DSP toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1).     

Prorocentrum lima (Dinophyceae) in northeastern USA coastal waters: II: Toxin load in the epibiota and in shellfish

The seasonal variation in diarrhetic shellfish poisoning (DSP)-type toxins was followed in the epibiotic community and in shellfish between 41° and 44°N in coastal waters of the northwest Atlantic during a 2-year period. Low levels of okadaic-acid equivalents were detected at all stations in the <90 μm fraction of the collected epibiota as measured by the protein phosphatase inhibition assay, but only 3.5% of the samples had values greater than 100 ng (g dry weight of epibiota)⁻¹. No seasonal pattern could be detected due to differences in intensity, duration and timing of toxin content in the epibiota between the 2 years and between stations. Nevertheless, the concentration of DSP-type toxins in the epibiota correlated weakly but significantly with the abundance of Prorocentrum lima, when data from all stations were considered. A very limited toxin uptake by shellfish was measured at only one station in October and November 2001 and in June and July 2002 at times of maximum cell concentration of P. lima in the epibiota. Toxin levels in shellfish remained well below regulatory limits that would have required quarantine or bans on harvesting. Results from our 2-year survey suggest that, at this time, the threat of DSP events appears minimal. However, the presence of a known toxin producer and its demonstrated ingestion by shellfish would argue for further studies to better understand conditions leading to DSP outbreaks generated by an epiphytic dinoflagellate.     

Diversity and plastid types in Dinophysis acuminata complex (Dinophyceae) in Scottish waters

Dinophysis acuminata produces lipophilic shellfish toxins (LSTs) that have economic and ecological impact on marine invertebrates in NE Atlantic where aquaculture farming is prevalent. Identification of D. acuminata can be complex. Cells exhibit a variety of morphotypes that overlap between species making identification using routine light microscopy difficult. These cells are mixotrophic and their population size is influenced by hydrographic conditions and prey populations. Dinophysis cells are able to acquire and temporarily keep prey plastids from a variety of photosynthetic unicellular sources. The Dinophysis community in Scottish waters tend to be dominated by cells with morphologies that appear to be variants of D. acuminata/norvegica complex particularly during late spring/early summer. To determine the identity of these morphotypes, DNA barcoding was performed on 32 single cell isolates from sites around the Scottish coast using the ribosomal internal transcribed spacer 1 (ITS1) and a partial cytochrome oxidase I (COI) fragment on the same single cells. Although the cells exhibited a variety of morphotypes, most were restricted to one cluster containing D. acuminata and three grouped with Dinophysis ovum. This is the first molecular confirmation of the presence of D. ovum in Scottish waters. Two isolates showed considerable divergence – one was unidentifiable from the public databases, whilst the other matched a Dinophysis cf. acuta isolate from Canada. To investigate prey plastids, molecular analysis of these Dinophysis single cells was conducted with a partial fragment of the plastid ribosomal marker (16S). Most cells harboured plastids from the cryptophyte Teleaulax – the most commonly reported plastid type, however one cell harboured a Rhodomonas/Storeatula derived plastid. This finding increases the range and variety of cryptophyte plastids found in Dinophysis and increases the range of prey-types.     

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.     

Toxicity of Dinophysis spp. in relation to population density and environmental conditions on the Swedish west coast

The aim of this study in the field was to investigate whether there are differences between the outer archipelago (Gullmar Fjord) and a semi-enclosed fjord system (Koljö Fjord) in occurrences of D. acuta and D. acuminata as well as in their content of diarrheic shellfish toxin (DST) per cell. When all data pairs of cell toxicity of D. acuminata and the corresponding number of cells l⁻¹ from the two sites were tested in a regression analysis, a statistically significant negative correlation became evident and was apparent as a straight line on a log–log plot (p < 0.0001). Obviously, there was an overall inverse relationship between the population density of D. acuminata and the toxin content per cell. Plotted on a linear scale, all data-pairs of cell toxicity and cell number made up a parabolic curve. On this curve the data-pairs could be separated into three groups: (i) D. acuminata occurring in numbers of fewer than approximately 100 cells l⁻¹, and with a toxin content per cell above 5 ρg cell⁻¹; (ii) cell numbers between 100 and approximately 250 cells l⁻¹ with a cell toxin content from 5 to 2 ρg cell⁻¹; (iii) when the population became greater than 250 cells l⁻¹, the toxicity, with few exceptions, was less than 2 ρg cell⁻¹. By applying this subdivision, some clear patterns of the distribution of the differently toxic D. acuminata became evident. When comparing the cell toxicity of the two sites, it was obvious that the D. acuminata cells from all depths from the Gullmar Fjord as a mean were significantly more toxic compared to the Koljö Fjord samples. The results have demonstrated that approximately 100 high-toxicity cells in a low-density population at surface may lead to the same accumulation of DST in a mussel as the ingestion of 1500 low-toxicity cells from a high-density pycnocline population.     

DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

For decades, many aspects of Dinophysis biology have remained intractable due to our inability to maintain these organisms in laboratory cultures. Recent breakthroughs in culture methods have opened the door for detailed investigations of these important algae. Here, for the first time, we demonstrate toxin production in cultures of North American Dinophysis acuminata, isolated from Woods Hole, MA. These findings show that, despite the rarity of Dinophysis-related DSP events in North America, D. acuminata from this area has the ability to produce DSP toxins just as it does in other parts of the world where this species is a major cause of DSP toxicity. In our cultures, D. acuminata cells were observed feeding on Myrionecta rubra using a peduncle. Culture extracts were analyzed using LC–MS/MS, providing unequivocal evidence for the toxin DTX1 in the Dinophysis cultures. In addition, a significant amount of an okadaic acid diol ester, OA-D8, was detected. These results suggest that this Dinophysis isolate stores much of its OA as a diol ester. Also, toxin PTX-2 and a hydroxylated PTX-2 with identical fragmentation mass spectrum to that of PTX-11, but with a different retention time, were detected in this D. acuminata culture. This demonstration of toxin production in cultured North American Dinophysis sets the stage for more detailed studies investigating the causes of geographic differences in toxicity. It is now clear that North American Dinophysis have the ability to produce DSP toxins even though they only rarely cause toxic DSP events in nature. This may reflect environmental conditions that might induce or repress toxin production, genetic differences that cause modifications in toxin gene expression, or physiological and biochemical differences in prey species.     

Microsatellite and mitochondrial DNA analyses of the genetic structure of blacktip shark (Carcharhinus limbatus) nurseries in the northwestern Atlantic, Gulf of Mexico, and Caribbean Sea

Abstract We investigated the genetic structure of blacktip shark (Carcharhinus limbatus) continental nurseries in the northwestern Atlantic Ocean, Gulf of Mexico, and Caribbean Sea using mitochondrial DNA control region sequences and eight nuclear microsatellite loci scored in neonate and young-of-the-year sharks. Significant structure was detected with both markers among nine nurseries (mitochondrial PhiST = 0.350, P < 0.001; nuclear PhiST = 0.007, P < 0.001) and sharks from the northwestern Atlantic, eastern Gulf of Mexico, western Gulf of Mexico, northern Yucatan, and Belize possessed significantly different mitochondrial DNA haplotype frequencies. Microsatellite differentiation was limited to comparisons involving northern Yucatan and Belize sharks with nuclear genetic homogeneity throughout the eastern Gulf of Mexico, western Gulf of Mexico, and northwestern Atlantic. Differences in the magnitude of maternal vs. biparental genetic differentiation support female philopatry to northwestern Atlantic, Gulf of Mexico, and Caribbean Sea natal nursery regions with higher levels of male-mediated gene flow. Philopatry has produced multiple reproductive stocks of this commercially important shark species throughout the range of this study.