Relationships between occurrences of toxic Dinophysis species (Dinophyceae) and small phytoplanktons in Japanese coastal waters

Fluctuations of the genus Dinophysis, which contained several toxic species of diarrhetic shellfish poisoning (DSP), were investigated during blooms in Hiroshima Bay, Mutsu Bay and Ise Bay, Japan. The co-occurrences of small phytoplanktons (cryptophytes, other nanophytoplanktons, cyanobacteria and eukaryotic picophytoplanktons) were investigated to search for relationships with mixotrophic Dinophysis. Cryptophytes were divided into three size-groups based on length of their chloroplasts (>10, 5–10 and <5 μm) during counting by epifluorescence microscopy. Clear relationships were not found between the occurrences of Dinophysis spp. and nanophytoplanktons, cyanobacteria and eukaryotic picophytoplanktons. However, the fluctuations of small-sized cryptophytes (<5 μm) showed a close relationship with that of D. acuminata in Hiroshima Bay. In Mutsu Bay, small-sized cryptophytes also accompanied the first occurrence peak of Dinophysis spp. In Ise Bay, peaks of the occurrences of middle- and small-sized cryptophytes were observed 2–3 weeks before the peak of D. acuminata. These cryptophytes decreased rapidly with increase in D. acuminata. These results suggest the possibility that small-sized cryptophytes may be food organisms for mixotrophic Dinophysis, with the abundance of Dinophysis dependent on these cryptophytes.     

Occurrence ofDinophysis spp. and toxic shellfish in the Northern Adriatic

The dynamics of the toxicity of the musselMytilus galloprovincialis was compared between two different shellfish farms, 5 km apart, but using the same cultivation technique. The main differences concerned the freshwater influx and the open aspect to the Gulf of Trieste. It is suggested that a deep closed bay and abundant fresh water inflow are the two main conditions for the low toxicity levels in mussels and for shorter periods of danger. A detailed study of the phytoplankton samples revealed the presence of eight species ofDinophysis in the area of both shellfish farms. During the period of the DSP outbreak in Slovenia (autumn and winter 1989).D. fortii andD. acuminata were the most frequentDinophysis species. There was a high positive correlation between the onset of mussel toxicity and the appearance ofDinophysis spp.     

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.     

Large subunit ribosomal RNA gene variation and sequence heterogeneity of Dinophysis (Dinophyceae) species from Scottish coastal waters

The purpose of the study was to investigate the genetic diversity of Dinophysis species from around the Scottish coast, with a view to an improved understanding of the dynamics and identification of this genus in Scottish waters. Single-cell PCR amplification with direct sequencing was performed on a total of 441 Dinophysis cells isolated from both live and Lugol's fixed plankton net samples. Universal eukaryotic primers were used to amplify the large subunit (LSU) ribosomal RNA (rRNA) gene of the Dinophysis isolates, with a frequency of PCR success of 26% for non-fixed and 48% for fixed samples. From this a total of 30 isolates were selected for this study and the D1–D2 region of the LSU-rRNA gene sequenced for phylogenetic analysis. No significant correlation could be made between geographical location and LSU sequence, although some regional sequence heterogeneity was observed within the Dinophysis acuta species. LSU sequence data was used to design Dinophysis genus specific and Dinophysis clade-specific primers primarily to ensure clean sequences from universal D1–D2 amplicons without a requirement for cloning. Three clade-specific primers designed to a region within the D2 hypervariable region of the LSU-rRNA gene allowed discrimination of Dinophysis acuminata/norvegica from Dinophysis tripos/caudata and Dinophysis fortii/acuta. In two isolates, SC359 (D. tripos) and LC58 (D. acuta), nested PCR products were observed with both the expected clade-specific primer, and Dasd-R2, the D. acuminata/norvegica clade-specific primer. Cloning and sequence analysis suggested that these amplicons were genuine “D. acuminata-like” sequences and their presence, albeit at a low frequency within different Dinophysis species, indicated that individual Dinophysis cells possess heterologous copies of the LSU-rRNA gene that are similar to LSU sequences normally associated with D. acuminata. The nature of the process that generated these hybrid cells, the frequency of such events and their importance is as yet unknown, but may provide a cautionary note for the development of PCR-based species specific detection methods.     

SMALL CELL AND INTERMEDIATE CELL FORMATION IN SPECIES OF DINOPHYSIS (DINOPHYCEAE, DINOPHYSIALES)

Observations of two distinct size classes with similar shape in natural populations of Dinophysis Ehrenberg were first reported by Jorgensen in 1923 and intermediate forms exhibiting a continuum between the typical vegetative cell and a putative small cell by Wood in 1954. Focused attention on Dinophysis spp. associated with diarrhetic shellfish intoxications in the last decade has provided new examples of small cells in the genus, sometimes with contours dissimilar from the corresponding vegetative cells; dimorphic individuals; and large/small cell couplets. This work was based on in situ observations during intensive sampling for cell cycle studies of Dinophysis acuminata Claparéde et Lachmann, Dinophysis acuta Ehrenberg, Dinophysis caudata Saville-Kent, and Dinophysis tripos Gourret; on laboratory incubations of D. acuminata; and on a thorough search of documented information on morphological variability of Dinophysis spp. During in situ division, most dividing cells exhibit a normal longitudinal fission, but some (1%–10%) undergo a “depauperating” fission, leading to pairs of dimorphic cells with dissimilar moieties. After separation and sulcal list regeneration, these dimorphic cells become D. skagii Paulsen, D. dens Pavillard, D. diegensis Kofoid, and D. diegensis Kofoid var. curvata-like individuals, which can also be observed forming couplets D. acuminata/D. skagii, D. acuta/D. dens, and D. caudata/D. diegensis attached by their ventral margins. Small cells can grow again to large size, as shown in laboratory incubations of D. acuminata, thus partly explaining observations of thecal intercalary bands, and intermediate forms. The sexual nature of the small cells will not be unequivocally demonstrated until controlled germination of the alleged cyst forms is achieved, and some intermediate forms may correspond to undescribed stages after cyst germination. These observations suggest common patterns in the life cycle of Dinophysis spp. Intraspecific morphological variability of Dinophysis spp. in a given geographic area can largely be attributed to small cell formation, as a response to changing environmental conditions, and may be a part of the sexual cycle of these species. Small cells seem to be able to enlarge, leading to intermediate cell and further vegetative cell formation as part of a three-looped life history pattern in Dinophysis.     

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.     

PHYLOGENETIC EVIDENCE FOR THE CRYPTOPHYTE ORIGIN OF THE PLASTID OF DINOPHYSIS (DINOPHYSIALES,DINOPHYCEAE)1

Photosynthetic members of the genus Dinophysis Ehrenberg contain a plastid of uncertain origin. Ultrastructure and pigment analyses suggest that the two‐membrane‐bound plastid of Dinophysis spp. has been acquired through endosymbiosis from a cryptophyte. However, these organisms do not survive in culture, raising the possibility that Dinophysis spp. have a transient kleptoplast. To test the origin and permanence of the plastid of Dinophysis, we sequenced plastid‐encoded psbA and small subunit rDNA from single‐cell isolates of D. acuminata Claparède et Lachman, D. acuta Ehrenberg, and D. norvegica Claparède et Lachman. Phylogenetic analyses confirm the cryptophyte origin of the plastid. Plastid sequences from different populations isolated at different times are monophyletic with robust support and show limited polymorphism. DNA sequencing also revealed plastid sequences of florideophyte origin, indicating that Dinophysis may be feeding on red algae.     

PREY SPECIFICITY AND FEEDING OF THE THECATE MIXOTROPHIC DINOFLAGELLATE FRAGILIDIUM DUPLOCAMPANAEFORME1

In summer to autumn of 2008, a recently described thecate mixotrophic dinoflagellate, Fragilidium duplocampanaeforme Nézan et Chomérat, occurred in Masan Bay, Korea, where it frequently contained bright‐orange fluorescent inclusions. Using cultures of F. duplocampanaeforme isolated from Masan Bay, we investigated feeding, digestion, and prey specificity of this mixotroph. F. duplocampanaeforme fed exclusively on Dinophysis spp. when offered a variety of prey including dinoflagellates, a raphidophyte, a cryptophyte, a ciliate, and diatoms separately. In addition, F. duplocampanaeforme had allelopathic effects on other organisms, including cell immobilization/motility decrease (in Dinophysis acuminata, D. caudata, D. fortii, D. infundibulus, Gonyaulax polygramma, Heterocapsa triquetra, and Prorocentrum triestinum), breaking of cell chains (in Cochlodinium polykrikoides), cell death (in Prorocentrum minimum), and temporary cyst formation (in Scrippsiella trochoidea). F. duplocampanaeforme engulfed whole Dinophysis cells through the sulcus. About 1 h after ingestion, F. duplocampanaeforme became immobile and shed all thecal plates. The ecdysal cyst persisted for ～7 h, during which the ingested prey was gradually digested. These observations suggest that F. duplocampanaeforme may play an important role in the Dinophysis population dynamics in the field.     

MOLECULAR STUDY OF THE TOXIC ALGAE DINOPHYSIS SPP. FROM THE FRENCH COAST

Dinoflagellates of the genus Dinophysis are agents of Diarrhetic Shellfish Poisoning (DSP). They occur along the French coast and affect shellfish exploitation during most of the year (during spring, summer and autumn). Because this species is difficult to cultivate, very little is known about this organism. The first problem is the species‐delineation within this genus which is sometimes unclear based upon the solely on morphological features, in particular for the complex D. acuminata (D. cf. acuminata,, D. cf. norvegica, D. cf.sacculus, and D. skagii) or the complex D. sacculus (D. sacculus and D. pavillardii). The second problem is its detection in natural samples. French Dinophysis blooms have been reported to be toxic under 100 cells L^?1, a concentration which corresponds to less than 1 cell 10‐mL^?1, as determined by the Utermöhl method of enumeration. Molecular tools may help to resolve these two kind of problems. During one year (spring 1999 to spring 2000), more than 100 fixed samples containing Dinophysis spp. cells were collected along the French coast by the French monitoring network (or REPHY; http://www. ifremer.fr). The genetic diversity of Dinophysis spp. was studied by sequencing and analysis of ribosomal DNA genes. We found that sequences were hightly conserved between species or within the D. acuminata or D. sacculus complex. Two oligonucleotide probes, specific to these complex groups, were designed. Their specificity and sensitivity are actually tested on natural samples by a PCR‐based assay. Furthur investigation will include the development of standard molecular diagnostics due to their rapid and sensitive detection in natural samples.     

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.     

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.     

Dinophysis norvegica (Dinophyceae), more a predator than a producer?

Several studies have proved that some Dinophysis species are capable of ingesting particulate organic matter besides of being photosynthetic, a form of nutrition termed mixotrophy. Phagotrophy may be an important aspect of the life history of the genus Dinophysis and the key to understand its ecology. We used modern techniques coupling flow cytometry and acidotropic probes to detect and score food vacuolated Dinophysis norvegica cells in natural samples. In addition, feeding experiments were conduced under controlled conditions to observe if D. norvegica would grow feeding on the cryptophyte Teleaulax amphioxeia. The results of the field observations showed a frequency of phagotrophy between 25 and 71% in a natural D. norvegica population from the Baltic Sea, which is higher than previous reports (1–20%). Although molecular methods have proved that the kleptoplastids of the D. norvegica from the Baltic Sea are from T. amphioxeia, the laboratory experiments showed that the presence of T. amphioxeia in the cultures did not enhance the survival rate of D. norvegica neither in phototrophic nor in heterotrophic conditions. We suggest that the D. norvegica Kleptoplats are obtained through a heterotrophic or mixotrophic protist, which have been feeding on cryptophytes, as it has recently been shown for Dinophysis acuminata. Our main conclusion is that D. norvegica, and probably all other species from the genus Dinophysis, is mainly phagotrophic and feeds on a larger prey than T. amphioxeia. Autotrophy through kleptoplastidy would be a secondary feature used as a complementary or short-term survival strategy.     

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Pectenotoxin and okadaic acid-based toxin profiles in Dinophysis acuta and Dinophysis acuminata from New Zealand

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

Real-time PCR Detection of Dinophysis Species in Irish Coastal Waters

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time polymerase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta, were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C, while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugol's preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by light microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.     

Diarrheic toxins in field-sampled and cultivated Dinophysis spp. cells from southern Brazil

In southern Brazil, mixotrophic dinoflagellates belonging to the Dinophysis acuminata complex have recently been involved in diarrheic shellfish poisoning episodes through the production of lipophilic toxins such as okadaic acid (OA) and dinophysistoxin-1 (DTX-1). The present investigation used a combination of laboratory cultures and field surveys at three large estuarine systems in that region to examine toxin retention in Dinophysis spp. cells under optimum or growth-limiting conditions. This study represents the first successful culture of a Dinophysis isolate from the Atlantic South America region. Starved D. acuminata complex cells reached 5.6-fold higher cellular OA quotas (up to 18 pg cell^?1) than Mesodinium rubrum-fed cultures 20 days after inoculation in the laboratory. Moreover, in field samples, light-limited cells at the bottom of a stratified water column were less abundant, yet 6.6- to 11-fold more toxic (up to 26.4 pg OA and 1.7 pg DTX-1 cell^?1) than those located at the illuminated surface. This phenomenon of toxin retention by slow-dividing cells may partially explain the enormous variation in cell toxin quota found within Dinophysis spp. populations from a single location, and it may have serious implications for cell count-based monitoring program in bivalve aquaculture areas. In fact, only low to moderate OA levels were detected in the digestive glands of oysters Crassostrea spp. (up to 17.8 ng g^?1) and the guts and livers of filter-feeding fish (44.7 ng g^?1) during the present study, despite the relatively high Dinophysis cell densities (up to 19,500 cells L^?1) found in the field.     

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.     

Are the mitochondrial cox1 and cob genes suitable markers for species of Dinophysis Ehrenberg?

The identification of Dinophysis species with similar morphology but different toxic (Diarrhetic Shellfish Poisoning, DSP) potential is a crucial task in harmful algae monitoring programmes. The taxonomic assignment of Dinophysis species using molecular markers is a difficult task due to extremely low interspecific variability within their nuclear ribosomal genes and intergenic regions. Mitochondrial cox1 gene has been proposed as a better specific marker for Dinophysis species based on its higher resolution for two morphologically related species (Dinophysis acuminata and Dinophysis ovum) of the “Dinophysis acuminata complex”. In this study, the potential of two mitochondrial genes (mt cox1 and cob) to discriminate among six Dinophysis species (field isolates and cultures) associated with DSP events was explored. Neither mt cox1 nor cob genes provided enough resolution for all species of Dinophysis. The cob gene showed very poor resolution and grouped all Dinophysis spp. in a common clade. In contrast, the cox1 phylogeny distinguished 5 clades in the Dinophysiales – the “acuminata complex”, the “caudata group”, “acuta + norvegica” and Phalacromaspp. However, within the “D. acuminata complex” mtcox1 is so far the unique marker that differentiates D. acuminata from other species: isolates of D. ovum and Dinophysis sacculus had almost identical sequences (only four mismatches), but they were well separated from D. acuminata. D. acuminata and Dinophysis skagii (considered a life cycle stage of the former) showed identical cox1 sequences. Probes towards this gene can be useful in Mediterranean and Western Iberia sites where the co-occurrence of close morphotypes of D. acuminata and D. sacculus pose a problem for monitoring analyses. This is the first report on cultures of D. sacculus and its phylogenetic relation with other species of the D. acuminata complex.