THE FINE STRUCTURE OF TWO PHOTOSYNTHETIC SPECIES OF DINOPHYSIS (DINOPHYSIALES,DINOPHYCEAE)1

Two closely related, photosynthetic species belonging to the genus Dinophysis were examined, D. acuminata Claparède et Lachmann and D. fortii Pavillard. Typical dinoflagellate features include the amphiesmal covering enclosing the cells and the structure of the nucleus and mitochondria. Many other characteristics seem to be specific to the order Dinophysiales. Many rhabdosomes are present, and complex mucocysts are found beneath the amphiesma. The thecal pores are unusual with the base of the pore occluded by a thin disc that is continuous with the main amphiesmal plate. The structure of the apical pore is also distinctive. Chloroplasts are grouped together in chromatospheres, enclosed by a double membrane, and contain paired thylakoids with electron dense contents in the lumen. The two pusules are extensive, each branching off the flagellar canal, and consisting of a large antechamber and a number of convoluted sacs. The entrance of each antechamber, and site of an emerging flagellum, is surrounded by a striated fibrous collar. Near the flagellar pore is a prominent microtubular/microbody complex which penetrates deep into the cell cytoplasm. Consideration is given to taxonomic position of the Dinophysiales and also to the nature and origins of the chloroplasts.     

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.     

Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.     

Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark?

In situ growth rates of the toxin-producing dinoflagellate Dinophysis norvegica collected in the central Baltic Sea were estimated during the summers of 1998 and 1999. Flow cytometric measurements of the DNA cell cycle of D. norvegica yielded specific growth rates (μ) ranging between 0.1 and 0.4 per day, with the highest growth rates in stratified populations situated at 15–20 m depth. Carbon uptake rates, measured using ¹⁴C incubations followed by single-cell isolation, at irradiances corresponding to depths of maximum cell abundance were sufficient to sustain growth rates of 0.1–0.2 per day. The reason for D. norvegica accumulation in the thermocline, commonly at 15–20 m depth, is thus enigmatic. Comparison of depth distributions of cells with nutrient profiles suggests that one reason could be to sequester nutrients. Measurements of single-cell nutrient status of D. norvegica, using nuclear microanalysis, revealed severe deficiency of both nitrogen and phosphorus as compared to the Redfield ratio.It is also possible that suitable prey or substrate for mixotrophic feeding is accumulating in the thermocline. The fraction of cells containing digestive vacuoles ranged from 2 to 22% in the studied populations. Infection by the parasitic dinoflagellate Amoebophrya sp. was observed in D. norvegica in all samples analysed. The frequency of infected cells ranged from 1 to 3% of the population as diel averages, ranging from 0.2 to 6% between individual samples. No temporal trends in infection frequency were detected. Estimated loss rates based on observed infection frequencies were 0.5–2% of the D. norvegica population daily, suggesting that these parasites were not a major loss factor for D. norvegica during the periods of study.     

PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE,DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES1

Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

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.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Genetic analyses of Dinophysis spp. support kleptoplastidy

The question of whether the toxin-producing and bloom-forming dinoflagellate genus Dinophysis contains plastids that are permanent or contains temporary so-called kleptoplastids is still unresolved. We sequenced plastid 16S rRNA gene, the complete trnA gene and the intergenic transcribed spacer region located between the trnA gene and the 23S rRNA gene, and performed diagnostic PCR on cells of the genus Dinophysis. Dinophysis spp. were collected from five different geographical regions: the Baltic Sea, the North Sea, the Greenland Sea and the Norwegian fjord Masfjorden. In most cases the sequence analysis showed that the sequences were identical to each other and to sequences from the cryptophyte Teleaulax amphioxeia SCCAP K0434, regardless of the place of sampling or the species analyzed. The exception was some cells of Dinophysis spp. from the Greenland Sea. These contained a 16S rRNA gene sequence that was more closely related to the cryptophyte Geminigera cryophila. The cells of Dinophysis contained either one of the 16S rRNA gene sequences or both in the same cell. Our results challenge the hypothesis that the plastids in Dinophysis are permanent and suggest that they are more likely to be kleptoplastids.     

A Dinoflagellate Amylax triacantha with Plastids of the Cryptophyte Origin: Phylogeny,Feeding Mechanism,and Growth and Grazing Responses

The gonyaulacalean dinoflagellates Amylax spp. were recently found to contain plastids of the cryptophyte origin, more specifically of Teleaulax amphioxeia. However, not only how the dinoflagellates get the plastids of the cryptophyte origin is unknown but also their ecophysiology, including growth and feeding responses as functions of both light and prey concentration, remain unknown. Here, we report the establishment of Amylax triacantha in culture, its feeding mechanism, and its growth rate using the ciliate prey Mesodinium rubrum (= Myrionecta rubra) in light and dark, and growth and grazing responses to prey concentration and light intensity. The strain established in culture in this study was assigned to A. triacantha, based on morphological characteristics (particularly, a prominent apical horn and three antapical spines) and nuclear SSU and LSU rDNA sequences. Amylax triacantha grew well in laboratory culture when supplied with the marine mixotrophic ciliate M. rubrum as prey, reaching densities of over 7.5 × 10³ cells/ml. Amylax triacantha captured its prey using a tow filament, and then ingested the whole prey by direct engulfment through the sulcus. The dinoflagellate was able to grow heterotrophically in the dark, but the growth rate was approximately two times lower than in the light. Although mixotrophic growth rates of A. triacantha increased sharply with mean prey concentrations, with maximum growth rate being 0.68/d, phototrophic growth (i.e. growth in the absence of prey) was ?0.08/d. The maximum ingestion rate was 2.54 ng C/Amylax/d (5.9 cells/Amylax/d). Growth rate also increased with increasing light intensity, but the effect was evident only when prey was supplied. Increased growth with increasing light intensity was accompanied by a corresponding increase in ingestion. In mixed cultures of two predators, A. triacantha and Dinophysis acuminata, with M. rubrum as prey, A. triacantha outgrew D. acuminata due to its approximately three times higher growth rate, suggesting that it can outcompete D. acuminata. Our results would help better understand the ecophysiology of dinoflagellates retaining foreign plastids.