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Highlights: [•] The pfiesteriacean dinoflagellate Luciella masanensis was found to be a fish killer. [•] Pfiesteria shumwayae and Luciella masanensis caused massive fish kills in two RAS (Recirculation Aquaculture Systems) fish farms in Denmark. [•] Pfiesteria shumwayae and Luciella masanensis showed considerable similarity in details of the food uptake process to the freshwater species Tyrannodinium edax. [•] Based on the similarity in feeding, it is possible and likely that several more species of the Pfiesteriaceae are fish killers. [•] Pfiesteriaceans are difficult to identify using light microscopy, and they are almost certainly misidentified during monitoring. [Copyright &y& Elsevier]

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future. [Copyright &y& Elsevier]

On the basis of morphological (light and electron microscopy) as well molecular data, we show that the widely distributed freshwater dinoflagellate presently known as Peridiniopsis berolinensis is a member of the family Pfiesteriaceae, an otherwise marine and estuarine family of dinoflagellates. P. berolinensis is a close relative of the marine species, which it resembles in morphology, mode of swimming, food-uptake mechanism, and partial LSU rRNA sequences. It differs from all known genera of the family in plate tabulation. P. berolinensis is only distantly related to the type species of Peridiniopsis, P. borgei, and is therefore transferred to the new genus Tyrannodinium as T. berolinense comb. nov. T. berolinense is a very common freshwater flagellate that feeds vigorously on other protists and is able to consume injured metazoans much larger than itself. Production of toxins has not been reported. [ABSTRACT FROM AUTHOR]

Conventional methods to identify the fish-killing dinoflagellate Karlodinium veneficum rely on rDNA sequence and scanning electron microscopy analyses that are not suitable for processing a large number of samples. To overcome this difficulty, SYTO9 and TaqMan format real-time PCR probes were developed for the internal transcribed spacer rDNA region for rapid detection and quantification of K. veneficum in the marine environment. Assay specificity, sensitivity, and accuracy were confirmed by testing against related organisms, sequencing PCR amplicons from field samples, and comparing real-time PCR results with microscopic counts. The SYTO9-based assay produced highly reproducible DNA melting curves over a broad range of starting DNA template. When the SYTO9- and TaqMan-based assays were compared in the quantitative measurements, they showed comparable results and reasonable reproducibility. The K. veneficum-specific assays were used in conjunction with Pseudopfiesteria shumwayae (Dinophyceae)-, Pfiesteria piscicida (Dinophyceae)-, and Cryptoperidiniopsis brodyi (Dinophyceae)-specific real-time PCR probes to investigate temporal changes in abundances of these four species in Chinhae Bay, South Korea. The field survey revealed that K. veneficum was dominant (maximum 970 cells ml21) among those species; whereas, Pfiesteria species rarely occurred. These findings suggest that the SYTO9- and TaqMan-based assays are specific and sensitive for detecting and quantifying K. veneficum in the environment. The wide distribution of K. veneficum also indicates potential fish kills by this organism in Chinhae Bay. [ABSTRACT FROM AUTHOR]

A semi-defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae. Maximum cell yields and division rates in the semi-defined medium ranged from 0.1 × 105 to 4.0 × 105 cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi-defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae, needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate. [ABSTRACT FROM AUTHOR]

Pfiesteria shumwayae Glasgow et J. M. Burkh. [= Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (104 · mL−1) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements. [ABSTRACT FROM AUTHOR]

Based on morphological and molecular data, calcareous dinoflagellates (Thoracosphaeraceae, Peridiniales) are a monophyletic group comprising the three major clades Ensiculifera/ Pentapharsodinium, Thoracosphaera/ Pfiesteria, and Scrippsiella sensu lato. We used stratigraphically well-documented first occurrences of particular archeopyle types to constrain relaxed Bayesian molecular clocks applied to nuclear rRNA sequences of 18 representatives of the three main clades. By comparing divergence estimates obtained in differently calibrated clocks with first stratigraphic occurrences of taxa not themselves used as constraints, we identified plausible divergence times for several subclades of calcareous dinoflagellates. The initial diversification of extant calcareous dinoflagellates probably took place in the Late Jurassic, with the three main clades all established by the Cretaceous. The two mesoepicystal operculum types observed in calcareous dinoflagellates probably evolved independently from simple apical archeopyles. Based on our taxon sample, the K/T boundary had relatively little effect on the diversity of the group, with several lineages dating to before 65 mya (million years ago). The first stratigraphic occurrences of key taxa, such as Thoracosphaera and Calciodinellum (not themselves used as constraints), are in agreement with the molecular time estimates. Conflicts that involve “ Calciodinellum” levantinum, Leonella, Pentapharsodinium, Pernambugia, and the Scrippsiella trochoidea species complex may be due to inaccurate assignment of fossils because of high morphological homoplasy and insufficient knowledge of the extant diversity of calcareous dinoflagellates. [ABSTRACT FROM AUTHOR]

Abstract: Cryptoperidiniopsis brodyi is a common heterotrophic dinoflagellate known to often co-occur with Pfiesteria species in eastern U.S. estuaries. In this study, C. brodyi from Australia and Pfiesteria piscicida from ballast water from Indonesia were characterized by morphological and genetic analyses. Two P. piscicida strains originating from ballast water samples showed little genetic differences compared to P. piscicida from other countries and their morphology was identical. This finding indicates a potential inflow of P. piscicida into Australian estuaries via ballast water. Nine cultures of C. brodyi were established from Tasmania, South Australia and Western Australia. All C. brodyi cultures exhibited identical thecal plate patterns and could not be discriminated from other non-Australian strains. In contrast, two distinct genotypes could be identified by rDNA sequence analyses which were distinct from the U.S. genotype of C. brodyi. A previous survey using PCR-based methods reported a wide distribution of Pfiesteria shumwayae in Australia. However, the present study demonstrated that SSU rDNA-based P. shumwayae-specific primers produce false-positive PCR reactions with Australian C. brodyi. These results suggest that genetic variants of C. brodyi are widely distributed in Australia and Australian genotypes of C. brodyi had previously been misidentified as P. shumwayae. This finding also indicates that previous Australian distribution studies of P. shumwayae using SSU rDNA-based primers are potentially erroneous and need to be revisited. [Copyright &y& Elsevier]

To explore the feeding ecology of the Pfiesteria-like dinoflagellate (PLD) Luciella masanensis (GenBank Accession no. AM050344, previously Lucy), we investigated the feeding behavior and the kinds of prey species that L. masanensis fed on and determined its growth and ingestion rates of L. masanensis when it fed on the dinoflagellate Amphidinium carterae and an unidentified cryptophyte species (equivalent spherical diam., ESD=5.6 μm), which were the dominant phototrophic species when L. masanensis and similar small heterotrophic dinoflagellates were abundant in Masan Bay, Korea in 2005. Additionally, these parameters were also measured for L. masanensis fed on blood cells of the perch Lateolabrax japonicus and the raphidophyte Heterosigma akashiwo in the laboratory. Luciella masanensis fed on prey cells by using a peduncle after anchoring the prey with tow filament, and was able to feed on diverse prey such as cryptophytes, raphidophytes, diatoms, mixotrophic dinoflagellates, and the blood cells of fish and humans. Among the prey species tested in the present study, perch blood cells were observed to be the optimal prey for L. masanensis. Specific growth rates of L. masanensis feeding on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte, either increased continuously or became saturated with increasing the mean prey concentration. The maximum specific growth rate of L. masanensis feeding on perch blood cells (1.46/day) was much greater than that of A. carterae (0.59/day), the cryptophyte (0.24/day), or H. akashiwo (0.20/day). The maximum ingestion rate of L. masanensis on perch blood cells (2.6 ng C/grazer/day) was also much higher than that of A. carterae (0.32 ng C/grazer/day), the cryptophyte (0.44 ng C/grazer/day), or H. akashiwo (0.16 ng C/grazer/day). The kinds of prey species which L. masanensis is able to feed on were the same as those of Pfiesteria piscicida, but very different from those of another PLD Stoeckeria algicida. However, the maximum growth and ingestion rates of L. masanensis on perch blood cells, A. carterae, H. akashiwo, and the cryptophyte were considerably lower than those of P. piscicida. Therefore, these three dinoflagellates may occupy different ecological niches in marine planktonic communities, even though they have a similar size and shape and the same feeding mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: Researchers examining the mechanisms of ichthyotoxicity of Pfiesteria shumwayae have come to different conclusions about the role of toxin in this process. Some attribute fish mortality solely to direct attack by these pedunculate dinoflagellates on exposed fish tissue while others have provided evidence for a role of a soluble toxin. Detection of toxin, especially in low concentrations, is a function of the sensitivity of the selected bioassay methods and the various groups addressing this question have utilized different methods. One notable difference in fish bioassay methods utilized to detect Pfiesteria-associated toxin (PfTx) is the species of fish tested. Studies that have not detected PfTx in bioassays generally have used Cyprinodon variegatus (sheepshead minnow) as the test fish while those that have detected toxin generally used Oreochromis spp. (Tilapia). In this study response of these two fish species was compared to determine their relative sensitivity to physical attack by P. shumwayae and to PfTx. The results indicate that Oreochromis niloticus is more susceptible to P. shumwayae and its associated toxin than C. variegatus and implicate differences in the ability these species to osmoregulate as a contributing factor for this phenomenon. Salinity stress enhanced susceptibility of O. niloticus to PfTx and thus improved the sensitivity of the bioassay. The observation that salinity stress enhances toxicity to O. niloticus provides additional information regarding the mechanism of PfTx toxicity although the conditions utilized are not representative of the natural habitat of these freshwater fish. [Copyright &y& Elsevier]

Abstract: The dinoflagellate species originally described as Pfiesteria shumwayae Glasgow et Burkholder, recently transferred to a new genus, Pseudopfiesteria Litaker et al., is reclassified into the redefined genus Pfiesteria Steidinger et Burkholder, as Pfiesteria shumwayae within the order Peridiniales. This change is based upon consideration of a compilation of previous and new morphological analyses and molecular phylogenetic analyses. Morphological analysis with scanning and transmission electron microscopy supports previous findings except in the sulcal area. In the cells examined, the sulcus is partly concealed by the peduncle cover plate (p.c.), which originates at the right side of the sulcus along the left side of the 6c and 5‴ plates. The fine structure of the p.c. appears similar to that of other thecal plates. The 1″ plate can also extend slightly over the sulcus. Transmission electron microscopy revealed that Pfiesteria shumwayae can have at least six sulcal plates; the number remains uncertain and may vary. The sulcal plates of this small, delicately thecate species have not been clearly discerned by scanning electron microscopy of membrane-stripped and/or suture-swollen cells. The Kofoidian thecal plate formula for the genus Pfiesteria is Po, cp, X, 4′, la, 5–6″, 6c, p.c., ?s, 5‴, 0p, 2‴′. The monophyletic grouping of “pfiesteria-like” taxa within the order Peridiniales, as well as the grouping of Pfiesteria piscicida and Pfiesteria shumwayae within the same genus, is also supported by the preponderance of previous molecular evidence, and by the phylogenetic trees contributed in the present analysis. Pfiesteria appears to be closely related to as-yet informally described cryptoperidiniopsoids and calcareous dinoflagellates such as Thoracosphaera; thus, the family classification requires revision that is beyond the scope of this study. [Copyright &y& Elsevier]

Abstract: A semi-idealized marine ecosystem model, designed as a heuristic tool for exploring the population dynamics of non-inducible versus toxic forms of Pfiesteria is described. The model is based on empirical evidence suggesting that these differing functional types of Pfiesteria also differ substantially in terms of what they eat and how they utilize it to optimize their growth. Non-inducible strains are similar to other mixotrophic dinoflagellates, whereas toxic strains may consume organic matter and detritus, produce toxins and attack fish. In our model formulation we represent these differences in a simplified way: the non-inducible strain is kleptochloroplastidic and it can take up DIN, but it cannot utilize DON, whereas the toxic strain is heterotrophic, it cannot utilize DIN, but it can utilize DON directly. These differences give rise to very different impacts on prey and nutrient concentrations in our model. Under high DIN/DON ratio conditions, the non-inducible cells grew much faster and were therefore more likely to bloom, but this advantage is substantially mitigated when the DIN/DON ratio is low. A turbulence parameterization was also incorporated into our model. The effect of this was to reduce the grazing rate of Pfiesteria when turbulence levels are high. According to our model, increased turbulence is more detrimental to the toxic functional type because it grows more slowly. The further imposition of microzooplankton grazing in the model showed that top-down control effects can be very significant, which is consistent with both laboratory and field studies and the general idea that plankton blooms can only happen in the absence of substantial grazing control. In general, our model results suggest that non-toxic blooms are more likely to occur in more turbulent inorganic-nutrient rich conditions, which are often found in more open coastal and estuarine waters that are subject to high inorganic loading. In contrast, toxic blooms are more likely to occur in calm, organic-nutrient rich conditions, which are often found in shallow, protected tributaries that are subject to high organic nutrient loading. Our model results also support the idea that the absence of strong grazing pressure is a prerequisite to bloom formation for both non-inducible and toxic strains of Pfiesteria. These results are generally consistent with observed patterns of toxic Pfiesteria blooms in Chesapeake Bay, the Neuse River of North Carolina and many other coastal and estuarine environments. [Copyright &y& Elsevier]

Abstract: The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function. [Copyright &y& Elsevier]

Abstract: Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species. [Copyright &y& Elsevier]

Abstract: The toxic dinoflagellate, Pfiesteria piscicida, is a common constituent of the phytoplankton community in the Delaware Inland Bays, USA. In this study, molecular methods were used to investigate the distributions of benthic stages (cysts) of P. piscicida in sediment cores from the Delaware Inland Bays. Cores from 35 sites were partitioned into nephloid and anoxic layers and analyzed for P. piscicida by nested amplification of the 18S rDNA gene using P. piscicida-specific primers. The presence of inhibitory substances in the PCR reaction was evaluated by inclusion of an exogenous control DNA in the extraction buffer, thus eliminating samples that may yield false-negative results. Our results indicate a patchy distribution of P. piscicida in sediments of the Delaware Inland Bays, with distinct differences between each of the three bays. Overall, P. piscicida was found more frequently in sediments from Rehoboth Bay compared to Indian River and Little Assawoman Bays. These differences suggest (i) that populations of P. piscicida may be more widely distributed in Rehoboth Bay, (ii) that populations of P. piscicida may have been introduced to Rehoboth Bay at an earlier time, (iii) that past blooms of P. piscicida in Rehoboth Bay estuaries may have seeded the sediments with higher numbers of cysts, and/or (iv) that Rehoboth Bay sediments may be more resistant to clearing due to storm turbulence. [Copyright &y& Elsevier]

A new genus and species of heterotrophic dinoflagellate, Cryptoperidiniopsis brodyi gen. et sp. nov., are described. This new species commonly occurs in estuaries from Florida to Maryland, and is often associated with Pfiesteria piscicida Steidinger et Burkholder, Pseudopfiesteria shumwayae (Glasgow et Burkholder) Litaker et al., and Karlodinium veneficum (Ballantine) J. Larsen, as well as other small (<20 μm) heterotrophic and mixotrophic dinoflagellates. C. brodyi gen. et sp. nov. feeds myzocytotically on pigmented microalgae and other microorganisms. The genus and species have the enhanced Kofoidian plate formula of Po, cp, X, 5′, 0a, 6″, 6c, PC, 5+s, 5″′, 0p, and 2″″ and are assigned to the order Peridiniales and the family Pfiesteriaceae. Because the Pfiesteriaceae comprise small species and are difficult to differentiate by light microscopy, C. brodyi gen. et sp. nov. can be easily misidentified. [ABSTRACT FROM AUTHOR]

BACKGROUND: Exposure to the dinoflagellate Pfiesteria has, under certain circumstances, been associated with deficits in human learning and memory. However, uncertainties remain about the health risk of chronic, low-level exposures (as seen among occupationally exposed commercial fishermen), particularly in light of studies suggesting that Pfiesteria strains are widespread in the estuarine environment in the U.S. mid-Atlantic region. METHODS: We selected an initial cohort of 152 persons, including 123 persons with regular, occupational exposure to the Chesapeake Bay; 107 of the cohort members were followed for the full four summer ‘seasons’ of the study. Cohort members were questioned biweekly about symptoms, and data were collected about the areas of the bay in which they worked. These latter data were matched with data on the presence or absence of Pfiesteria in each area, based on polymerase chain reaction analysis of > 3,500 water samples. Cohort members underwent neuropsychological testing at the beginning and end of each summer season. RESULTS: No correlation was found between work in an area where Pfiesteria was identified and specific symptomatology or changes on neuropsychological tests. CONCLUSIONS: Although high-level or outbreak-associated exposure to Pfiesteria species (or specific strains within a species) may have an effect on health, routine occupational exposure to estuarine environments in which these organisms are present does not appear to pose a significant health risk. [ABSTRACT FROM AUTHOR]

Information on the abundance of Pfiesteria piscicida in the natural environment is needed for understanding the ecological roles of this dinoflagellate. In this study, a real-time polymerase chain reaction (PCR) assay was developed using mitochondrial cytochrome b upstream region and 18S rDNA (PpmtDNA and Pp18S), and the geographic and temporal distribution of P. piscicida was investigated in several locations. Both PpmtDNA and Pp18S generally gave similar results, indicating that P. piscicida was present at all studied regions along the American coast (from Maine to North Carolina along the US Atlantic coast and Los Lagos along the Chilean Pacific coast). Despite its widespread distribution, P. piscicida was only detected in 36% of the 431 water samples analyzed, and its abundance was generally low (<1.0–1.5 cells mL−1). Populations detected at the five stations in the Neuse River (North Carolina) and two stations in Chesapeake Bay (Maryland) were genetically homogeneous, whereas those from other locations appeared to be genetically diverse. It can be concluded that (i) the PpmtDNA–Pp18S real-time PCR assay is sensitive and specific for detecting and quantifying P. piscicida in the natural environment and (ii) P. piscicida is widespread along the American coasts, but normally only as a minor component of plankton even in the high-risk estuaries (Neuse River, Chesapeake Bay). [ABSTRACT FROM AUTHOR]

Abstract: Eight novel pfiesteria-similar dinoflagellates are described that form a genetically well-defined group with distinct morphological features, and are capable of feeding myzocytotically on cryptophytes and other algae. Taxonomically, there was concordance between the genetic analysis and plate tabulation features among these taxa. It is suggested that these dinoflagellates are important grazers of other phytoplankton in the environment, and their total ecological significance has been underestimated. [Copyright &y& Elsevier]

Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and 18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5'-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the US. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml-1), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of F. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay). [ABSTRACT FROM AUTHOR]

Abstract: The phylogenetic relationships of calcareous dinoflagellates (i.e., Calciodinellaceae and Thoracosphaera) are investigated. Molecular data from the ribosomal 5.8S rRNA and highly conserved motifs of the ITS1 show Calciodinellaceae s.l. to be monophyletic when few non-calcareous taxa are included. They segregate into three monophyletic assemblages in a molecular analysis that considers the 5.8S rRNA and both the Internal Transcribed Spacer regions ITS1 and ITS2: a clade comprising species of Ensiculifera and Pentapharsodinium (E/P-clade), Scrippsiella s.l. (including fossil-based taxa such as Calciodinellum and Calcigonellum), and a heterogeneous group (T/P-clade) of calcareous (e.g., Thoracosphaera) and non-calcareous taxa (e.g., the highly toxic Pfiesteria). The potential to produce calcareous structures is considered as apomorphic within alveolates, and non-calcareous taxa nesting with calcareous dinoflagellates may have reduced calcification secondarily. Molecular results do not contradict general evolutionary scenarios provided by previous morphological (mainly paleontological) investigations. [Copyright &y& Elsevier]

Pfiesteria shumwayaeGlasgow et Burkholder is assigned to a new genusPseudopfiesteriagen. nov. Plate tabulation differences betweenPfiesteriaandPseudopfiesteriagen. nov. as well as a maximum likelihood phylogenetic analysis based on rDNA sequence data warrant creation of this new genus. The Kofoidian thecal plate formula for the new genus is Po, cp, X, 4′, 1a, 6′′, 6c, PC, 5+s, 5′′′, 0p, 2′′′′. In addition to having six precingular plates,P. shumwayaecomb. nov. also has a distinctive diamond or rectangular-shaped anterior intercalary plate. BothPfiesteriaandPseudopfiesteriagen. nov. are reassigned to the order Peridiniales based on an apical pore complex (APC) with a canal (X) plate that contacts a symmetrical 1′, four to five sulcal plates, and the conservative hypothecal tabulation of 5′′′, 0p, and 2′′′′. These morphological characters and the life histories ofPfiesteriaandPseudopfiesteriaare consistent with placement of both genera in the Peridiniales. Based on the plate tabulations forP.shumwayae, P. piscicida, and the closely related“cryptoperidiniopsoid” and“lucy” groups, the family Pfiesteriaceae is amended to include species with the following tabulation: 4-5′, 0-2a, 5-6′′, 6c, PC, 5+s, 5′′′, 0p, and 2′′′′ as well as an APC containing a pore plate (Po), a closing plate (cp), and an X plate; the tabulation is expanded to increase the number of sulcal plates and to include a new plate, the peduncle cover (PC) plate. Members of the family have typical dinoflagellate life cycles characterized by a biflagellated free-living motile stage, a varying number of cyst stages, and the absence of multiple amoeboid stages. [ABSTRACT FROM AUTHOR]

Evaluates mRNA detection capabilities for identification of viable cysts of the dinoflagellate, Pfiesteria piscicida, in natural sediment samples. Transcripts for cytochrome c oxidase subunit 1 as targets; Expression confirmed in laboratory cultures and compared with natural sediment samples; Indication that methods targeting mRNA transcripts may be useful for detection of viable cysts in natural sediment samples.

Abstract: The mechanism by which Pfiesteria shumwayae (Glasgow and Burkholder) kills fish is controversial. Several studies have implicated a Pfiesteria-associated exotoxin in fish mortality while other studies indicate that physical attack of dinoflagellates on fish (micropredation) and not exotoxin is responsible. We examined the ichthyotoxicity of two strains of P. shumwayae (CAAE 101272 and CCMP 2089) in a bioassay system that exposed test fish to the dinoflagellates both with and without direct contact in the same aquarium at the same time. Dinoflagellate-free supernatants from both strains were also tested for toxicity. The results showed that direct contact between P. shumwayae and fish significantly enhanced fish mortality with both strains (P < 0.001). About 87.5% and 100% of fish died when exposed directly to CAAE 101272 and CCMP 2089, respectively. When protected from direct contact with Pfiesteria cells, 19% of the fish exposed to CAAE 101272 and 6% of those exposed to CCMP 2089 died. No deaths were observed in controls. Supernatant killed fish when obtained from cultures of CAAE 101272 but not when obtained from CCMP 2089. Analysis of variance showed that, for both strains, fish mortality in Pfiesteria-inoculated bioassays was significantly higher than control bioassays both with and without direct contact (P < 0.001). Differences between strains were not significant (P = 0.3). These results indicate that both strains are associated with exotoxin production. However, the dominant and most consistent mechanism of fish mortality observed in this study required physical contact between fish and Pfiesteria cells. [Copyright &y& Elsevier]

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters. [Copyright &y& Elsevier]

Discusses the identification of amoebae implicated in the life cycle of Pfiesteria and Pfiesteria-like dinoflagellates. Isolations of amoebae and dinoflagellates from water and sediment samples from five tributaries of the Chesapeake Bay; Reproduction of amoebae from bioassay aquaria; Use of electron microscopy and small subunit rRNA gene sequence analysis.

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 µM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 µM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MESH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates. [ABSTRACT FROM AUTHOR]

In this case, students speculate on what may have caused a major fish kill in an estuary in North Carolina. In the process they explore how land runoff and excess nutrients affect aquatic communities and learn about the complex life cycle of the dinoflagellate Pfiesteria. [ABSTRACT FROM AUTHOR]

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid-Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC-MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α-methyl-substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present. [ABSTRACT FROM AUTHOR]

The extraordinary chemical diversity seen in the cyanobacteria (blue-green algae) is especially pronounced in the ubiquitous tropical marine species, Lyngbya majuscula. The gene clusters responsible for the production of some of the secondary metabolites have recently been elucidated. The dinoflagellates, which are lower eukaryotic algae, also demonstrate chemical diversity and produce unique polycyclic ethers of polyketide origin. A new mechanism for the formation of the truncated polyketide backbones has recently been proposed. The toxicogenicity of dinoflagellates of the genus Pfiesteria has been the focus of controversy — are they ‘killer organisms’, as alleged? A recent investigation of Pfiesteria genes seems to rule out the presence of polyketide synthase, which is the gene responsible for the production of most dinoflagellate toxins. [Copyright &y& Elsevier]

It is not possible to determine the causal relationship between fishkills and the toxic dinoflagellate Pfiesteria piscicida in the Neuse River Estuary, North Carolina, using observational data on these two variables alone. However, consideration of a third variable, indicating the presence of a broader class of more easily measured Pfiesteria-like organisms (PLOs), leads to causal model structures from which the nature of this relationship can be distinguished. We use reported field data to evaluate alternative models and find that the suggestion that toxic Pfiesteria cause fishkills is inconsistent with observation. The data are more indicative of a model in which PLOs (including potentially toxic Pfiesteria) are stimulated to become actively toxic by the presence of already dead or dying fish. Laboratory experiments performed to date do not provide evidence contradictory to this finding. However, neither can the existence of a common cause of both Pfiesteria toxicity and fishkills be ruled out. The differing implications for ecosystem management suggest that these causal associations should be further investigated through additional modeling and data collection efforts. Graphical methods of model construction and evaluation can assist in this process. Corresponding author; e-mail: cstow@duke.edu --> [ABSTRACT FROM AUTHOR]

Since its identification, the dinoflagellate Pfiesteria piscicida has been implicated in fish kills and fish disease in the southeastern United States. Adverse health effects have been reported in researchers working with the organism and in watermen following exposure to a fish kill in Maryland. A bioactive secretion is postulated as the cause of these effects but has not yet been isolated and chemically characterized. The biology and toxicology of this organism remain the topic of debate and research. [Copyright &y& Elsevier]

Examines the incidence of toxic Pfiesteria, a dinoflagellate, in the estuaries of North Carolina, in light of outbreaks in the Chesapeake Bay. Detection of the organism at estuarine fish kills; Basic biology, ecology, and toxicity of Pfiesteria as of 1997; Benign and toxic strains; Impacts on fish; Research and socioeconomic concerns in North Carolina.

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ‘Pfiesteria-like’ dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster. [ABSTRACT FROM AUTHOR]

The article examines the cognitive effects of the occupational exposure to the dinoflagellate Pfiesteria, a common inhabitant of estuarine waters in the U.S. mid-Atlantic region in the summer and fall. The results show that the subjects, commercial fishermen, exposed of Pfiesteria likely do not face significant health risks from routine occupational exposure to the organism. Accordingly, the study is identical with similar studies in North Carolina and Virginia in providing reassurance of the health risks of watermen against Pfiesteria.

Focuses on a study by David Oldach and colleagues, which dealt with a technique for identifying ribosomal gene sequences from Pfiesteria species. Toxic dinoflagellate called Pfiesteria; Development of polymerase chain reaction assays for Pfiesteria species; Inability to pinpoint how many Pfiesteria cells are in the water or sediment.