Discovery of the toxic dinoflagellate Pfiesteria in northern European waters.

Several dinoflagellate strains of the genus Pfiesteria were isolated by culturing techniques from sediment samples taken in the Oslofjord region of Norway. Pfiesteria piscicida, well known as a fish killer from the Atlantic coast of America, was identified by genetic methods and light microscopy. The related species Pfiesteria shumwayae was attracted from the sediment by the presence of fish, and has proved toxic. This present survey demonstrates the wide distribution of these potentially harmful species, but so far they have not been connected with fish kills in Europe.     

KLEPTOPLASTIDY IN THE TOXIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)

The ichthyotoxic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder has a complex life cycle with several heterotrophic flagellated and amoeboid stages. A prevalent flagellated form, the nontoxic zoospore stage, has a proficient grazing ability, especially on cryptophyte prey. Although P. piscicida zoospores lack the genetic capability to synthesize chloroplasts, they can obtain functional chloroplasts from algal prey (i.e. kleptoplastidy), as demonstrated here with a cryptophyte prey. Zoospores grown with Rhodomonas sp. Karsten CCMP757 (Cryptophyceae) grazed the cryptophyte population to minimal densities. After placing the cultures in near darkness where cryptophyte recovery was restricted and further prey ingestion did not occur, the time-course patterns in growth, prey chloroplast content·zoospore⁻¹, and prey nucleus content·zoospore⁻¹ were followed. Ingested chloroplasts were selectively retained in the dinoflagellate, as indicated by the decline and, ultimately, near absence of cryptophyte nuclei in plastid-containing zoospores. Chloroplasts retained inside P. piscicida cells for at least a week were photosynthetically active, as indicated by starch accumulation and microscope-autoradiographic measurements of bicarbonate uptake. Recognition that P. piscicida can function as a phototroph broadens our perspective of the physiological ecology of the dinoflagellate because it suggests that, at least during part of its life cycle, P. piscicida 's growth and survival might be affected by photoregulation and nutritional control of photosynthesis.     

Predator/Prey Interaction between <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> and <Emphasis Type="Italic">Rhodomonas</Emphasis> Mediated by a Marine Alpha Proteobacterium

The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.     

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Characterization of ichthyocidal activity of Pfiesteria piscicida: dependence on the dinospore cell density

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.     

THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

MIXOTROPHY AND NITROGEN UPTAKE BY PFIESTERIA PISCICIDA (DINOPHYCEAE)

The nutritional versatility of dinoflagellates is a complicating factor in identifying potential links between nutrient enrichment and the proliferation of harmful algal blooms. For example, although dinoflagellates associated with harmful algal blooms (e.g. red tides) are generally considered to be phototrophic and use inorganic nutrients such as nitrate or phosphate, many of these species also have pronounced heterotrophic capabilities either as osmotrophs or phagotrophs. Recently, the widespread occurrence of the heterotrophic toxic dinoflagellate, Pfiesteria piscicida Steidinger et Burkholder, has been documented in turbid estuarine waters. Pfiesteria piscicida has a relatively proficient grazing ability, but also has an ability to function as a phototroph by acquiring chloroplasts from algal prey, a process termed kleptoplastidy. We tested the ability of kleptoplastidic P. piscicida to take up ¹⁵N-labeled NH     , NO     , urea, or glutamate. The photosynthetic activity of these cultures was verified, in part, by use of the fluorochrome, primulin, which indicated a positive relationship between photosynthetic starch production and growth irradiance. All four N substrates were taken up by P. piscicida , and the highest uptake rates were in the range cited for phytoplankton and were similar to N uptake estimates for phagotrophic P. piscicida . The demonstration of direct nutrient acquisition by kleptoplastidic P. piscicida suggests that the response of the dinoflagellate to nutrient enrichment is complex, and that the specific pathway of nutrient stimulation (e.g. indirect stimulation through enhancement of phytoplankton prey abundance vs. direct stimulation by saprotrophic nutrient uptake) may depend on P. piscicida 's nutritional state (phagotrophy vs. phototrophy).     

Chemotaxis of Silicibacter sp. strain TM1040 toward dinoflagellate products

The alpha-proteobacteria phylogenetically related to the Roseobacter clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP). Silicibacter sp. strain TM1040, isolated from a DMSP-producing Pfiesteria piscicida dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate. TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with P. piscicida through a chemotactic response to compounds produced by its dinoflagellate host. A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040. These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80 degrees C. To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants. The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates. Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates. The implication of Silicibacter sp. strain TM1040 chemotaxis in establishing and maintaining its interaction with P. piscicida is discussed.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

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.     

Detection and quantification of Pfiesteria piscicida by using the mitochondrial cytochrome b gene

Mitochondrial cytochrome b was isolated from the dinoflagellate Pfiesteria piscicida, and the utility of the gene for species identification was examined. One of the primer sets designed was shown to be highly specific for P. piscicida. A time step PCR protocol was used to demonstrate the potential of this primer set for quantification of this species.     

Feeding preferences and grazing rates of Pfiesteria piscicida and a cryptoperidiniopsoid preying on fish blood cells and algal prey

The grazing rates and feeding preferences of the dinoflagellates Pfiesteria piscicida and a cryptoperidiniopsoid on the alga Rhodomonas sp. and fish blood cells were calculated at different ratios of the two food types and at different total food densities. Data from 6 h grazing periods within microcosms were used to calculate grazing rates. Grazing rates of both dinoflagellates increased linearly with an increased ratio of blood cells to Rhodomonas, and P. piscicida had a higher maximum grazing rate than the cryptoperidiniopsoid. The grazing rate of P. piscicida on Rhodomonas also increased with increased Rhodomonas densities relative to the blood cells, but increased densities of Rhodomonas did not increase the grazing rate of the cryptoperidiniopsoid, suggesting a lower feeding threshold for this species. Both dinoflagellates demonstrated a preference for fish blood cells over Rhodomonas cells, with no significant difference in the index of preference between the two species. Total food abundance affected the degree of preference differently for each dinoflagellate species. A higher index of feeding preference was attained by P. piscicida when resource levels were high, while the cryptoperidiniopsoid did not show this response. A preference for fish blood cells occurred at all food ratios for both dinoflagellates, including when blood cells were scarce relative to the alternate food type (15% of total available food). These results suggest that these strains of P. piscicida and the cryptoperidiniopsoid share similar feeding preferences for the prey types tested, although cryptoperidiniopsoids have not been associated with fish kills.     

Response of two zooptankton grazers to an ichthyotoxic estuarine dinoflagellate

The dinoflagellate Pfiesteria piscicida (gen. et sp. nov.).a toxic ‘ambush predator’, has been implicated asa causative agent of major fish kills in estuanne ecosystemsof the southeastern USA. Here we report the first experimentaltests of interactions between P.piscicida and estuarine zooplanktonpredators. specifically the rotifer Brachionus plicatilis andthe calanoid copepod Acartia tonsa. Short-term (10 day) exposureof adult B.plicatilis to P.piscicida as a food resource, aloneor in combination with the non-toxic green algae Nannochlorisand Tetraselmis. did not increase rotifer mortality relativeto animals that were given only non-toxic greens Similarly,short-term (3 day) feeding trials using adult A.tonsa indicatedthat the copepods survived equally well on either P.piscicidaor the non-toxic diatom Thalassiosira pseudonana. Copepods giventoxic dinoflagellates exhibited erratic behavior, however, relativeto animals given diatom prey. The fecundity of B.plicatiliswhen fed the toxic dinoflagellate was comparable to or higherthan that of rotifers fed only non-toxic greens We concludethat, on a short-term basis, toxic stages of P.piscicida canbe readily utilized as a nutritional resource by these commonestuarine zooplankters. More long-term effects of P.piscicidaon zooplankton, the potential for toxin bioaccumulation acrosstrophic levels, and the utility of zooplankton as biologicalcontrol agents for this toxic dinoflagellate. remain importantunanswered questions.     

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.     

Human health effects of exposure to Pfiesteria piscicida: a review

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.     

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

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 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM 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.     

Trophic Controls on Stage Transformations of a Toxic Ambush-Predator Dinoflagellate1

ABSTRACT. The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush-predator” and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.     

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL^?1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD₅₀ of 108 zoospores·mL^?1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.