A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

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.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Development of an immunofluorescence technique for detecting Pfiesteria piscicida

While several DNA-based methods have been developed for the putatively toxic dinoflagellate Pfiesteria piscicida Burkholder et Steidinger, an independent detection method such as immunofluorescence can be a useful alternative. In this study, P. piscicida-specific antisera were developed, and an immunofluorescence (IF) procedure was optimized. A total of six antisera were raised using whole cells (WCA) and the insoluble cellular fraction (ICF) as antigens, respectively, and their titer and specificity were examined using dot blot analysis and whole cell IF. Results showed that the two antisera produced from the ICF antigen had a markedly higher titer (1500) than the other four yielded from the WCA (200). In addition, the two ICF-derived antisera exhibited much higher species specificity, showing no cross-reaction with P. shumwayae, Cryptoperidiniopsis sp., Karlodinium micrum, and other more distant algae tested, and very low background for field collected samples. In evaluation of the IF technique using a P. piscicida-specific polymerase chain reaction (PCR) technique, results from both methods generally agreed well for both field samples (from eastern Long Island Sound) spiked with cultured P. piscicida and those containing naturally occurring P. piscicida (from Chesapeake Bay tributaries).     

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid‐Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps. shumwayae and P. piscicida was examined by PCR–RFLP analysis using cultures obtained from the Provasoli‐Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues’ collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps. shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps. shumwayae strains. Conversely, PCR–RFLP examination of 14 strains of P. piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P. piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.     

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free‐living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high‐resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida‐specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free‐living marine dinoflagellates and lacks any amoeboid or other specious stages.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Distribution of Pfiesteria piscicida cyst populations in sediments of the Delaware Inland Bays, USA

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.     

Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond

A dinoflagellate bloom was found associated with a fish kill event in a South Carolina brackish water retention pond. A multi-analytical approach was used to confirm the identity of the bloom dinoflagellate and evaluate its potential toxicity. Karlodinium micrum was confirmed through light microscopy, pigment profile comparisons, species-specific PCR, and gene sequence data. Necropsy findings on several fish were suggestive of an acute kill event. Toxicity of filtrate from bloom samples was tested by a hemolytic assay using rainbow trout (Oncorhynchus mykis) erythrocytes and an ichthyotoxicity assay using larval zebrafish (Danio rerio). Hemolytic activity was measurably high (>80% hemolysis) in both whole filtrate and fractionated filtrate (from the 80% MeOH C₁₈ column elution). This fraction also demonstrated high ichthyotoxic activity as exposed fish experienced rapid death. These results implicate toxic K. micrum as a causative factor in fish death in a non-aquaculture brackish pond associated with a housing development, and extend recent findings linking this species to fish kills in aquaculture ponds.     

Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum

The harmful dinoflagellate Prorocentrum minimum has different effects upon various species of grazing bivalves, and these effects also vary with life-history stage. Possible effects of this dinoflagellate upon mussels have not been reported; therefore, experiments exposing adult blue mussels, Mytilus edulis, to P. minimum were conducted. Mussels were exposed to cultures of toxic P. minimum or benign Rhodomonas sp. in glass aquaria. After a short period of acclimation, samples were collected on day 0 (before the exposure) and after 3, 6, and 9 days of continuous-exposure experiment. Hemolymph was extracted for flow-cytometric analyses of hemocyte, immune-response functions, and soft tissues were excised for histopathology. Mussels responded to P. minimum exposure with diapedesis of hemocytes into the intestine, presumably to isolate P. minimum cells within the gut, thereby minimizing damage to other tissues. This immune response appeared to have been sustained throughout the 9-day exposure period, as circulating hemocytes retained hematological and functional properties. Bacteria proliferated in the intestines of the P. minimum-exposed mussels. Hemocytes within the intestine appeared to be either overwhelmed by the large number of bacteria or fully occupied in the encapsulating response to P. minimum cells; when hemocytes reached the intestine lumina, they underwent apoptosis and bacterial degradation. This experiment demonstrated that M. edulis is affected by ingestion of toxic P. minimum; however, the specific responses observed in the blue mussel differed from those reported for other bivalve species. This finding highlights the need to study effects of HABs on different bivalve species, rather than inferring that results from one species reflect the exposure responses of all bivalves.     

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.     

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.     

Prorocentrum minimum blooms: potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth

The cosmopolitan dinoflagellate Prorocentrum minimum is a recurrent bloom forming species in the Chesapeake Bay and its tributaries, generally observed at its highest levels in late spring and summer. Laboratory studies were conducted to assess potential bloom impacts on diel oxygen concentrations in shallow littoral zones as well as settlement success and post-set growth of the eastern oyster Crassostrea virginica. Using light–dark and dark cultures and periodic diel sub-sampling, bloom levels of P. minimum produced supersaturated oxygen levels at the end of each day while darkened cultures were typified by rapid decreases in dissolved oxygen (DO) (1.1–1.3 mg L⁻¹ h⁻¹) to hypoxic and anoxic levels within 4 days. These data suggest shallow, poorly flushed systems and the biota in them will experience rapid and large diel variations in oxygen, implying recurrent P. minimum blooms need be considered as short-term oxygen stressors for Bay oyster spat and other living resources. Direct effects of P. minimum impacts on oysters were not as expected or previously reported. In one experiment, pre-bloom isolates of P. minimum were grown and then exposed to polyvinyl chloride (PVC) settlement plates to see whether dinoflagellate preconditioning of the hard substrate might affect oyster sets. No differences were noted between set on the PVC with P. minimum exposure to set recorded with filtered seawater, Instant Ocean^®, or Isochrysis. In the second oyster experiment, spat on PVC plates were exposed to field collected P. minimum blooms and a commercial mixture of several other food types including Isochrysis. Oyster growth was significantly higher in P. minimum exposures than noted in the commercial mix. These results, compared to results with other isolates from the same region, indicate substantial positive impact from some of the P. minimum blooms of the area while others separated in space, time, or nutrient status could severely curtail oyster success through toxin production induced by nutrient limitation.     

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.     

POTENTIAL UTILITY OF MITOCHONDRIAL CYTOCHROME B AND ITS MRNA EDITING IN RESOLVING CLOSELY RELATED DINOFLAGELLATES: A CASE STUDY OF PROROCENTRUM (DINOPHYCEAE)1

Difficulties often occur in separating closely related dinoflagellate species. In this study, the potential utility of mitochondrial cytochrome b (cob) gene sequence and mRNA editing characteristics was assessed using Prorocentrum Ehrenberg as a model. The cob sequences and the patterns of their mRNA editing were analyzed for several Prorocentrum taxa. Results revealed little difference in cob sequence and mRNA editing characteristics between geographic populations of P. minimum (Pavillard) Schiller, while a notable difference was detected between different species (P. minimum and P. micans Ehrenberg). Furthermore, these P. minimum populations consistently formed a tight cluster on phylogenetic trees inferred from cob sequences as well as mRNA editing characteristics, whereas different Prorocentrum species were well separated, with a genetic distance of 0.0042±0.0024 for the former and 0.0141±0.0012 for the latter (P<0.01; two‐tailed t‐test). When the analysis was applied to the case of P. donghaiense Lu et Goebel and CCMP1517 strain of P. dentatum Stein, no differences were detected between these two taxa with respect to cob mRNA editing pattern and only small differences equivalent to those between P. minimum populations were detected in terms of cob sequence. On the cob sequence‐ and editing‐based phylogenetic trees, P. donghaiense and P. dentatum CCMP1517 consistently clustered together at a position sister to P. minimum. The results suggest that cob, combined with its mRNA editing, can potentially be a useful delineator of Prorocentrum species, and that P. donghaiense and P. dentatum CCMP1517 are most likely the same species and both are closely related to P. minimum.     

Relative contribution of exotoxin and micropredation to icthyotoxicity of two strains of Pfiesteria shumwayae (Dinophyceae)

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.     

Detection of a novel ecotype of <Emphasis Type="Italic">Pfiesteria piscicida</Emphasis> (Dinophyceae) in an Antarctic saline lake by real-time PCR

The heterotrophic dinoflagellate Pfiesteria piscicida was detected in Ace Lake in the Vestfold Hills, eastern Antarctica by using real-time PCR based on 18S rDNA sequences. Antarctic water samples collected in 2004 were tested by species-specific real-time PCR assays for the identification of P. piscicida and P. shumwayae. Positive results were shown with P. piscicida-specific real-time PCR, and PCR products were examined by sequence analysis for confirmation. A phylogenetic tree made from partial 18S rDNA sequences showed that the Antarctic clone clustered with P. piscicida. This result suggests that P. piscicida is present in the extreme conditions of an Antarctic saline lake which has not contained fish for thousands of years.     

Prorocentrum minimum (Dinophyceae) in the Baltic Sea: morphology, occurrence—a review

The potentially toxic dinoflagellate Prorocentrum minimum (Pavillard) Schiller has successfully established in the Baltic Sea in the last two decades. A review of the invasion history is presented as well as new data on the spatial and inter-annual variability of this species and its relation to salinity, temperature, and nutrient concentrations. A short literature review of the morphological characters of the Baltic P. minimum is also included.From 1993 to 2002, P. minimum was a regular component of the summer and autumn plankton flora of the Baltic Sea proper and the Gulf of Finland. Its abundance varied considerably inter-annually and did not show any clear trends during the period. Abundance of P. minimum was significantly higher in the nutrient-enriched Bay of Mecklenburg (German coast) and the southern Baltic proper than in the central and northern Baltic proper and the Gulf of Finland, where its abundance was mostly sparse. In coastal waters P. minimum occasionally reached densities of several million cells per litre and dominated phytoplankton biomass (>90%).Abundance of the Baltic P. minimum was generally not related to salinity or temperature. It could be a dominant species at both high and low salinity (over 15 and 4.8 PSU), and its temperature range was broad (from 2.7 to 26.4 °C). However, dense populations usually occurred from July to October at temperatures above 10 °C.Further, there appears to be a positive correlation between the success of P. minimum in the Baltic Sea and high concentrations of total phosphorus and nitrogen.This tolerant and morphologically variable dinoflagellate seems to be a morphospecies without subtaxa, which can expand its range in the Baltic Sea, especially in nutrient-rich coastal waters.     

Grazing activity of Pfiesteria piscicida (Dinophyceae) and susceptibility to ciliate predation vary with toxicity status

Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in <40% of actively toxic cells for ≤16 h (high light) and ≤23 h (low light). These results suggest a relatively high reliance on phagotrophic carbon assimilation and more rapid response to algal prey availability in Pfiesteria cells with lower toxicity. Grazing by two euplotid benthic ciliates (Euplotes vannus and E. woodruffi) on P. piscicida also varied among functional types. Grazing on actively toxic P. piscicida cells did not occur, whereas net positive ingestion rates were calculated for the other prey cultures. These results support concurrent experimental findings that a natural assemblage of microzooplankton displayed lower grazing potential on actively toxic P. piscicida than on cultures with reduced toxicity. In summary, pronounced differences in trophic interactions were found between actively toxic cultures and those with reduced or undetectable toxicity, providing additional evidence of the importance of cellular toxicity in the trophic ecology of Pfiesteria.     

Ingestion of the dinoflagellate, Pfiesteria piscicida, by the calanoid copepod, Acartia tonsa

The dinoflagellate, Pfiesteria piscicida, can form harmful algal blooms in estuarine environments. The dominant copepod species usually found in these waters is Acartia tonsa. We tested the ability of A. tonsa to graze the non-toxic zoospore stage of P. piscicida and thus serve as a potential biological control of blooms of this algal species. A. tonsa grazed the non-toxic zoospore stages of both a non-inducible P. piscicida strain (FDEPMDR23) and a potentially toxic strain (Tox-B101156) at approximately equal rates. Ingestion of P. piscicida increased with cell concentration and exhibited a saturated feeding response. Both the maximum number of cells ingested (I_max) and the slope of the ingestion curve (α) of A. tonsa feeding on P. piscicida were comparable to these ingestion parameters for A. tonsa fed similar-sized phytoplankton and protozoan species. When these laboratory ingestion rates were combined with abundance estimates of A. tonsa from the Pocomoke Estuary and Chesapeake Bay, we found that significant grazing control of the non-toxic zoospore stage of P. piscicida by A. tonsa would only occur at high copepod abundances (>10 copepods L⁻¹). We conclude that under most in situ conditions the potential biological control of blooms of P. piscicida is exerted by microzooplankton grazers. However, in the less saline portions of estuaries where maximum concentrations of copepods often occur with low abundances of microzooplankton, copepod grazing coefficients can be similar to the growth rates of P. piscicida.     

Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae)—a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility

The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC₅₀ for hemolysis of a sonicated cell suspension was 2.4×10⁴ cells ml⁻¹, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC₅₀ for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml⁻¹ while the LC₅₀ of the second, smaller, fraction (Tox B) was 600 ng ml⁻¹. For comparison, the LC₅₀ for the standard hemolysin saponin was 3203 ng ml⁻¹. At concentrations of 800 and 2000 ng ml⁻¹, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml⁻¹ Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml⁻¹. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.