Morphological and molecular genetic characterization of Cryptoperidiniopsis brodyi (Dinophyceae) from Australia-wide isolates

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.     

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 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.     

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.     

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.     

Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences

The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h⁻¹) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH₄⁺ > NO₃⁻. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.     

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 μg 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.     

Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA

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 454 bp 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.     

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.     

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.     

Screening capacity of UV-absorbing compounds in spores of Arctic Laminariales

The functional significance of phlorotannins as ultraviolet radiation screens in brown algae is presented. Spectral analysis of zoospore suspensions of the three Arctic Laminariales Saccorhiza dermatodea, Alaria esculenta and Laminaria digitata showed strong absorption in the UV waveband, characteristic of phlorotannins. An induction in the synthesis of the UV-absorbing compound in zoospore suspensions of S. dermatodea and A. esculenta was observed as an increase in absorbance in the UV region after 8 h exposure to the whole light spectrum. Transmission of UVR was also negatively correlated with zoospore density in both these species but not in L. digitata. ‘Biofilters’ constructed from UV-transparent acrylic sheet, containing zoospore suspensions or solutions of phloroglucinol showed varying capacity to protect zoospore cultures from the lethal effects of ultraviolet radiation. Phloroglucinol protects the zoospores from damage by screening out the much harmful shorter UV-B spectra (280-290 nm). Cultured spores of A. esculenta and L. digitata after exposure to the whole light spectrum covered by filters containing phloroglucinol showed high rates of germination, unlike controls covered by seawater-only filters that showed 100% mortality. Biofilters containing zoospore suspensions act as buffers and showed variable UV-protection properties on the germination of its conspecies. At highest zoospore density (∼ 4 × 10⁶ spores ml^− 1), zoospores were observed to screen UV radiation maintaining viability among shielded spores in all species investigated. The protective function of zoospore film is, however, density-dependent in L. digitata. At lower spore density, UV-screening function in S. dermatodea and A. esculenta is attributed to their capacity to accumulate and release UV-absorbing compounds into the medium. Ultraviolet radiation transmission by zoospore suspensions of Saccorhiza and Alaria decreased during exposure to the whole light spectrum which is consistent with the earlier observation of enlarged phenolic vesicles following UVR exposure. The increase in vesicle size and the corresponding increase in UV-absorbing capacity may contribute to greater tolerance of UVR exposure in both species.     

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.     

Growth responses of the mixotrophic dinoflagellates, Cryptoperidiniopsis sp. and Pfiesteria piscicida, to light under prey-saturated conditions

Recent research emphasis on the ecology of Pfiesteria spp. (Dinophyceae) has led to recognition of several morphologically similar heterotrophic dinoflagellates that often co-occur with Pfiesteria spp. in estuaries along the United States Atlantic coast. These include cryptoperidiniopsoid dinoflagellates, which resemble Pfiesteria spp. in having complex life cycles that include zoospores capable of kleptoplastidy. To examine and compare the role of kleptoplastidy in Cryptoperidiniopsis sp. and Pfiesteria piscicida, we tested the effects of irradiance on growth under prey-saturated (Storeatula major, Cryptophyceae) conditions. Growth of Cryptoperidiniopsis was strongly influenced by light intensity while no major effects were observed in P. piscicida. In Cryptoperidiniopsis, highest cell numbers and specific growth rates, but lowest specific cryptophyte consumption rates, were found at the highest light intensity tested (100 μmol photons m⁻² s⁻¹). A growth model was developed and used to estimate that the average half-life of chloroplasts ingested by Cryptoperidiniopsis decreased 3.4-fold from 12.6 h at high light to 3.7 h in the dark. These results show that light strongly enhances specific growth rate and growth efficiency of Cryptoperidiniopsis feeding on cryptophytes, and suggest that retained kleptochloroplasts may play a quantitatively significant role in carbon and energy metabolism of this organism. Differences in the effects of light between Cryptoperidiniopsis and P. piscicida may reflect different nutritional strategies, and allow these closely related dinoflagellates to occupy different niches and co-exist.     

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.     

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.     

Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.     

Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species

A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 10³ cells ml⁻¹, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 10³ cells ml⁻¹) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 10³ cells ml⁻¹). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.     

Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in Environmental Samples

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter⁻¹, with higher abundance (maximum, 112 cells liter⁻¹) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter⁻¹. P. shumwayae was not detected during the survey.     

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).