Pfiesteria Species Research Articles

Toxigenic Pfiesteria species—Updates on biology, ecology, toxins, and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden ( Brevoortia tyrannus), mostly at densities of ≥4(3) × 10 2 to 10 3 (rarely, 10 4) flagellate cells mL −1. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL −1), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.

Development of a SYTO9 based real-time PCR probe for detection and quantification of toxic dinoflagellate Karlodinium veneficum (Dinophyceae) in environmental samples

T.-G. Park, Y.-T. Park and Y. Lee. 2009. Development of a SYTO9 based real-time PCR probe for detection and quantification of toxic dinoflagellate Karlodinium veneficum (Dinophyceae) in environmental samples. Phycologia 48: 32–43. DOI: 10.2216/08-52.1.Conventional methods to identify the fish-killing dinoflagellate Karlodinium veneficum rely on rDNA sequence and scanning electron microscopy analyses that are not suitable for processing a large number of samples. To overcome this difficulty, SYTO9 and TaqMan format real-time PCR probes were developed for the internal transcribed spacer rDNA region for rapid detection and quantification of K. veneficum in the marine environment. Assay specificity, sensitivity, and accuracy were confirmed by testing against related organisms, sequencing PCR amplicons from field samples, and comparing real-time PCR results with microscopic counts. The SYTO9-based assay produced highly reproducible DNA melting curves over a broad range of starting DNA template. When the SYTO9- and TaqMan-based assays were compared in the quantitative measurements, they showed comparable results and reasonable reproducibility. The K. veneficum–specific assays were used in conjunction with Pseudopfiesteria shumwayae (Dinophyceae)–, Pfiesteria piscicida (Dinophyceae)–, and Cryptoperidiniopsis brodyi (Dinophyceae)–specific real-time PCR probes to investigate temporal changes in abundances of these four species in Chinhae Bay, South Korea. The field survey revealed that K. veneficum was dominant (maximum 970 cells ml−1) among those species; whereas, Pfiesteria species rarely occurred. These findings suggest that the SYTO9- and TaqMan-based assays are specific and sensitive for detecting and quantifying K. veneficum in the environment. The wide distribution of K. veneficum also indicates potential fish kills by this organism in Chinhae Bay.

Recent evolutionary diversification of a protist lineage

Here, we have identified a protist (dinoflagellate) lineage that has diversified recently in evolutionary terms. The species members of this lineage inhabit cold-water marine and lacustrine habitats, which are distributed along a broad range of salinities (0-32) and geographic distances (0-18 000 km). Moreover, the species present different degrees of morphological and sometimes physiological variability. Altogether, we analysed 30 strains, generating 55 new DNA sequences. The nuclear ribosomal DNA (nrDNA) sequences (including rapidly evolving introns) were very similar or identical among all the analysed isolates. This very low nrDNA differentiation was contrasted by a relatively high cytochrome b (COB) mitochondrial DNA (mtDNA) polymorphism, even though the COB evolves very slowly in dinoflagellates. The 16 Maximum Likelihood and Bayesian phylogenies constructed using nr/mtDNA indicated that the studied cold-water dinoflagellates constitute a monophyletic group (supported also by the morphological analyses), which appears to be evolutionary related to marine-brackish and sometimes toxic Pfiesteria species. We conclude that the studied dinoflagellates belong to a lineage which has diversified recently and spread, sometimes over long distances, across low-temperature environments which differ markedly in ecology (marine versus lacustrine communities) and salinity. Probably, this evolutionary diversification was promoted by the variety of natural selection regimes encountered in the different environments.

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.

Pfiesteria in Estuarine Waters: The Question of Health Risks

The conclusion of Morris et al. (2006) that “Exposure to Pfiesteria Species in Estuarine Waters Is Not a Risk Factor for Illness” is unsupported because a) a description of Pfiesteria-related fish kills in the Chesapeake estuaries during 1999–2002 was omitted; b) quantitative data on Pfiesteria were not collected; c) data on visual contrast sensitivity (VCS) were collected but not reported; d) a comprehensive list of other results was not presented; and e) data were lost due to a 30% attrition rate. These data are needed to justify or negate the conclusion.

Remember Pfiesteria ?: Occupational Exposure Unlikely to Cause Cognitive Effects

Vol. 114, No. 7 EnvironewsOpen AccessRemember Pfiesteria?: Occupational Exposure Unlikely to Cause Cognitive Effectsis accompanied byOccupational Exposure to Pfiesteria Species in Estuarine Waters Is Not a Risk Factor for Illness Tanya Tillett Tanya Tillett Search for more papers by this author Published:1 July 2006https://doi.org/10.1289/ehp.114-a429bAboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit Case reports have suggested that exposure to the dinoflagellate Pfiesteria may contribute to deficits in human learning and memory. Until now, however, there has been no clear, objective documentation of health effects associated with regular occupational exposure to this organism. The results of the first systematic, multiyear study of Pfiesteria’s human health effects now demonstrate that commercial fishermen (“watermen”) likely do not face significant health risks from routine occupational exposure to the organism [EHP 114:1038–1043; Morris et al.].Pfiesteria is a common inhabitant of estuarine waters in the U.S. mid-Atlantic region in the summer and fall. In 1997, watermen working along the Pocomoke River, an estuary off Chesapeake Bay, experienced a pattern of neuropsychological deficits in association with fish kills linked to Pfiesteria outbreaks. Researchers studying Pfiesteria in a lab environment had reported similar memory and learning deficits.Using a cohort of 88 healthy watermen with regular occupational exposure to Chesapeake Bay waters and 19 controls with minimal contact to the waters (matched to the watermen by zip code, age, and educational level), a team of Maryland researchers collected data over four summers, from 1999 through 2002. They questioned the subjects biweekly about symptoms like those reported in the 1997 episode and about their exposure to the waters and to known chemical toxicants. Subjects were tested at the beginning and end of each summer season on sensory and motor functions, attention and concentration, memory, visual functions, and verbal functions. In addition, the research team analyzed more than 3,500 water samples taken from Chesapeake Bay to monitor the presence of Pfiesteria and other harmful species.P. piscicida was found in water samples drawn from a number of locations in all four years of the study, and P. shumwayae (recently renamed Pseudopfiesteria shumwayae) was found in the last two years. However, the investigators found no decline in neurological function among the watermen in any year of the study.The scientists note that unique, isolated instances of Pfiesteria outbreaks or unusually toxic strains of the dinoflagellate may have been associated with the marked, reversible health effects documented in the past. They point out that the present study is congruent with similar studies in North Carolina and Virginia in providing reassurance that in the absence of these conditions, watermen do not appear to face significant health risks from routine occupational exposure to estuarine waters that contain Pfiesteria.Safe from PfiesteriaNew data suggest commercial fishermen need not fear routine exposure to the organism.FiguresReferencesRelatedDetailsRelated articlesOccupational Exposure to Pfiesteria Species in Estuarine Waters Is Not a Risk Factor for Illness18 April 2006Environmental Health Perspectives Vol. 114, No. 7 July 2006Metrics About Article Metrics Publication History Originally published1 July 2006Published in print1 July 2006 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

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.

Occupational Exposure to Pfiesteria Species in Estuarine Waters Is Not a Risk Factor for Illness

BackgroundExposure to the dinoflagellate Pfiesteria has, under certain circumstances, been associated with deficits in human learning and memory. However, uncertainties remain about the health risk of chronic, low-level exposures (as seen among occupationally exposed commercial fishermen), particularly in light of studies suggesting that Pfiesteria strains are widespread in the estuarine environment in the U.S. mid-Atlantic region.MethodsWe selected an initial cohort of 152 persons, including 123 persons with regular, occupational exposure to the Chesapeake Bay; 107 of the cohort members were followed for the full four summer “seasons” of the study. Cohort members were questioned biweekly about symptoms, and data were collected about the areas of the bay in which they worked. These latter data were matched with data on the presence or absence of Pfiesteria in each area, based on polymerase chain reaction analysis of > 3,500 water samples. Cohort members underwent neuropsychological testing at the beginning and end of each summer season.ResultsNo correlation was found between work in an area where Pfiesteria was identified and specific symptomatology or changes on neuropsychological tests.ConclusionsAlthough high-level or outbreak-associated exposure to Pfiesteria species (or specific strains within a species) may have an effect on health, routine occupational exposure to estuarine environments in which these organisms are present does not appear to pose a significant health risk.

Contaminant-free cultivation of Pfiesteria shumwayae (Dinophyceae) on a fish cell line

Geographically distinct strains of the heterotrophic dinoflagellate Pfiesteria shumwayae were cultivated on a fish cell line in the apparent absence of bacteria and other microbial conta- minants. Cultures were established with a high rate of success by inoculating single purified P. shumwayae cells into fish cell cultures containing a simple saltwater medium suitable for both cell types, and resulting isolates were serially cultivated on fish cells for months without visible signs of abnormality or reduced viability. P. shumwayae fed phagocytically on the fish cells and exhibited higher cell production than reported using other culturing methods. Compared to previous methods of studying the interaction between Pfiesteria spp. and fishes, this system enabled closer and more direct observation of the dinoflagellates and was also more economical and sustainable as a culturing method. The absence of bacteria and other contaminating microorganisms should facilitate important physiological and biochemical investigations. The methods used were inadequate for cultivating strains of P. piscicida, suggesting a possible difference in nutritional requirements between the 2 Pfiesteria 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.

Are Pfiesteria species toxicogenic? Evidence against production of icthyotoxins by Pfiesteria shumwayae

Are Pfiesteria species toxicogenic? Evidence against production of icthyotoxins by Pfiesteria shumwayae

Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification

Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.

The standardized fish bioassay procedure for detecting and culturing actively toxic Pfiesteria, used by two reference laboratories for atlantic and gulf coast states.

In the absence of purified standards of toxins from Pfiesteria species, appropriately conducted fish bioassays are the "gold standard" that must be used to detect toxic strains of Pfiesteria spp. from natural estuarine water or sediment samples and to culture actively toxic Pfiesteria. In this article, we describe the standardized steps of our fish bioassay as an abbreviated term for a procedure that includes two sets of trials with fish, following the Henle-Koch postulates modified for toxic rather than infectious agents. This procedure was developed in 1991, and has been refined over more than 12 years of experience in research with toxic Pfiesteria. The steps involve isolating toxic strains of Pfiesteria (or other potentially, as-yet-undetected, toxic Pfiesteria or Pfiesteria-like species) from fish-killing bioassays with natural samples; growing the clones with axenic algal prey; and retesting the isolates in a second set of fish bioassays. The specific environmental conditions used (e.g., temperature, salinity, light, other factors) must remain flexible, given the wide range of conditions from which natural estuarine samples are derived. We present a comparison of information provided for fish culture conditions, reported in international science journals in which such research is routinely published, and we provide information from more than 2,000 fish bioassays with toxic Pfiesteria, along with recommendations for suitable ranges and frequency of monitoring of environmental variables. We present data demonstrating that algal assays, unlike these standardized fish bioassays, should not be used to detect toxic strains of Pfiesteria spp. Finally, we recommend how quality control/assurance can be most rapidly advanced among laboratories engaged in studies that require research-quality isolates of toxic Pfiesteria spp.

Report from the NOAA Workshops to Standardize Protocols for Monitoring Toxic Pfiesteria Species and Associated Environmental Conditions

Report from the NOAA Workshops to Standardize Protocols for Monitoring Toxic Pfiesteria Species and Associated Environmental Conditions

Report from the NOAA workshops to standardize protocols for monitoring toxic Pfiesteria species and associated environmental conditions.

Long-term monitoring of water quality, fish health, and plankton communities in susceptible bodies of water is crucial to identify the environmental factors that contribute to outbreaks of toxic Pfiesteria complex (TPC) species. In the aftermath of the 1997 toxic Pfiesteria outbreaks in North Carolina and Maryland, federal and several state agencies agreed that there was a need to standardize monitoring protocols. The National Oceanic & Atmospheric Administration convened two workshops that brought together state, federal, and academic resource managers and scientific experts to a) seek consensus on responding to and monitoring potential toxic Pfiesteria outbreaks; b) recommend standard parameters and protocols to characterize water quality, fish health, and plankton at historical event sites and potentially susceptible sites; and c) discuss options for integrating monitoring data sets from different states into regional and national assessments. Workshop recommendations included the development of a three-tiered TPC monitoring strategy: Tier 1, rapid event response; Tier 2, comprehensive assessment; and Tier 3, routine monitoring. These tiers correspond to varying levels of water quality, fish health, and plankton monitoring frequency and intensity. Under the strategy, sites are prioritized, depending upon their history and susceptibility to TPC events, and assigned an appropriate level of monitoring activity. Participants also agreed upon a suite of water quality parameters that should be monitored. These recommendations provide guidance to state and federal agencies conducting rapid-response and assessment activities at sites of suspected toxic Pfiesteria outbreaks, as well as to states that are developing such monitoring programs for the first time.

Use of molecular probes to assess geographic distribution of Pfiesteria species.

We have developed multiple polymerase chain reaction (PCR)-based methods for the detection of Pfiesteria sp. in cultures and environmental samples. More than 2,100 water and sediment samples from estuarine sites of the U.S. Atlantic and gulf coasts were assayed for the presence of Pfiesteria piscicida Steidinger & Burkholder and Pfiesteria shumwayae Glasgow & Burkholder by PCR probing of extracted DNA. Positive results were found in about 3% of samples derived from routine monitoring of coastal waters and about 8% of sediments. The geographic range of both species was the same, ranging from New York to Texas. Pfiesteria spp. are likely common and generally benign inhabitants of coastal areas, but their presence maintains a potential for fish and human health problems.

Chemosensory attraction of zoospores of the estuarine dinoflagellates, Pfiesteriapiscicida and P. shumwayae, to finfish mucus and excreta

Toxic strains of the estuarine dinoflagellates, Pfiesteria piscicida and P. shumwayae, can cause fish death and disease, whereas other estuarine ‘lookalike’ species such as cryptoperidiniopsoids have not been ichthyotoxic under ecologically relevant conditions. Chemosensory attraction of three functional types of these Pfiesteria spp. were separately evaluated for their attraction to fresh fish mucus and excreta. Clonal cultures of actively toxic (TOX-A, engaged in killing fish) and temporarily nontoxic (tested as toxic but without access to live fish for >1 week to 5 months (in most experiments, ≤3 months) as ‘short-duration’ TOX-B; and without access to live fish for ≥1.5 years as ‘long-duration’ TOX-B) functional types of P. piscicida and P. shumwayae were derived from the same clones whereas the non-inducible cultures (NON-IND, tested as incapable of toxic activity in the presence of fish), of necessity, were from different clonal isolates. NON-IND cultures previously had been grown on algal prey for 3–8 months, and had repeatedly been tested as incapable of causing fish distress, disease or death via toxic activity. Attraction to fish materials was based on the number of zoospores that entered microcapillary tubes containing sterile-filtered 15-ppt water (controls), vs. entry into tubes with sterile-filtered mucus and excreta (collected in 15-ppt water) that had been collected from live tilapia, bluegill, hybrid striped bass, and Atlantic menhaden (tested separately within 3 h of removal from live fish). TOX-A zoospores of both Pfiesteria species exhibited the strongest attraction to the fish mucus and excreta, with comparable response to the materials from all four test fish species. Short-duration TOX-B zoospores showed an intermediate response that apparently depended on the duration of mucus separation from the live fish: the shorter the separation period, the stronger the zoospore attraction to the fish materials. In contrast to TOX-A and short-duration TOX-B zoospores, NON-IND and long-duration TOX-B zoospores generally showed little or no response to the fish materials. Zoospores of the cryptoperidiniopsoid demonstrated a moderate attraction that did not appear to depend on the time of isolation from fish. TOX-A zoospores were also tested for attraction to sterile-filtered vs. non-filtered fish mucus (time separated from the live animal, 3–96 h). These zoospores, which initially had been actively attracted, were no longer attracted to the unfiltered fish materials after 48 h, whereas attraction to the sterile-filtered fish mucus and excreta persisted throughout the duration of the experiment. Thus, the attractant signal in the materials was degraded or effectively blocked by the bacterial community within hours of isolation from live fish. This study indicates the importance of functional type or toxicity status, and the importance of the history of exposure to live fish, in the behavioral ecology of Pfiesteria spp. Initial attraction to fish materials strongly depended on the functional type, and on the history of toxic activity. Non-inducible and long-duration TOX-B cultures of Pfiesteria spp., unlike actively toxic and short-duration TOX-B (potentially toxic) strains, initially were virtually unresponsive to fish mucus.

Overview and present status of the toxic Pfiesteria complex (Dinophyceae)

This paper reviews the Pfiesteria issue and Pfiesteria science and presents new information on variation in toxicity among Pfiesteria strains, culture effects on their toxicity, the trophic interactions of Pfiesteria spp ., and impacts on fish and mammals. We also assess Pfiesteria spp. impacts on fish in comparison to certain other estuarine dinoflagellates of similar appearance. Species of the toxic Pfiesteria complex (TPC) thus far include P. piscicida and P. shumwayae. These species share morphological and genetic similarities, and both have toxic strains that (1) show strong attraction to live fish;(2) exhibit toxicity that is triggered by live fish or their fresh tissues and excreta; and (3) produce toxin(s) that cause fish stress, disease and death under ecologically relevant conditions (the standardized fish bioassay process involves testing live Pfiesteria cells at similar densities to those encountered during Pfiesteria-related fish kill/disease events). Both Pfiesteria species also have a complex life cycle with multiple amoeboid, flagellated and cyst stages, several of which are ichthyotoxic. TPC species are eurythermal and euryhaline, with prey spanning the estuarine food web, from bacteria to mammalian tissues. They can be stimulated directly or indirectly by nitrogen and phosphorus enrichment. Toxic strains can be either actively or potentially toxic (the TOX-A and TOX-B functional types, respectively); in addition, c.40% of randomly isolated clones have been found to be benign [the noninducible or NON-IND functional type, which apparently lacks the ability to produce bioactive substances (toxins) that cause fish disease or death]. These functional types differ significantly in response to algal prey, predators, nutrients and fish. Moreover, as an apparent artifact of culture conditions, toxic strains generally lose their ability to cause fish death and disease and become NON-IND within weeks to months. At low cell densities, toxic strains can be causative agents of acute and/or chronic diffuse and focal lesions and of other fish diseases, as demonstrated in fish bioassays. A partially purified, water-soluble Pjiesteria toxin disrupts calcium metabolism in rat pituitary cells and mimics an adenosine triphosphate neurotransmitter that targets P2X7 purinoreceptors found predominantly on immune cells. Respiratory, visual, and neurological impacts have been sustained by people exposed to aerosols from fish-killing Pfiesteria cultures or to water and aerosols during estuarine fish kills associated with toxic Pfiesteria. Neurocognitive impacts from exposure to toxic Pfiesteria have been replicated experimentally in small mammals. Toxic strains of Pfiesteria species have been confirmed from mid-Atlantic and Gulf Coast estuaries in the United States and from northern Europe and New Zealand, indicating that these toxic dinoflagellates are cosmopolitan in distribution.

A second species of ichthyotoxic Pfiesteria (Dinamoebales, Dinophyceae)

A second toxic species within the family Pfiesteriaceae, Pfiesteria shumwayae Glasgow & Burkholder sp. nov., is described from the New River Estuary and the Neuse Estuary of the Albemarle-Pamlico Estuarine Ecosystem, USA. The species is polymorphic and multiphasic, with flagellated, amoeboid and cyst stages. The flagellated zoospores (diameter 8–24 μm) have permanently condensed chromosomes (mesokaryotic nucleus); a chrysophyte-like cyst (diameter 6–25 μm)with organic scales and bracts; and thin thecal plates arranged in a Kofoidian series of Po, cp, X, 4′, la, 6″, 6c, 4s, 5″′, 2″″. The benthic filopodial (filose), lobopodia1 (lobose) and rhizopodial amoeboid stages (5–250 μm) have an outer covering that ranges from rough to smooth in texture, depending on the stage of origin and the prey source. Pfiesteria shumwayae amoebae have a normal eukaryote nucleus and cysts of multiple sizes (diameter 4–25 μm) with a reticulate outer covering. Toxic strains of the two Pfiesteria species have overlapping distributions in the mid-Atlantic and southeastern United States and Scandinavia, with toxic P. shumwayae also having been verified from New Zealand. Pfiesteria shumwayae is similar to P. piscicida in its complex life cycle, general nutrition, attraction to live fish prey, and ichthyotoxic activity that is stimulated by the presence of live fish or their fresh tissues and excreta. However, it can be distinguished from P. piscicida morphologically by having six precingular plates and a four-sided la plate, as well as genetically, on the basis of its ISS ribosomal DNA sequence.