Howard B. Glasgow

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

ABSTRACT 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 Pfiesteriaspecies, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugols 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.

PFIESTERIA PISCICIDA GEN. ET SP. NOV. (PFIESTERIACEAE FAM. NOV.), A NEW TOXIC DINOFLAGELLATE WITH A COMPLEX LIFE CYCLE AND BEHAVIOR

The newly described toxic dinoflagellate Pfiesteria piscicida is a polymorphic and multiphasic species with flagellated, amoeboid, and cyst stages. The species is structurally a heterotroph; however, the flagellated stages can have cleptochloroplasts in large food vacuoles and can temporarily function as mixotrophs. The flagellated stage has a typical mesokaryotic nucleus, and the theca is composed of four membranes, two of which are vesicular and contain thin plates arranged in a Kofoidian series of Po, cp, X, 4′, 1a, 5″, 6c, 4s, 5″′, and 2″″. The plate tabulation is unlike that of any other armored dinoflagellate. Nodules often demark the suture lines underneath the outer membrane, but fixation protocols can influence the detection of plates. Amoeboid benthic stages can be filose to lobose, are thecate, and have a reticulate or spiculate appearance. Amoeboid stages have a eukaryotic nuclear profile and are phagocytic. Cyst stages include a small spherical stage with a honeycomb, reticulate surface and possibly another stage that is elongate and oval to spherical with chrysophyte‐like scales that can have long bracts. The species is placed in a new family, Pfiesteriaceae, and the order Dinamoebales is emended.

KLEPTOPLASTIDY IN THE TOXIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)

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

WATER QUALITY TRENDS AND MANAGEMENT IMPLICATIONS FROM A FIVE‐YEAR STUDY OF A EUTROPHIC ESTUARY

The Neuse River and Estuary, a major tributary of the second largest estuary on the United States mainland, historically has sustained excessive blooms of algae and toxic dinoflagellates, hypoxia, and fish kills. Previous attempts have been made to use short-term databases of 2–3 years, or data sets from infrequent (monthly) sampling, to assess whether nutrient inputs to the Neuse are increasing and supporting higher algal production. These previous efforts also have relied on single-point-determined flow velocity data, at upstream sites remote from the estuary, to estimate the volume of flow in quantifying nutrient loading to the estuary. We completed a five-year study of the Neuse, including a comparative inventory of nutrients to the watershed from point sources and from concentrated animal operations (CAOs) as recent nonpoint sources, as well as an intensive assessment of water quality over time in the mesohaline estuary. Estimates of nutrient loads were based on volume of flow data from shore-to-shor...

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

Abstract 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.

Trophic controls on stage transformations of a toxic ambush-predator dinoflagellate.

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.

MIXOTROPHY AND NITROGEN UPTAKE BY PFIESTERIA PISCICIDA (DINOPHYCEAE)

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

Discovery of the “Phantom” dinoflagellate in Chesapeake Bay

Since its discovery in natural estuarine habitat of North Carolina in 1991, the widespread impact of the toxic dinoflagellate, Pfiesteria piscicida (gen. et sp. nov.), popularly called the “phantom” dinoflagellate, on North Carolina fish stocks has been established, yet little is known about its influence outside of North Carolina estuaries. Here, we document the presence of P. piscicida in Chesapeake Bay. A fish kill was observed after inoculating an aquarium containing mummichogs with sediment samples from Jenkins Creek, a brackish creek (salinity 11‰) of the Chesapeake Bay system. P. piscicida was the cause of the kill, as supported by morphological, physiological, and histological evidence. The appearance and behavior of the algae and symptoms associated with fish mortality were consistent with those previously observed in P. piscicida-associated aquaria fish kills in North Carolina. The discovery of P. piscicida in Chesapeake Bay supports the speculation that these toxic dinoflagellates have a dramatic and far-reaching impact on fish stocks in shallow, eutrophic estuaries along the eastern United States.

A second species of ichthyotoxic Pfiesteria (Dinamoebales, Dinophyceae)

Abstract 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.

History of Toxic Pfiesteria in North Carolina Estuaries from 1991 to the Present

T fish kills for which Pfiesteria became well known began in North Carolina, when very little research had been conducted on this unusual dinoflagellate. North Carolina’s Albemarle-Pamlico Estuarine System, the epicenter of toxic Pfiesteria outbreaks, is the second largest estuary in area on the US mainland and the most important fish nursery ground on the US Atlantic Coast (Burkholder and Glasgow 1997, Mallin et al. 2000). As a conservative estimate, the state had sustained 48 toxic Pfiesteria outbreaks by 1997, involving more than a billion fish in an area more than 100 km2 (Burkholder et al. 2001a). These events had occurred nearly every summer beginning in 1991, when the organism was first recognized as an estuarine, fish-killing agent (Burkholder et al. 1992, 2001a, Burkholder and Glasgow 1997). In 1997, 50,000 fish died in a small area of the Chesapeake Bay from toxic Pfiesteria , and press coverage of the event was like an explosion. In the same summer, 1.2 million fish died during toxic Pfiesteria outbreaks in Albemarle-Pamlico estuaries, a 4-hour drive south of Washington, DC; those deaths went virtually unmentioned. North Carolina was the first state to encounter toxic Pfiesteria, and the knowledge gained there, especially about toxic Pfiesteria outbreaks and health impacts on laboratory workers (Burkholder et al. 1992, 1995, Burkholder and Glasgow 1995, 1997, Glasgow et al. 1995), benefited Maryland officials, who were challenged to act quickly and decisively. They evaluated and rapidly verified the role of toxic Pfiesteria in the Chesapeake Bay outbreak (MDNR 1998). Maryland was the first state from which people who reported neurocognitive, respiratory, and other symptoms from environmental exposure were clinically evaluated within a short period (1–3 weeks) after being exposed (Grattan et al. 1998). Maryland was also the first state to address the Pfiesteria problem by making significant advances in legislation for protection of water quality (State of Maryland 1998). Congressional attention following the toxic Pfiesteria outbreaks in Maryland led to the appropriation of many millions of dollars to federal agencies to research and manage toxic Pfiesteria outbreaks, resulting in excellent progress in some areas and setbacks in others. Here we examine the history of toxic Pfiesteria outbreaks before the four in Chesapeake Bay and a few aspects of the aftermath, from the perspective and experience of our research in the Albemarle-Pamlico Estuarine System with high toxic Pfiesteria activity. An important part of the Pfiesteria story is how North Carolina—with 98% of the Pfiesteria problem—subsequently moved to strengthen water resource protection, environmental education, and support of Pfiesteria research, actions that would not have been possible without the events that unfolded in Chesapeake Bay.