Holly A. Bowers

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.

Seven Gene Phylogeny of Heterokonts

Nucleotide ssu and lsu rDNA sequences of all major lineages of autotrophic (Ochrophyta) and heterotrophic (Bigyra and Pseudofungi) heterokonts were combined with amino acid sequences from four protein-coding genes (actin, beta-tubulin, cox1 and hsp90) in a multigene approach for resolving the relationship between heterokont lineages. Applying these multigene data in Bayesian and maximum likelihood analyses improved the heterokont tree compared to previous rDNA analyses by placing all plastid-lacking heterotrophic heterokonts sister to Ochrophyta with robust support, and divided the heterotrophic heterokonts into the previously recognized phyla, Bigyra and Pseudofungi. Our trees identified the heterotrophic heterokonts Bicosoecida, Blastocystis and Labyrinthulida (Bigyra) as the earliest diverging lineages. A separate analysis of the phototrophic lineages, by adding the rbcL gene, further resolved the Ochrophyta lineages by increased support for several important nodes. Except for the positioning of Chrysophyceae, Eustigmatophyceae, Raphidophyceae and Pinguiophyceae, all main branches of Ochrophyta were resolved. Our results support the transfer of classes Dictyochophyceae and Pelagophyceae from subphylum Phaeista to Khakista. Based on all our trees, in combination with current knowledge about ultrastructure of heterokonts we suggest that a more advanced flagellar apparatus originated at one occasion in the ancestor of Phaeista whereas, Khakista independently reduced their flagellar apparatus and gained chlorophyll c(3).

Discovery of the toxic dinoflagellate Pfiesteria in northern European waters

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

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay.

Nuclear and chloroplast‐encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5′–3′ exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.

Identical ribosomal DNA sequence data from Pfiesteria piscicida (Dinophyceae) isolates with different toxicity phenotypes

Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.

Occupational exposure to pfiesteria species in estuarine waters is not a risk factor for illness.

Background Exposure 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. Methods We 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. Results No correlation was found between work in an area where Pfiesteria was identified and specific symptomatology or changes on neuropsychological tests. Conclusions Although 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.

Real-Time PCR Monitoring of Estuarine Water Samples for Pfiesteria piscicida: A Dinoflagellate Associated with Fish Kills and Human Illness

Pfiesteria piscicida, a heterotrophic estuarine dinoflagellate, has been associated with fish kills in North Carolina and Maryland [2, 3]. The rivers most affected in Maryland are tidal tributaries of the Chesapeake Bay, where many residents make their living on the water, either as commercial fishermen or in the recreation industry. During the summer of 1997, Maryland experienced a series of fish kills, mostly affecting Atlantic menhaden (Brevoortia tyrannus), with dramatic impact on the seafood and tourism industries of the State [5]. In addition, numerous persons that came into contact with water containing Pfiesteria piscicida developed skin rashes, flu-like symptoms, and cognitive problems [4, 7]. Due to the potential human health risks, extensive loss of fish, and economic impacts related to fish kills, the Maryland Department of Natural Resources (DNR) developed a comprehensive water quality monitoring program, initiated in 1998 and fully deployed in 1999 and 2000, to determine ecological parameters associated with Pfiesteria piscicida blooms. As part of this effort, water samples are collected monthly from stations throughout tributaries of the Chesapeake Bay and analyzed by our laboratory for the presence of the organism.