Parke A. Rublee

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.

Insidious effects of a toxic estuarine dinoflagellate on fish survival and human health

The estuarine dinoflagellate Pfiesteria piscicida gen. et sp. nov. produces exotoxin(s) that can be absorbed from water or fine aerosols. Culture filtrate (0.22 microns porosity filters, > 250 toxic flagellated cells/ml) induces formation of open ulcerative sores, hemorrhaging, and death of finfish and shellfish. Human exposure to aerosols from ichthyotoxic cultures (> or = 2000 cells/ml) has been associated with narcosis, respiratory distress with asthma-like symptoms, severe stomach cramping, nausea, vomiting, and eye irritation with reddening and blurred vision (hours to days); autonomic nervous system dysfunction [localized sweating, erratic heart beat (weeks)]; central nervous system dysfunction [sudden rages and personality change (hours to days), and reversible cognitive impairment and short-term memory loss (weeks)]; and chronic effects including asthma-like symptoms, exercise fatigue, and sensory symptoms (tingling or numbness in lips, hands, and feet; months to years). Elevated hepatic enzyme levels and high phosphorus excretion in one human exposure suggested hepatic and renal dysfunction (weeks); easy infection and low counts of several T-cell types may indicate immune system suppression (months to years). Pfiesteria piscicida is euryhaline and eurythermal, and in bioassays a nontoxic flagellated stage has increased under P enrichment (> or = 100 micrograms SRP/L), suggesting a stimulatory role of nutrients. Pfiesteria-like dinoflagellates have been tracked to fish kill sites in eutrophic estuaries from Delaware Bay through the Gulf Coast. Our data point to a critical need to characterize their chronic effects on human health as well as fish recruitment, disease resistance, and survival.

Seasonal distribution of bacteria in salt marsh sediments in North Carolina

The number and size of bacteria at four depths (0-1, 5-6, 10-11, and 20-21 cm) in a North Carolina salt marsh were minotored by direct counts for 13 months. The number of bacteria reached a maximum of about 1•4 × 1010 cells cm-3 at the sediment surface in October, corresponding to the period of Spartina alterniflora die-back. Cell numbers were lowest and most consistent throughout the year at the 20 cm depth of sediment. Cell volumes averaged 0•2 µm3 at the marsh surface and decreased with depth. Mean standing crop of bacteria to a depth of 20 cm of sediment was about 14 g bacterial carbon m-2. In surface sediments bacteria contribute up to 15% and algae up to 10% of total living microbial biomass as estimated by adenosine triphosphate (ATP). Bacteria were the major biomass component at sediment depths of 5, 10 and 20 cm. At all depths the microbial community contributes < 4% total organic carbon and < 8 % of total nitrogen.

A method to determine in situ zooplankton grazing rates on natural particle assemblages

In situ zooplankton grazing rates on natural particle assemblages were stimated by measuring zooplankton uptake of labelled autotrophic (with Na14CO3) and heterotrophic (with [methyl-3H]-thymidine) particulate matter in 1-h incubations in clear, Plexiglas, Haney chambers. The in situ grazing rates are in the same range as those measured for zooplankton in the laboratory using standard particle counting techniques. A negative selection coefficient for 3H-labelled particles indicated a lower filtration efficiency or avoidance of these particles by zooplankton.

Potential invasion of microorganisms and pathogens via ‘interior hull fouling’: biofilms inside ballast water tanks

Surfaces submerged in an aquatic milieu are covered to some degree with biofilms – organic matrices that can contain bacteria, microalgae, and protozoans, sometimes including disease-causing forms. One unquantified risk of aquatic biological invasions is the potential for biofilms within ships’ ballast water tanks to harbor pathogens, and, in turn, seed other waters. To begin to evaluate this vector, we collected biofilm samples from tanks’ surfaces and deployed controlled-surface sampling units within tanks. We then measured a variety of microbial metrics within the biofilms to test the hypotheses that pathogens are present in biofilms and that biofilms have higher microbial densities compared to ballast water. Field experiments and sampling of coastwise and oceangoing ships arriving at ports in Chesapeake Bay and the North American Great Lakes showed the presence of abundant microorganisms, including pathogens, in biofilms. These results suggest that ballast-tank biofilms represent an additional risk of microbial invasion, provided they release cells into the water or they are sloughed off during normal ballasting operations.

Direct Counts of Bacteria in the Sediments of a North Carolina Salt Marsh

The number of bacteria in sediments of a North Carolina salt marsh was determined by direct counts with epifluorescent illumination and acridine orange stain. Cell number decreased from 8.36–10.90×109 cells/cm3 of sediment at the surface to 2.19–2.58×109 cells/cm3 of sediment at a depth of 20 cm. No significant difference was found among four stations located on a transect which crossed the marsh and spanned subtidal to intertidal sediments.

The Limnology of Toolik Lake

The scientific study of the Arctic is recent with the exception of various collections and cataloging of plants and animals in the 19th century. The limnological investigation of the Arctic is even more recent with the first review paper of arctic limnology listing only seven papers that dealt with arctic lakes (Rawson, 1953). However, after World War II, research stations developed in arctic Europe, Greenland, and at Point Barrow in Alaska. There was considerable research activity at the Naval Arctic Research Laboratory (NARL) in Barrow (Livingstone et al., 1958; Hobbie, 1964, Chapter 2; Stross and Kangas, 1969), much of which is reviewed by Hobbie (1973).

Decomposition of turtlegrass (Thalassia testudinum Konig) in flowing sea-water tanks and litterbags: Compositional changes and comparison with natural particulate matter

Thalassia testudinum Konig litter was incubated up to 3 yr in flowing sea-water tanks and litterbags in the field. Weight loss of litter was evident within 1 wk and 50% loss of ash free dry weight took < 35 days. Carbon and nitrogen were lost from litter, but the C: N ratio (by weight) changed only slightly over the first 180 days of incubation. Protein, carbohydrate, and lipid content of litter decreased with time. Number of bacteria on litter increased from ˜ 9 to 37-60 × 109 cells • g dry wt-1. Lipid synthesis activity suggested a succession in the litter microbial community. Thalassia detritus from long-term tank incubations (2 to 3 yr) exhibited similarities in biochemical composition with particulate matter in natural water.

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.

Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: a review of detection methods and geographic distribution.

Abstract. Molecular methods, including conventional PCR, real‐time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets.