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.G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
1. Introduction
Estuarine and laboratory studies have identified dinoflagellates of the toxic Pfiesteria complex TPC; thus far including Pfiesteria piscicida, Steidinger et al., 1996; and P.
shumwayae sp. nov., Glasgow, 2000 with production of toxins associated with fish kill events and human illness Burkholder et al., 1992; Glasgow et al., 1995; Burkholder and
Glasgow, 1997; Grattan et al., 1998. The most frequent and extensive occurrences of these fish deaths have been in North Carolina’s Albemarle–Pamlico Estuarine System,
with a lesser degree of incidence and fish mortality in tributaries of the eastern shore of Chesapeake Bay Lewitus et al., 1995; Burkholder and Glasgow, 1997; Burkholder et
al., 1999; Magnien et al., 1999. TPC species have also been found in other estuaries in benign forms, from New York through the Gulf Coast Burkholder and Glasgow, 1997;
Rublee et al., 1999.
These studies indicate that Pfiesteria spp. exist as nontoxic i.e., non-toxin producing heterotrophs feeding on algae and other organisms, and that under certain environmental
conditions, the cells become toxic in the presence of fish. Pfiesteria spp. have been further described by Burkholder and Glasgow 1995, 1997, Burkholder et al. 1999,
and Burkholder in press as existing in several functional types based on their capability of toxin production Woods Hole Oceanographic Institution WHOI, 2000. Active
toxin-producing cells in the presence of live fish are considered the Toxic A functional type TOX-A, which become temporarily nontoxic TOX-B functional type when
removed from access to live fish and fed algae, or other prey. These TOX-B cells have the potential for toxin production and become TOX-A, or actively toxic, when re-
introduced to live fish. In addition, there are strains of Pfiesteria that do not have, or apparently have permanently lost, the toxin production response that is triggered by the
presence of live fish. This third functional type is called non-inducible NON-IND, and on the basis of present understanding, cannot be induced to produce bioactive substances
that cause fish disease and death WHOI, 2000; Burkholder, in press.
Toxic strains of TPC species demonstrate the following traits: they show strong attraction toward live fish or their fresh tissues and secreta excreta attraction measured
using motion analysis techniques as in Kamykowski et al., 1992; Burkholder and Glasgow, 1997; they produce bioactive substances or toxins that cause fish disease and
death Fairey et al., 1999; and they are stimulated to produce these substances in the presence of live fish or their fresh tissues separated from the live animal , 2 h;
Burkholder and Glasgow, 1995, 1997. The two toxic Pfiesteria spp. known thus far Glasgow, 2000 are heterotrophic and lack chloroplasts, but can be mixotrophic by
retaining kleptochloroplasts from algal prey Lewitus et al., 1999. If kleptochloroplasts are present, they are always contained within an epithecal food vacuole Burkholder and
Glasgow, 1995; Lewitus et al., 1999; Glasgow, 2000. Pfiesteria spp. also have a complex life cycle with many stages or forms, which vary in response to the changing
environmental conditions present in estuaries especially prey availability; Burkholder et al., 1992, 1995b; Burkholder and Glasgow, 1995, Glasgow, 2000. These life forms
include multiple amoeboid and cyst stages as well as flagellate stages zoospores, anisogamous gametes, planozygote. Earlier reports of such diverse life stages in
dinoflagellates, with arrays of amoebae, include Pascher 1916, Bursa 1970a,b,
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
53
´ ´
´ Pfiester and Popovsky 1979, Pfiester and Lynch 1980, Popovsky 1982, Popovsky
and Pfiester 1990, and Buckland-Nicks et al. 1990, among others. The ecological and habitat relationships that stimulate Pfiesteria development and
toxin production have been discussed by Burkholder et al. 1992, 1995a, 1999, Burkholder and Glasgow 1997, Magnien et al. 1999, and others. The laboratory and
field studies that have associated fish mortality with P . piscicida have been reported over
a broad salinity range and at various water temperatures. However, the toxic events have most commonly occurred at low to mid-salinity values 5–15 psu and temperatures
above 268C. Most of these events have been in nutrient-enriched waters of shallow, poorly flushed estuaries Burkholder and Glasgow, 1997; Burkholder et al., 1997;
Magnien et al., 1999. These conditions also favor the development of the algal prey that Pfiesteria may utilize as a food resource in the absence of abundant live fish Burkholder
and Glasgow, 1995, 1997.
In Virginia estuaries, intensive investigations over the past 3 years have documented numerous ‘pfiesteria-like organisms’ PLOs; Marshall, 1999; WHOI, 2000. These
organisms superficially resemble Pfiesteria spp. in general appearance under light microscopy LM, but they mostly have been tested as benign or incapable of producing
bioactive substances that cause fish stress, disease or death this study; Burkholder, in press; J. Burkholder and H. Glasgow, North Carolina State University, Raleigh, NC,
unpublished data. PLOs have similar size and morphological features as Pfiesteria spp., and most cannot be distinguished from Pfiesteria spp. using only LM. Therefore, we
have used scanning electron microscopy SEM of both suture-swollen cells Glasgow, 2000, and membrane stripping protocols by K. Steidinger pers. commun. and Truby
1997 to determine the plate tabulation and plate characteristics of these PLOs. The toxin-producing capability is assessed by fish bioassays as in Burkholder et al. 1995b,
1999; Burkholder and Glasgow 1997; and Burkholder in press. Pfiesteria-like organisms in Virginia estuaries have included at least two cryptoperidiniopsoids
Cryptoperidiniopsis sp. [gen. nov.; Dr. K. Steidinger, Florida Department of En- vironmental Protection — Florida Marine Research Institute, St. Petersburg, FL, pers.
commun.] and Cryptoperidiniopsis brodyi [gen. et sp. nov.; Steidinger, pers. commun.], along with certain Gymnodinium spp. and Gyrodinium spp. e.g., Gyrodinium
galatheanum Braarud [ 5 Gymnodinium galatheanum Braarud]. These PLOs are widely distributed in Virginia estuaries and in other areas of the Chesapeake Bay Marshall et
al., 1999; Seaborn et al., 1999. Many of the smaller Virginia estuaries along the Potomac River and several of the smaller inlets along the mid-western shoreline of the
21
Chesapeake Bay also have supported at times high concentrations . 200 cells ml of
these cells Marshall et al., 1999; H. Marshall, unpublished data. Little is known about the comparative ecology of TPC species versus PLOs.
Comparative growth studies with a TOX-B temporarily nontoxic P . piscicida, a
cryptoperidiniopsoid species, and G . galatheanum using Cryptomonas sp. and two
diatoms as a food source, showed that the cryptoperidiniopsoid had the highest growth rate Seaborn et al., 1999. This characteristic may over time give this dinoflagellate a
competitive advantage in abundance over Pfiesteria during long term and or seasonal periods of development. G
. galatheanum is an auxotroph and mixotroph, and photo- synthesizes with its own chloroplasts that are located in the cell cytoplasm Tomas,
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.G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
1996. Thus, it can be discerned from Pfiesteria spp. under light microscopy, especially
4 21
with epifluorescence. In high cell densities . 10 zoospores ml , several isolates of
G . galatheanum have also been reported as toxic to fish Steidinger, 1993; Nielsen,
1993; Burkholder, 1998. However, isolates from North Carolina, Maryland, and Florida waters thus far have shown neither attraction to live fish nor ichthyotoxicity in repeated
bioassays Glasgow, 2000; Burkholder, in press. The objectives of the present study were to compare P. piscicida and a cryptoperidiniopsoid dinoflagellate in terms of their
basic stage morphologies and potential toxicity to fish, toward strengthening insights about distinguishing characteristics of co-occurring Pfiesteria versus pfiesteria-lookalike
species.
2. Materials and methods
