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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
2.1. Cultures Cultures of two pfiesteria-like species were developed from water Gyrodinium
galatheanum and sediment Cryptoperidiniopsis sp. samples taken from Virginia estuaries between 1997 and 1999 Seaborn et al., 1999. Unialgal cultures of these
species were established after numerous individual cell isolations and subsequent dilutions. Scanning electron microscope examination of the cryptoperidiniopsoid species
indicated that it was different from C
. brodyi gen. et sp. nov.; Steidinger, pers. commun.. This species [DEQ002 and G. galatheanum were monitored daily and
routinely checked for contaminants and other dinoflagellates through light microscopy. Subsequent DNA sequencing analysis by Dr. D. Oldach U.MD indicated the
cryptoperidiniopsoid to be a Cryptoperidiniopsis sp. gen. nov. that was separate from C
. brodyi gen. et sp. nov, but closely related to P. piscicida. The identity of G. galatheanum was confirmed by K. Steidinger pers. commun. with SEM and by D.
Oldach through a Heteroduplex mobility assay Oldach et al., 2000. These cultures were maintained in falcon flasks in f 2-Si medium at 15 psu, under ambient light and
room temperature, and were given Cryptomonas sp. CCMP [767, Bigelow Labora- tory as a food source. Prior to using the Cryptomonas culture, it was routinely
examined for contaminants, with cells routinely isolated to establish a series of sub-cultures.
A toxic Pfiesteria piscicida culture [271A-1 was provided by Burkholder and Glasgow NCSU. This culture had been isolated from the Neuse Estuary, North
Carolina, cloned in uni-dinoflagellate culture with algal prey cryptomonads as a food source at NCSU. Pfiesteria spp. have not been cultured successfully without a prey
source e.g., Burkholder and Glasgow, 1995; Steidinger et al., 1996, and it has not been possible to induce toxin production unless live fish are added Burkholder and Glasgow,
1997. Thus, a clonal culture contains an isolate of a uni-dinoflagellate P
. piscicida or P
. shumayae sp. nov., with its associated endosymbiont bacterial consortium Steiding- er et al., 1995 and its prey Burkholder, in press. The culture was allowed to graze
algal prey to residual levels , 10 cells ml, and then added to fish bioassays see
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
55
Section 2.3, below to test for ichthyotoxic activity five fish per replicate culture vessel, n 5 3; Burkholder et al., 1995a,b; Burkholder and Glasgow, 1997; Glasgow, 2000;
Burkholder, in press. The control fish remained healthy while the Pfiesteria-exposed fish all died, and accompanying tests indicated no difference between control and test
culture vessels in presence abundance of bacteria or other microflora fauna that could act as fish pathogens. Culture [271A-1 was thus evaluated as a toxic strain, was
identified to species by H. Glasgow and J. Burkholder SEM of suture-swollen zoospores; Burkholder and Glasgow, 1995; Glasgow, 2000, and made available for our
studies.
The culture [271A-1 was shipped to our laboratory as TOX-A or actively toxic P. piscicida, taken from the fish-killing cultures. However, since toxic strains of Pfiesteria
spp. are known to rapidly cease toxin production in the absence of live fish within hours; Burkholder and Glasgow, 1997, this strain was received in TOX-B temporarily
nontoxic status. We initially maintained the culture for 4 weeks with Cryptomonas as the food source. We also identified this isolate using SEM analysis Leo model 435VP
SEM and, as had been done in the Burkholder and Glasgow laboratory, we again cross-confirmed the species identification as P. piscicida by two independent laboratories
Dr. P. Rublee of UNC-Greensboro, and Dr. D. Oldach of U. MD using gene sequencing techniques Rublee et al., 1999; Oldach et al., 2000. This P
. piscicida culture was used in comparative growth studies Seaborn et al., 1999 and in our initial
fish bioassay studies Fish bioassay I with this species. A second TOX-A P. piscicida isolate [2200, Neuse Estuary that had been similarly prepared was supplied by the
Burkholder and Glasgow laboratory, and was used for additional fish bioassays and morphological comparisons with the cryptoperidiniopsoid Cryptoperidiniopsis sp. gen.
nov.. Prior to the bioassays, the identity of this species was also cross-confirmed by the two laboratories mentioned above. This isolate was maintained with fish in culture
facilities in a TOX-A mode see Section 2.3 fish bioassays, below, without the addition of algal prey.
2.2. Morphology of life stages A Leo Model 435VP scanning electron microscope SEM at Old Dominion
University was used for the plate tabulation of these dinoflagellates, following the suture-swollen cell approach of Glasgow 2000. In addition, an environmental scanning
electron microscope Phillips XL30 ESEM-FEG, at the University of Miami, Rosenstiel School of Marine and Atmospheric Science, Miami, FL, was also used to characterize
several life stages of Pfiesteria piscicida and the cryptoperidiniopsoid, and to conduct elemental X-ray analysis on the cyst stages of these two species. Previous reports by
Burkholder et al. 1992, Burkholder and Glasgow 1995, 1997, and Steidinger et al. 1996 have indicated P. piscicida is known to have chrysophyte-like scales. Our intent
was to compare scales we observed on the cysts of these species and the likelihood of using morphological differences among these scales as a basis for species identification.
In addition, we wanted to determine if these scales were siliceous and or organic, as found in chrysophytes.
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.G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
2.3. Fish bioassays The Pfiesteria piscicida cultures used in these bioassays were previously identified as
the toxin producing source of fish deaths in the Burkholder and Glasgow laboratory. Their bioassay procedure to identify toxin producing P
. piscicida has been briefly summarized in various publications and used in their bioassay studies e.g., Burkholder
et al., 1995a,b, 1999; Burkholder and Glasgow, 1997; Glasgow, 2000, with a more detailed description provided by Burkholder in press. This approach evaluates the
presence of toxic agents in relation to fish deaths that may occur across a range of environmental conditions low to high nutrients, salinities, organics, toxic substances of
other types, bacterial concentrations, protozoan densities, etc.. The Pfiesteria cultures that were provided for our studies by the Burkholder and Glasgow laboratory were
identified as toxic strains based on bioassay results coming from the procedure outlined in Fig. 1. This cause effect relationship is based on the presence of toxic rather than
infectious agents to determine ichthyotoxic activity of P
. piscicida. The bioassay steps include: a confirmation of the presence of Pfiesteria piscicida at potentially lethal
densities at an in-progress fish kill event, identified by SEM examination and gene sequencing analysis; b isolation of P
. piscicida from a water sample taken from the in-progress kill, in positive fish bioassays, and development of clonal cultures of these
cells uni-dinoflagellate and axenic except for endosymbiont bacteria; containing residual
21
axenic algal or other benign prey, such as ca. five to 10 cryptomonads ml ; c
addition of the clonal Pfiesteria isolate to healthy fish cultures, resulting in fish deaths, while fish in control cultures remained healthy; and d re-isolation and re-cloning of the
organism from the second set of positive fish bioassays where deaths occurred, with subsequent species identification verified by SEM and gene sequencing, plus cross-
confirmation of toxicity by an independent laboratory experienced in culturing toxic Pfiesteria.
Fish bioassays were used to test for ichthyotoxic activity of P. piscicida versus the cryptoperidiniopsoid dinoflagellate and G. galatheanum. The toxins of Pfiesteria spp. are
incompletely characterized Fairey et al., 1999. Thus, at present, properly conducted fish bioassays are the ‘gold standard’ — i.e., the only reliable technique available — for
assessing toxin-producing capability of the known toxic Pfiesteria spp. and potentially toxic pfiesteria-like dinoflagellates Burkholder and Glasgow, 1997; Burkholder et al.,
1999, Glasgow, 2000; Burkholder, in press. The technique requires maintaining healthy fish in aerated culture vessels that are amenable to cell production and toxic activity of
TPC species, then adding the cloned dinoflagellate population to a subgroup of replicate fish cultures, while maintaining another set as replicate controls. Actively toxic,
fish-killing strains of TPC species have been associated with serious human health impacts in laboratory and field exposures, apparently through production of aerosolized
neurotoxins Glasgow et al., 1995; Grattan et al., 1998; Duke University Medical Center records, Durham, NC. Therefore, we constructed a custom-designed biohazard III
containment system at ODU modified from the biohazard III containment system in the Burkholder and Glasgow laboratory at NCSU, and fish bioassays to detect and culture
toxic Pfiesteria were conducted within that system.
Hybrid tilapia Oreochromis sp. Aqua Safra, Bradenton, FL total length, 3–6 cm
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
57
Fig. 1. Standardized steps of the Burkholder Glasgow fish bioassay procedure used to evaluate the role of Pfiesteria in fish kills, and grow toxic Pfiesteria Burkholder et al., 1992, 1995a,b, 1999; Burkholder and
Glasgow, 1995, 1997; Glasgow, 2000; modified from Burkholder, in press. These represent a modified approach of Koch’s postulates steps III, IV regarding toxic rather than infectious organisms. The toxin
producing Pfiesteria piscicida zoospores TOX-A functional type were identified and isolated through this procedure by the Burkholder and Glasgow laboratory, with cultures of these zoospores provided for the
bioassays used in this study.
were used as the standard test fish species. All fish were initially maintained in a holding facility at another ODU laboratory, separate from the biohazard III laboratory containing
toxic and potentially toxic Pfiesteria. Environmental conditions were similar to those described in Burkholder and Glasgow 1997 and Burkholder et al. 1995a,b. The fish
bioassays were conducted at ca. 248C, under ambient light conditions, in 15 psu water made with Instant Ocean salts. Each of the fish cultures was covered and aerated. When
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.G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
Fig. 1. continued
fish died, they were replaced with live fish. No sediment was introduced into the culture vessels. Triplicate 1-ml water samples were taken from each series I–IV of bioassay
and control vessels at 2–3-day intervals, preserved in acidic Lugols’s solution and examined in a Palmer–Maloney counting cell at 3 400 magnification for Pfiesteria
zoospore density Marshall et al., 1999. Samples were also checked for discernable differences in other microflora fauna in the control versus test fish culture vessels.
Protozoan ciliates were rarely detected throughout the bioassays. Oxygen and ammonia were measured at 2-day intervals. Oxygen was determined using an oxygen electrode
YSI model 5300 Biological Oxygen Monitor, Yellow Springs Instr., Yellow Springs,
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
59
OH. Ammonia was measured colorimetrically using an ammonia test kit Aquarium Pharmaceuticals, Chalfont, PA.
In our initial bioassay study I, the TOX-B functional type of P. piscicida isolate [
271-A was inoculated into three bioassay holding systems initial concentration ca.
21
50–60 zoospores ml , each containing three tilapia. Three similar control fish systems
with three tilapia each were also maintained. In a second set of fish bioassays II, we used TOX-A P
. piscicida isolate [2200 and inoculated three replicate bioassay systems [1, [2, [3 containing 10 tilapia each
2l
initial concentration ca. 50–60 zoospores ml . Three similar systems each with 10 tilapia were also maintained as controls without P. piscicida
. These bioassays were otherwise conducted similarly as the first set. When fish died, they were replaced to
re-establish the original total of 10 fish. Actively toxic status of the P . piscicida isolate
was maintained by continual replacement of the dead fish with live fish. When fish deaths occurred during the fourth day in replicate [2 of this series II, a third
experimental series III of three bioassays [4, [5, [6, plus three additional controls, was established, each containing10 tilapia. An inoculant from the surface waters of
replicate [2 was introduced into [4, [5, and [6 initial concentration ca. 50–75
2l
zoospores ml . A similar pattern of replacing dead fish with live fish, and recording Pfiesteria and bacterial concentrations was followed and the practice continued for
another 10 days. After 10 days, a similar inoculant was transferred from [6 to three additional replicate bioassay systems series IV with test fish [7, [8, [9; initial
2l
concentration ca. 50–60 zoospores ml . Three additional control fish bioassay systems without P
. piscicida were also maintained. The cryptoperidiniopsoid Cryptoperidiniopsis sp. [gen. nov.] [CB002 and G
. galatheanum, were introduced to separate bioassay facilities holding three to six tilapia
each, with control sets with three to six fish also established to determine their toxicity to fish over time. These assays were continued for 10 and 14 weeks, respectively, for G
. galatheanum and the cryptoperidiniopsoid species.
2.4. Fish autopsies Autopsies were performed on fish in the test bioassay systems to assess the presence
and quantity of bacteria within the blood of the fish. These fish appeared stressed after exposure to P
. piscicida by their sluggish and spastic pattern of movement. Fish during the bioassays often rested on or near the bottom of the bioassay vessels prior to death.
From each of the three replicate bioassays in experimental series II–IV, we randomly selected 10 of these moribund fish and 10 dead fish for autopsy. The surface of each fish
was disinfected prior to autopsy with laboratory disinfectant Conflikt, Fisher Scientific. Disinfectant was removed by rinsing with sterile Instant Ocean and wiping with a sterile
swab, and an incision was made below the dorsal fin with a sterile scalpel. Blood was removed from the incision with a sterile swab and applied to bacteriological medium
TCBS, Difco, Detroit, MI and incubated at room temperature for 24 h. The resulting growth was streak plated onto Trypticase Soy agar Difco, Detroit, MI for purification
´ and identification using the API 20E identification system Meriux Vitek, Hazelwood,
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.G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
MD. This system consists of 23 standard biochemical tests for the identification of Enterobacteriaceae and other Gram-negative bacteria. Triplicate 1-ml water samples
were also taken from each series I–IV of bioassay and control vessels at 2–3-day intervals, for determining bacterial abundance in the water by acridine orange direct
count Hobbie et al., 1977.
3. Results
