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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
3.1. Morphology of life stages Seven life stage morphologies of Cryptoperidiniopsis sp. gen. nov. were identified
during this study. These included a cyst, a resting stage, zoospores, gametes, planozygotes, lobose amoebae, and a rhizopodal amoeba with slender, retractable,
pointed pseudopodia Fig. 2a–g. These pseudopodia were non-branching, but when joined by other similar amoebae, would form a network around algal prey Fig. 2b. In
P. piscicida we noted motile zoospores, rhizopodal amoebae, lobose amoebae, filose star amoebae, and a cyst stage. These P
. piscicida life stages are similar to those described by Burkholder et al. 1992 and Burkholder and Glasgow 1995, 1997. In the
motile zoospore stage, both P. piscicida and Cryptoperidiniopsis sp. gen. nov. used a peduncle to feed on microalgal prey. The peduncle was extended from behind a hinged
plate on the ventral surface of these cells, and attached to the prey. A swarming action around the attacked cell by several zoospores was common, and resulted in the transfer
of the interior algal cell contents through the peduncle into the food vacuole located in
¨ the zoospore epitheca process called myzocytosis; Schnepf and Elbrachter, 1992. This
feeding process resulted in a swelling of the zoospore and extension of the food vacuole into the hypotheca sometimes occurred following high feeding activity. Examples of this
type of feeding behavior in dinoflagellates have been described earlier by Spero 1982,
¨ Gaines and Elbrachter 1987, Glasgow et al. 1998, Lewitus et al. 1999, and others.
Gyrodinium galatheanum also isolated and cultured from Virginia estuaries, exhibited similar feeding behavior with extended peduncle when fed Cryptomonas
In the original description of the dinoflagellate that came to be formally named P. piscicida Burkholder et al., 1992; Steidinger et al., 1995, it was noted that a cyst stage
of this species possesses scales that resemble those found on chrysophytes. Burkholder 1999 also illustrated several types of Pfiesteria cysts that differ in their origin, size,
and the type of scales present. The cyst we have identified from this Cryptoperidiniopsis sp. gen. nov. has elongated spine-like projections resting upon a surface scale,
somewhat similar to one of the P
. pfiesteria cysts described by Burkholder 1999. This dinoflagellate has two types of scales covering the cyst. There is an inner layer of
surface scales Fig. 2f external to the cell membrane that are flat, and slightly panduriform 1.8 3 3.4 mm. They have dissimilar surfaces, containing small circular
nodules, with a ridge on the outer surface and a corresponding depression on their undersurface. The second type of scale is external to this first layer, with three to four of
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61 Fig. 2. Cryptoperidiniopsis sp. life stages and scales: a lobose amoeba and a Cryptomonas cell; b rhizopodal amoebae with Cryptomonas
cells; c cyst; d amoebae and cyst; e spine-like scale from cyst; f surface scales from cyst; g motile zoospore.
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these loosely attached to the surface of the other scales. They consist of a flattened bottom portion that rests on these inner scales, with each possessing an elongated and
bluntly pointed spine Fig. 2e. The bottom portion of these scales is cordiform in shape 0.9–1.9 3 0.6–2.0 mm. The shaft of the spine is hollow length of 6.6–10.7 mm, mean
diameter 0.25 mm; n 5 15 spines measured. SEM X-ray analysis revealed that silicon is a component of both the cyst Fig. 3a and scale types. The examination of cysts derived
from the TOX-A P. piscicida using SEM X-ray analysis indicated these cysts also contained silicon Fig. 3b.
3.2. Fish bioassays In the first set of fish bioassays I, a strain of TOX-B Pfiesteria piscicida potentially
toxic, but previously fed algal prey, [271-A was added to three replicate test fish
21
bioassay systems initial Pfiesteria density ca. 50–60 zoospores ml . There was no
immediate or recognizable reaction from the fish for the first 15 days of exposure to P .
piscicida. The first fish death occurred after 16 days. The dead fish were replaced with three live tilapia which died within 24 h. Thereafter, fish deaths occurred on an
intermittent basis in all three replicate bioassays. There were no fish deaths in the controls.
In the second set of fish bioassays II, a strain of TOX-A P . piscicida previously
given live fish, rather than algal prey, [2200 was added to the initial three test fish
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bioassay systems initial Pfiesteria density ca. 50–60 zoospores ml . After 72 h of
exposure to P. piscicida, six of 10 fish had died in replicate bioassay system [2, and
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zoospores had increased ca. 10-fold to ca. 600 cells ml Fig. 4. The six dead fish and
four remaining live fish were removed and replaced with 10 live fish. Within 24 h, all 10
21
fish had died, and the zoospore density had increased to ca. 1180 cells ml . Further
daily replacement of dead with live fish over the next 4 days resulted in death of all fish,
21
with zoospore concentrations reaching ca. 5000 cells ml . Fish deaths in replicate
bioassay system [1 did not occur until day 5 of exposure to P. piscicida zoospores at
21 21
ca. 460 cells ml , with all fish dead by day 9 zoospores at 10 000 cells ml
. In contrast to these results in the first two replicates, fish did not die in replicate
21
bioassay [3 even when Pfiesteria concentrations were . 30 000 cells ml day 9. At
day 14, the still-live fish in replicate [3 were transferred to replicate [2, and all fish were dead within 24 h. When 10 replacement fish were added to replicate [3, no fish
deaths occurred over an additional 14-day period. In replicates [1 and [2, toxicity was maintained by replacing dead with live fish, with the shortest time to fish death occurring
within 1.5 h of exposure to TOX-A P. piscicida. Throughout the experimental period, none of the 30 fish in the three replicate control bioassay systems without exposure to P.
piscicida died, and all appeared healthy. These toxic P
. piscicida have since been continuously maintained in our laboratory by providing them with live fish.
On the fifth day of the above bioassays, another series of inoculations bioassay III from replicate [2 with P
. piscicida were transferred to three additional replicates, [4,
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[ 5, and [6 initial P
. piscicida density at 50–75 zoospores ml . Fish deaths began to
occur on the fourth day of exposure to P. piscicida in replicate [4, on the seventh day in replicate [5, and on the fifth day in replicate [6 Fig. 5. Cell concentrations for P
.
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
63
Fig. 3. Results of elemental X-ray analysis. Note presence of silicon: a Cryptoperidiniopsis sp. cyst; b Pfiesteria piscicida cyst.
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Fig. 4. Results of fish bioassay II. Bars indicate percent of fish 10 deaths over time in reference to
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concentrations of Pfiesteria piscicida zoospores ml in culture vessels [1–3.
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piscicida during the initial fish deaths were ca. 500–1200 zoospores ml . By the eighth
day, death of all 10 fish had occurred in replicates [4 and [6, with P . piscicida at
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8000–11 000 zoospores ml . Replicate [5 required a longer time interval for total fish
death by the 12th day. Toxicity was maintained in replicate test bioassays with P .
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
65
Fig. 5. Results of fish bioassay III. Bars indicate percent of fish 10 deaths over time in reference to
21
concentrations of Pfiesteria piscicida zoospores ml in culture vessels [4–6.
piscicida by replacing dead with live fish throughout an 18-day period. Over the test period, one of the 30 fish in the three replicate controls died, and examination revealed
no dinoflagellates present in the water of that control vessel. On the 18th day, fish bioassays [7, [8, [9 10 fish per replicate were inoculated
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from replicate [6 initial P. piscicida density ca. 50–60 zoospores ml . Control
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bioassay systems of fish without exposure to P. piscicida were maintained in triplicate as well, with no deaths occurring in the controls over the 15-day experimental period. In
contrast, fish deaths occurred in the replicate bioassay systems containing P . piscicida at
11 days [9, 12 days [8, and 15 days [7 Fig. 6, corresponding to zoospore
Fig. 6. Results of fish bioassay IV. Bars indicate percent of fish 10 deaths over time in reference to
21
concentrations of Pfiesteria piscicida zoospores ml in culture vessels [7–9.
H .G. Marshall et al. J. Exp. Mar. Biol. Ecol. 255 2000 51 –74
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concentrations of 1000–16 000 cells ml . The results of the entire series II, III, IV are
illustrated in Fig. 7. In each of the test replicates with P. piscicida within all three of the experimental
series, the fish exhibited noticeable stress prior to death. They moved with irregular, spastic motion, and had little forward advancement in their attempts to swim to the
surface, after which they slowly settled to the bottom of the bioassay systems. This pattern was repeated until the fish died. All oxygen measurements taken during fish
deaths indicated there were comparable oxygen levels in both the bioassay and the
21
controls . 4 mg dissolved oxygen l , above levels generally considered to stress fish
Meyer and Barclay, 1990. Ammonia levels also were similar within the test bioassay
21
systems and the controls. Ammonia was generally , 0.25 mg l . In one control
21
replicate, ammonia was 8 mg l on one date, but no fish died in this replicate; and all
replicate test bioassay systems with P . piscicida had ammonia concentrations , 0.05 mg
21
l .
Fig. 7. Comparison of serial transfer inoculations for fish bioassays II–IV, indicating fish deaths in the bioassay and culture vessels, each containing 10 fish.
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All fish bioassays conducted with the Cryptoperidiniopsoid sp. gen. nov. were negative. This set of bioassays was conducted over a 14-week period, with cell
21
abundance reaching ca. 600–750 zoospores ml . None of the fish died in either the
bioassay, or the control vessels, and all appeared healthy. Similar results were found for the fish bioassays using G
. galatheanum. Over a 10-week period of exposure with cell
21
concentrations reaching ca. 700–800 zoospores ml , no fish deaths occurred and all
fish appeared healthy. 3.3. Fish autopsies and bacteria analysis
In nine of the 10 moribund fish taken from the fish bioassays II–IV, the blood contained no bacteria-forming colonies on TCBS medium. Autopsy of the 10th
previously moribund fish revealed the presence of oxidase-positive, non-fermentative, gram-negative rods in the blood. In contrast, the 10 fish that were randomly sampled for
autopsy after death all contained bacteria in their blood that grew on TCBS medium, including several Vibrio and Aeromonas spp.
In experimental series II of the fish bioassays, the mean bacterial concentrations in the water column of both the test replicates fish bioassays [1, [2, [3, with P
. piscicida and the controls increased over the first 5 days before slightly decreasing by the seventh
day Fig. 8. Mean bacterial abundance in test fish bioassays were significantly different from that of controls on only one of four dates analyzed 7 Feb., Student’s t-test,
P 5 0.023. In analyses from experimental series II [4, [5, [6 with P. piscicida, there were no significant differences in mean bacterial abundance from test fish bioassay
versus control water. Of eight dates for series III [7, [8, [9 with P
. piscicida, the bacterial abundance was similar in the test bioassays and the controls, except for one
date 24 Feb., P 5 0.011. The bacterial cell concentrations in this last group were the lowest of the three sets, but these results were also the most comparable and extended
over a 16-day period. Overall, there were no significant differences in the bacterial abundance between the fish bioassays and the control vessels for 14 of the 16
comparison dates.
4. Discussion
