Download effects of light on photosynthesis, grazing, and population dynamics

J. Phycol. 38, 659–669 (2002)
EFFECTS OF LIGHT ON PHOTOSYNTHESIS, GRAZING, AND POPULATION DYNAMICS OF
THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1
Timothy N. Feinstein, Ryan Traslavina
Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340, USA
Ming-Yi Sun
Department of Marine Sciences, University of Georgia, Athens, Georgia 30605, USA
and
Senjie Lin2
Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340, USA
In studying how environmental factors control the
population dynamics of Pfiesteria piscicida Steidinger
et Burkholder, we examined the influence of light regime on kleptoplastidic photosynthesis, growth, and
grazing. Prey (Rhodomonas sp.)-saturated growth rate
of P. piscicida increased (0.67 0.03 d1 to 0.91 0.11 d1) with light intensity varying from 0 to 200 mol
photonsm2s1. No significant effect was observed on
grazing, excluding the possibility that light enhanced P.
piscicida growth through stimulating grazing. Lightgrown P. piscicida exhibited a higher gross growth efficiency (0.78 0.10) than P. piscicida incubated in
the dark (0.32 0.16), and photosynthetic inhibitors
significantly decreased growth of recently fed populations. These results demonstrate a role of kleptoplastidic photosynthesis in enhancing growth in P.
piscicida. However, when the prey alga R. sp. was depleted, light’s stimulating effect on P. piscicida growth
diminished quickly, coinciding with rapid disappearance of Rhodomonas-derived pigments and RUBISCO
from P. piscicida cells. Furthermore, the effect of light
on growth was reversed after extended starvation, and
starved light-grown P. piscicida declined at a rate significantly greater than dark-incubated cultures. The
observed difference in rates of decline appeared to
be attributable to light-dependent cannibalism. Using
a 5-chloromethylfluorescein diacetate staining technique, cannibalistic grazing was observed after 7 days
of starvation, at a rate four times greater under illumination than in the dark. The results from this study
suggest that kleptoplastidy enhances growth of P. piscicida only in the presence of algal prey. When prey
is absent, P. piscicida populations may become vulnerable to light-stimulated cannibalism.
Abbreviations: CHN, Carbon, hydrogen and nitrogen
content; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; DCMU, Dichloro 1,3-dimenthyl urea; FASW,
0.45 m-filtered autoclaved 15%; seawater; GGE, gross
growth efficiency; Pfiest, Pfiesteria piscicida; Rhod,
Rhodomonas sp.
Pfiesteria piscicida Steidinger et Burkholder is an
“ambush” predator dinoflagellate implicated in major
fish kills in North Carolina and Maryland estuaries
(Burkholder et al. 1992, 2001). Pfiesteria piscicida has
been found in coastal regions from Florida to New
York and may exist in coastal regions of Northern Europe and New Zealand (Burkholder et al. 2001). This
dinoflagellate has a complex trophic mode, known to
consume fish tissue and several types of phytoplankton (Glasgow et al. 1998). Although putative toxin
production seems to depend strictly on the presence
of live fish, P. piscicida populations also graze algal
food and respond to enrichments of inorganic nutrients (Burkholder et al. 1998, Glasgow et al. 1998).
Earlier reports demonstrated that P. piscicida may benefit from the selective retention and use of chloroplasts from its cryptophyte prey (“kleptoplastidy”)
(Lewitus et al. 1999a,b). Although kleptoplastidy was
suggested to play a role in surviving a shortage of food
(Lewitus et al. 1999b), the extent to which stolen
chloroplasts support P. piscicida growth remains unclear. One way to address this issue is to examine effects of light. If kleptoplastidy plays a significant role
in supporting the growth of P. piscicida, then the benefit conferred on growth by photosynthesis should depend on light regime in addition to nutrient enrichment.
Possible effects of light on heterotrophic processes
would also need to be investigated. Illumination has
been shown to stimulate grazing and growth in some
mixotrophic dinoflagellates (Li et al. 2000) and enhances growth in the kleptoplastidic dinoflagellates
Gymnodinium “gracilentum” and Amphidinium poecilochroum Larsen (Skovgaard 1998, Jakobsen et al. 2000)
and in the kleptoplastidic ciliate Lohmanniella sp.
Key index words: cannibalism; grazing; growth; kleptoplastidy; light; Pfiesteria piscicida
1
2
Received 14 September 2001. Accepted 19 March 2002.
Author for correspondence: e-mail [email protected].
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TIMOTHY N. FEINSTEIN ET AL.
(Chen and Chang 1999). Light can increase the rate
at which mixotrophic ciliates respire stored photosynthate (Putt 1990) and can stimulate growth in herbivorous protozoans through photodegradation of ingested material and photostimulation of digestive
enzymes (Moran and Zepp 1997, Strom 2001).
As part of an effort to understand how environmental factors regulate population dynamics of P. piscicida, we examined the influence of light on photosynthesis, growth, and grazing. Results were used to
propose a growth model for P. piscicida in the fluctuating estuarine environment.
materials and methods
Pfiesteria cultures. Stock cultures of P. piscicida (strain CCMP1831, Guillard Center for the Culture of Marine Phytoplankton, Bigelow Laboratory, Maine, USA) were maintained at 20 0.5 C in a 12:12-h light:dark (LD) cycle at a photon flux of 50
mol photonsm2s1 and fed Rhodomonas sp. (strain CCMP768)
weekly. Light intensity for experimental cultures was 100 mol
photonsm2s1 unless specified otherwise. Filtered (0.45 m)
and autoclaved 15‰ salinity seawater (FASW) with and without amendment with f/2 nutrients was used for Rhodomonas sp.
and P. piscicida cultures, respectively. Pfiesteria piscicida identity
was regularly confirmed with PCR using established 18S rRNA
(Oldach et al. 2000) and mitochondrial cytochrome b primers
(Zhang and Lin 2002), as well as through immunostaining using a recently developed species-specific polyclonal antibody
against P. piscicida cell surface antigens (Lin et al. unpublished
data).
Cell counts in all experiments were obtained from 1-mL
samples collected immediately after mild agitation to homogenize the culture. Samples were fixed in 2% neutral Lugol’s solution (Utermohl 1958) and enumerated using Sedgwick-Rafter
counting chambers on a Zeiss Universal microscope (Zeiss,
Germany).
Light regimes (L experiments). Three experiments were conducted to examine the influence of continuous illumination,
varying light intensities, 12:12-h LD cycles, and continuous darkness on grazing and growth. The first two experiments involved
well-fed and starved then re-fed cultures, and were performed
at a continuous illumination of 100 mol photonsm2s1 and
darkness. In experiment L1, P. piscicida, maintained in log-phase
growth in a 750-mL culture flask in a 12:12-h LD cycle at 100 mol
photonsm2s1, was allowed to graze down algal prey and subsequently was divided into six 50-mL subcultures in 75-mL flasks,
three of which were wrapped with aluminum foil. All cultures
were fed initial concentrations of approximately 10 RhodPfiest1.
Subsequently, daily feeding was performed by adding centrifugeconcentrated R. sp. Concentrated R. sp. was resuspended in 5-mL
of experimental P. piscicida culture, and the 5 mL was repipetted into experimental flasks to maintain a constant culture volume for the course of the experiment. This procedure was carried out gently to prevent mechanical damage of P. piscicida
cells. Roughly half of R. sp. appeared to recover from this procedure, yielding about 5 RhodPfiest1, a reasonably high concentration of food. Daily cell counts were taken for 7 days until
the density of P. piscicida in experimental cultures made it impractical to maintain an excess of prey. Pfiesteria piscicida growth
rates were calculated using the exponential growth equation
ln N t – ln N 0
µ = ------------------------------t
(1)
where Nt and N0 are cell concentrations at time t and time 0,
and t is the time interval. Predator-specific grazing rates were
calculated as in Frost (1972), modified for predator growth following Heinbokel (1978).
In the second experiment (L2), P. piscicida was allowed to
graze R. sp. until prey concentrations reached low levels ( 0.1
RhodPfiest1) and was then maintained for 48 h without feeding. The culture was divided into six 50-mL aliquots with P. piscicida concentrations of 7500 cellsmL1. Three of the six cultures were wrapped in aluminum foil, and R. sp. was added to
the six experimental and to six R. sp. control flasks at a concentration of 45,000 cellsmL1. Cell counts were taken at 0, 6, 12,
and 24 h. For each treatment and time point a 1-mL sample was
fixed in 2% Lugol’s, and at least 60 cells were immediately measured for estimation of cell volume using a digital camera
(DVC) and Northern Eclipse image analysis software (Empix
Imaging, Inc., Mississauga, Ontario, Canada). Gross growth efficiencies (GGEs) (biomass of P. piscicida produced/biomass of
cryptophyte grazed) were estimated using measured volumespecific C and N values for R. sp. and P. piscicida (see below).
In the third experiment (L3), different light intensities were
compared. Aliquots of 300 mL P. piscicida were acclimatized
over 3 days to light intensities of 0, 50, 100, 200, and 400 mol
photonsm2s1 on a 12:12-h LD cycle. On the fourth day each
culture was diluted with FASW and fed with R. sp. to reach an
initial concentration of 8000 PfiestmL1 and 60,000 RhodmL1
in a 50-mL volume. Controls consisted of triplicate 50-mL aliquots of 60,000 RhodmL1 for each light intensity. A second
feeding of equal amounts of R. sp. was carried out at 24 h, when
the mean R. sp. concentration in all treatments had only decreased from about 7-fold to about 5-fold higher than the concentrations of P. piscicida. Pfiesteria piscicida has been observed
to grow at near-maximum rates at this ratio of predator-to-prey
(Lin et al. unpublished data), so growth was not considered to
be prey limited during this period.
CHN analyses. To determine GGE in experiment L2, C and
N content of P. piscicida and R. sp. was measured. Because volume-specific C and N may vary between light-grown and wellfed cells and dark-grown and starved cells, R. sp. samples were
collected from cultures maintained in a 12:12-h LD regimen at
100 mol photonsm2s1 and from cultures maintained under continuous darkness for 24 h. Pfiesteria piscicida samples
were taken at 0, 2, and 7 days after most R. sp. in heterotrophic
culture had been grazed. Rhodomonas sp. maintained under a
12:12-h LD regimen was sampled at the midpoint of the light
period. Although some experiments were performed under
continuous illumination (L1 and L2), R. sp. used for feeding in
these experiments were grown under a 12:12-h LD regimen,
and this regimen was considered to more accurately represent
the prey on which the P. piscicida fed in both cases. Samples of
30 to 40 mL were filtered onto precombusted GF/F filters, and
cell abundances and volumes were measured concurrently with
sampling. Filters were then dried, and CHN analysis was carried
out using a Perkin-Elmer 2400 Series 2 CHNS/O analyzer.
Inhibition of photosynthesis. This experiment was designed to
determine whether photosynthesis contributes to growth or reduces population decline over 7 days after feeding. To eliminate the possibility of secondary effects due to the influence of
inhibitors on prey, P. piscicida were allowed to graze down algal
prey to very low levels immediately before the addition of inhibitors. Preliminary experiments with R. sp. indicated appropriate levels of three compounds that were inhibitory to photosynthetic growth of R. sp. but not immediately toxic, that is, did
not cause a significant decline in cell concentrations relative to
dark-incubated controls (results not shown): methyl viologen
0.5 mM (Sigma, St. Louis, MO, USA), DCMU 10 M, and antimycin A 20 M (Sigma). Pfiesteria piscicida maintained in exponential growth phase grazed R. sp. prey to very low concentrations (0.1 RhodPfiest1) within 24 h, at which time the
culture was split into 50-mL aliquots and inhibitors were added
to the final concentrations shown above. By day 1, R. sp. had
been grazed down to the detection limit and remained at or below this level for the remainder of the experiment. Because P.
piscicida appear to require a 3-fold higher concentration of prey
to prevent population decline (S. Lin, unpublished data), the
effect of residual R. sp. at such low levels would be negligible.
All treatments were prepared in triplicate for both the light
661
LIGHT EFFECT ON PFIESTERIA PISCICIDA
(continuous illumination of 150 mol photonsm2s1) and
darkness (continuous darkness except for unavoidable short
[1-min] exposure to approximately 6 mol photonsm2s1
when samples were taken for enumeration). Cell counts were
taken daily between 1200 h and 1500 h for 10 days.
Persistence of chloroplast activity (P experiments). Experiment P1
was designed to investigate any long-term influence of kleptoplastidy on growth and survival. Pfiesteria piscicida was kept without feeding for 7 days at a constant illumination of 150 mol
photonsm2s1 in a 750-mL culture flask, and the culture was
then split into six 75-mL culture flasks. Three subcultures were
wrapped with aluminum foil, whereas three remained under
continuous illumination. Daily cell counts were taken for 7 days,
during which time no R. sp. was detected in any flask (detection
limit, 20–40 R. sp.mL1).
In experiment P2, samples containing around 10 6 P. piscicida cells from 2-L cultures were collected after 2, 5, and 7 days
of starvation, when there were few (roughly 0.05 RhodPfiest1),
very few (0.01 RhodPfiest1), or undetectable levels of prey
cells remaining, respectively. Persistence of Rhodomonas-derived
RUBISCO in starved P. piscicida cells was examined using Western blotting. Samples were centrifuged at 3000 g and 4 C,
and the cell pellets were resuspended in Laemmli buffer
(Laemmli 1970) and stored at 80 C until all samples were
collected. Samples were thawed at room temperature and homogenized using a micropestle (Fisher Scientific, Springfield,
NJ, USA) in Eppendorf tubes. Cell homogenate was boiled for
3 min and then cleared by centrifugation. SDS-PAGE and Western blotting were conducted following Lin et al. (1994), with
each lane containing proteins representing 5 105 cells. For a
positive control, a sample of R. sp. was prepared in the same
way and an amount of crude protein extract equivalent to 5 105 cells was loaded to one lane. Anti-Isochrysis RUBISCO serum
(Falkowski et al. 1989) was used at a dilution of 1:5000. After
immunodetection of RUBISCO, the protein blot was stripped
of anti-RUBISCO and used again for detection of -tubulin to
indicate amount of total proteins loaded to each lane (Lin et al.
1994).
In experiment P3, the persistence of R. sp.-derived pigments
in P. piscicida cells was estimated. Cultures of 600 mL of P. piscicida were maintained in log-phase growth with daily feeding until cell concentrations reached 70,000 cellsmL1 and R. sp was
grazed to 15,000 cellsmL1, at which time feeding was discontinued and the first set of samples was taken. No R. sp. prey was
detected in experimental cultures on any subsequent day. Samples were taken daily for 5 days for epifluorescence microscopic
examination of phycoerythrin autofluorescence and for HPLC
analysis of chl a. Five-milliliter samples for phycoerythrin examination were spun for 10 min at 3000 g and 4 C. Pelleted
cells were resuspended in 1 mL 0.5% paraformaldehyde and
stored overnight at 4 C. Aliquots of 200 L of the fixed samples were rinsed by centrifugation in PBS, spun onto poly-lysine–
coated slides (Sigma), and counter-stained with 0.2 gmL1
4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Stained
slides were rinsed in PBS, dried, and mounted with Gel/Mount
(Biomeda Corp., Foster City, CA, USA). The percentage of cells
retaining phycoerythrin-derived autofluorescence (orange) under green light excitation was recorded using an Olympus
BX51 epifluorescence microscope.
Triplicate samples for HPLC analysis containing 7.8–12.7 106 P. piscicida cells (roughly 110 mL each) were centrifuged
for 40 min at 3000 g and 4 C, resuspended in 1 mL of culture medium, transferred to 15-mL centrifuge tubes, and spun
again for 10 min. For a control, an aliquot of R. sp. equal to the
concentration in experimental cultures on day 0 (1 day before
sampling was begun; approximately 150,000 cellsmL1) was
grown in medium identical to that in experimental cultures,
and one sample of 11–38 106 R. sp cells was taken each day
for pigment analysis. The supernatant was aspirated, and the
pellet was resuspended in 2.5 mL acetone and stored in the
dark at 20 C. Pigment concentrations in the acetone extracts
were determined using ion-pairing reverse-phase HPLC (Mantoura and Llewellyn 1983). The HPLC system consisted of a
Hewlett-Packard 1100 series with a quaternary pump and a variable wavelength detector and a 5-m C-18 (ODS) Alltech column (250 4.6 mm i.d.). Detection was accomplished by measuring absorbance at a wavelength of 420 nm. Mobile phases
used in the gradient elution included a primary eluant (80%
methanol:20% aqueous solution of 0.5 mM tetrabutyl ammonium acetate and 10 mM ammonium acetate) and a secondary
eluant (20% acetone in methanol). After injection (250 L extract), a gradient program ramped from 100% A to 100% B in
15 min with a hold for 50 min, providing sufficient resolution
of all pigments of interest. Identification of chl a in the extracts
was confirmed by coelution with an authentic standard. Authentic chl a (Sigma) was quantified spectrophotometrically
(Shimadzu UV-2501 spectrophotometer) using an extinction
coefficient of 68,700 at 440 nm (Mantoura and Llewellyn
1983). HPLC peak areas from the P. piscicida and R. sp. samples
were converted to concentrations using a response factor calculated from the authentic standard. Replicate HPLC measurements of pigment standard varied by 5%. Chl a measured by
HPLC was normalized to ng chl acell1.
Cannibalism. Preliminary experiments showed that cannibalism may occur in P. piscicida and may respond to illumination, and this prompted us to study its role in the feeding ecology of P. piscicida. Six 600-mL cultures of mid-log phase P.
piscicida were allowed to graze down algal prey, after which time
three were wrapped in aluminum foil and cannibalism was estimated for all cultures at days 1 and 7 after the grazing of most
algal prey. As with other experiments, prey cell concentration
was low (0.1 RhodPfiest1) on day 1 and undetectable on
subsequent days. For estimation of cannibalism, aliquots from
each culture were stained for 2 h in the dark with 3 M 5-chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR,
USA), a general stain of living cells. Stained aliquots were
rinsed with FASW by gravity filtration on 5-m filters and then
resuspended in FASW, enumerated, and recombined with unstained aliquots from the same culture to yield an equal number of P. piscicidamL1 between treatments and a 1:1 ratio of
stained:unstained cells within treatments. Recombined cultures
were incubated for 4 h in the dark or in the light (75 mol photonsm2s1) and then fixed overnight at 4 C in 0.5% paraformaldehyde. Fixed samples were rinsed in PBS by centrifugation,
spun onto poly-L-lysine–coated slides (Sigma), and counterstained
with 0.2 gmL1 DAPI (Sigma) for 10 min at 4 C (Lin and Carpenter 1996). Slides were rinsed in PBS, air dried, mounted with
Gel/Mount, and examined using an Olympus BX51 epifluorescence microscope with blue light excitation and a long pass emission filter.
Use of 5-chloromethylfluorescein diacetate-prestained cells
allowed better identification of ingested cells than nuclear
staining with DAPI alone, because the presence of multiple nuclei could result from cell cycle-related processes. When using
the live-staining method, only one of four possible cannibalism
events could be enumerated—when an unstained cell feeds
myzocytotically on a stained cell. This was observed as an unstained cell containing more than one nucleus and in which
one of the nuclei is contained within a stained food vacuole
(see Fig. 8). In the other three types of encounter (unstained
cell ingesting unstained cell, stained cell ingesting unstained or
stained cell), the fluorescent signal would either be absent or
masked. We assumed that no selection took place between
stained and unstained prey (Kamiyama 2000). At least 250 cells
were counted in each sample and placed into one of three categories: uni-nucleated cells, multinucleated cells, and unstained
multinucleated cells containing a stained food vacuole, although only the results from the first and last categories are
shown here. Hourly cannibalism rate was thus estimated as
c
C = --------- × 4
n⋅t
(2)
where C is the hourly cannibalism rate in Pfiest ingestedPfiest1
h1, c is the number of cannibalism events counted (unstained
ingests stained), n is the total number of cells counted, and t is
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TIMOTHY N. FEINSTEIN ET AL.
the time of incubation in hours, 4 is a factor to account for the
fact that only one of the four possible cannibalism events was
enumerated (unstained cell preys on stained cell). To reduce
the chance that herbivorous grazing may be recorded as a cannibalism event, cannibalism was only recorded when the
stained food vacuole contained a clearly distinguishable dinoflagellate nucleus (see Fig. 8). Given that any cells that cannibalize more than once during the 4-h incubation would be
counted as a single event, it is likely that this technique presents
an underestimation of the actual cannibalism rate and the underestimation would increase with the observed rate of cannibalism. Because this would act to reduce the difference between measured cannibalism rates, we considered any difference
measured by this procedure to be conservative.
Data analysis. Statistical analyses were performed by repeated-measures analysis of variance (ANOVA) unless stated
otherwise; the most appropriate repeated-measures ANOVA
was chosen by goodness of fit according to Akaike’s information criterion unless stated otherwise. Values representing replicate measurements are presented as means SD.
results
Light effects on growth rates. In experiment L1, logphase P. piscicida fed R. sp. daily grew under continuous illumination of 150 mol photonsm2s1 at a
rate roughly double the growth rate of cultures maintained in the dark ( 0.42 0.03 d1 and 0.25 0.04 d1, respectively; Fig. 1A; P 0.001). Results of
experiment L2 demonstrated that the difference in P.
Fig. 1. Pfiesteria piscicida growth under light and dark conditions. Values are means SD of triplicate measurements. (A) Growth
of P. piscicida in continuous light (100 mol photonsm2s1) and in darkness over a 7-day period with daily additions of centrifugeconcentrated R. sp. prey. (B–D) Growth of 48-h starved P. piscicida over 24 h after the addition of an excess of R. sp. prey. (B) Cell
abundances of P. piscicida and R. sp. (C) Pfiesteria piscicida mean cell volume. (D) P. piscicida biomass.
LIGHT EFFECT ON PFIESTERIA PISCICIDA
piscicida growth between light and dark treatments
started to manifest within 24 h in cells that had been
starved for 48 h before feeding (Fig. 1, B–D). Although light’s influence on cell numbers was not significant within 24 h of feeding (P 0.33 in both treatments, means for t 0 vs. t 24 h compared using
LSD ANOVA; Fig. 1B), cell volume and carbon-based
biomass increased over the period of the experiment
to a greater magnitude in the light than in the dark
(Fig. 1, C and D). Mean cell volumes (Fig. 1C) increased by roughly 2-fold in the first 6 h in both treatments, after which cell volumes did not change in the
dark treatment. In the light treatment, cell volumes
continued to increase from 6 to 12 h to a value
roughly 3-fold over the initial volume, after which
time cell volume remained nearly constant. The cell
volume was significantly higher in the light than in
the dark at 12 and 24 h (P 0.001 for both time
points, single-factor ANOVA) but not at 0 and 6 h
(P 0.3 and P 0.1, respectively). Biomass, the
product of cell number, cell volume, and volume-specific cell carbon from CHN analysis, increased in the
dark treatment for the first 6 h and remained unchanged thereafter (Fig. 1D). In the light treatment,
biomass increased at a continuous rate from 0 to 24 h
after the addition of prey. During the first 12 h of the
experiment the biomass increase was attributable to
increased cell volume, whereas the increase between
12 and 24 h after the addition of prey was due entirely
to an increase in cell numbers. The increase in biomass was significant in the light treatment (P 0.001)
but not in the dark treatment (P 0.79).
Among the five light intensities examined in experiment L3, the growth rate of the food-saturated P. piscicida culture peaked at a light intensity of 100 mol
photonsm2s1, with a growth rate of 0.91 0.11
d1 (Fig. 2). The overall significance of the effect of
light on growth was established by LSD ANOVA (P 0.013). Growth at 100 mol photonsm2s1 was significantly greater than growth at 0 and 400 mol photonsm2s1 (Fisher’s protected LSD ANOVA, P 0.01
and 0.0015, respectively) but was not significantly different from growth at 200 and 50 mol photonsm2s1
(P 0.3 and 0.2, respectively).
Light effects on grazing rates. Pfiesteria piscicida maintained under continuous illumination of 100 mol
photonsm2s1 and in darkness (experiment L2)
grazed R. sp. at similar rates (P 0.1 in all cases; Fig.
3). In both treatments most grazing occurred within
the first 6 h after the addition of R. sp., after which
time grazing was not significantly different from zero
(single-factor t-test, P 0.05 in all cases; Fig. 3).
CHN and GGE. Overall, values for R. sp. and 1-day
starved P. piscicida agreed closely with previously published estimates for phytoplankton (Montagnes et al.
1994, Menden-Deuer and Lessard 2000). Volume-specific carbon content for P. piscicida was 0.209 .014
pg Cm3 at 1 day since feeding and 0.343 .028 pg
Cm3 at 3 days since feeding (P 0.02, two-tailed
t -test). A decrease in total volume compensated for
663
Fig. 2. Effect of light intensities on growth of Pfiesteria piscicida. Cultures were acclimated to experimental light intensities
(0 to 400 mol photonsm2s1) for 3 days before measurement
began. Values are means SD of triplicate measurements.
the small increase in volume-specific carbon content,
so that cell-specific carbon values were not significantly different (P 0.34, two-tailed t-test) and averaged 206 17 pg Ccell1. Cell-specific and volumespecific carbon content for R. sp. was not significantly
different between 12:12-h LD and 24 h of darkness (P 0.17 and 0.77, respectively, two-tailed t-test), with a
mean value of 0.263 0.034 pg Cm3 and 48.2 7.3 pg Ccell1. Based on measured cell volumes and
carbon values, P. piscicida that had been starved for
48 h before feeding (experiment L2) grew over a 24-h
Fig. 3. Effect of light on grazing in Pfiesteria piscicida over a
24-h period. Measurement was made to cultures grown under
continuous light at 100 mol photonsm2s1 and continuous
darkness. Values are means SD of triplicate measurements.
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TIMOTHY N. FEINSTEIN ET AL.
period with a GGE of 0.78 0.10 under continuous illumination of 100 mol photonsm2s1 and a GGE
of 0.32 0.16 in continuous darkness (P 0.027; single-factor ANOVA). Similar results would be obtained
if published volume-specific carbon values for dinoflagellates and cryptophytes (Montagnes et al. 1994,
Menden-Deuer and Lessard 2000) were used.
Growth of starved and photosynthetically inhibited cultures. During the first day after the removal of prey,
moderate, albeit not significant (P 0.3), growth was
observed in the light incubation ( 0.286 0.11
d1; P 0.15) and to a lesser degree in the dark incubation ( 0.0806 0.032 d1; P 0.097), whereas
no growth was observed in light-incubated photosynthetically inhibited samples (Fig. 4). After day 2 all
cultures declined monotonically, and no difference
was observed in the rate of decline between any of the
treatments (mean 0.403 0.094 d1; P 0.085, single-factor ANOVA). Overall, repeated-measures ANOVA indicated a difference between control
and inhibitor treatments but not between control and
dark treatments (P 0.001 and P 0.2, respectively).
Population decline during extended starvation. Negative
growth (i.e. population decline) was consistently observed under extended starvation. In the experiment
shown in Figure 5, dark-incubated P. piscicida declined
at a similar rate to that of light-incubated P. piscicida
from day 7 of starvation until day 10 ( 0.55 0.03 d1 in the dark and 0.50 0.07 d1 in the light;
P 0.2; two-tailed paired t -test). From day 10 to day
14, however, the rate of decline under continuous
illumination ( 0.90 0.06 d1) was significantly
greater than the rate of decline in continuous darkness ( 0.58 0.05 d1; P 0.001, two-tailed t -test).
Fig. 4. Response of starved Pfiesteria piscicida to photosynthesis inhibitors and darkness. Inhibitors used included DCMU
(10 M), methyl viologen (0.5 mM), and antimycin A (20 M).
A culture maintained at 150 mol photonsm2s1 was used as
a control. Rhodomonas sp. prey was below detectable levels (20–
40 cellsmL1) throughout the experiment.
Fig. 5. Effects of light on Pfiesteria piscicida population during extended starvation. Cultures were maintained under continuous illumination (150 mol photonsm2s1) or darkness
over a 7-day period after 7 days of starvation under continuous
illumination (150 mol photonsm2s1). Rhodomonas sp. prey
was below detectable levels (20–40 cellsmL1) in both light
and dark cultures during the course of the experiment. Values
are means SD of triplicate measurements.
RUBISCO and pigments. Analysis by Western blot
showed that RUBISCO was abundant in R. sp. used as
a control (Fig. 6). In experimental cultures that recently grazed several-fold higher concentrations of R.
sp. than that used in the controls, RUBISCO was
barely detectable at 2 days after the removal of prey
and undetectable at 5 and 7 days (Fig. 6). In the cultures from which samples were taken for autofluorescence and for quantitation of chl a by HPLC, R. sp.
was low (about 0.1 RhodPfiest1) on day 1 and below
detectable levels (detection limit 20–40 cellsmL1)
on subsequent days. As determined by epifluorescence microscopy, P. piscicida exhibiting phycoerythrin autofluorescence declined rapidly from 48.1 2.5% on day 1 to 5.51 1.2% on day 2 and was not
observed on subsequent days (Fig. 7A). Analysis by
HPLC indicated that chl a, abundant in R. sp. used
for controls (2.22 0.59 ngcell1), disappeared rapidly after the removal of prey (Fig. 7B). On a per cell
basis (inclusive of P. piscicida and R. sp), chl a in P. piscicida cultures that had recently (within 48 h) grazed
several-fold higher concentrations of R. sp. cells was
lower than that in the control (Fig. 7B). Taking into
account the small amount of prey cells present in the
cultures on day 1, there was no chl a that can be attributed to P. piscicida. On day 2 and on subsequent
days, chl a was not detected in any of the experimental cultures (Fig. 7B).
Cannibalism was observed in P. piscicida using a
live-staining technique as a stained P. piscicida cell ingested by a nonstained cell (Fig. 8). Within 24 h of
feeding, only one positive cannibalism event was observed out of 875 cells counted in the light treatment
and no cannibalism events were observed in the dark
LIGHT EFFECT ON PFIESTERIA PISCICIDA
Fig. 6. Immunoblot for RUBISCO (A) and -tubulin (B).
Amount of proteins loaded to each lane was adjusted so that
each lane contained the same number (5 105) of Pfiesteria piscicida cells in the experimental samples and R. sp in the control. -Tubulin (B) was used as an indicator of total proteins.
Lanes 1 to 6 are P. piscicida samples (P) collected 2 (lane 5), 5
(lane 2), and 7 days (lanes 1, 3, 4, and 6) after feeding was discontinued. Lane 7 (R) is Rhodomonas sp. used as a positive control. Light (; 150 mol photonsm2s1) or dark () conditions of the cultures are indicated at the top of the figure. On
the left are markers for molecular weight. Arrows on the right
point to the bands of RUBISCO (A) and -tubulin (B).
treatment. After 7 days of starvation, hourly cannibalism rates were estimated conservatively as 6.6 1.1 102 and 1.5 1.2 102 Pfiest ingestedPfiest1h1
in the light and in the dark, respectively (P 0.036;
two-tailed t-test). Cannibalism was observed only in
cultures in which no free-living R. sp. was detected,
further reducing the probability that herbivorous
grazing was counted as a cannibalism event.
discussion
Results presented here indicate that light and algal
prey significantly influence growth in populations of
P. piscicida. The light-induced differences in growth
rates observed here appear to be due to a GGE benefit conferred by kleptoplastidic photosynthesis rather
than to a difference in grazing. When prey is depleted
kleptoplastidic photosynthesis appears to diminish
665
Fig. 7. Chl a content and phycoerythrin (PE) autofluorescence in recently fed Pfiesteria piscicida maintained under a
12:12-h LD regime (100 mol photonsm2s1). (A) Percentage of P. piscicida cells that displayed discernible PE autofluorescence over 5 days after grazing. (B) Chl a detected by HPLC
in a Rhodomonas sp.-only control (black bars) and in P. piscicida
cultures after feeding was discontinued (gray bars). Values
were normalized to the total number of R. sp. and P. piscicida
present in the sample. Numbers on the x-axis are time since
feeding in days (d) and the relative proportion of P. piscicida
(P) and R. sp. (R) present in the sample (Ratio). Total cell
counts (Pfiest Rhod) for samples taken for HPLC ranged
from 95 to 127 106 cells. Data shown are means SD from
triplicate cultures.
within 1–2 days, and when starvation is prolonged
light promotes cannibalistic grazing, probably due to
demand for metabolic carbon.
Light and growth. The stimulatory effects of light on
growth of P. piscicida has been consistently observed
in this study as a markedly higher growth rate under
continuous or intermittent (12:12-h LD) illumination
than that in the darkness. The difference between
light- and dark-incubated cultures appears to require
a short time to manifest, with significant differences
in biomass manifesting within 24 h. Darkness reduced
666
TIMOTHY N. FEINSTEIN ET AL.
Fig. 8. Cannibalism in Pfiesteria piscicida. Images shown are from a sample collected after 7 days of starvation and were made using transmitted light (A, D), 4,6-diamidino-2-phenylindole dihydrochloride epifluorescence (B, E), and 5-chloromethylfluorescein
diacetate epifluorescence (C, F). Experimental incubations contained equal numbers of stained and unstained P. piscicida (A–C; bottom right and top left, respectively). (D–F) A positive cannibalism event in which an unstained cell contains multiple unequal nuclei
(E), with the smaller nucleus contained within a 5-chloromethylfluorescein diacetate-stained food vacuole (F). Arrows point to the
5-chloromethylfluorescein diacetate-stained P. piscicida cell that was ingested. Scale bar, 10 m.
growth rate in all experiments. The enhanced growth
under light could result from either light-stimulated
grazing and digestion (Moran and Zepp 1997, Strom
2001) or from photosynthesis (e.g. Skovgaard 1998,
Lewitus et al. 1999a,b).
Light and grazing. No effect of light was observed in
any experiments performed in this study. It is interesting to note, however, that when an excess of R. sp.
prey was added to a starved population, P. piscicida
grazed 2.9 0.75 RhodPfiest1 within the first 6 h after addition of prey; thereafter, ingestion rate was
nearly zero. Taken together with the finding that
most increase in cell numbers occurred between 12
and 24 h after feeding, it may be proposed that a hungry P. piscicida cell ingests a threshold amount of prey
(roughly 3 RhodPfiest1 in this case, or approximately 100% starved body carbon) and then enters a
nonfeeding stage lasting 12–24 h before division occurs. Based on our experimental results that grazing
rate was not different between light and dark cultures,
the role of grazing can be excluded as a factor in
light’s stimulation of growth.
Kleptoplastidy and GGE. The influence of illumination on growth appears to be largely attributable to kleptoplastidy, although the possibility of light-enhanced
digestion was not specifically explored here. Pfiesteria
LIGHT EFFECT ON PFIESTERIA PISCICIDA
piscicida has been shown to be capable of kleptoplastidic photosynthesis (Lewitus et al. 1999a,b), and results from this study reinforced the previous finding
using different approaches. First, photosynthesis inhibition reduced growth and prevented a short-term increase in population numbers that was observed in
light-incubated cultures from which prey had recently
been removed. Second, if no benefit of photosynthesis was considered, then the GGE of light-grown P. piscicida was unusually high. Reported GGEs for strictly
heterotrophic dinoflagellates range from 12% to 53%
(Strom 1991, Hansen 1992, Buskey et al. 1994, Naustvoll
1998, Strom and Morello 1998), whereas the mixotrophic dinoflagellate Gymnodinium “gracilentum” had a
GGE of 59%–64%, depending on light intensity (Skovgaard 1998). Our measurement of dark-incubated GGE
(32%) falls within expected values for heterotrophic dinoflagellates, whereas the light-incubated value of 78%
is unusually high and may be an underestimate because
it was derived from recently starved cultures. One striking observation made in this study was that kleptoplastic
photosynthesis occurred while prey alga was still abundant or only recently depleted, thus suggesting that
P. piscicida supplements ingested carbon with photosynthesis.
Furthermore, results from this present study suggest that kleptoplastic photosynthesis in P. piscicida is
ephemeral. Lewitus et al. (1999a,b) suggested that
kleptoplastidy persists in P. piscicida for as long as a
week after feeding and may serve as a survival strategy
in the absence of prey, which would agree with the estimated time frame (7–14 days) for kleptoplastidy in
the ciliate Mesodinium rubrum Lohmann (Gustafson et
al. 2000). Our findings more closely agree with Skovgaard (1998) and Stoecker and Silver (1990), in
which the growth benefit from kleptoplastidy was
found to persist for less than 2 days in the dinoflagellate G. “gracilentum” and in the ciliate Strombidium capitatum Kahl, respectively. The ephemeral nature of
kleptoplastidy may be due to aging of the enslaved
chloroplast (Stoecker and Silver 1990). In accordance, Rhodomonas-derived RUBISCO diminished
within 2 days and completely disappeared within 5
days of the removal of prey. Furthermore, most
Rhodomonas-derived chl a was decomposed within 2
days of feeding, as determined using HPLC, and similarly, Rhodomonas-derived phycoerythrin autofluorescence in P. piscicida cells disappeared within 2 days.
Several possibilities may account for the apparent discrepancy between our results and those of Lewitus et
al. (1999a). The strain used in our experiments
(CCMP 1831) was confirmed as P. piscicida (Zhang
and Lin 2002) but was isolated independently of the
strain used in the previous study (Lewitus et al.
1999a), and difference may exist between the populations. In experiments conducted to examine persistence of kleptoplastidy, prey was still detectable in the
previous study, whereas R. sp was below the detection
limit of 20 cellsmL1 in the present study. Nevertheless, given that prey alone can support P. piscicida
667
growth in the dark and that the presence of the prey
alga is indispensable for photosynthesis, the role of
kleptoplastidic photosynthesis may be limited to that
of a nutritional supplement. This is in contrast to mixotrophic dinoflagellates in which photosynthesis appears to be the main carbon source and phagotrophy
only provides supplements of inorganic nutrients
(Stoecker et al. 1997), organic carbon (Skovgaard
1996, Jeong et al. 1999), or both (Li et al. 1999, 2000,
Skovgaard 2000). Additionally, P. piscicida starved for
more than 7 days responds to light intensity in a way
that would be counterintuitive if kleptoplastidic photosynthesis remained an important contributor to
growth. The negative effect of light on population
numbers is consistent, however, with the presence of a
greater metabolic demand for carbon that is not replenished by external means and which appears to be
provided by cannibalism. The observation of cannibalism after 7 days of starvation (see below) suggests
that kleptoplastidic photosynthesis is less important
than cannibalistic grazing in sustaining populations of
P. piscicida after the removal of food.
Cannibalism. Cannibalistic feeding has been observed
in the dinoflagellate genus Protoperidinium and in the
ciliates Stylonichia sp. and Euplotes versatilis n. sp. and
may be much more widespread among the protozoa
(Giese and Alden 1938, Buskey et al. 1994, Latz and
Jeong 1996, Tuffrau et al. 2000). Cannibalism has not
yet been reported in P. piscicida despite some attempts
(Burkholder et al. 2001), and in our research cannibalism was not observed in living culture and was tentatively observed only once in Lugol-preserved cells
when fluorescent staining was not used. Our results
with a novel 5-chloromethylfluorescein diacetate
staining technique demonstrate that cannibalism
does occur in P. piscicida and may play an important
role in its ecology.
Pfiesteria piscicida does not cannibalize in the presence of cryptophyte prey, and the presence of cannibalism during starvation suggests that it may play a
role as a survival strategy when other carbon sources
are not available. Light has been found to stimulate
cannibalism in the larval stages of distantly related
metazoans: the Australian giant crab (Pseudocarcinus
gigas Lamarck) (Gardner and Maguire 1998), the dorada fish (Brycon moorei Steindachner) (Baras et al.
2000), and the vundu catfish (Heterobranchus longifilis
Valenciennes) (Baras et al. 1998). Although the mechanisms connecting illumination to cannibalism may
not be comparable, a similar relationship has been
found in cannibalizing populations of P. piscicida.
The stimulatory effect of light, apparently in conflict with lack of an effect on grazing of R. sp., may be
due to increased metabolic demand as mentioned
earlier. If cannibalism serves only to replenish carbon
for basal metabolism rather than for growth (Latz and
Jeong 1996) and kleptoplastidy does not significantly
contribute carbon to starved cells, then the rate of cannibalistic grazing could be tightly coupled with basal
metabolic rate. Interestingly, the 4.4-fold increase in
668
TIMOTHY N. FEINSTEIN ET AL.
cannibalism rate would agree qualitatively with Putt
(1990), in which moderate levels of illumination were
found to stimulate metabolic rate in the kleptoplastidic protozoan Laboea strobila 6-fold over darkness.
A model of light’s influence on Pfiesteria piscicida. The
work described here suggests a modification of a growth
model proposed previously (Lewitus et al. 1999b) for
the influence of light and kleptoplastidic photosynthesis on “herbivorous” populations of P. piscicida. When
appropriate algal prey (cryptophyte) is present, kleptoplastidy significantly enhances population growth rate, in
a light- and presumably nutrient-dependent manner.
When prey is removed, however, the growth-supporting
role of kleptoplastidy quickly diminishes and P. piscicida
populations decline until algal prey is replenished. If lack
of algal prey is prolonged, P. piscicida populations will
continue to decline and will be maintained through cannibalism, the extent of which may be dictated by light’s
influence on basal metabolic rate. Under such circumstances, P. piscicida would benefit from a dark environment such as the epibenthic zone of eutrophic estuaries.
We thank Keri Costa for assistance in the laboratory, Drs. Paul
Renaud and Jeffrey Terwin for assistance with the statistics, and
Carol Rosetta for assistance with the 5-chloromethylfluorescein diacetate staining. This research was supported by ECOHAB-NOAA grant NA860P0491. Connecticut Sea Grant and
the University of Connecticut Department of Marine Sciences
provided support for travel to present results at the 55th annual
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