Journal of Eukaryotic Microbiology ISSN 1066-5234
ORIGINAL ARTICLE
Growth, Grazing, and Starvation Survival in Three
Heterotrophic Dinoflagellate Species
Sean R. Anderson & Susanne Menden-Deuer
Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island 02882
Keywords
Starvation; food web; ingestion;
phytoplankton patchiness; population
dynamics; predation; protist.
Correspondence
S. Menden-Deuer, Graduate School of
Oceanography, University of Rhode Island,
215 South Ferry Road, Narragansett, RI
02882, USA
Telephone number: +1 (401) 874-6608;
FAX number: +1 (401) 874-6728;
e-mail: [email protected]
Received: 16 June 2016; revised 29 July
2016; accepted July 31, 2016.
doi:10.1111/jeu.12353
ABSTRACT
To assess the effects of fluctuating prey availability on predator population
dynamics and grazing impact on phytoplankton, we measured growth and
grazing rates of three heterotrophic dinoflagellate species—Oxyrrhis marina,
Gyrodinium dominans and Gyrodinium spirale—before and after depriving them
of phytoplankton prey. All three dinoflagellate species survived long periods
(> 10 d) without algal prey, coincident with decreases in predator abundance
and cell size. After 1–3 wks, starvation led to a 17–57% decrease in predator
cell volume and some cells became deformed and transparent. When reexposed to phytoplankton prey, heterotrophs ingested prey within minutes
and increased cell volumes by 4–17%. At an equivalent prey concentration,
continuously fed predators had ~2-fold higher specific growth rates (0.18 to
0.55 d1) than after starvation (0.16 to 0.25 d1). Maximum specific predator
growth rates would be achievable only after a time lag of at least 3 d. A delay
in predator growth poststarvation delays predator-induced phytoplankton mortality when prey re-emerges at the onset of a bloom event or in patchy prey
distributions. These altered predator-prey population dynamics have implications for the formation of phytoplankton blooms, trophic transfer rates, and
potential export of carbon.
CARBON cycling in the ocean is mediated by predation on
primary producers and the transfer of organic matter
through planktonic food webs (Worden et al. 2015). Most
predation loss of primary production is due to the grazing
activity of microzooplankton (e.g. protists < 200 lm) (Sherr
and Sherr 2002). These organisms, which are ubiquitous in
marine ecosystems, have been estimated to consume
~67% of daily primary production, and typically exert a
grazing impact on phytoplankton biomass exceeding that
of copepods and other mesozooplankton (Calbet and
Landry 2004; Campbell et al. 2009; Putland 2000; Sherr
and Sherr 2002, 2007). Of these protists, heterotrophic
dinoflagellates are particularly important, often accounting
for more than half of the biomass of the microzooplankton (Kim and Jeong 2004; Lessard 1991; Sherr and
Sherr 2007).
The survival of herbivorous protists depends on the
rates at which prey is encountered, which may be
delayed by spatial and seasonal fluctuations in phytoplankton abundance or mismatches in predator and prey
€fer et al. 2007). Patchiness
types present (Fig. 1; Paffenho
of phytoplankton can occur over wide spatial (micrometers-meters) and temporal (minutes-weeks) scales
(McManus et al. 2003; Montagnes 1996). Moreover, phytoplankton abundance can fluctuate seasonally and
remain low for weeks to months of the year in both
polar and temperate regions (Sherr et al. 2009; Winder
and Cloern 2010). While heterotrophic protists use foraging strategies and chemical cues (e.g. Menden-Deuer
€nbaum 2006; Montagnes et al. 2008) to locate
and Gru
patches of high prey density, physical and temporal
constrains may limit encounter. Based on realistic swimming rates (150–350 lm/s) and assuming phytoplankton
are aggregated within a single layer within a 30 m water
column (Menden-Deuer 2008), grazers would require
1–3 d to reach a prey layer. Therefore, even under optimal
swimming conditions, grazers may be starved for a few
days before prey is encountered. Additionally, changes in
prey type, quality or size may affect prey availability, since
many heterotrophic protists are selective feeders (Buskey
1997; Jakobsen and Hansen 1997; Montagnes et al. 2008).
Patchy prey coupled with predator selection means suitable
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
1
Poststarvation Feeding in Heterotrophic Dinoflagellates
prey may be rare at times and once prey has been
exploited, protistan predators may be faced with prolonged
periods of starvation, extending days to weeks depending
on seasonality, patchiness, and phytoplankton community
composition.
The responses of diverse heterotrophic protists to
famine-like conditions have been assessed in a variety
of laboratory studies (Calbet et al. 2013; Fenchel 1982,
1989; Strom 1991). Heterotrophic dinoflagellates are
known to survive starvation for longer periods than
ciliates, which die within a matter of days when starved
(Jackson and Berger 1984; Jeong and Latz 1994; Montagnes 1996; Strom and Morello 1998). Common
dinoflagellates such as those in the genus Protoperidinium can survive beyond 20 d of starvation and up to
71 d at algal prey concentrations < 1 lg C/liter (MendenDeuer et al. 2005), and Gymnodinium may survive for
30 d (Strom 1991).
Once starved, predator responses may involve increased
feeding activity coupled with fast digestion times or
increased food storage (Fenchel 1989; Menden-Deuer et al.
2005; Meunier et al. 2012). Upon commencing starvation,
some heterotrophic grazers may undergo a residual cell
division, producing smaller and faster daughter cells (termed swarmers) (Fenchel 1982, 1989). Possible secondary
responses include the formation of temporary cysts, a
reduction in metabolism, or reallocation of cellular reserves
such as autophagy of the mitochondria (Calbet et al. 2013;
Fenchel 1989; Hansen 1992; Menden-Deuer et al. 2005).
Grazers may also utilize other food sources between
blooms or when preferred prey is absent (bacteria or other
heterotrophic protists) or adopt mixotrophy (Hansen 1992;
Kim and Jeong 2004; Uchida et al. 1997). These survival
responses to starvation are accompanied by energetic
trade-offs that balance energy allocation to long-term survival vs. maintenance of prey ingestion capacity.
Starved ciliates respond to newly added prey by immediate prey ingestion, though predator growth rate may lag
depending on the duration of starvation (Fenchel 1982,
1989). In heterotrophic dinoflagellates, there has been only
one study on starvation and subsequent re-feeding in the
species Oxyrrhis marina and Gyrodinium dominans (Calbet
et al. 2013). Calbet et al. (2013) reported both species were
able to survive up to 12 d when starved, though significant
decreases in cell size, respiration and fatty acid content
were measured; both O. marina and G. dominans
responded to prey poststarvation by increasing cell volume,
though ingestion rates were initially depressed (Calbet et al.
2013). In the field, Saito et al. (2006) reported an increased
abundance of Gyrodinium sp. over a period of days during
an iron-enriched diatom bloom. These studies suggest that
after relatively short starvation periods, heterotrophic
dinoflagellates can rapidly resume grazing and exert predation pressure on phytoplankton. However, little is known
about poststarvation predator population growth.
Determining heterotrophic protist growth and feeding
responses to poststarvation prey encounter is critical to
understanding predator population dynamics and feeding
impact on phytoplankton primary production and potential
2
Anderson & Menden-Deuer
algal biomass accumulation. A significant delay in feeding
or predator growth after starvation may have direct implications for how grazers survive when prey availability is
low and adaptations to starvation may affect grazing success once prey is encountered (Sherr et al. 2003). Moreover, delays in the resumption of predator growth, and
thus delay in the escalation of grazing pressure, may also
help explain phytoplankton biomass accumulation at the
initiation of a bloom event and could be a critical factor
controlling carbon flux during this early-bloom period
(Sherr and Sherr 2009).
Despite the well-documented ability of heterotrophic
dinoflagellates to survive starvation (Menden-Deuer et al.
2005; Strom 1991) and suggestions that predators can
feed after starvation when prey is re-introduced (Calbet
et al. 2013; Schmoker et al. 2011), there are, to our
knowledge, no reports of heterotrophic dinoflagellate
growth rates after starvation. The focus of this study was
to compare the functional and numerical responses of
three
cosmopolitan
heterotrophic
dinoflagellates
(O. marina, Gyrodinium spirale and G. dominans) in
response to different food conditions (continuously-fed vs.
starved) and over different time scales (hours vs. days).
We assessed whether rapid ingestion by heterotrophic
dinoflagellates after starvation results in immediate growth
and thus by implication an escalation of predation pressure, or if there is a time lag before growth is resumed,
as suggested by the work of Fenchel (1989) on ciliates. A
better understanding of how heterotrophic dinoflagellates
respond to prolonged periods of starvation and quantification of their growth with recurring prey will allow assessment of the impact of poststarvation predation rates
under conditions when prey availability is limited in the
marine environment (Fig. 1).
MATERIALS AND METHODS
Culture maintenance
Predators
Clonal cultures of G. spirale (PA300413), G. dominans
(SPMC 103), and O. marina (SPMC 107) were established
by single cell isolation. G. spirale originated from Narragansett Bay, RI (2013), while strains of G. dominans and
O. marina are identical to the ones used in Strom et al.
(2013). Oxyrrhis marina cultures used for starvation experiments were non-axenic, though algal prey was depleted in
starvation experiments. All heterotroph cultures were
maintained in 1-liter polycarbonate (PC) bottles on a
12 h:12 h light–dark cycle at 14.5 °C, salinity of ~30 psu,
and a light intensity of 8–15 lmol photons/m2s. Unless
specified otherwise, heterotrophs were fed twice per
week with the prey culture Heterocapsa triquetra to a final
concentration of ~2,000 cells/ml and refreshed with filtered seawater (FSW).
Prey
Three phytoplankton species were cultured for use in feeding experiments. The mixotrophic dinoflagellate H. triquetra
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
Anderson & Menden-Deuer
Poststarvation Feeding in Heterotrophic Dinoflagellates
Figure 1 Prey availability to herbivorous predators can fluctuate due to (A) spatial heterogeneity in prey distribution, (e.g. plankton layers) that
may not always be overcome despite predator motility and foraging strategies, (B) seasonal variations of phytoplankton biomass and (C) a mismatch between predators and phytoplankton type present. Graph in panel (B) is based on averaged monthly surface chlorophyll (2004–2014)
taken from the Narragansett Bay long term plankton times series at the Graduate School of Oceanography at the University of Rhode Island.
(CCMP 448), the diatom Skeletonema marinoi (CCMP
1332), and the prymnesiophyte Isochrysis galbana (CCMP
1323) were cultured in f/2 medium and transferred every
3–5 d to maintain exponential growth (Guillard 1975). All
prey cultures were maintained in 250 or 500-ml PC bottles
on a 12 h:12 h light-dark cycle at 15 °C, salinity of ~30 psu,
and a light intensity of 70–80 lmol photons/m2s.
chains in suspension, otherwise experiments were conducted as described above. Growth rates (l, d1) were
calculated as the slope of the natural log of abundance
over time for samples collected daily. The numerical
response was determined by fitting Eq. 1 to the growth
rate data (Montagnes and Berges 2004),
l¼
Cell abundance and biomass
To determine predator and prey abundance, samples were
fixed with Lugol’s iodine (1%) and enumerated using a
1-ml Sedgewick-Rafter chamber. To determine biomass,
predator, and prey biovolumes were determined for ≥ 40
cells, assuming they were prolate spheroids with volume
(V) calculated as V = 4/3PI r12 * r2 where r1 is the cell
width and r2 is the cell length, with the assumption that
r2 ≥ r1. General size measurements were made on fixed
cells, whereas post-starvation measurements were made
on live cultures. Carbon biomass (ng C/cell) was determined using published C to volume conversion equations
(Menden-Deuer and Lessard 2000).
Growth and grazing experiments
Growth and ingestion rates of the predators were determined at 15 concentrations of the prey H. triquetra
(50–6,000 cells/ml equivalent to 18–2.1 9 103 ng C/ml).
Prey were added to duplicate 150-ml bottles of known
initial predator concentration (80–300 cells/ml). Duplicate
prey-only controls were established to measure prey
growth. Predators were starved for 2–3 d prior to grazing
experiments to ensure no residual prey remained at the
onset of experiments (this was verified microscopically).
The feeding response of G. spirale on S. marinoi was also
tested. Feeding experiments with S. marinoi were placed
on a rotating wheel, to maintain the nonmotile diatom
lmax ðx x 0 Þ
kl þ ðx x 0 Þ
ð1Þ
where lmax is the maximum growth rate, x is the prey
concentration (cells/ml or ng C/ml), x 0 is the threshold prey
concentration where l = 0, and kl is a constant dictating
the shape of the response as it approaches the asymptote. The numerical response function was fitted to data
using SigmaPlot. For O. marina, a threshold prey concentration was not detected, and thus x 0 was not accounted
for in the numerical response.
Ingestion rates (I, cells predator1 d1 or ng C predator1 d1) were calculated using the equations of Frost
(1972) and Heinbokel (1978) to account for predator
growth. Incubation time used to calculate these rates was
the same as for estimating growth rates. The functional
response model was fitted to ingestion rates at each
concentration using Eq. 2:
I¼
Imax ðxÞ
kI þ ðxÞ
ð2Þ
where Imax is the maximum ingestion rate of the predator,
x is the prey concentration (cells/ml or ng C/ml), and kI is
a constant.
Short-term starvation and re-feed experiments
We tested the capacity of starved predators to exploit a
prey pulse after a 1 to 3-week period of prey withdrawal.
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
3
Poststarvation Feeding in Heterotrophic Dinoflagellates
Predators fed with H. triquetra were transferred to triplicate 500-ml PC bottles, with abundance and biomass
determined daily. Predators were starved for 1–3 wks until
a reduction in cell number and/or cell size was detected,
indicating a negative impact of algal prey withholding on
the predator population. Poststarvation, triplicate predator
cultures were exposed to a pulse of prey by spiking the
starved cultures with H. triquetra or I. galbana. Prey type
offered was chosen for logistical reasons to ensure appropriate distinction between predator and prey size measurements on a MultisizerTM 3 Coulter Counter (version 3.53,
Beckman Coulter, Indianapolis, IN). Oxyrrhis marina and
G. dominans were fed ~7.5 9 104 cells/ml (~1 9 103 ng
C/ml) of I. galbana, which induces positive growth of
O. marina (Kimmance et al. 2006). The larger G. spirale
was fed ~4 9 103 cells/ml (~1.4 9 103 ng C/ml) of H. triquetra, which supports maximum growth (this study).
Both predator and prey-only controls were included in triplicate to quantify predation-independent changes in predator cell abundance and volume. These starved and then
re-fed cultures were subjected to both short-term (min-h)
and long-term (up to 3 d) analysis of predator abundance
and volume. For short-term measurements, cell volume
and abundance of triplicate predator cultures was measured at intervals of 5–30 min for the first 2 h and again
after 3 h (Coulter Counter). The Coulter Counter provided
a more rapid sampling approach than microscopy and
allowed repeat measurements at minute intervals. Microscope samples were taken and preserved throughout the
re-feed experiment to verify Coulter Counter measurements and to image re-fed predators; all images were
taken on a light microscope at 20X magnification. To measure poststarvation daily growth and grazing response,
re-fed heterotroph cultures were incubated for up to 3 d.
Growth and ingestion rates of all three heterotrophs were
measured as described above. Cell volume measurements
were used to determine changes in predator biomass.
Anderson & Menden-Deuer
Table 1. Nonlinear curve fits of the numerical (Eq. 1) and functional
(Eq. 2) response of the three heterotrophic dinoflagellate species (see
Fig. 2)
HTD
Numerical response
Oxyrrhis marina
Gyrodinium spirale
Gyrodinium dominans
Functional response
O. marina
G. spirale
G. dominans
Parameter
Parameter
value
SE
Adjust. r2
lmax
kl
x0
lmax
kl
x0
lmax
kl
x0
0.60
79
NA
0.46
474
188
0.22
191
107
0.03
16
NA
0.07
97
24
0.03
31
17
0.82
Imax
kI
Imax
kI
Imax
kI
2.29
1081
6.06
1208
1.79
1053
0.29
254
1.15
415
0.28
311
0.92
0.71
0.66
0.85
0.89
Standard error (SE) and adjusted r2 are shown for each parameter.
HTD, heterotrophic dinoflagellate; lmax, maximum growth rate (d1);
Imax, maximum ingestion rate (ng C predator1 d1); kl/kI, half-saturation constant of numerical or functional response (ng C/ml); x 0 , threshold prey concentration (ng C/ml).
volume measurements were made on ≥ 24 predator cells
at discrete time points to assess changes in cell size.
Duplicate 350-ml tissue cultures of the same starved
predator stock were included in parallel to allow for visual
inspection of live predator morphology and apparent swimming behavior under a dissecting microscope.
RESULTS
Long-term starvation experiments
Growth rates
The response of each predator species to prolonged starvation—on the order of weeks to months—was tested. To
starve predators, triplicate 2-liter PC bottles were inoculated with predator cultures and fed H. triquetra
(~2 9 103 cells/ml). Starvation for G. spirale and G. dominans began once H. triquetra reached threshold levels at
which predator growth equaled zero (l = 0) as determined
from the numerical response (see Table 1). The no-netgrowth prey threshold concentration corresponded to
188 ng C/ml and 107 ng C/ml for G. spirale and G. dominans, respectively. Oxyrrhis marina consumed all H. triquetra, and starvation experiments began once all prey
was removed, typically within 2 d, but other potential prey
sources, including bacteria remained (see Discussion). The
bottles were maintained in low light to minimize H. triquetra growth. Samples for counts and cell size analysis were
taken every 1–2 d, but after 2–3 wks, frequency
decreased to twice per week. Samples were fixed and
predator abundance was counted microscopically. Cell
When continuously fed, all three heterotrophic dinoflagellate species were able to ingest and achieve positive
growth on H. triquetra (Fig. 2A). Of the dinoflagellate species, O. marina exhibited the highest maximum specific
growth rate of 0.6 d1 at 0.3 9 103 ng C/ml. Oxyrrhis
marina growth rates were positive, even at the lowest
prey concentration, resulting in a good approximation of
the numerical response (Table 1).
The specific growth rate of G. spirale feeding on H. triquetra increased with increasing prey concentration,
reaching a maximum of 0.46 d1 at 1.4 9 103 ng C/ml
(Table 1
and
Fig. 2A).
At
prey
concentrations
< 0.2 9 103 ng C/ml, G. spirale exhibited negative growth.
Remarkably, the magnitude of the negative growth was
proportional to the prey concentration, with near linear
increases in predator growth rate as prey concentration
increased. Gyrodinium dominans had the lowest maximum
growth rate of the three dinoflagellate species when fed
with H. triquetra (0.22 d1), and negative growth rates
4
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
Anderson & Menden-Deuer
Poststarvation Feeding in Heterotrophic Dinoflagellates
Figure 2 Growth and ingestion rates of heterotrophic dinoflagellates continuously-fed Heterocapsa triquetra. (A) Specific growth and (B) ingestion
rates of three heterotrophic dinoflagellate species as a function of prey concentration (cells/ml) and prey biomass (ng C/ml). (A) Growth and (B) ingestion rates at each of up to fifteen prey concentrations were fitted as individual replicates to the numerical and functional response models (Eqs. 1, 2).
Table 2. Comparison of growth (d1) and ingestion (ng C predator1 d1) rates of the dinoflagellates Gyrodinium spirale, Gyrodinium dominans
and Oxyrrhis marina cultured with diverse phytoplankton prey
HTD
G.
G.
G.
G.
G.
G.
G.
G.
O.
O.
O.
O.
spirale
spirale
spirale
spirale
dominans
dominans
dominans
dominans
marina
marina
marina
marina
Prey
lmax
Imax
References
Heterocapsa triquetra (DN)
H. triquetra (DN)
Skeletonema marinoi (DIA)
Prorocentrum minimum (DN)
H. triquetra (DN)
H. triquetra (DN)
P. minimum (DN)
Chattonella antiqua (RA)
H. triquetra (DN)
Heterosigma akashiwo (RA)
Isochrysis galbana (PRM)
Amphidinium carterae (DN)
0.46
0.58
0.20
0.45
0.22
0.27
0.65
0.30
0.60
0.81
0.45
0.66
6.06
7.24
3.90
7.59
1.79
2.75
0.69
1.29
2.29
0.72
3.98
1.58
This study
Hansen (1992)
This study
Kim and Jeong (2004)
This study
Nakamura et al. (1995)
Kim and Jeong (2004)
Nakamura et al. (1992)
This study
Jeong et al. (2003)
Goldman et al. (1989)
Jeong et al. (2001)
For comparison, rates are adjusted to 14.5 °C using Q10 = 2.8 (Hansen et al. 1997).
lmax, maximum growth rate (d1); Imax, maximum ingestion rate (ng C predator1 d1); HTD, heterotrophic dinoflagellate; DN, dinoflagellate; DIA,
diatom; RA, raphidophyte; PRM, prymnesiophyte.
were observed at prey concentrations < 0.07 9 103 ng
C/ml (Fig. 2A). Growth rate-prey abundance relationships
were well-approximated by the numerical model for both
G. dominans and G. spirale (Table 1). Gyrodinium spirale
grew poorly on the chain-forming diatom S. marinoi.
Specific growth rates of G. spirale on S. marinoi were
negative at all prey concentrations except at 1 9 103 ng
C/ml (4.5 9 104 cells/ml), where the predator grew at
0.20 d1 (Table 2).
Ingestion rates
Ingestion rates of O. marina continuously-fed with H. triquetra reached a maximum rate of 2 ng C grazer1 d1
(6 cells grazer1 d1) at 2 9 103 ng C/ml (Fig. 2B). Ingestion rates of O. marina were well approximated by the
functional response model (Table 1).
The functional response of G. dominans was similar to
O. marina and ingestion rates on H. triquetra steadily
increased without saturation (Fig. 2B). Gyrodinium dominans reached a maximum ingestion rate of 1.8 ng C
grazer1 d1 (5 cells grazer1 d1) at 2 9 103 ng C/ml
(Table 1). Ingestion rates of G. spirale on H. triquetra
increased steadily with increasing prey concentration, saturating > 1 9 103 ng C/ml. The maximum ingestion rate
of G. spirale on H. triquetra was 6 ng C grazer1 d1 (17
cells grazer1 d1) (Table 1).
Though G. spirale did not sustain positive growth on
S. marinoi, ingestion was seen at all prey concentrations.
Ingestion rates on S. marinoi increased with increasing
prey concentration, reaching a maximum ingestion rate of
4 ng C grazer1 d1 (155 cells grazer1 d1) (Table 2). This
may indicate that G. spirale was unable to digest ingested
S. marinoi over the sampling duration (3 d) and may have
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
5
Poststarvation Feeding in Heterotrophic Dinoflagellates
Figure 3 Heterotrophic dinoflagellate abundance (cells/ml) as a function of time (d) in starvation experiments where algal prey was either
absent (Oxyrrhis marina) or at sub-threshold prey concentrations at initial sampling. Symbols represent triplicate treatment means 1 standard deviation. Note log scale of y-axis.
required a longer incubation time to acclimate to that food
source.
Starvation
All three dinoflagellate species were able to survive
extended periods of time (at least 18 and up to 118 d) in
the absence of phytoplankton or at very low phytoplankton
prey concentrations (below threshold levels; Fig. 3).
The population abundance of G. spirale decreased by
more than half (59%) in the first 13 d of starvation (Fig. 3).
Gyrodinium spirale biomass (ng C/ml) decreased further
(78%) within 2 weeks of starvation, while biomass
matched abundance changes in the other two dinoflagellate species. The population size of G. dominans
decreased by 76% in the first 12 d, a more pronounced
decrease than G. spirale over the same period (Fig. 3).
Figure 4 Change in cell volume (%) of heterotrophic dinoflagellate
species during the initial starvation period of 3 wks. Error bars of 1
standard deviation are contained within symbols.
6
Anderson & Menden-Deuer
Population abundance of G. dominans decreased steadily
after 2 wks of starvation and remained at cell concentrations 0.5–1 cells/ml for 63 d, while G. spirale remained at
concentrations 0.5–1 cells/ml for 18 d.
Cell volumes of G. dominans decreased substantially
(31%) within 24 h of starvation and even further (57%) after
10 d (Fig. 4). Average cell volume of G. spirale initially
increased after 2 d (10%) but decreased after 6 d (40%).
Live inspection of G. dominans indicated that cells
appeared to swim faster within the first 2 weeks of starvation. In contrast, G. spirale appeared to swim faster
within the first week of starvation, but appeared sluggish
after 10 d. Throughout the starvation period, G. spirale
cells became increasingly deformed in cell shape, often
appearing more elongated.
In contrast with Gyrodinium spp., population size of
O. marina remained stable within the first 15 d of starvation and even increased slightly (10%). After 32 d, cell
abundance of O. marina decreased by 77% from initial
abundance (Fig. 3). Cell abundance continued to gradually
decrease over time and O. marina was able to survive for
118 d (> 1 cells/ml). No apparent changes in O. marina
swimming speed were noticed at the onset of starvation.
Cell volume of O. marina increased after 2 d and
decreased at a slower rate (17%) than Gyrodinium spp.
over the first 15 d (Fig. 4). Oxyrrhis marina and G. dominans became increasingly transparent throughout their
respective starvation periods.
Re-feed short-term cell volume response
Cell volumes of starved O. marina and G. dominans
increased after addition of a single pulse of I. galbana prey
(~7.5 9 104 cells/ml or 1 9 103 ng C/ml) (Fig. 5). Oxyrrhis
marina was re-fed after cultures were starved for 16 d,
while G. dominans was re-fed after 8 d of starvation. Average cell volume of starved O. marina and G. dominans
were similar at the beginning of the re-feed. Average cell
volume of O. marina increased by 9% after 5 min of feeding and increased by > 15% over the next 3 h (Fig. 5A). Cell
volumes of the unfed O. marina control that was kept
starved did not change over the same time period.
Starved G. dominans that received the same prey pulse
as O. marina did not respond as quickly to added prey
compared to O. marina (Fig. 5B). Average cell volume of
G. dominans increased after 45 min of incubation by ~3%
and a total of ~7% after 3 h. Control cultures of G. dominans that were kept under starvation conditions remained
smaller and identical in cell size to those at the initiation of
the re-feed.
The largest increase in G. spirale cell volume (~8%)
occurred within the first 5 min of the short-term re-feed
experiment with a pulse of H. triquetra (~4 9 103 cells/ml
or 1.4 9 103 ng C/ml) after 6 d of starvation (Fig. 5C). This
increase in cell volume was not sustained and fluctuated
over the sampling period. Gyrodinium spirale that were
unfed, showed similar fluctuations in cell volume, and
thus, it was impossible to attribute changes in cell size to
a feeding response.
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
Anderson & Menden-Deuer
Poststarvation Feeding in Heterotrophic Dinoflagellates
Re-feed daily growth and grazing rates
Figure 5 Cell volume (lm3) of three species of heterotrophic
dinoflagellates that were fed poststarvation (solid) or continuously
starved and not re-fed (open). (A) Oxyrrhis marina, (B) Gyrodinium
dominans and (C) Gyrodinium spirale over a 3-h period after addition
of a single pulse of Isochrysis galbana (~7.5 9 104 cells/ml or
1 9 103 ng C/ml; A, B) or Heterocapsa triquetra (4 9 103 cells/ml or
1.4 9 103 ng C/ml; C). Note difference in y-axis ranges due to differences in predator size.
Observed changes in cell volume of previously starved
and then re-fed O. marina and G. dominans were due to
prey ingestion, as revealed by microscope observations
(Fig. 6, 7A, B). Though re-fed G. spirale did not exhibit
measurable increases in cell volume during the short-term
re-feed, microscopy suggested rapid ingestion of H. triquetra (Fig. 7C, D).
Tracking of previously starved and then re-fed predator biomass and abundance over 3 d revealed substantive
increases in predator cell size and thus biomass. Cell volume of all three starved dinoflagellate species continued to
increase after the short-term sampling period (Fig. 8). Oxyrrhis marina reached a maximum cell volume after 48 h, representing a 56% increase from the initial starved predator
cell size. Average cell volume of G. dominans and G. spirale
reached a maximum increase at 51% and 35% respectively
from the initial starved cell volume after 72 h (Fig. 8).
After a pulsed re-feeding, all starved heterotrophic
dinoflagellate species ultimately resumed growth, but
responses were not rapid and predators were unable to
reach the maximum growth rates of continuously-fed cultures over the 3-d period (Fig. 9A). After 24 h, O. marina refed I. galbana did not show positive growth (l = 0.10 d1).
The specific growth rate of O. marina reached 0.04 d1 after
48 h and a substantive, positive growth rate of 0.25 d1 was
measured after 72 h of incubation. Specific growth rate of
G. dominans was similar to that of O. marina after the first
48 h of incubation and G. dominans also reached its maximum growth after 72 h (0.09 d1). Gyrodinium spirale
growth rate remained negative, even after 72 h
(l = 0.16 d1). Growth rates of continuously-fed
O. marina and G. dominans at a similar prey biomass
(~1 9 103 ng C/ml) were ~2-fold higher at 0.55 and
0.18 d1, respectively compared to growth rates of starved
predators (Fig. 9A). The maximum growth rate of continuously-fed G. spirale was > 2-fold higher (0.45 d1) compared
to starved cultures fed an equal amount of H. triquetra.
Unlike growth rates determined from cell abundance,
biomass of starved O. marina and G. dominans quickly
increased after the re-feed (Fig. 9B) because cell volumes
increased substantially prior to cell division. The biomassbased growth rate of O. marina was 0.37 d1 after 24 h
and slightly increased to 0.42 d1 after 72 h. The biomass
growth rate of G. dominans was 0.23 d1 after 24 h and
reached a maximum after 72 h (0.27 d1). In contrast,
growth of G. spirale remained negative after 72 h, irrespective of whether cell abundance or biomass was considered to determine growth (0.05 d1; Fig. 9B). Thus,
although cell volume of the surviving G. spirale increased
over the 3 days upon re-feed, cell abundance decreased
leading to an overall negative population growth rate. This
discrepancy between individual cell sizes and population
abundance points to a noteworthy heterogeneity in the
cell-specific responses to prey exposure, suggesting that
a few cells could feed and increase in volume, but their
numbers were insufficient to support an overall increase
in population abundance.
The maximum ingestion rate of starved O. marina refed I. galbana was 0.3 ng C grazer1 d1 (34 cells
grazer1 d1) after 48 h and ingestion became saturated
after 72 h. Ingestion rates of G. dominans also increased
throughout the re-feed period, reaching a maximum rate
of 0.4 ng C grazer1 d1 (37 cells grazer1 d1) after 72 h.
Ingestion rates of continuously-fed O. marina and
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
7
Poststarvation Feeding in Heterotrophic Dinoflagellates
Anderson & Menden-Deuer
Figure 6 Starved Oxyrrhis marina (A) before and (B–D) after re-feed. (A) O. marina cells after 20 d of starvation. After re-feed (B) 1–3, (C) 4–6
and up to (D) 8–12 prey cells were distinguishable within O. marina, verifying rapid ingestion of prey. Scale bars represent 20 lm.
G. dominans were > 2-fold higher (~1 ng C grazer1 d1)
than starved cultures fed an equivalent prey concentration.
Unlike the other two dinoflagellate species, positive ingestion was not detectable for re-fed G. spirale and thus rates
are not presented.
DISCUSSION
Heterotrophic protists are considered particularly important
to the structure and function of marine food webs. The
growth rates of heterotrophic protists are assumed to be
on par with the growth of their phytoplankton prey, which
may result in considerable biogeochemical impacts (Sherr
and Sherr 2009; Worden et al. 2015). Here we quantified
the maximum specific growth and grazing rates of prominent dinoflagellate predator species in response to
8
fluctuations in the presence and quantity of prey. Our
experiments sought to mimic the conditions in a heterogeneous ocean, with frequent spatial or seasonal fluctuations
in prey abundance and type.
After periods of 1–3 wks, which seem reasonable for
in situ conditions (e.g. between blooms; low winter-time
prey abundances), starvation negatively impacted grazers,
as evidenced by decreases in dinoflagellate abundance,
alterations in cell morphology and delays in predator population growth. This finding of lower growth potential by grazers after starvation has implications for how we view
predator-prey dynamics in a heterogeneous ocean and especially at the onset of seasonal phytoplankton blooms. Heterotrophic dinoflagellates are one of the dominant groups of
phytoplankton grazers and as a result can be potentially
important conduits of carbon transport in marine food webs.
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
Anderson & Menden-Deuer
Poststarvation Feeding in Heterotrophic Dinoflagellates
Figure 7 Starved Gyrodinium spp. before (left panels) and after (right panels) re-feed. Left panels represent (A) Gyrodinium dominans and
(C) Gyrodinium spirale cells after 8 d of starvation. Right panels show ingestion of (B) Isochrysis galbana or (D) Heterocapsa triquetra by Gyrodinium spp. (within 2-h period). Scale bars represent 20 lm. Prey cells are clearly visible within re-fed predators.
Although predator growth rates are commonly assumed
to be on par with that of their prey, we found a significant delay in the resumption of predator growth after
starvation. Poststarvation, all three heterotrophic dinoflagellate species investigated were unable to reach their
maximum daily growth and ingestion rates after 3 d
despite an abundance in suitable prey. Our results confirm the already well-established ability of heterotrophic
dinoflagellates to withstand extended periods of starvation (Hansen 1992; Menden-Deuer et al. 2005; Strom
1991) and report, to our knowledge, the first observations
of significantly delayed predator growth poststarvation.
Starvation-dependent delays in predator growth have
implications for our understanding and forecasting of
predator impacts on primary and export production. Since
fluctuations in prey availability are predictable and detectable by autonomous or remote sensing technology
de et al. 2015), incorporation of starvation effects
(Sauze
of predation on net primary production appears attainable.
Immediate poststarvation ingestion
Predators maintained the ability to immediately ingest and
remove phytoplankton biomass upon re-encountering prey
after starvation. This agrees with the one prior study investigating re-feeding capacity in dinoflagellates, in which cell
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
9
Poststarvation Feeding in Heterotrophic Dinoflagellates
Anderson & Menden-Deuer
Figure 8 Increase in daily cell volume (%) of previously starved heterotrophic dinoflagellate species for 3 d after re-feed with new prey.
Error bars represent 1 standard deviation of triplicates.
volume of re-fed O. marina increased by ~72%, while
G. dominans increased by ~38%, over 10 h (Calbet et al.
2013). In both re-feed studies (Calbet et al. 2013; this
study), rapid increases in cell volume of heterotrophic
dinoflagellates suggested grazers were able to immediately,
within minutes, ingest prey poststarvation. Using photomicrographs, we documented that these rapid cell volume
changes after starvation were indeed a result of the predator ingesting algal prey and could visually observe that
G. spirale is also capable of poststarvation predation,
despite the lack of direct feeding measurements. The ability
of starved heterotrophic dinoflagellates to immediately
ingest encountered prey and increase in biomass implies
cells are not in an inert state after starvation and no transition period to resumption of feeding is necessary.
Lag time for population growth
A predator’s ability to resume cell division after starvation is
critical for survival and has been quantified in a collection of
ciliates (Fenchel 1982, 1989), but not in heterotrophic
dinoflagellates. Thus, we incorporated measurements of
predator population growth rates after starvation to address
this gap in knowledge. Only after 2–3 d did re-fed O. marina
and G. dominans populations show positive growth, while
G. spirale was unable to attain positive growth or measurable ingestion throughout the re-feed period. Even after
3 d, all three grazers were unable to reach their maximum
growth rates attained before prey deprivation.
The observed time lag before commencement of positive population growth may reflect a type of energetic
trade-off between starvation and ability to resume predation, as proposed by Fenchel (1989). When a predator
undergoes starvation, over time metabolic rates decrease,
the extent of this reduction is determined by how long the
predator starves (Fenchel 1989). After 5 d of starvation,
Calbet et al. (2013) recorded decreases in respiration rates
of O. marina and G. dominans strains by up to 70% and
50%, respectively. Heterotrophic dinoflagellates in our
10
Figure 9 Daily specific growth rates of each re-fed heterotrophic
dinoflagellate species over 3 d. Growth rate estimates based on
(A) cell abundance and (B) biomass. Error bars represent 1 standard
deviation of triplicates. Dotted lines in (A) correspond to the maximum
specific growth rate of a continuously-fed predator culture growing at
an equivalent prey biomass. Maximum specific growth rates of continuously-fed predators (dotted lines) are the average of duplicate samples (standard deviation = 0.01–0.02).
study were subjected to longer starvation times (6–16 d)
before new prey was added and thus significant
decreases in respiration and in turn metabolism were
likely to have occurred prior to the re-feed, though no
measurements were made. We did measure reductions in
cell volume and deformations to cell morphology in
starved heterotrophic dinoflagellates, which may have signified a shift in metabolism (Fenchel 1989). Thus, although
grazer cells could remove prey and increase in cell volume
(and overall biomass) immediately after starvation,
increases in predator ingestion and predator population
abundance were limited initially and only materialized after
several days.
Response to prolonged starvation
It has been argued that food particles are rarely exhausted
in the ocean and protist grazers never experience complete
withholding of algal prey as simulated in our study
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
Anderson & Menden-Deuer
€fer et al. 2007). However, phytoplankton are rare
(Paffenho
or absent over wide spatio-temporal scales (McManus
et al. 2003) and grazers have been shown to be selective
in the prey they consume (e.g. Jakobsen and Hansen
1997; Montagnes et al. 2008). Thus, grazers are likely to
be subject to periods of starvation, and foraging and
€nbaum
chemotactic strategies (e.g. Menden-Deuer and Gru
2006; Stoecker et al. 1984) may not be sufficient to overcome periods of starvation. In our study, O. marina and
G. dominans exhibited a tremendous ability to survive
without algal prey, which may offer a competitive advantage over other heterotrophic dinoflagellate species like
G. spirale, and certainly heterotrophic protists with lower
starvation capacity (e.g. ciliates). Grazers that can adapt to
prey fluctuations will be better equipped when a new prey
patch is encountered or when prey abundances increase
seasonally.
All three heterotrophic dinoflagellate species survived for at
least 18 d and up to 118 d in the case of O. marina. Long starvation periods have been observed previously for dinoflagellates (Menden-Deuer et al. 2005; Strom 1991) but not for
ciliates, which die rapidly within hours to days (Jackson and
Berger 1984; Jakobsen and Hansen 1997). Though ciliates
may exhibit faster rates of ingestion and cell division compared to heterotrophic dinoflagellates (Jeong et al. 2010;
Strom and Morello 1998), they are less equipped for long periods of starvation.
Heterotrophic dinoflagellates may allocate part of their
energy into storage products to prolong survival (MendenDeuer et al. 2005). The difference in survival vs. feeding
between these two grazer types may follow an r- vs.
k-strategy, as suggested by Weisse et al. (2016). Generally, heterotrophic dinoflagellates (k-strategy) are slower to
divide, but can survive longer under starvation. In contrast,
ciliates (r-strategy) have fast division times, but are unable
to survive beyond 2–3 d without prey. In order to accurately compare the starvation and feeding response of
these different grazer types and their respective predation
impact on phytoplankton biomass, measures of both feeding and survival ability must be considered.
Extensive periods of algal prey withholding (> 100 d) had
negative impacts on O. marina populations (e.g. severely
diminished abundance and cell volume decrease), which
signified predator cells were in a starved state despite cultures being non-axenic. Though no evidence of bacterial
ingestion was found, it may be reasonable to assume that
starved O. marina were ingesting bacteria, as cells continued to divide during the first 2 wks of starvation. O. marina
does have a slow digestion time (Klein et al. 1986) and
large capacity for food storage which allows cell division of
the organism, even when algal prey is absent (Calbet et al.
2013; Flynn and Mitra 2009; this study). While cannibalism
has been shown to occur in O. marina populations, the
probability even at high predator abundance is low (≤ 2%)
and even less likely as predator abundance decreases
(Montagnes et al. 2010). Moreover, O. marina has been
shown to uptake dissolved organic molecules (Lowe et al.
2010; Roberts et al. 2010) and utilize proteorhodopsins
(Hartz et al. 2011), which may aid in navigation and
Poststarvation Feeding in Heterotrophic Dinoflagellates
metabolism when prey abundance is low. Together, these
adaptations may have aided in the ability of O. marina to
survive months of starvation in culture.
As starvation time increased, all three heterotrophic
dinoflagellate species became increasingly transparent,
especially in the case of O. marina and G. dominans.
These two heterotrophs survived much longer, suggesting
that transparency likely indicates the use of cellular
reserves, a response also seen in starving dinoflagellates
of the genus Protoperidinium (Menden-Deuer et al. 2005).
While no cyst production was observed, there were signs
of swarmer production in the two species of Gyrodinium
as evidenced by decreased cell volumes > 50% and an
appearance of faster swimming cells at the onset of starvation. These morphological changes may represent a
strategy to maximize swimming at the onset of starvation
to help expedite the search for new prey, particularly as
Gyrodinium spp. reside in open waters with patchy prey.
Overall, some generalizations can be made in regards to
the response of heterotrophic dinoflagellates to starvation,
which include the ability to avoid cyst production, become
smaller in size, and more transparent over time.
Responses of dinoflagellates to starvation may be the
result of a strategy to preserve cellular metabolic demands
in the absence of food, enabling long starvation survival
while maintaining the ability to consume prey immediately
upon encounter.
Implications of delayed grazer population growth
A starvation period negatively impacted heterotrophic
dinoflagellate population dynamics, as indicated by the
sustained depression of population growth rates. Pastprey conditions (e.g. starvation; Li et al. 2013) as well as
prey type and strain (Table 2) can have strong influences
on the numerical and functional response of the predator.
Depressed and even negative population growth is contrary to the common assumption that protistan grazers
undergo rapid increases in predator population growth to
match growth of their prey (Sherr et al. 2003). Instead,
the species included here showed initial increases in cell
volume rather than cell division and population increase,
suggesting ingestion and replenishment of storage
reserves may be a strategy to survive future starvation
and may be a preferred strategy over reproduction. Predator population abundance and grazing impact is not
expected to increase rapidly once new prey is encountered after starvation, which could indirectly result in the
enhancement of phytoplankton biomass growth, for
instance at the initiation of a bloom event.
There have been a number of hypotheses as to why
grazing is low at the initiation of a phytoplankton bloom,
relating to predator defenses by the prey (Irigoien et al.
2005; Tillmann 2004), or temperature constraints (Rose
and Caron 2007). Sherr and Sherr (2009) proposed that
low phytoplankton concentrations at the beginning of a
bloom cannot support grazer growth, and predators are in
a continued starved state with low predation impact on
the prey populations, even as conditions favor enhanced
© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists
Journal of Eukaryotic Microbiology 2016, 0, 1–13
11
Poststarvation Feeding in Heterotrophic Dinoflagellates
prey growth. While threshold prey concentrations exist for
predator growth (e.g. Montagnes 1996) and have been
documented in situ (Lessard and Murrell 1998), to our
knowledge, there had been no poststarvation feeding
measurements prior to this study to support this proposed
delay in feeding at the onset of a bloom. The lag time in
grazer population growth observed in this study does support the hypothesis by Sherr and Sherr (2009) and thus
could be a determining factor in driving phytoplankton biomass accumulation rates and ultimately the fate of organic
carbon. Therefore, incorporating measures of growth and
ingestion coupled with an understanding of survival capacity in heterotrophic dinoflagellates will be critical in assessing the impact of predation in a heterogeneous
environment and especially during seasonal or spatial variations in phytoplankton prey availability.
ACKNOWLEDGMENTS
We thank technician Amanda Montalbano and undergraduate
student Cynthia Michaud for assistance with this research.
Comments by David Montagnes improved an earlier version
of this manuscript. This work is based on work supported by
the National Science Foundation award #1142107 through
the Office of Polar Program, National Aeronautics and Space
Administration (award #NNX15AL2G), and in part by the
National Science Foundation EPSCoR Cooperative Agreement (award #EPS-1004057). Research was conducted in
the EPSCoR supported Marine Science Research Facility
(MSRF) at the University of Rhode Island, Graduate School of
Oceanography.
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