Harmful Algae 49 (2015) 1–9
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Harmful Algae
journal homepage: www.elsevier.com/locate/hal
Feeding by heterotrophic protists on the toxic dinoflagellate Ostreopsis
cf. ovata
Yeong Du Yoo a, Hae Jin Jeong b,c,**, Sung Yeon Lee b,*, Eun Young Yoon c, Nam Seon Kang b,
An Suk Lim b, Kyung Ha Lee b, Se Hyeon Jang b, Jae Yeon Park c, Hyung Seop Kim a
a
Department of Marine Biotechnology, Kunsan National University, Kunsan 573-701, Republic of Korea
School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea
Environment, Energy, Resource Institute, Advanced Institutes of Convergence Technology, Seoul National University-Gyeonggi Province, Suwon 443-270,
Republic of Korea
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 4 May 2015
Received in revised form 6 August 2015
Accepted 6 August 2015
Available online
Ostreopsis cf. ovata is a toxic dinoflagellate with a wide distribution, from tropical to temperate waters.
This species is primarily an epiphytic benthic dinoflagellate, but it also lives in the water column and
sometimes forms harmful red tides. The taxonomy, physiology, and distribution of O. cf. ovata have been
extensively investigated, and toxin production by this species is well documented. However, data
regarding potential predators of this toxic dinoflagellate are lacking. In particular, no studies of
heterotrophic protistan grazers have been conducted. To investigate feeding by the heterotrophic
dinoflagellates (HTDs) and a ciliate on O. cf. ovata, whether the common HTDs Gyrodinium dominans,
Gyrodinium moestrupii, Gyrodinium spirale, Oxyrrhis marina, Pfiesteria piscicida, Polykrikos kofoidii,
Protoperidinium bipes, and Stoeckeria algicida and the naked ciliate Strobilidium sp. are able to feed on
O. cf. ovata was tested. In addition, the growth and ingestion rates of G. moestrupii and P. kofoidii on O. cf.
ovata as a function of prey concentration were measured because this prey supported positive growth of
only these two predators. Furthermore, these growth and ingestion rates were compared with those on
the other algal prey for exploring comparative nutritional value of this prey. G. dominans, G. moestrupii,
O. marina, P. piscicida, and P. kofoidii were able to feed on O. cf. ovata; in contrast, G. spirale, P. bipes,
S. algicida, and Strobilidium sp. were unable to feed on this prey species. The maximum growth rates of
G. moestrupii and P. kofoidii on O. cf. ovata were 0.86 and 0.73 day1, respectively, while the maximum
ingestion rates were 6.2 and 33.3 ng C predator1 day1, respectively. The maximum ingestion rates of G.
moestrupii and P. kofoidii on O. cf. ovata were higher than the previously reported values for these two
predators on any other dinoflagellate prey; on the other hand, the maximum specific growth rates of
G. moestrupii and P. kofoidii feeding on O. cf. ovata were intermediate to the previously reported values for
these two predators on any other dinoflagellate prey. With the exception of the small dinoflagellate
Prorocentrum minimum, the maximum swimming speed of O. cf. ovata was lower than that of any other
dinoflagellate prey. The results of the present study suggest that O. cf. ovata is an easily edible prey
because of its slow swimming speed; however, this species is not a nutritional prey for growth of
G. moestrupii or P. kofoidii.
ß 2015 Elsevier B.V. All rights reserved.
Keywords:
Gyrodinium
Harmful algal bloom
Ingestion
Polykrikos
Red tide
Toxin
1. Introduction
Ostreopsis spp. are primarily benthic dinoflagellates with a
diverse range of habitats (Faust et al., 1996; Pocsidio and Dimaano,
* Corresponding author: Tel.: +82 2 880 6746; fax: +82 2 874 9695.
** Corresponding author at: School of Earth and Environmental Sciences, College
of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Tel.: +82 2 880 6746; fax: +82 2 874 9695.
E-mail addresses: [email protected] (H.J. Jeong), [email protected] (S.Y. Lee).
http://dx.doi.org/10.1016/j.hal.2015.08.001
1568-9883/ß 2015 Elsevier B.V. All rights reserved.
2004; Penna et al., 2005; Aligizaki and Nikolaidis, 2006; Kim et al.,
2011; Laza-Martinez et al., 2011). However, they have frequently
been observed in the water column (Nikolaides and MoustakaGouni, 1990; MacKenzie, 1991; Faust et al., 1996; Chang et al.,
2000, 2003; Gallitelli et al., 2005; Totti et al., 2010; Accoroni et al.,
2011; Vila et al., 2012; Gadea et al., 2013; Godrijan et al., 2013; Fani
et al., 2014; Reñé et al., 2014). Gallitelli et al. (2005) observed
Ostreopsis populations blooming with high abundance
(>1000 cells ml1) in the water column off the coast of Bari,
southern Italy.
2
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
Ostreopsis ovata is the most commonly found species in the
genus Ostreopsis. However, there is considerable debate regarding
the taxonomy of O. ovata for the following reasons: (1) strains have
been found in various locations; (2) marked intra-specific variation
exists among these strains; and (3) these strains have a very wide
size range. In contrast, the shape and plate pattern of these strains
show very little variation. Many published literature reports
regarding the morphology of O. ovata lack supporting genetic
characterization. Thus, the name O. cf. ovata has been proposed for
O. ovata (e.g., Penna et al., 2010).
Ostreopsis cf. ovata (previously O. ovata) is a toxic dinoflagellate
that is known to produce diverse toxins such as putative palytoxin
(pPLTX), ovatoxins (OVTXs), and Ostreol A (Ciminiello et al., 2006,
2008, 2012; Hwang et al., 2013). This species has a wide
distribution, from tropical to temperate waters (e.g., Kang et al.,
2013). O. cf. ovata is a primary epiphytic benthic dinoflagellate, but
it also lives in the water column and sometimes forms red tides
(e.g., Guerrini et al., 2010; Totti et al., 2010). Ciminiello et al. (2006)
reported that the abundance of O. ovata during a red tide in the
coastal waters of Genoa, Italy was 1800 cells ml1. Totti et al.
(2010) subsequently reported the occurrence of O. ovata in the
water column at all the stations of the Conero Riviera, located in
the northern Adriatic Sea; during the period of bloom, the
maximum abundance of this species was 25.2 cells ml1 in the
water column and 13,500 cells ml1 in the re-suspended mat.
Ostreopsis spp. is generally known to become abundant when they
are re-suspended because of stormy weather (e.g., Totti et al.,
2010). This variation in cell abundance of a dinoflagellate species is
a function not only of its growth rate, but also of its mortality rate
caused by predation. Therefore, the bloom dynamics of this species
are affected by its predators. The growth dynamics of O. cf. ovata
have been extensively investigated, but data regarding the
potential predators of this toxic dinoflagellate are lacking; in
particular, no studies of heterotrophic protistan grazers have been
conducted (e.g., Pistocchi et al., 2011; Nascimento et al., 2012;
Scalco et al., 2012; Furlan et al., 2013; Tanimoto et al., 2013).
Heterotrophic dinoflagellates (HTDs) and ciliates are major
components of marine ecosystems (Porter et al., 1985;Stoecker
and Capuzoo, 1990; Jeong, 1999; Jeong et al., 2010b; Yoo et al.,
2013a). These organisms are ubiquitous and some genera are
cosmopolitan (Lessard, 1984; Rublee et al., 2004; Jeong et al.,
2010b). Many HTDs and ciliates are known to be effective grazers
on a diverse range of algal prey, and they sometimes control prey
populations (Watras et al., 1985; Jeong and Latz, 1994; Tillmann,
2004; Jeong et al., 2010b, 2014, 2015; Yoo et al., 2010, 2013c).
Some HTD species are known to feed on toxic algal prey; on the
other hand, many HTDs and most ciliates do not feed on toxic algal
prey (Hansen, 1989; Matsuyama et al., 1999; Jeong et al., 2001a,
2003, 2007; Yoo et al., 2013b). Thus, the presence and absence of
algal prey toxins influences feeding by many HTDs and ciliates. The
ability of some species to feed on toxic prey may be an evolutionary
trait.
Recently, Ostreopsis cf. ovata was isolated from the coastal
waters of Jeju Island, Korea. A clonal culture of this strain was
established and was shown to produce the newly described toxin
Ostreol A (Hwang et al., 2013). This toxic dinoflagellate may be a
source of differential feeding by HTDs and ciliates. In the present
study, the feeding capabilities of the common HTDs Gyrodinium
dominans, Gyrodinium moestrupii, Gyrodinium spirale, Oxyrrhis
marina, Pfiesteria piscicida, Polykrikos kofoidii, Protoperidinium
bipes, and Stoeckeria algicida, and the naked ciliate Strobilidium
sp. on O. cf. ovata were investigated. In addition, the growth and
ingestion rates of G. moestrupii and P. kofoidii as a function of prey
concentration were determined. The nutritional value of O. cf.
ovata as prey was also evaluated by comparing these growth and
ingestion rates with those previously reported in the literature for
G. moestrupii and P. kofoidii on other algal prey. Finally, the
swimming speed of O. cf. ovata was determined and compared this
with the data previously reported in the literature for other
dinoflagellate prey.
The findings in the present study provide a basis for
understanding the interactions between Ostreopsis spp. and
heterotrophic protists and the population dynamics of these
species in marine food webs.
2. Materials and methods
2.1. Preparation of experimental organisms
For the isolation and culture of Ostreopsis cf. ovata, macroalgal
samples (Gelidium amansii) were collected by divers at a depth of
ca. 3 m from waters off Chaguido, Jeju Island, Korea. The samples
were collected during May 2008, when the water temperature and
salinity were 18.6 8C and 31.2, respectively. A clonal culture of O. cf.
ovata was established by using two serial single-cell isolations
(GenBank accession number HE793379; Kang et al., 2013). As the
concentration of O. cf. ovata increased, the culture was transferred
to 50-ml, 125-ml, and 500-ml polycarbonate (PC) bottles containing fresh f/2-Si seawater medium. The bottles were filled to
capacity with freshly filtered seawater, capped, and incubated at
20 8C under an illumination intensity of 20 mE m2 s1 with cool
white fluorescent light and a 14-h light:10-h dark cycle. When
dense cultures of O. cf. ovata were obtained, the cells were
transferred at approximately 3-week intervals to new 500-ml PC
bottles containing fresh f/2-Si seawater media before the feeding
experiments were conducted.
For the isolation and culture of the HTD predators Gyrodinium
dominans, Gyrodinium moestrupii, Gyrodinium spirale, Oxyrrhis
marina, Polykrikos kofoidii, Pfiesteria piscicida, Stoeckeria algicida,
and Protoperidinium bipes, plankton samples were collected by
using water samplers, from the coastal waters off Masan,
Saemankeum, Keum River Estuary, Incheon or Shiwha, Korea
during 2001–2010. A clonal culture of each species was established
by using two serial single-cell isolations (Table 1).
For the isolation and culture of the ciliate Strobilidium sp. (cell
length, 30–40 mm), plankton samples were collected by using a
water sampler, from the waters of Shiwha Bay, Korea. The samples
were collected during August 2011, when the water temperature
and salinity were 27.0 8C and 15.0, respectively (Table 1). A clonal
culture of Strobilidium sp. was established by using two serial
single-cell isolations.
The carbon contents of Ostreopsis cf. ovata (2.29 ng C per cell,
n = 50), the investigated HTDs, and the ciliate were estimated from
the cell volume, according to the procedure of Menden-Deuer and
Lessard (2000). The cell volumes of the predators were estimated
using the methods of Kim and Jeong (2004) and Yoon et al. (2012)
for Gyrodinium dominans, Gyrodinium moestrupii, and Gyrodinium
spirale; Jeong et al. (2008) for Oxyrrhis marina; Jeong et al. (2001b)
for Polykrikos kofoidii; Jeong et al. (2007) for Pfiesteria piscicida and
Stoeckeria algicida; Jeong et al. (2004) for Protoperidinium bipes; and
Jeong et al. (2011) for Strobilidium sp.
2.2. Feeding capability
Experiment 1 was designed to examine the feeding capabilities
of Gyrodinium dominans, Gyrodinium moestrupii, Gyrodinium spirale,
Oxyrrhis marina, Pfiesteria piscicida, Polykrikos kofoidii; Protoperidinium bipes, Stoeckeria algicida, and Strobilidium sp. on Ostreopsis
cf. ovata (Table 2).
Approximately, 1.6 105 Ostreopsis cf. ovata cells were added
to each of two 80-ml PC bottles containing Gyrodinium spirale or
Polykrikos kofoidii (100–150 cells ml1). Next, each of the other
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
3
Table 1
Isolation and maintenance conditions for the experimental organisms. Sampling location and time; water temperature (T, 8C); salinity (S, practical salinity units) for isolation;
and prey species and concentrations (cells ml1) for maintenance. HTD, heterotrophic dinoflagellate; CIL, ciliate; PTD, phototrophic dinoflagellate. Feeding capability: Y, able
to feed; N, not able to feed.
Organism
Location
Time
T
S
Prey species
Concentration
Feeding
Gyrodinium dominans (HTD)
Gyrodinium moestrupii (HTD)
Gyrodinium spirale (HTD)
Oxyrrhis marina (HTD)
Polykrikos kofoidii (HTD)
Strobilidium sp. (CIL)
Pfiesteria piscicida (HTD)
Stoeckeria algicida (HTD)
Protoperidinium bipes (HTD)
Ostreopsis cf. ovata (PTD)
Masan Bay
Off Saemankeum
Masan Bay
Keum Estuary
Shiwha Bay
Shiwha Bay
Off Incheon
Masan Bay
Shiwha Bay
Off Jeju
April 2007
October 2009
May 2009
May 2001
March 2010
Aug 2011
July 2005
May 2007
November 2008
March 2008
15.1
21.2
19.7
16.0
20.2
27.0
24.0
20.9
13.0
18.6
33.4
31.0
31.0
27.7
32.2
15.0
25.4
30.1
28.5
31.2
Amphidinium carterae
Alexandrium minutum
Prorocentrum minimum
Amphidinium carterae
Lingulodinium polyedrum
Heterocapsa rotundata
Amphidinium carterae
Heterosigma akashiwo
Skeletonema costatum
30,000–40,000
8000–10,000
20,000–30,000
8000
4000
50,000–60,000
20,000–30,000
30,000
50,000
Y
Y
N
Y
Y
N
Y
N
N
HTDs (1000–5000 cells ml1) and the ciliate (50 cells ml1) were
added (Table 2). In each bottle, the final Ostreopsis prey
concentration was ca. 2000 cells ml1. One control bottle (without
prey) was set up for each experiment. The bottles were placed on a
plankton wheel rotating at 0.9 rpm and were incubated at 20 8C
under an illumination intensity of 20 mE m2 s1 with cool white
fluorescent light and a 14-h light:10-h dark cycle.
After 1 h, 2 h, 6 h, 24 h, and 48 h of incubation, 5-ml aliquots
were removed from each bottle and were transferred into 6-well
plate chambers (or slide glasses). Approximately, 200 cells were
observed under a dissecting microscope (or inverted microscope)
at a magnification of 20–90 (or 100–630) to determine the
feeding capability of each predator on Ostreopsis cf. ovata.
2.3. Growth and ingestion rates
Ostreopsis cf. ovata supported positive growth of only two of the
investigated HTDs, namely, Gyrodinium moestrupii and Polykrikos
kofoidii. Hence, experiments 2 and 3 were designed to determine
the growth and ingestion rates of G. moestrupii and P. kofoidii as a
function of prey concentration (Table 2).
The day before experiments 2 and 3 were conducted, dense
cultures of Gyrodinium moestrupii (or Polykrikos kofoidii) growing
on algal prey were transferred into 500-ml PC bottles containing
two different concentrations of the target prey (ca. 50 cells ml1
and ca. 300 cells ml1). This transfer procedure was conducted to
minimize possible residual growth resulting from the ingestion of
prey during batch culture. The bottles were filled to capacity with
freshly filtered seawater, capped, and placed on plankton wheels
rotating at 0.9 rpm. The cultures were incubated at 20 8C under an
illumination intensity of 20 mE m2 s1 with cool white fluorescent light and a 14-h light:10-h dark cycle. To monitor the
conditions and interaction between G. moestrupii (or P. kofoidii) and
Ostreopsis cf. ovata, the cultures were periodically removed from
the rotating wheels, examined visually through the surface of the
capped bottles by using a dissecting microscope, and then returned
to the rotating wheels. When the target prey cells were no longer
detectable, triplicate 1-ml aliquots from each bottle were counted
under a light microscope to determine the cell concentrations of
G. moestrupii (or P. kofoidii). The cultures were subsequently used
in experiments 2 and 3.
For each experiment, the initial concentrations of Gyrodinium
moestrupii (or Polykrikos kofoidii) and Ostreopsis cf. ovata were
established by using an autopipette to deliver predetermined
volumes of known cell concentrations to the bottles. Triplicate
42-ml PC experimental bottles (containing mixtures of predator
and prey) and triplicate control bottles (prey only) were set up for
each predator–prey combination. In addition, triplicate control
bottles containing only G. moestrupii (or only P. kofoidii) were
established for a single predator concentration. To obtain similar
water conditions, the water of a predator culture was filtered
through a 0.7-mm GF/F filter and added to the prey control bottles
in the same amount as the volume of the predator culture added to
the experimental bottles for each predator–prey combination. All
the bottles were then filled to capacity with freshly filtered
seawater and capped. To determine the actual predator and prey
densities at the start of the experiment, a 5-ml aliquot was
removed from each bottle, fixed with 5% Lugol’s solution, and
examined under a light microscope. The predator and prey
abundance were determined by counting the cells in triplicate
1-ml Sedgewick–Rafter chambers (SRCs). The bottles were refilled
to capacity with freshly filtered seawater, capped, and placed on
rotating wheels under the conditions described above. Dilution of
the cultures via refilling the bottles was considered when
Table 2
Experimental design. The numbers in the prey and predator columns are the initial densities (cells ml1) of prey and predator. The values within parentheses in the predator
column are the predator densities in the control bottles.
Expt. no.
Prey
Species
Density
Species
Density
1
Ostreopsis cf. ovata
2
3
Ostreopsis cf. ovata
Ostreopsis cf. ovata
2000
2000
2000
2000
2000
2000
2000
2000
2000
20, 90, 230, 790, 1970, 4155, 0
40, 110, 290, 1150, 2000, 3690, 0
Gyrodinium dominans
Gyrodinium moestrupii
Gyrodinium spirale
Oxyrrhis marina
Polykrikos kofoidii
Protoperidinium bipes
Pfiesteria piscicida
Stoeckeria algicida
Strombilidium sp.
Gyrodinium moestrupii
Polykrikos kofoidii
1000
1000
150
3000
100
1000
5000
5000
50
7, 12, 21, 30, 45, 107, (130)
4, 7, 6, 12, 13, 32, (25)
Predator
4
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
calculating growth and ingestion rates. After 48 h of incubation, a
10-ml aliquot was removed from each bottle and fixed with 5%
Lugol’s solution. The abundances of G. moestrupii (or P. kofoidii) and
O. cf. ovata were determined by counting all the cells (or where
appropriate >200 cells) in triplicate 1-ml SRCs. Prior to removing
the subsamples, the cultures of G. moestrupii (or P. kofoidii) and
O. cf. ovata were examined visually under a dissecting microscope
as described above.
The specific growth rate of Gyrodinium moestrupii (or Polykrikos
kofoidii), m(day1) was calculated as follows:
mðday1 Þ ¼
½LnðPt =P0 Þ
t
(1)
where P0 and Pt = concentration of G. moestrupii (or P. kofoidii) at
time (t) 0 h and 48 h, respectively.
The calculated growth rate was fitted to a Michaelis–Menten
equation as follows:
m¼
mmax ðx x0 Þ
K GR þ ðx x0 Þ
(2)
where mmax = maximum specific growth rate (day1); x = prey
concentration (cells ml1 or ng C ml1), x0 = threshold prey concentration (i.e., the prey concentration where m = 0), and
KGR = prey concentration sustaining 0.5 mmax. The data were
iteratively fitted to the model by using DeltaGraph1 (Delta Point).
The ingestion and clearance rates were calculated by using the
equations of Frost (1972) and Heinbokel (1978). The incubation
time used to calculate the ingestion and clearance rates was the
same as that used to estimate the growth rate. The ingestion rate
data were fitted to a Michaelis–Menten equation as follows:
IR ¼
Imax ðxÞ
K IR þ ðxÞ
(3)
where Imax = the maximum ingestion rate (cells predator1 day1
or ng C predator1 day1); x = prey concentration (cells ml1 or
ng C ml1), and KIR = the prey concentration sustaining 0.5 Imax.
2.4. Swimming speed
A culture of Ostreopsis cf. ovata (ca. 1000 cells ml1) was
transferred into a 500-ml PC bottle. An aliquot from the bottle was
added to a 50-ml cell culture flask and allowed to acclimate for
30 min. The video camera focused on a single field seen as a circle
in a cell culture flask under a dissecting microscope at 20 8C.
Swimming of O. cf. ovata cells was recorded at a magnification of
40 by using a video analyzing system (Samsung, SV-C660, Seoul,
Korea) and a CCD camera (Hitachi, KP-D20BU, Tokyo, Japan). The
mean and maximum swimming velocities were calculated for all
swimming cells observed during the first 10 min. The average
swimming speed (n = 20) was calculated based on the linear
displacement of cells during 1 s in single-frame playback.
The swimming speed of the mixotrophic dinoflagellate
Gymnodinium impudicum (which was not previously been reported
in the literature) was determined in the same manner as described
above (Table 4).
Fig. 1. Feeding by heterotrophic dinoflagellates on the toxic dinoflagellate Ostreopsis
cf. ovata. (A) An unfed O. cf. ovata cell. (B) Gyrodinium dominans with an ingested
O. cf. ovata cell, (C) Gyrodinium moestrupii with two ingested O. cf. ovata cells, (D)
Oxyrrhis marina with an ingested O. cf. ovata cell, (E) Pfiesteria piscicida (Pp) feeding
on O. cf. ovata (Ov) by using peduncle. (F) Polykrikos kofoidii with several ingested
O. cf. ovata cells. Scale bars are 10 mm. Arrows indicate ingested prey cells. All
photographs were taken with an inverted microscope.
cell. Regarding the predators that were able to feed on O. cf. ovata,
the prey species supported positive growth of G. moestrupii and
P. kofoidii, but not of G. dominans, O. marina, or P. piscicida.
The specific growth rates of Gyrodinium moestrupii on Ostreopsis
cf. ovata increased rapidly with increasing mean prey concentration up to ca. 70 ng C ml1 (30 cells ml1), but became saturated at
higher concentrations (Fig. 2). When the data were fitted to Eq. (2),
the maximum specific growth rate (mmax) of G. moestrupii on O. cf.
ovata was 0.862 day1 (Table 3). The feeding threshold prey
concentration for growth of G. moestrupii (i.e., the concentration at
which the species showed no growth) was 8.5 ng C ml1
(4 cells ml1).
The specific growth rates of Polykrikos kofoidii on Ostreopsis cf.
ovata increased rapidly with increasing mean prey concentration
up to ca. 430 ng C ml1 (190 cells ml1) and then increased
gradually at higher concentrations (Fig. 3). When the data were
3. Results
3.1. Feeding occurrence and growth rate
Gyrodinium dominans, Gyrodinium moestrupii, Oxyrrhis marina,
Pfiesteria piscicida, and Polykrikos kofoidii were able to feed on
Ostreopsis cf. ovata; in contrast, Gyrodinium spirale, Protoperidinium
bipes, Stoeckeria algicida, and Strobilidium sp. were unable to feed
on this prey species (Fig. 1). Furthermore, G. spirale, P. bipes,
S. algicida, and Strobilidium sp. did not try to attack an O. cf. ovata
Fig. 2. Specific growth rates of the heterotrophic dinoflagellate Gyrodinium
moestrupii on Ostreopsis cf. ovata as a function of mean prey concentration (x).
Symbols represent treatment means 1 SE. The curve is fitted by a Michaelis–
Menten equation [Eq. (2)] using all treatments in the experiment. Growth rate (GR,
day1) = 0.862 [(x 8.5)/(26.3 + (x 8.5))], r2 = 0.966.
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
5
Table 3
Growth and grazing data for the heterotrophic dinoflagellates Gyrodinium moestrupii and Polykrikos kofoidii on Ostreopsis cf. ovata. Parameters are for numerical and/or
functional response from Eqs. (2) and (3) as presented in Figs. 2–5. PDV: Predator’s volume (103 mm3). mmax (maximum growth rate, day1), KGR (prey concentration
sustaining 0.5 mmax, ng C ml1), x0 (threshold prey concentration, ng C ml1), Imax (maximum ingestion rate, ng C predator1 day1), KIR (prey concentration sustaining 0.5
Imax, ng C ml1), and Cmax (maximum clearance rate, ml predator1h1).
Figures
Predator
PDV
mmax
KGR
x0
r2
Imax
KIR
r2
Cmax
Figs. 2 and 4
Figs. 3 and 5
Gyrodinium moestrupii
Polykrikos kofoidii
1.2–10.2
57–297
0.862
0.725
26
250
8.5
61.4
0.966
0.969
6.2
33.3
33.0
250.0
0.929
0.975
6.1
6.3
fitted to Eq. (2), the mmax of P. kofoidii on O. cf. ovata was
0.725 day1 (Table 3). The feeding threshold prey concentration for
growth of P. kofoidii was 61.4 ng C ml1 (26 cells ml1).
3.2. Ingestion and clearance rates
The ingestion rates of Gyrodinium moestrupii on Ostreopsis cf.
ovata increased rapidly with increasing mean prey concentration
up to ca. 320 ng C ml1 (140 cells ml1), but became saturated at
higher concentrations (Fig. 4). When the data were fitted to Eq. (3),
the maximum ingestion rate (Imax) of G. moestrupii on O. cf. ovata
was 6.2 ng C predator1 day1 (2.7 cells predator1 day1). The
maximum clearance rate of G. moestrupii on O. cf. ovata was
6.1 ml predator1 h1.
The ingestion rates of Polykrikos kofoidii on Ostreopsis cf. ovata
increased rapidly with increasing mean prey concentration up to
ca. 430 ng C ml1 (190 cells ml1) and then increased gradually at
higher concentrations (Fig. 5). When the data were fitted to Eq. (3),
the Imax of P. kofoidii on O. cf. ovata was 33.3 ng C predator1 day1
(14.5 cells predator1 day1) (Table 3). The maximum clearance
rate of P. kofoidii on O. cf. ovata was 6.3 ml predator1 h1.
3.3. Swimming speed
The average (SE, n = 20) and maximum swimming speeds of
Ostreopsis cf. ovata under the experimental conditions used were
140 16 and 255 mm s1, respectively (Table 4). In addition, the
average (SE, n = 20) and maximum swimming speeds of Gymnodinium impudicum under the experimental conditions used were
328 32 and 684 mm s1, respectively (Table 4).
4. Discussion
4.1. Feeding capability and predator type
Fig. 3. Specific growth rates of the heterotrophic dinoflagellate Polykrikos kofoidii on
Ostreopsis cf. ovata as a function of mean prey concentration (x). Symbols represent
treatment means 1 SE. The curve is fitted by a Michaelis–Menten equation [Eq. (2)]
using all treatments in the experiment. Growth rate (GR, day1) = 0.725 [(x 61.4)/
(250.0 + (x 61.4))], r2 = 0.969.
Fig. 4. Ingestion rates of the heterotrophic dinoflagellate Gyrodinium moestrupii on
Ostreopsis cf. ovata as a function of mean prey concentration (x). Symbols represent
treatment means 1 SE. The curve is fitted by a Michaelis–Menten equation [Eq. (3)]
using all treatments in the experiment. Ingestion rate (IR, ng C predator1 day1) = 6.2
[x/(33.0 + x)], r2 = 0.929.
This study is the first report regarding feeding by heterotrophic
protists on Ostreopsis spp. The common HTDs Gyrodinium
dominans, Gyrodinium moestrupii, Oxyrrhis marina, and Polykrikos
kofoidii were able to feed on Ostreopsis cf. ovata by engulfment,
whereas Pfiesteria piscicida fed by using a peduncle. Thus, these
HTDs should be considered as predators of O. cf. ovata. In contrast,
the other investigated heterotrophic protists, namely, Gyrodinium
spirale, Protoperidinium bipes, Stoeckeria algicida, and Strobilidium
sp., did not feed on O. cf. ovata. G. spirale and Strobilidium sp. are
known to feed on prey by engulfment (Kim and Jeong, 2004; Jeong
Fig. 5. Ingestion rates of the heterotrophic dinoflagellate Polykrikos kofoidii on
Ostreopsis cf. ovata as a function of mean prey concentration (x). Symbols represent
treatment means 1 SE. The curve is fitted by a Michaelis–Menten equation [Eq. (3)]
using all treatments in the experiment. Ingestion rate (IR, ng C predator1 day1) = 33.3
[x/(250.0 + x)], r2 = 0.975.
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
6
Table 4
Comparison in growth and grazing data for Gyrodinium moestrupii (A) and Polykrikos kofoidii (B) on phototrophic dinoflagellate prey. ESD (equivalent spherical diameter, mm),
mmax (maximum growth rate, day1), Imax (maximum ingestion rate, ng C predator1 day1), MSS (maximum swimming speed, mm s1). Rates are corrected to 20 8C using
Q10 = 2.8 (Hansen et al., 1997). T: toxic. NT: non-toxic.
A. Gyrodinium moestrupii predator
Prey species
ESD
Main toxins
mmax
Imax
MSS
Referencea
Ostreopsis cf. ovata, T
Symbiodinium voratum, NT
Prorocentrum minimum, NT
Biecheleria cincta, NT
Alexandrium minutum, T
Karenia brevis, T
Scrippsiella trochoidea, NT
Alexandrium tamarense, T
Alexandrium tamarense, NT
26.4
11.1
12.1
12.2
21.9
22.0
22.8
24.3
24.3
Ostreol A, ovatoxins, putative palytoxin
–
–
–
Gonyautoxin
Brevetoxins
–
Gonyautoxin, saxitoxin
–
0.86
0.11
1.07
0.09
1.60
0.80
1.50
0.68
0.71
6.2
1.5
1.4
0.1
2.6
1.9
3.0
2.1
1.3
255
287
194
691
474
368
348
406
406
(1,
(2,
(3,
(4,
(3,
(3,
(3,
(3,
(3,
8, 11)
12)
7)
12)
5 9)
6, 10)
7)
5, 9)
5)
B. Polykrikos kofoidii predator
Prey species
ESD
Main toxins
mmax
Imax
MSS
Referenceb
Ostreopsis cf. ovata, T
Amphidinium carterae, T
Symbiodinium voratum, NT
Prorocentrum minimum, NT
Biecheleria cincta, NT
Gymnodinium aureolum, NT
Gymnodinium impudicum, NT
Scrippsiella trochoidea, NT
Prorocentrum micans, NT
Ceratium furca, NT
Gymnodinium catenatum, T
Lingulodinium polyedrum, T
26.4
9.8
11.1
12.1
12.2
19.4
17.8
22.8
26.6
29.0
33.9
38.2
Ostreol A, ovatoxins, putative palytoxin,
Amphidinol, carteraol
0.73
0.10*
0.04*
0.03*
0.25*
0.11
0.06*
0.97
0.06*
0.35
1.12
0.83
33.3
255
244
287
194
691
576
684
348
380
403
247
510
(1,
(2,
(3,
(2,
(5,
(4,
(1,
(2,
(2,
(2,
(2,
(2,
Gonyautoxins
Yessotoxins
10.0*
0.6*
2.3*
5.4*
16.6
4.6
9.8
17.1
24.4
15, 16)
6, 12)
17)
7)
17)
8)
2)
7)
7)
9)
10, 14)
8, 11, 13)
a
1. This study; 2. Jeong et al. (2014); 3. Yoo et al. (2013b); 4. Yoo et al. (2013c); 5. Lewis et al. (2006); 6. MacKay et al. (2006); 7. Jeong et al. (1999); 8. Hwang et al. (2013); 9.
Carreto et al. (2001); 10. Pierce et al. (2003); 11. Kang et al. (2013); 12. Our unpublished data.
b
1. This study; 2. Jeong et al. (2001b); 3. Jeong et al. (2014); 4. Yoo et al. (2010); 5. Yoo et al. (2013c); 6. Kamykowski and McCollum (1986); 7. Jeong et al. (1999); 8. Jeong
et al. (2010a); 9. Baek et al. (2009); 10. Fraga et al. (1989); 11. Jeong (1994a); 12. Huang et al. (2009); 13. Paz et al. (2004); 14. Oshima et al. (1993); 15. Hwang et al. (2013); 16.
Kang et al. (2013); 17. Our unpublished data.
*
The maximum value among the mean growth and/or ingestion rates measured at the given prey concentrations.
et al., 2011), whereas P. bipes feeds by using a pallium and
S. algicida feeds by using a peduncle (Jeong et al., 2004, 2005). The
body sizes of P. bipes and S. algicida are similar to that of P. piscicida
(Jeong et al., 2004, 2005, 2006). Therefore, predator feeding
mechanisms and sizes may not influence feeding occurrence by
heterotrophic protists on O. cf. ovata. This prey species is reported
to produce three major toxins (Ciminiello et al., 2006, 2008, 2012;
Hwang et al., 2013). G. dominans, G. moestrupii, O. marina,
P. piscicida, and P. kofoidii are known to feed on toxic prey species
such as Amphidinium carterae, Alexandrium minutum, Alexandrium
tamarense, and Gymnodinium catenatum (Jeong et al., 2001a, 2003,
2006; Yoo et al., 2013b). Thus, these HTDs are likely to possess
enzymes that are involved in detecting, capturing, ingesting, and
digesting toxic dinoflagellates, including O. cf. ovata. However,
S. algicida is known to feed only on the raphidophyte Heterosigma
akashiwo; hence, it may not possess enzymes other than those
involved in feeding on this prey species (Jeong et al., 2005; Lim
et al., 2014). Yoo et al. (2013b) reported that, when incubated with
the toxic dinoflagellate A. minutum, the body of G. spirale dissolved.
In the present study, the body of G. spirale did not dissolve when
incubated with O. cf. ovata; however, G. spirale did not try to attack
O. cf. ovata. Thus, G. spirale may not possess enzymes that are
involved in detecting O. cf. ovata. Jeong et al. (2004) reported that
P. bipes is able to feed on the common diatom Skeletonema
costatum. However, feeding by P. bipes on prey species other than S.
costatum has not previously been reported in the literature. Species
belonging to the genus Protoperidinium are able to feed on prey of
size similar to or larger than themselves (e.g., Jeong, 1994b; Jeong
et al., 1997). Thus, prey size is unlikely to be a critical factor
influencing feeding by predator species belonging to this genus.
The results of the present study suggest that P. bipes does not feed
on dinoflagellates. Several tintinnid and naked ciliate species are
known to ingest toxic dinoflagellates. In contrast, the feeding and
swimming behaviors of other ciliates are deleteriously affected by
toxic dinoflagellates; moreover, ciliates are sometimes killed by
toxic dinoflagellates [reviewed by Turner (2006)]. In the present
study, Strobilidium sp. did not try to attack O. cf. ovata. Therefore,
this ciliate may not possess enzymes involved in detecting O. cf.
ovata. In addition, the presence of O. cf. ovata toxins may be
partially responsible for the absence of attack by this ciliate.
4.2. Growth and ingestion rates
The maximum ingestion rate (Imax) of Gyrodinium moestrupii on
Ostreopsis cf. ovata was the highest among the investigated
dinoflagellate prey species; on the other hand, the maximum
specific growth rate (mmax) of G. moestrupii on O. cf. ovata was
intermediate among the investigated dinoflagellate prey species
(Table 4). O. cf. ovata has the largest body size among the potential
edible prey species for G. moestrupii (Table 4). However, the Imax
value of G. moestrupii on autotrophic/mixotrophic prey was not
significantly correlated with prey size (p > 0.1, linear regression
ANOVA; Fig. 6A). Therefore, the high Imax value of G. moestrupii on
O. cf. ovata may not solely be derived from the large body size of
O. cf. ovata. With the exception of the small dinoflagellate
Prorocentrum minimum, the maximum swimming speed of O. cf.
ovata (255 mm s1) was the lowest among the potential edible
prey species for G. moestrupii (Table 4). Thus, it is likely that
G. moestrupii is able more easily to capture and ingest the large and
slow-swimming prey O. cf. ovata than the other potential edible
dinoflagellate prey; this trait may be responsible for the high Imax
value of this predator on O. cf. ovata. In addition, the mmax value of
G. moestrupii on autotrophic/mixotrophic prey was not significantly correlated either with prey size or with Imax (p > 0.1; Fig. 6B
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
Fig. 6. Maximum ingestion rate (Imax, A) and maximum specific growth rate (mmax,
B) of Gyrodinium moestrupii on autrotrophic/mixotrophic algal prey as a function of
prey size (ESD: equivalent spherical diameter, mm) and the mmax as a function of the
Imax (C) as in Table 4. The p values in (A), (B), and (C) were all p > 0.1 (linear
regression ANOVA). Am, Alexandrium minutum; At (T), Alexandrium tamarense toxic
strain; At (NT), Alexandrium tamarense non-toxic strain; Bc, Biecheleria cincta; Kb,
Karenia brevis; Ost, Ostreopsis cf. ovata; Pm, Prorocentrum minimum; St, Scrippsiella
trochoidea; Sv, Symbiodinium voratum (clade E).
and C). With the exception of the small dinoflagellate Symbiodinium voratum (clade E), the ratio of mmax to Imax obtained for G.
moestrupii on O. cf. ovata was the lowest among the investigated
prey species (Fig. 6C; Table 4). Therefore, it is proposed that the
nutritional value of O. cf. ovata for G. moestrupii is lower than that
of the other investigated prey species, except for S. voratoum.
The Imax value of Polykrikos kofoidii on the autotrophic/
mixotrophic dinoflagellates was positively correlated with prey
size (p < 0.05, linear regression ANOVA; Fig. 7A). However, the Imax
value of P. kofoidii on Ostreopsis cf. ovata was the highest among the
investigated prey species, despite the fact that the body size of O. cf.
ovata was intermediate to those of the potential edible prey species
for P. kofoidii (Table 4). The Imax value of P. kofoidii on O. cf. ovata
was markedly higher than the previously reported Imax values for P.
kofoidii on Prorocentrum micans or Ceratium furca, both of which
have a similar body size to O. cf. ovata. In contrast, the maximum
swimming speed of O. cf. ovata was markedly lower than the
previously reported values for P. micans and C. furca. Therefore, the
high Imax value of P. kofoidii on O. cf. ovata may be partially derived
from the slower swimming speed of O. cf. ovata compared to P.
micans and C. furca. Furthermore, the Imax value of P. kofoidii on O.
cf. ovata was almost two times higher than that of the chainforming Gymnodinium catenatum, despite the similar maximum
swimming speeds of these two prey species (Table 4). It is likely
that P. kofoidii more easily captures and ingests single O. cf. ovata
cells than the chain-forming G. catenatum. In addition, the mmax
value of P. kofoidii on the autotrophic/mixotrophic dinoflagellates
was positively correlated with prey size (p < 0.01, linear regression
ANOVA; Fig. 7B). Thus, the intermediate mmax value of P. kofoidii is
probably derived from the intermediate body size of O. cf. ovata. On
7
Fig. 7. (A) Maximum ingestion rate (Imax) and (B) maximum specific growth rate (mmax)
of Polykrikos kofoidii on autotrophic/mixotrophic algal prey as a function of prey size
(ESD, equivalent spherical diameter, mm). (C) mmax as a function of Imax (see Table 4). (A)
p < 0.05; (B) p < 0.01; (C) p > 0.1 (linear regression, ANOVA). The equations of the linear
regressions were as follows: (A) Imax (ng C predator1 day1) = 0.770 (ESD) 6.31,
r2 = 0.467; (B) mmax (day1) = 0.037 (ESD) 0.449, r2 = 0.580. Ac, Amphidinium
carterae; Bc, Biecheleria cincta; Cf, Ceratium furca; Ga, Gymnodinium aureolum; Gc,
Gymnodinium catenatum; Gi, Gymnodinium impudicum; Lp, Lingulodinium polyedrum;
Ost, Ostreopsis cf. ovata; Pmc, Prorocentrum micans; Pm, Prorocentrum minimum; St,
Scrippsiella trochoidea; Sv, Symbiodinium voratum (clade E).
the other hand, the mmax value of P. kofoidii was not significantly
correlated with the Imax (p > 0.1; Fig. 7C). With the exception of
Gymnodinium impudicum and P. micans, the ratio of mmax to Imax
obtained for P. kofoidii on O. cf. ovata was the lowest among the
investigated prey species (Fig. 7C). Therefore, it is proposed that
the nutritional value of O. cf. ovata for P. kofoidii is lower than that
of the other investigated prey species, except for G. impudicum and
P. micans.
The Imax values of Gyrodinium moestrupii and Polykrikos kofoidii
on the toxic dinoflagellates Alexandrium minutum (which produces
gonyautoxin as the main toxin), Alexandrium tamarense (main
toxins, gonyautoxin and saxitoxin), Gymnodinium catenatum (main
toxin, gonyautoxin), Karenia brevis (main toxin, brevetoxin),
Lingulodinium polyedrum (main toxin, yessotoxin), or Ostreopsis
cf. ovata (main toxins, putative palytoxin, ovatoxin, and Ostreol A)
were not markedly lower than the previously reported literature
values for these predator species on other non-toxic dinoflagellate
prey (Table 4). Therefore, it is proposed that feeding by
G. moestrupii and P. kofoidii is not affected by these diverse toxins
and therefore these predator species may have a potential grazing
impact on populations of toxic prey species in natural environments.
It was impossible to estimate the grazing impact of Gyrodinium
moestrupii and Polykrikos kofoidii on Ostreopsis cf. ovata because
data regarding the abundances of co-occurring O. cf. ovata and
8
Y.D. Yoo et al. / Harmful Algae 49 (2015) 1–9
G. moestrupii or P. kofoidii have not yet been obtained. However,
heterotrophic Gyrodinium spp. and/or Polykrikos spp. have been
observed in the waters of the Adriatic Sea and Balearic Sea, the
Algerian coastal waters in the Mediterranean Sea, and the New
Zealand coastal waters where Ostreopsis spp. has been reported to
occur in the water column (Nikolaides and Moustaka-Gouni, 1990;
MacKenzie, 1991; Chang et al., 2000, 2003; Gallitelli et al., 2005;
Totti et al., 2010; Accoroni et al., 2011; Vila et al., 2012; Gadea et al.,
2013; Godrijan et al., 2013; Fani et al., 2014; Reñé et al., 2014).
Thus, further studies to determine the abundances of co-occurring
O. cf. ovata and P. kofoidii or G. moestrupii in natural environments,
and thereby to estimate their grazing impact on O. cf. ovata, are
required.
Acknowledgements
This paper was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MSIP) (NRF2015-M1A5A1041806), and Management of marine organisms
causing ecological disturbance and harmful effect Program of
Korea Institute of Marine Science and Technology Promotion
(KIMST) award to H.J. Jeong and Developing the method of
converting food wastes to bioenergy using mass cultured marine
protozoa, funded by the Ministry of Oceans and Fisheries, Korea
award to J.Y. Park. [SS]
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