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Eukaryotic Microbiology Society of
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Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Stress-related Responses in Alexandrium tamarense Cells

Exposed to Environmental Changes
Cecile Jauzein & Deana L. Erdner
Marine Science Institute, University of Texas, Port Aransas, Texas

Keywords
Apoptosis; caspase-like enzymes; cell cycle;
dinoflagellate; metacaspases; physico-
chemical challenge; reactive oxygen
species; temporary cyst.
Correspondence
D.L. Erdner, Marine Science Institute,
University of Texas, 750 Channel View
Drive, Port Aransas, Texas 78373, USA
Telephone number: +1 361 749 6719;
FAX number: +1 361 749 6777;
e-mail: derdner@utexas.edu
Received: 12 December 2012; revised
4 April 2013; accepted May 2, 2013.
doi:10.1111/jeu.12065
ABSTRACT
Organisms tend to be sensitive to drastic changes in environmental conditions.
For unicellular microorganisms, variations in physico-chemical conditions are par-
ticularly challenging and may result in acclimation, entrance into quiescence, or
death through necrotic or autocatalytic pathways. This study focuses on the the-
cate dinoflagellate Alexandrium tamarense. Cellular responses to oxidative, ther-
mal, and nutrient stress were characterized using stress indicators, such as
pigment content, efficiency of photosystem II or production of reactive oxygen
species (ROS), as well as hallmarks of apoptosis including activity of caspase-
like enzymes and expression of a metacaspase gene homolog. The formation of
temporary cysts, a survival strategy of short-term quiescence, was also moni-
tored. Cellular responses appeared to depend on multifactorial influences where
type and intensity of stimulus as well as position in cell cycle may act in combi-
nation. Sequences of events observed implicate ROS production as a key deter-
minant of stress-related pathways, playing potential roles in intracellular
signaling, formation of temporary cysts, or cellular damage. Variations observed
in caspase-like activities and metacaspase gene expression did not appear to be
associated with programmed cell death pathways; our results suggest a wider
range of functions for these proteases in phytoplankton cells, including roles in
survival pathways and cell cycle progression.

LIVING organisms have been able to exploit a wide diver-
sity of potential habitats by adapting their metabolism or
cellular structures (Cavicchioli et al. 2011). On shorter time-
scales, organisms tend to be sensitive to drastic changes
in environmental variables, even in a range of conditions
suitable for survival. Variations in physico-chemical condi-
tions are particularly challenging for unicellular microorgan-
isms, which are in perpetual and direct contact with their
surrounding environment. While nutrient depletion can indi-
rectly affect cellular functions through alteration of metabo-
lism, acute changes in temperature, salinity, pH, or
irradiance can, for their part, directly affect biomolecules
and cellular integrity, thereby disrupting cell homeostasis.
In the face of environmental challenge, stress-related
responses of unicellular microalgae may result in acclima-
tion, entrance into quiescence, or death (Bidle and Falkow-
ski 2004; Bravo et al. 2010; Thamatrakoln et al. 2012). At
the molecular level, stress-related responses involve
complex signaling pathways that may overlap under
multi-stress conditions, leading to potential activation of
‘cross-tolerance’ mechanisms (Zuppini et al. 2010).
Environmental stressors, such as nutrient deprivation,
oxidative stress, heat shock, UV exposure, and excessive
salt concentrations, have all been reported to trigger
programmed cell death (PCD) in unicellular phytoplankton
(Moharikar et al. 2006; Segovia et al. 2003; Thamatrakoln
et al. 2012; Vardi et al. 1999; Zuppini et al. 2009, 2010).
While genes and molecular pathways leading to cell
death have been extensively characterized in metazoans
(Kanduc et al. 2002), PCD in microorganisms, including
microalgae, is still poorly understood (Franklin et al.
2006). In metazoans, key executioners of PCD are a fam-
ily of cysteine proteases, named caspases (Nicholson and
Thornberry 1997). Caspases are essential for apoptosis, a
particular type of PCD originally characterized by specific
alterations in cell morphology (Kerr et al. 1972) and
marked by, for instance, DNA fragmentation or inversion
of phosphatidylserine (PS) from the internal to the exter-
nal side of cell membrane (Leist and Nicotera 1997). As
the identification by Uren et al. (2000) of caspase ortho-
logues (paracaspases and metacaspases) in various king-
doms, including fungi and protozoa, characterization of
PCD in microorganisms has received increased attention
in microbiology and phytoplankton ecology (Franklin et al.
2006).
Evolutionary reasons for retention, maintenance, and
activation of cell death-related genes in phytoplankton
genomes are still matter of intense debate (Bidle and

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526 Journal of Eukaryotic Microbiology 2013, 60, 526–538
Jauzein & Erdner
Falkowski 2004; Franklin et al. 2006). Because PCD
results in the complete loss of the organism, cell death
machinery seems to provide a mechanism for negative
selection pressure. During the last decade, hypotheses
defining benefits of PCD at the population scale have dri-
ven most of the research performed on stress and death
pathways in phytoplankton. These concepts include
removal of aging and/or damaged cells, limitation of virus
multiplication, or maximization of genetic fitness of coop-
erating communities (Bidle and Falkowski 2004; Franklin
et al. 2006). In contrast, another hypothesis, put forth by
Bidle and Falkowski (2004), has received much less
attention: the retention of death-related genes because
of their housekeeping and regulatory functions. Recent
studies have brought to light nondeath roles of caspases
in metazoans and metacaspases in plants, fungi, and pro-
tozoa. As noted by Tsiatsiani et al. (2011), “during the
last 10 years, the pendulum has shifted from na€ıve hopes
that metacaspases are responsible for cell death events
and associated caspase-like activities in plants and other
nonmetazoans to well-grounded view that metacaspases
[…] can regulate (similar to caspases) various cell biologi-
cal processes, including PCD.” This idea has just started
to emerge in phytoplankton ecology. Thamatrakoln et al.
(2012) very recently reported the first nondeath role of
death-related genes in phytoplankton, showing their impli-
cation in stress acclimation of Thalassiosira pseudonana
to Fe-limitation.
The aim of this study was to gain insight into
stress-related pathways in the toxic thecate dinoflagellate
Alexandrium tamarense. Cellular responses to different
physico-chemical stressors were investigated, using
cellular stress indicators as well as classical markers of
apoptosis. The formation of temporary cysts, a strategy
that allows A. tamarense cells to survive unfavorable con-
ditions via short-term quiescence (Bravo et al. 2010), was
also monitored. In addition to providing new insights on
regulation and transduction of stress-related pathways in
this phytoplankton species, this study widens our knowl-
edge of the potential nondeath roles of PCD executioners
in protists.
MATERIALS AND METHODS
Cultures maintenance
Two strains were used in this study, CCMP 1493 and
GTM 253, isolated from the China Sea (Bay West of Hong
Kong Island, China) in 1991 and from the east coast of the
USA (Mitchell River, MA, USA) in 1980, respectively. Tem-
perature at their isolation sites ranges from 14 °C to 30 °C
for CCMP 1493 and 1–22 °C for GTM 253. Stock cultures
were nonaxenic and maintained in f/2 medium (minus Si)
(Guillard and Ryther 1962), at 16 °C, under ~100 lmol
photons/m2/s with a 12:12 h light:dark cycle. To avoid any
stress due to turbulence, cultures were maintained with-
out bubbling and agitation was limited to a gentle stirring
every day when samples were taken to check their health
and estimate growth rates.
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Stress-related Responses in A. tamarense
Experimental setup
Cultures in mid-log phase, maintained under optimal
growth conditions, were submitted to physico-chemical
stresses: oxidative stress, heat shock, and nitrogen limita-
tion. Cellular responses were characterized using mea-
sures related to stress and death pathways.
Oxidative stress was induced by addition of H2O2
(0.4 mM final concentration) to an exponentially growing
culture (strain CCMP 1493) split in two 500 ml-replicates.
An aliquot of the mother culture (without H2O2 addition)
was used for control. Cellular responses were monitored
during 6 d. Cell membrane status and caspase-like activi-
ties (caspase 2, 3, 8, and 9) were determined twice a day
during the first 48 h after stress induction (day 0 and day
1), then once a day during the next 4 d (days 2–5). Ecdy-
sis (shedding of thecal plates and flagella leading to cyst
formation, Taylor 1987), was assessed at 10 and 30 min
after H2O2 addition, then on the same sampling frequency
as described above. Cell density was determined on a
daily basis.
Temperature stress was applied to the two strains, GTM
253 and CCMP 1493, by transferring two replicates of expo-
nentially growing cultures from 16 °C to 28 °C (D = +12 °C)
or 32 °C (D = +16 °C). To obtain four cultures in similar
growth conditions, a 1.2 liter culture in exponential phase
was used to inoculate four flasks of 3.5 liter at an initial con-
centration of 340 cells/ml. These cultures were grown for
~1 wk in optimal growth conditions. When the cell density
reached 1,500–3,500 cells/ml, 200 ml were taken for the
estimation of control values, then two cultures were trans-
ferred to 28 °C and the two others were transferred to
32 °C. Cultures were sampled twice during the first 24 h
after stress induction, once during the light phase and once
at the beginning of the dark phase, then once a day during
several days. As GTM 253 cultures reacted strongly to heat
shock, analyses for this strain focused on changes in cell
membrane status, intracellular production of reactive oxy-
gen species (ROS), formation of temporary cysts, and varia-
tions in cell density. The response to temperature stress
was studied in more details in CCMP 1493 cultures: the
same parameters as GTM 253 were monitored, as well as
measurements of caspase 3-like enzyme activity, expres-
sion of metacaspase gene transcripts, cellular chlorophyll a
content, and efficiency of photosystem II (Fv/Fm ratio). The
cell membrane status of CCMP 1493 cells was restricted to
measurements of membrane integrity as the analysis of
changes in membrane asymmetry in response to heat
shock gave inconclusive results for GTM 253.
Nutrient stress consisted of induction of nitrogen starva-
tion in batch cultures of GTM 253. An exponentially grow-
ing stock culture under N-replete conditions was used to
inoculate three 10 liter-flasks filled with 7 liter of modified
f/2 medium. For inoculation, A. tamarense cells were col-
lected by gravity filtration using a 20 lm mesh size net
and were resuspended at an initial concentration of
450 cells/ml. The three 10 liter-flasks allowed the testing
of three nutritive conditions corresponding to initial nitrate
(NO3-) concentrations of 44 lM (f/40), 11 lM (f/160), and
527
Stress-related Responses in A. tamarense
0 lM. Concentration of other nutrients followed f/2 med-
ium composition. Re-suspended cultures were maintained
during 14–20 d, with slight bubbling to avoid any carbon
limitation. Cultures were regularly sampled, with at least
one sampling every 2 d after the NO3- concentration
dropped below 6 lM and until the end of the log phase of
growth. Monitored cytomorphological and physiological
parameters were the same as those analyzed during heat
shock on CCMP 1493 cultures. NO3- concentration in the
culture medium was also measured.
Microscopic observations
Cell abundances were estimated in replicate, from culture
samples preserved in Lugol’s iodine. For each sample, a
minimum of 600 cells were counted under the microscope
using a Sedgewick Rafter counting chamber. Cell concen-
trations were used to calculate net exponential growth
rate (l, d 1) as defined by Guillard (1973).
Three cellular parameters were microscopically
determined by fluorescent labeling: ecdysis, cell mem-
brane status, and ROS production. Results were obtained
from observations of more than 400 cells, in replicate,
using a Sedgewick Rafter chamber under epifluorescent
illumination.
Ecdysis
One drop of formaldehyde and one drop of Calcofluor
(Sigma-Aldrich, St. Louis, MO, USA) were added to 2 ml
samples. The blue labeling of the thecal plates by Calcoflu-
or allowed the enumeration of ecdysal cysts.
Cell membrane status
Loss of membrane asymmetry by the externalization of
PS was assayed using the phospholipid-binding protein
Annexin V. The protocol used was adapted from manu-
facturer recommendations. Alexandrium tamarense cells
were pelleted by centrifugation (2,000 g, 5 min, RT,
room temperature) and resuspended in 100 ll of Annex-
in V buffer (Biolegend, San Diego, CA, USA). Thirty
minute incubations were performed in dark at RT after
addition of 5 ll of Annexin-V-FITC (Biolegend) and
stopped by the addition of 700 ll of Annexin V buffer.
Integrity of the cell membrane was examined using
SYTOX-orange dye (0.5 lM final concentration; Invitro-
gen, Life Technologies, Grand Island, NY, USA), which is
a high-affinity nucleic acid stain that only penetrates cells
with compromised plasma membranes. Observations of
SYTOX-stained cells were performed after 15 min of
dark incubation at RT.
ROS production
Intracellular ROS production was assayed with
carboxy-H2DCFDA (C400, Invitrogen), using a working
probe solution prepared in 100% ethanol and according to
manufacturer’s specifications. Alexandrium tamarense
cells were collected by centrifugation (2,000 g, 5 min, RT)
and resuspended in PBS containing the probe at a final
concentration of 5 lM. Green fluorescence coming from
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
528
Jauzein & Erdner
the oxidation of carboxy-H2DCFDA was monitored after
30 min of incubation in the dark at RT.
Metacaspase gene expression
Samples of 200 ml to 1 liter were collected, containing
3.5 9 105 to 106 cells per sample. Cells were concentrated
by gravity using a 20 lm mesh size net and resuspended
in 5–10 ml of 0.2 lm filtered seawater. Cells were then
pelleted by centrifugation (2,400 g, 7 min, 7 °C), and each
pellet was resuspended in 1 ml of TRIzol reagent (Invitro-
gen) and kept at 80 °C until analysis. Thecae and cellular
membranes were broken by vortexing for 1 min in the
presence of 0.5 mm silica-zirconium beads. Extraction and
washing of total RNA was performed according to the TRI-
zol reagent protocol. mRNA was extracted from total RNA
using the MicroPoly (A) Purist kit (Ambion, Life Techno-
logies, Grand Island, NY, USA), and mRNA was quantified
using RiboGreen (Molecular Probes, Life Technologies,
Grand Island, NY, USA). One lg of mRNA per 20 ll of total
reaction volume was used to generate cDNA using Super-
Script II reverse transcriptase (Invitrogen) according to the
manufacturer’s instructions. Following cDNA synthesis,
RNA was digested by adding 2 ll of RNAse H per 1 lg of
mRNA in the reaction and incubating for 20 min at 37 °C.
The cDNA was used as a template for quantitative real-
time PCR of metacaspase transcript levels; reactions con-
tained 4 ll of cDNA and 1.5 ll of each primer in a total vol-
ume of 25 ll. Reaction conditions included 40 cycles of
primer annealing and DNA replication at 60 °C and 72 °C,
respectively, with 30 s for each step. Quantification was
performed at the end of the extension step. The PCR prim-
ers were designed from the EST sequence of an A. tama-
rense metacaspase (AtMC) homolog (GenBank accession
CF947457, A. tamarense cDNA clone UI-D-GC1-aad-n-06-0-
UI). Threshold cycle (Ct) values were determined by the
software.
To determine the level of expression of the AtMC gene
in each sample, a stock solution of the target sequence
was created from PCR amplicons of A. tamarense cell
lysate using AtMC primers. The AtMC amplicon was
quantified using the Quant-it dsDNA HS kit (Invitrogen)
after purification of PCR products (Ultra Clean PCR Clean-
up DNA purification kit; Mo Bio Laboratories, Carlsbad,
CA, USA). Standard solutions covered a concentration gra-
dient ranging from 102 to 106 copies/reaction.
Activity of caspase-like enzymes
Algal cells were pelleted by centrifugation (2,400 g, 7 min,
7 °C) and resuspended in PBS . Thecae and cellular mem-
branes were broken by vortexing for 1 min in the presence
of 0.5 mm silica-zirconium beads. Cell debris was removed
by centrifugation (14,000 g, 8 min, 6 °C). Measurements of
caspase-like protein activity as well as protein content were
performed using supernatant of cell lysates and commercial
kits based on fluorescence measurements. Caspase 3-like
protein activity was determined from incubation with
DEVD-substrate (Apo-ONEâ Homogeneous Caspase 3/7
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Jauzein & Erdner Stress-related Responses in A. tamarense

assay kit, Promega, Madison, WI, USA); the SensoLyteâ

AFC caspase sampler kit (Anaspec, Fremont, CA, USA) was
used to estimate cleavage rates of AFC-substrates for casp-
ases 8, 9, and 2; protein content of cell lysates was esti-
mated using the Nano-orangeâ protein quantitation kit
(Molecular Probes). All incubations were performed in the
dark, in microplates, following respective manufacturer kit
protocols. For enzymatic activity measurements, kinetic
analysis of substrate cleavage was performed over a 1 h 20
period at 20 min intervals using a fluorescence microplate
reader. Cleavage rates were normalized to total protein con-
centration.

Indicators of photosynthetic health and nutritive

conditions
Culture samples (10–20 ml in replicate) for the determina-
tion of cellular chlorophyll a content were filtered onto
Whatman GF/C filters and extracted in 100% methanol for
24 h at 4 °C. The fluorescence of the extracted pigments
was measured with a Turner Designs 10-AU fluorometer
(Turner Designs, Sunnyvale, CA, USA), and chlorophyll a
concentrations were determined using a calibration curve
and correction for phaeopigments.
The fluorescence-based maximum quantum yield of
photosystem II (Fv/Fm) was determined from estimations
of the initial (Fo) and the maximum (Fm) in vivo fluores-
cence of culture samples dark adapted for 30 min. Fo and
Fm corresponded to fluorescence measurements per-
formed before and 30 s after the addition of 3-(3,4-dichlor-
ophenyl)-1,1-dimethylurea (DCMU), respectively. These
measurements were performed on 3 ml-samples in tripli-
Figure 1 Stress-related responses in Alexandrium tamarense cells
cates, using a Turner Designs 10-AU fluorometer and a
(strain CCMP 1493) exposed to 0.4 mM H2O2. (A) Variations in total
final DCMU concentration of 20 lM. Fv/Fm ratios were
number of ecdysal cysts and cysts labeled with Calcofluor in particu-
calculated as (Fm Fo)/Fm (Vincent 1980).
lar. (B) Variations in cell density and proportion of cells labeled with
During the nutrient stress experiment, the determination
SYTOX and Annexin-V. Data points show average values from two
of nitrate concentrations in the culture medium was based replicate cultures, and vertical lines indicate the range of the data.
on the method of Jones (1984) where nitrate is reduced to
nitrite using cadmium granules. Nitrate concentrations were
derived from absorbance measurements performed at sampling, reaching 66  14% of the total cell number
543 nm with a spectrophotometer, using a calibration curve. after 5 d of exposure (Fig. 1A).
During the first 24 h of H2O2 exposure, the total cell
density increased by 11.8  3.6% (Fig. 1B). Only vegeta-
RESULTS
tive cells could be responsible for this increase, and they
represented 11.7  3.5% of the whole population at the
Oxidative stress
beginning of the dark phase. Thus, these variations
Addition of 0.4 mM H2O2 to the culture medium of expo- resulted in a cell division rate of vegetative cells higher
nentially growing cells of A. tamarense (strain CCMP than 0.63  0.09 d 1, i.e. more than two times, the
1493, l = 0.30 d 1) caused rapid and acute ecdysis. After growth rate in exponential phase. Cell death started to
less than 2 h of exposure to H2O2, ecdysal cysts already occur after 24 h of H2O2 exposure, as shown by the
represented 60.5  1.4% of the total number of cells, and increase in the proportion of SYTOX-positive cells and a
their proportion reached 94.3  1.4% after 24 h (Fig. 1A). slight decrease in cell density over the next 4 d (Fig. 1B).
These cysts were initially identified by their round shape After 5 d, 75.7  1.6% of A. tamarense cells were con-
and an absence of thecal plates (Fig. 2). They showed a sidered dead: 24.6  3.5% were lysed and 67.7  1.6%
progressive labeling of their pellicle with Calcofluor, easily of the remaining cells showed a permeable membrane
differentiated from labeling of vegetative cell theca where (SYTOX-positive cells, Fig. 1B). A concomitant increase in
sutures between plates appeared bright blue (Fig. 2). The the percentage of Annexin-V positive cells was observed
proportion of blue labeled cysts visible under the epifluo- with time, but this proportion remained lower than and
rescent microscope increased with time from the 24 h subsequent to membrane permeabilization (Fig. 1B).

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Journal of Eukaryotic Microbiology 2013, 60, 526–538 529
Stress-related Responses in A. tamarense
Figure 2 Microscopic observations of Alexandrium tamarense cells
(strain CCMP 1493) labeled with Calcofluor. (A) Chain of two vegeta-
tive cells with thecal plates labeled. (B) Figure of excystment where
the pellicle of the ecdysal cyst was labeled.
The monitoring of caspase-like activities revealed a
background activity for each of the four substrates tested.
No oxidative stress specific induction of caspase-like activ-
ity could be observed. The background activity level was
maintained during 36 h for caspases 8- and 9-like enzymes
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
530
Jauzein & Erdner
and 48 h for caspases 2- and 3-like enzymes before
decreasing (data not shown).
Temperature stress
Heat shock (increase of 12 °C and 16 °C) tested on two
strains (GTM 253 and CCMP 1493) induced responses
ranging from crash of cultures to recovery over a few
days. Experiments used exponentially growing cultures,
characterized by respective growth rates of 0.20  0.03
and 0.24  0.04 d 1 for the strains GTM 253 and CCMP
1493.
For the GTM 253 strain, temperature stress did not
induce ecdysis but resulted in acute oxidative stress
(Fig. 3A, B). Two hours after the heat shock, all the cells
transferred from 16 °C to 32 °C (D = + 16 °C) showed an
intracellular accumulation of ROS (Fig. 3A), while ROS pro-
duction was detected in 30.3  1.3% of the cells trans-
ferred to 28 °C (D = + 12 °C) (Fig. 3B). Oxidative stress
was apparent within the whole population at 28 °C in
28 h. Exposure to 32 °C (D = + 16 °C) induced cata-
strophic mortality. All of the cells displayed permeable
membranes (SYTOX-positive) after 14 h of treatment, and
29.1  6.4% of the cells were lysed in 24 h under these
stressful conditions (Fig. 3C). Exposure to 28 °C
(D = + 12 °C) also induced death pathways: although a
decrease of only 4.3  2.7% of the total cell density was
observed over 2 d, two thirds (66.6  1.3%) of the cells
showed compromised membrane integrity after 28 h
(Fig. 3D). As observed with oxidative stress, a concomi-
tant increase in the percentage of Annexin-V positive cells
was observed (Fig. 3C, D), but it remained lower than and
subsequent to SYTOX staining.
For the strain CCMP 1493, a transfer of cells from
16 °C to 32 °C (D = + 16 °C) resulted in stress and mortal-
ity but over a longer timescale than responses observed
for GTM 253. In contrast to strain GTM 253, heat shock
induced a low level of ecdysis in CCMP 1493 cultures dur-
ing the first 40 h, after which the proportion of ecdysal
cysts stabilized around 10–20% of total cells (Fig. 4A).
Production of ROS increased within the population over
the 4 d following heat shock (Fig. 4A). More than half of
the original cells were still viable 96 h after heat shock,
when one third (31.1  6.2%) of the remaining cells had a
permeable membrane (SYTOX-positive) and 19.1  5.0%
of the initial population had been lysed (Fig. 4C). Negative
impacts on photosynthetic abilities started to be observed
after 48 h under these stressful conditions, as shown by a
decrease in both chlorophyll a content per cell and Fv/Fm
(Fig. 4E).
A transfer of CCMP 1493 cells from 16 °C to 28 °C also
resulted in low-level ecdysis (Fig. 4B). This heat shock of
12 °C, however, only halted growth of this strain tempo-
rarily and allowed for a recovery after 48 h. Indeed, no
increase in the proportion of SYTOX-positive cells was
observed and, although cell concentration remained con-
stant during 48 h, cell density then increased again at a
growth rate of 0.22  0.04 d 1 (Fig. 4D). Furthermore,
only a part of the population (up to 33.4  6.1%) showed
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Jauzein & Erdner Stress-related Responses in A. tamarense

Figure 3 Stress-related responses in Alexandrium tamarense cells (strain GTM 253) transferred from 16 °C to 32 °C (A, C) and 28 °C (B, D). (A,
B) Variations in proportion of ecdysal cysts, cysts labeled with Calcofluor and cells showing overproduction of reactive oxygen species (ROS). (C,
D) Variations in cell density and proportion of cells labeled with SYTOX and Annexin-V. Data points show average values obtained from two repli-
cate cultures, and vertical lines indicate the range of the data.

an intracellular accumulation of ROS in response to heat
shock (Fig. 4B). No modification in chlorophyll a content
per cell or Fv/Fm was observed over the course of the
experiment (Fig. 4F).
Both heat shock conditions tested (increase of 12 °C or
16 °C) induced a similar pattern of variations of caspase
3-like activity for CCMP 1493 cells (Fig. 4G, H). As
reported above for oxidative stress, a background level of
caspase 3-like activity can be detected during growth of
A. tamarense (Fig. 4H). No activation of caspase 3-like
activity was observed during cell death processes induced
by heat shock (Fig. 4G). Peak values of enzyme activity
were recorded during the 4 d of monitoring, but they
were likely associated with the diel cycle; peak values cor-
responded to samples collected during the dark phase.
Metacaspase gene expression was not correlated with
caspase 3-like enzyme activity (Fig. 4G, H). A slight over-
expression of the metacaspase gene occurred during cell
death processes (Fig. 4G), while a net increase in meta-
caspase gene expression was measured in the cultures
transferred to 28 °C (Fig. 4H).
Nutrient stress
Cellular responses of A. tamarense to nutrient stress were
studied through time during NO3- depletion. The three cul-
tures monitored simultaneously allowed for successive
exhaustion of the initial NO3- pool; the initial concentra-
tions obtained after addition of 0, 11 and 44 lM NO3-
were exhausted in 3, 6, and 8 d, respectively (Fig. 5). In
all cultures, chlorophyll a concentration per cell declined
sharply from the day NO3- became exhausted in the med-
ium (Fig. 5), decreasing by half in about 5 d. Fv/Fm did not
reflect variations in nutrient conditions as values remained
high and stable throughout the experiment, averaging 0.64
( 0.03 SD), 0.66 ( 0.03 SD), and 0.65 ( 0.02 SD) for
the 0, 11 and 44 lM NO3- cultures, respectively (data not
shown).

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Journal of Eukaryotic Microbiology 2013, 60, 526–538 531
Stress-related Responses in A. tamarense Jauzein & Erdner

Figure 4 Stress-related responses in Alexandrium tamarense cells (strain CCMP 1493) transferred from 16 °C to 32 °C (A, C, E, G) and 28 °C (B,
D, F, H). (A, B) Variations in the proportion of ecdysal cysts, cysts labeled with Calcofluor and cells showing an overproduction of reactive oxygen
species (ROS). (C, D) Variations in cell density and proportion of cells labeled with SYTOX. (E, F) Variations in chlorophyll a cellular content and in
Fv/Fm ratio. (G, H) Variations in caspase 3-like activity and level of expression of metacaspase gene homolog. Data points show average values
obtained from two replicate cultures, and vertical lines indicate the range of the data.

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532 Journal of Eukaryotic Microbiology 2013, 60, 526–538
Jauzein & Erdner
Figure 5 Stress-related responses in Alexandrium tamarense cells
(strain GTM 253) during nitrate depletion. Variations in cell density,
chlorophyll a cellular content and nitrate concentration in culture med-
ium after resuspension of cells in medium containing 0 lM (A),
11 lM (B) and 44 lM (C) of nitrate. Data points show the average of
multiple measurements. A solid arrow shows when nitrate became
depleted in culture medium, i.e. when nitrate concentration dropped
under the detection limit. A dashed arrow indicates entrance into sta-
tionary phase, when cell density reached a plateau.
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Stress-related Responses in A. tamarense
The graded nutrient conditions tested allowed for sev-
eral cell division cycles, even in the culture where no
NO3- was initially added. The initial N-limitation gradient
was not reflected in the growth rate estimated during
the log phase (average value of 0.27  0.04 d 1 between
the three cultures) but in the maximal cell density
reached at the stationary phase (Fig. 5). Maximal cell
densities were 4.0, 5.5, and 9.0 times higher than the
starting density in the 0, 11 and 44 lM NO3- cultures,
respectively. No induction of mortality was noted over
the course of the experiment as the percentage of
SYTOX-positive cells remained below 2% in the three
conditions tested (data not shown). Only a slight increase
in the proportion of ecdysal cysts was observed with
time, and percentages remained below 7% even when
cultures reached stationary phase (data not shown).
These changes, as well as acute variations in pigment
content, were not driven by a production of ROS; no
trend was noted in the proportion of cells showing an
exacerbated ROS production and average values of 5.8%
( 2.6 SD), 4.8% ( 1.2 SD), and 6.9 ( 1.8 SD) were
obtained for the 0, 11 and 44 lM NO3- cultures, respec-
tively (data not shown).
Concerning apoptosis indicators, no induction of PS
inversion was observed during NO3- depletion as the per-
centage of Annexin-V-positive cells remained below 1.4%
in the three conditions tested (data not shown). However,
caspase enzyme activity and metacaspase transcript levels
did vary over the course of the experiment (Fig. 6). Varia-
tions were on the order of two to threefold and were
coherent between the three cultures. They consisted of
an increase in metacaspase transcript levels at the point
when specific growth rate began to decline, followed by a
decrease few days later. A concomitant increase in cas-
pase 3-like enzyme activity was observed for the 0 and
11 lM NO3- cultures. In the 44 lM NO3- culture, the
enzyme activity increased a few days after the increase in
metacaspase transcripts, then decreased sharply when
the culture entered stationary phase.
DISCUSSION
Several ways to respond to stress: influence of
intrinsic and extrinsic factors
Exposure of A. tamarense cells to oxidative, temperature
and nutrient challenges required highly stressful conditions
to induce cell death. Tolerance of this species to these
potential stressors revealed various strategies for coping
with sublethal conditions, including strain-specific
responses to the same stress. For instance, a similar stim-
ulus (temperature increase of 12 °C or 16 °C) resulted in
death of one A. tamarense strain, but recovery of the
other. Differences between strains regarding temperature
tolerance may be related to the existence of ecotypes,
adapted to environmental conditions prevailing in their ori-
ginal habitat (Kobiyama et al. 2010). Indeed, GTM 253
appeared to be more sensitive to high temperature
533
Stress-related Responses in A. tamarense
Figure 6 Stress-related responses in Alexandrium tamarense cells
(strain GTM 253) during nitrate depletion. Variations in caspase 3-like
activity and level of expression of metacaspase gene homolog in
resuspended cultures with initial nitrate concentrations of 0 lM (A),
11 lM (B) and 44 lM (C). Data points show the average of multiple
measurements. A solid arrow shows when nitrate became depleted
in culture medium, i.e. when nitrate concentration dropped under the
detection limit. A dashed arrow indicates entrance into stationary
phase, when cell density reached a plateau.
exposure (above 28 °C) than CCMP 1493; temperatures at
their isolation sites range from 1 °C to 22 °C and 14 °C to
30 °C, respectively. The response of A. tamarense cells to
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
534
Jauzein & Erdner
different physico-chemical challenges appears to depend
on both extrinsic and intrinsic factors.
ROS production as a key determinant of stress
response
Exposure of A. tamarense cells to an abrupt temperature
increase induced rapid and widespread ROS generation.
Over-production of ROS is commonly detected in marine
organisms exposed to various environmental stressors
such as heat shock or high levels of visible and ultraviolet
radiation (Lesser 2012). An excess of intracellular ROS can
irreversibly alter lipids, proteins, carbohydrates, and DNA,
and may ultimately result in cell death (Lesser 2012).
Although ROS are obvious agents of cellular damage, it
has also been shown that ROS can directly mediate stress
signaling in various organisms (Lesser 2012). For instance,
heat shock proteins (e.g., Hsp70) are involved in early
sensing of H2O2 production and transduction of thermal
shock signals in plants (Suzuki and Mittler 2006). Identifi-
cation of Hsp70 has recently been reported in A. tama-
rense (Kobiyama et al. 2010), suggesting the existence of
similar processes in this species. It is not known what
proportion of ROS was spontaneously generated as a
direct result of temperature stress vs. actively produced
for the specific purpose of signaling. Nonetheless, it has
been suggested that these two different pathways of
ROS production occur with different timing and subcellular
localizations (Suzuki and Mittler 2006).
Our findings show that sequences of events occurring
during temperature stress are largely correlated with inten-
sity of ROS production, supporting the hypothesis of a del-
icate balance between intracellular roles in signaling or
destruction (Suzuki and Mittler 2006). Indeed, amounts of
ROS detected during heat shock experiments were corre-
lated with three main sequences of events: (i) (CCMP
1493–28 °C) moderate and temporary increase in ROS
production; low level of ecdysal cyst formation; cells
recovered after 2 d without induction of death or irrevers-
ible damage on photosynthesis apparatus; (ii) (CCMP 1493
– 32 °C) strong and steady increase in ROS production;
reduced photosynthetic function; formation of pellicle
cysts or death; (iii) (GTM 253 – 28 °C and 32 °C) intense
and rapid ROS production; cells were committed to die,
skipping cyst formation. Comparable gradual and dose-
dependent sequences of events were reported by Haberk-
orn et al. (2011) for Alexandrium minutum cells exposed
to H2O2, with cellular responses ranging from cyst forma-
tion to death without encystment. Formation of cysts has
also been observed during H2O2 exposure performed on
A. tamarense cells in present work.
While most recent studies reporting ROS production in
microalgae focus on implication of ROS in PCD pathways
(Ding et al. 2012; Thamatrakoln et al. 2012; Zuppini et al.
2010), our experiments allow for a detailed characterization
of an alternative role of ROS for thecate dinoflagellate: the
formation of pellicle cysts after ecdysis. Surprisingly, these
cysts became labeled with Calcofluor after several hours
under oxidative stress, suggesting the development of a
cellulose-like layer in the pellicle. Synthesis of layers
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Jauzein & Erdner
containing cellulose or cellulose-like compounds in the cyst
wall has been described for some dinoflagellate species
(Okuda and Sekida 2007) and in particular for Alexandrium
ostenfeldii (Jensen and Moestrup 1997), but never for A.
tamarense. Pellicle cysts formed asexually by A. tama-
rense are usually identified according to descriptions of
Anderson and Wall (1978), where the cyst wall is thin, un-
ornamented and lacking cellulose. Formation of pellicle
cysts by A. tamarense was recently reported in situ
(Angles et al. 2012; species referred as Alexandrium fundy-
ense). Field observations are, however, required to assess
if multi-layered pellicle cysts, synthesizing a cellulose-like
layer, are laboratory artifacts due to harsh conditions or if
they occur in nature. Lastly, our findings showed that ecdy-
sal cysts formed under recent H2O2 exposure quickly
reverted to motile cells when transferred to fresh f/2 med-
ium (data not shown), suggesting that they are ‘temporary
cysts’ (Bravo et al. 2010), a quiescent form of A. tama-
rense life cycle.
Implication of cell cycle stage in stress responses
Formation of temporary cysts during thermal stress (only
observed for the strain CCMP 1493) solely occurred during
the first 40 h of exposure, suggesting that quiescence is a
transient response to thermal stress. After the number of
cysts reached a plateau, two different cell fates were
observed, depending on stress intensity: proliferation
restarted in cells exposed to moderate thermal stress
while cell mortality was observed in response to high tem-
perature stress. These sequences of events implicate
temporary encystment as a way to avoid stressful condi-
tions by entering into quiescent stage, as reported for A.
tamarense in response to different environmental stres-
sors (Bravo et al. 2010). Interestingly, the temporal win-
dow during which ecdysis occurred (40 h) was close to
the time needed for the whole population to go through a
complete cell cycle (2 d for an unsynchronized population
growing at 0.3 d 1). The position in the cell cycle when
stress-related responses are activated might then be a
factor in the “choice” between quiescence and death/
growth responses.
In this study, although most of cells exposed to H2O2
entered into quiescence in a few hours, the remaining
vegetative cells still underwent cell division during the
dark phase, with a surprisingly high specific division rate
(≥ 0.63 d 1) very close to one division per cell (0.69 d 1).
Cell division of A. tamarense is known to happen during
the dark phase, with G1/S (replication of organelles/synthe-
sis of DNA) and G2/M (second growth phase/mitosis) tran-
sitions occurring at the beginning and middle of the dark
phase, respectively (Taroncher-Oldenburg et al. 1997).
This further suggests an influence of cell cycle position on
A. tamarense stress responses, with postponement of
ecdysis for cells already committed to cell division. In
eukaryotic cells, a “point of no return” or restriction point
exists during the G1/S transition, beyond which the cell is
committed to divide. The restriction point is under control
of cyclin-dependent kinases (cdk) bound to their specific
regulatory subunits (cyclins) (Blagosklonny and Pardee
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Stress-related Responses in A. tamarense
2002). There is growing evidence that dinoflagellate cell
cycle progression is mediated by universal cell cycle regu-
lators, as homologues of cdk and cyclins have been
described in dinoflagellate species such as Karenia brevis
(Barbier et al. 2003; Van Dolah and Leighfield 1999). Exis-
tence of restriction point(s) in the A. tamarense cell cycle
is therefore highly conceivable. Our findings suggest that
A. tamarense stress responses depend on multi-factorial
influences, where extrinsic and intrinsic factors, such as
type and intensity of stimulus as well as position in cell
cycle, may act in combination.
Non apoptosis-related detection of usual PCD
hallmarks
Annexin-V staining of PS is one of the commonly used
hallmarks for PCD determination (Leist and Nicotera
1997). This labeling allows for the tracking of an early sign
of apoptosis: the loss of membrane asymmetry by translo-
cation of PS from the inner- to the outer-part of the
plasma membrane, which is thought to precede the break-
down in membrane integrity. Annexin-V stained cells of A.
tamarense were observed in the present work. However,
their proportion was always lower or similar to the propor-
tion of SYTOX-positive cells, suggesting that occurrence
of Annexin-V staining lagged loss of membrane integrity.
Our results mirror those of recent studies reporting
Annexin-V staining in phytoplankton (Moharikar et al.
2006; Segovia and Berges 2009; Thamatrakoln et al.
2012). We hypothesize that they do not support occur-
rence of PCD but are more likely associated with Annexin-
V binding to the inside of the cell membrane, without PS
translocation. Thus, Annexin-V staining does not appear to
be a reliable indicator of PCD in phytoplankton cells.
Increases in metacaspase gene expression and caspase
3-like activity were observed during nutrient challenge.
NO3- depletion did not, however, activate either death or
quiescence pathways. Actual induction of N-stress was
supported by the drop in cellular content of the N-rich
photosynthetic pigment chlorophyll a, even though Fv/Fm
ratio remained high and stable during the course of exper-
iments. Franklin et al. (2009) similarly noticed that high
values of photosystem II efficiency do not always reflect
presence of photosynthetically active cells and can be
even consistent with the presence of a large fraction of
quiescent or dead cells. In this study, part of the varia-
tions in expression of metacaspase transcripts and cas-
pase-like activity appear to be independent of cell death
and therefore may be related to other factors, as dis-
cussed below.
Multifunctionality of metacaspases and caspase-like
proteases
While progress has been recently made towards the identi-
fication of proteases responsible for caspase-like activities
in plants, these proteases are still unknown in microalgae
and are unlikely to be metacaspases (Bonneau et al. 2008).
Indeed, several publications have now demonstrated that
plant metacaspases are unable to cleave caspase-specific
535
Stress-related Responses in A. tamarense
substrates, and that their enzymatic activity is not affected
by caspase inhibitors (He et al. 2008; Vercammen et al.
2007). Such advanced results have not been obtained for
phytoplankton species, but Thamatrakoln et al. (2012) have
recently proposed several gene candidates for caspase-like
proteases in the diatom T. pseudonana. In this study, no
correlation was found between temporal variations in meta-
caspase gene expression and caspase-3-like activity for A.
tamarense cells exposed to heat shock and nutrient stress.
These results support the hypothesis that metacaspases in
unicellular organisms are not functional homologs of animal
caspases.
Metacaspases, as caspase-like proteases, can be
involved in the regulation of cell death directly, as execu-
tioner of cell death machinery, or indirectly by signaling
cascades leading to death (Segovia and Berges 2009; Tsia-
tsiani et al. 2011). In our experiments, an increase meta-
caspase transcript level in A. tamarense cells was
observed when density of cultures or nutrient availability
resulted in a decrease in growth rate, suggesting involve-
ment of metacaspases in the aging processes of A. tama-
rense cultures. This is concordant with the results of
Thamatrakoln et al. (2012), who reported greater expres-
sion of metacaspase genes in exponential vs. late expo-
nential cells of the diatom T. pseudonana. An increase in
metacaspase gene expression during logarithmic growth
when compared to stationary phase cultures has also
been reported for LmjMCA in the protozoan Leishmania
major (Ambit et al. 2008). Studies in yeast (Lee et al.
2010) and fungi (Richie et al. 2007) suggest that involve-
ment of metacaspases in aging processes during active
growth may be related, among other activities, to their
role in regulation of protein aggregates. Indeed, progres-
sive accumulation of damaged protein aggregates eventu-
ally contributes to cellular dysfunction and senescence
(Martinez-Vicente et al. 2005).
Different caspase-specific substrates (corresponding to
caspase-2, -3, -8 and -9) were tested in this study and a
baseline protease activity was observed for each of them.
The activity of caspase-3-like proteases was examined
more closely than others and revealed maintenance of this
background activity during exponential growth of A. tama-
rense. A similar constitutive activity of caspase 3-like prote-
ases has recently been described during exponential
growth of two other toxic dinoflagellates, K. brevis and
Karenia mikimotoi (Bouchard and Purdie 2011). This back-
ground activity could be responsible for the maintenance of
a low lysis rate in actively growing populations and/or it sup-
ports a role for these proteases in housekeeping functions,
as hypothesized by Segovia et al. (2003) for protein turn-
over. Housekeeping functions for caspase-like enzymes in
microalgal cell are supported by recent reports of nondeath
roles of caspases in metazoans, including involvement in
survival pathways and cell proliferation (Algeciras-Schimnich
et al. 2002; Launay et al. 2005). In this study, a potential
role of caspase 3-like protease in cell proliferation could
explain diel variations in enzyme activity observed during
thermal challenge of strain CCMP 1493: increases were
noted during the dark phase, when cell division occurs
© 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists
536
Jauzein & Erdner
(Taroncher-Oldenburg et al. 1997). In metazoans, a role of
caspases in cell division regulation could rely on cleavage of
negative regulators preventing cell cycle progression (Algec-
iras-Schimnich et al. 2002). Activation of caspase 3-like
enzyme was also observed along a growth curve, before
the entrance into stationary phase, during the nutrient
stress experiment. As discussed above for metacaspases,
such variations imply a role of the cysteine protease in the
slowing down of the growth rate and aging of cultures.
Our results suggest that cysteine proteases (metacasp-
ases and caspase-like enzymes) may play multiple roles in
phytoplankton cells, including potential involvement in cell
survival, cell division or aging processes. Housekeeping
and regulatory functions for death-related genes in microal-
gae were hypothesized by Bidle and Falkowski (2004), and
subsequent studies in diverse organisms have supported
this view of the multifunctionality of caspases and meta-
caspases (e.g. Ambit et al. 2008; Launay et al. 2005; Lee
et al. 2010; Thamatrakoln et al. 2012). This multifunctional-
ity could be explained by a dose-dependent effect: the pro-
tein quantity could mediate the balance between
proliferation (baseline level) and limitation of growth
(increase in activity), as proposed by Ambit et al. (2008)
and Zalila et al. (2011) for the metacaspase LmjMCA.
Another potential explanation could rely on subcellular local-
ization of proteases and/or the accessibility of substrates
(Algeciras-Schimnich et al. 2002; Bonneau et al. 2008).
Conclusion
This study highlights the complexity of regulatory pro-
cesses leading to “choices” between survival, quies-
cence, or death in phytoplankton cells, where type and
intensity of stimulus as well as intrinsic factors such as
cell cycle position could act in combination. Production of
ROS appeared as a key determinant of stress-related path-
ways, playing potential roles in intracellular signaling and
formation of temporary cysts but also as agents of cellular
damage. According to the observed sequences of events
under stressful conditions, PCD was not activated in A.
tamarense cells in response to oxidative, temperature, or
nutrient stress. This study emphasizes the fact that classi-
cal hallmarks of apoptosis have to be used with caution
for characterization of PCD pathways in phytoplankton. In
particular, present results suggest a wider range of roles
for cysteine proteases (caspase-like enzymes and meta-
caspases) in microalgae, including nondeath functions
such as cell cycle regulation or survival pathways. Non-
death roles of cysteine proteases might provide an addi-
tional explanation for the conservation of death-related
genes in genomes of unicellular algae.
ACKNOWLEDGMENTS
This article is a result of research funded by the National
Oceanic and Atmospheric Administration Center for Spon-
sored Coastal Ocean Research under award no.
NA09NOS4780166 to the University of Texas Marine Sci-
ence Institute. The authors are grateful to Dr. Yun hee
Journal of Eukaryotic Microbiology 2013, 60, 526–538
Jauzein & Erdner
Park for nutrient and pigment analyses. We also acknowl-
edge Dr. Ludovic “Chou” Donaghy for his constructive
comments. This is ECOHAB contribution no. 752.
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