The “Cheshire Cat” escape strategy of the
coccolithophore Emiliania huxleyi in response
to viral infection
Miguel Frada*†‡, Ian Probert*, Michael J. Allen§, William H. Wilson¶, and Colomban de Vargas*
*Station Biologique, Equipe EPPO-Evolution du Plancton et Pale ´oOce ´ans, Centre National de la Recherche Scientifique et Universite ´ Pierre et Marie Curie
(Unite ´ Mixte de Recherche 7144), Station Biologique, 29682 Roscoff, France;†Departamento de Geologia, Faculdade de Cie ˆncias, Universidade de Lisboa,
Edificio C6, Campo Grande, 1749-016 Lisbon, Portugal;¶Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, United Kingdom; and
§Bigelow Laboratory for Ocean Sciences, 180 McKown Point, P.O. Box 475, West Boothbay Harbor, ME 04575-0475
Communicated by Paul G. Falkowski, Rutgers, The State University of New Jersey, New Brunswick, NJ, August 6, 2008 (received for review June 9, 2008)
The coccolithophore Emiliania huxleyi is one of the most successful
eukaryotes in modern oceans. The two phases in its haplodiploid
life cycle exhibit radically different phenotypes. The diploid calci-
fied phase forms extensive blooms, which profoundly impact
global biogeochemical equilibria. By contrast, the ecological role of
the noncalcified haploid phase has been completely overlooked.
Giant phycodnaviruses (Emiliania huxleyi viruses, EhVs) have been
shown to infect and lyse diploid-phase cells and to be heavily
implicated in the regulation of populations and the termination of
blooms. Here, we demonstrate that the haploid phase of E. huxleyi
is unrecognizable and therefore resistant to EhVs that kill the
diploid phase. We further show that exposure of diploid E. huxleyi
to EhVs induces transition to the haploid phase. Thus we have
clearly demonstrated a drastic difference in viral susceptibility
between life cycle stages with different ploidy levels in a unicel-
lular eukaryote. Resistance of the haploid phase of E. huxleyi
provides an escape mechanism that involves separation of meiosis
from sexual fusion in time, thus ensuring that genes of dominant
diploid clones are passed on to the next generation in a virus-free
environment. These ‘‘Cheshire Cat’’ ecological dynamics release
host evolution from pathogen pressure and thus can be seen as an
opposite force to a classic ‘‘Red Queen’’ coevolutionary arms race.
In E. huxleyi, this phenomenon can account for the fact that the
selective balance is tilted toward the boom-and-bust scenario of
optimization of both growth rates of calcifying E. huxleyi cells and
infectivity of EhVs.
eukaryotic life cycle ? haplo-diploidy ? marine viruses ?
host-parasite interaction ? Red Queen
photosynthetic unicellular eukaryotes in modern oceans. Coc-
colithophores (Calcihaptophycidae, Haptophyta) produce com-
posite skeletons of minute calcite platelets (the coccoliths) and,
consequently, have been key contributors to both the oceanic
carbon pump and the counterpump, and thus to the flux of CO2
(1). In this context, the impact of predicted anthropogenically
induced ocean acidification on calcifying plankton is a subject of
intense debate (2, 3). Coccolith-bearing E. huxleyi cells period-
ically develop extensive blooms covering wide coastal and mi-
doceanic areas at high latitudes in both the northern and
southern hemispheres. Termination of these blooms is accom-
panied by massive release of organic and inorganic matter to the
and are readily detectable in satellite images (4). Over the last
decade, the role of large (?175 nm) lytic coccolithoviruses
(Phycodnaviridae), named E. huxleyi viruses (EhVs), in the
clearly established (5), to the extent that this system has become
a case study in marine virology (e.g., refs. 6–8). Both E. huxleyi
he coccolithophore Emiliania huxleyi (Lohmann) Hay and
Mohler is one of the most abundant and widely distributed
and EhV populations have been shown to be genetically diverse
(9), with host succession suggested to follow ‘‘kill the winner’’
dynamics (10). However, recent observations show that the same
E. huxleyi genotype blooms and is infected and decimated by the
same EhV genotype over multiannual time scales (11).
What, then, is the selective advantage for the ‘‘winner’’ E.
huxleyi clone(s) to bloom? Is there intense and permanent
selection pressure for resistance to viral infection [‘‘Red Queen’’
(RQ) dynamics]? And how is the high lytic virulence of EhVs
sustained? These questions are fundamental to understanding
the evolutionary ecology of this biogeochemically important
species. The answers may be related to sex and life cycling, basic
biologic features of unicellular eukaryotes that are typically
ignored in oceanographic models addressing the ecology of
planktonic functional groups. The fitness of many eukaryotic
species may be based on their potential for alternation between
variable life cycle phases and adaptation of each phase to
different ecological niches (12, 13), including in terms of bio-
logical interactions. Current evidence suggests that coccolith-
ophore life cycles are characterized by independent haploid and
diploid phases displaying radically different morphologies (14,
15) and distinct physiologies (refs. 16 and 17; our unpublished
data). In E. huxleyi, the life cycle comprises two main forms: the
diploid (2N), nonmotile, coccolith-bearing phase that forms
blooms, and the haploid (N) flagellated phase that possesses
nonmineralized organic scales overlying the cell membrane (18,
19). This motile, noncalcifying, haploid stage is not easily
amenable to identification by conventional microscope tech-
niques and has been almost completely overlooked by biological
oceanographers. Its ecological role and the importance of sexual
Flow cytometric surveys of mesocosm blooms of 2N E. huxleyi
have revealed the onset, after virus-mediated bloom demise, of
new active populations of cells with the same chlorophyll fluo-
rescence signature but lower light-scattering values than 2N
calcified cells (20, 21). We hypothesized that these new popu-
lations consisted of noncalcifying N cells, and that their presence
reflected their resistance to infection by the viruses responsible
for the decline of the 2N blooms.
In the present work, we explore the in vitro infectivity profiles
of both the coccolith-bearing diploid and noncalcifying haploid
life cycle phases of E. huxleyi by using multiple host and viral
Author contributions: M.F., I.P., and C.d.V. designed research; M.F. and I.P. performed
research; I.P. and C.d.V. contributed new reagents/analytic tools; M.F., I.P., M.J.A., W.H.W.,
and C.d.V. analyzed data; and M.F., I.P., and C.d.V. wrote the paper.
The authors declare no conflict of interest.
See Commentary on page 15639.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
October 14, 2008 ?
vol. 105 ?
strains and various experimental setups. The results show the
critical significance of life cycling in the survival and ecological
dynamics of E. huxleyi. They illustrate a previously unrecognized
type of ecological and evolutionary interaction that opposes
classical RQ host–pathogen dynamics and is potentially a fun-
damental force for the maintenance of sex and life cycling and
for untying the constraints imposed by pathogens in the evolu-
tion of eukaryotic microbes.
15 available EhV strains against both life cycle stages of three
different strains of E. huxleyi (RCC1216, RCC1249, and
RCC1213). All 2N E. huxleyi strains were sensitive to five of the
viral strains (EhV201, EhV202, EhV205, EhV207, and EhV208),
whereas none of the N strains were affected. As no obvious
differences in infectivity were observed between the E. huxleyi
and EhV strains tested, the host–virus combination
RCC1216:EhV201 was chosen for subsequent experiments.
We then monitored the growth (Fig. 1) and photosynthetic
activity [supporting information (SI) Fig. S1] of both infected
flow cytometry and fluorimetric measurements revealed 96%
and 98% decreases of cell density and photosynthetic activity,
respectively, within 3 days of virus inoculation into the growing
culture. There was a concurrent increase in viral particle con-
centration from 3.5 ? 105to 23 ? 107viral particles per ml from
days 8–14. In contrast, no differences were observed between
infected and noninfected N cultures throughout the experiment,
and virus concentration neither increased nor decreased. Visu-
ally, the 2N cultures became transparent, and deposits of cell
debris were observed at the bottom of the culture flasks 3 days
after infection (Fig. S2). To further verify the absence of
infection of the E. huxleyi haploid phase, various viral densities
and coexistence times were tested (Fig. S3). Haploid cultures
that were previously used for infection experiments and fresh N
cultures were incubated for 26 days with viruses at various
multiplicities of infection (MOI). All cultures displayed similar
growth curves, reaching typical concentrations of 15 ? 105to
20 ? 105cells per ml at day 10.
We then used transmission electron microscopy (TEM) and
(Fig. 2) to verify the absence of viral adsorption and production
by haploid E. huxleyi suggested by flow cytometry. No viral
capsids were detected inside or adsorbed to the N cells from
virus-infected cultures in any of the multiple TEM preparations,
whereas capsids were obvious in the cytoplasm of infected 2N
the MCP gene was easily detected by PCR of DNA extracts from
filtered and washed infected 2N cells, whereas the N cells in
contact with viruses never yielded positive amplifications
Finally, we set up a series of 50-day experiments to test
longer-term responses of 2N and mixed 2N–N E. huxleyi cultures
to viruses. The infected 2N cultures crashed 5 days after
infection (Fig. 3A). On day 6, a small new peak of cells (?4 ?
defined for N cells (Fig. S4). Microscope observations revealed
that these E. huxleyi cells possessed neither coccoliths nor
flagella, resembling moribund noncalcified diploid cells. Thirty
of these were sorted by flow cytometry into fresh, virus-free
culture media, but none of them established a new growing
population. Between days 7 and 23, cell density remained at a
background level of 10 to 103cells per ml. At day 24, however,
a new population of N cells appeared, as observed by both flow
cytometry (Fig. 3A) and light microscopy. The concentration of
these actively swimming N cells reached a plateau after ?10
days. In contrast, the noninfected 2N control culture declined
after the plateau phase, with no sign of the presence of N cells
Two additional long-term experiments were performed with
both life cycle stages mixed in the same culture vessel. In the first
of these experiments, the diploid and haploid stages were grown
together in the presence of viruses (Fig. 3C). The population
dynamics were similar to those in the experiment with 2N cells
only, with the same peak of drifting, noncalcified cells at day 6,
followed by a lag phase of very low cell densities, and the
Growth curves of both life cycle stages of E. huxleyi (strain RCC1216) and the
virus EhV201 are shown. The arrow indicates the day of virus addition (mul-
The impact of EhV on the growth of diploid and haploid E. huxleyi.
and/or on infected E. huxleyi haploid and diploid cells. (A) TEM: 1, healthy 2N
cell before infection; 2, 2N cell at day 1 after infection, displaying newly
formed viral particles (dashed circle); 3, N cell from an infected culture; and 4,
detail of the N cell periplast, showing the presence of organic scales attached
(B) PCR amplifications of the viral MCP gene at day 11 after infection (see Fig.
1). Agarose gel lanes: 1, positive control (EhV201 DNA extract); 2, N culture; 3,
2N culture; 4, N culture exposed to viruses; and 5, 2N culture exposed to
viruses. Cells were carefully filtered and washed several times to remove free
viral particles before DNA extractions.
Microscopy and genetic tests for the presence/absence of EhVs inside
Frada et al.
October 14, 2008 ?
vol. 105 ?
no. 41 ?
development of the N population by day 20. The population of
N cells attained a higher density than in the assay that started
with 2N cells only. In the last experiment, 2N and N cells were
grown together without viruses. The shape of the 2N growth
curve was identical to that of the culture without N cells (Fig.
3B), although of lower cell density. However, haploid cell density
remained low and constant (?104cells per ml) during the first
24 days and then decreased to minimum values of ?103cells per
ml in the second half of the experiment. Controls with pure
haploid cultures with or without viruses were conducted, and
these displayed typical haploid cell growth curves (data not
Our data demonstrate that the noncalcifying haploid phase of
the coccolithophore E. huxleyi is resistant to viruses that infect
and lyse the diploid calcifying phase of the same species. This
phenomenon was confirmed in vitro with multiple E. huxleyi and
EhV strains. In all experiments using haploid E. huxleyi cultures,
concentrations of free-floating viral particles were stable over
time, and neither viral capsids nor viral DNA was detected by
TEM or PCR (Fig. 2) within N cells in contact with viruses. This
adsorption of viruses in E. huxleyi haploid cells. Differential
susceptibility to viral infection has been reported between clones
of the same ploidy level in a number of microalgal taxa (22), but
there has been no previous clear demonstration of a drastic
difference in viral susceptibility between life cycle stages with
different ploidy levels in a marine protist. Our results further
indicate that viral infection may trigger meiosis, or at least a shift
from diploid to haploid populations, in E. huxleyi (Fig. 3). These
observations have important implications for the ecology and
protists with sexual life cycles.
Mechanisms of Viral Resistance of E. huxleyi Haploid Cells.Toinitiate
infection, viruses attach to host-specific cell surface receptors
(23). Obvious phenotypic differences exist in the nature of the
cell cover between life cycle phases in E. huxleyi (Fig. 2). One or
several loose layers of interlocking coccoliths surround diploid
cells, but these do not produce organic scales covering the cell
membrane (commonly known as body scales and typical for most
haptophytes). By contrast, haploid cells do not produce cocco-
liths, but their cell surface is covered by distinctively tightly
packed body scales organized in overlapping layers (18). This
haploid cell covering, characteristic of most noncalcifying mem-
bers of the order Isochrysidales, may efficiently prevent viruses
from coming into contact with putative receptor sites on the cell
membrane. Alternatively, plasmalemma molecules recognized
by EhV capsids may be modified or simply absent in haploid
cells. Modification or loss of receptor molecules is the most
common way in which bacteria develop resistance to bacterio-
phages (24), but these phenotypic differences typically result
from mutation(s) of the genotype rather than differential gene
expression, as would most likely be the case over the E. huxleyi
life cycle. Note that the fact that the three haploid E. huxleyi
strains tested proved resistant to viral infection makes it highly
improbable that resistance results from a simple Mendelian
allele segregation effect. The receptors to which viruses bind
typically serve primary metabolic functions in the host, and
physiologic differentiation. In this context, a notable difference
between E. huxleyi life cycle phases is calcium metabolism, which
is known to be particularly intense in the calcifying 2N phase
(25), but is presumably negligible in the noncalcifying N phase.
Calcium often promotes the physical interactions between vi-
ruses and host receptors through a direct effect on the confor-
mation of the viral capsid as, for example, in the hepatitis A virus
in humans (26).
Other mechanisms of viral avoidance reported in marine
protists include the extracellular production of viral inhibitors,
like the cell wall sulfated polysaccharide produced by the red
microalga Porphyridium sp. (27), the exudation of a protective
extracellular polysaccharidic layer or aggregates that may trap
viruses, as observed in the colony-forming haptophytes Phae-
ocystis globosa and Phaeocystis pouchetii (28, 29), or the secretion
of dimethyl sulfide and acrylic acid, which are known to inhibit
infection (30). However, such mechanisms do not seem to be
relevant in the case of the E. huxleyi life cycle, as EhVs infected
2N cells in mixed 2N–N cultures as efficiently as in pure 2N
Viral Infection and Life Cycle Phase Transition in E. huxleyi. Prelim-
inary indications suggest that life cycle phase switches in coccolith-
ophores may be regulated by chemical (31) or physical (14) prop-
erties of the medium. Our data show that biotic interactions with
viruses may also play a key role in directly triggering life cycle
changes. Oxidative stress in response to viral infection could be the
trigger for both the peak of dead E. huxleyi 2N cells at day 6 after
production of reactive oxygen species (ROS) in diploid E. huxleyi
cultures (32). ROS and viral infection were found to be associated
with the induction of metacaspases and programmed cell death
(PCD) in diploid E. huxleyi (33), a phenomenon also demonstrated
in other marine protists (34). However, cell death is not the
exclusive response to oxidative stress in single-celled eukaryotes.
PCD are alternative responses to increased oxidative stress in the
colonial green alga Volvox carteri (35), and developmental pro-
grams that lead to the concomitant formation of dead cells and
huxleyi. (A) 2N cells infected with EhV201 virus. (B) 2N cells without virus. (C)
Mixture (100:1) of 2N and N cells infected with EhV201. (D) Mixture (100:1) of
2N and N cells without virus. 2N naked cells (due to coccolith loss after viral
arrows indicate time of infection. Standard deviation bars are generally too
short to be visible.
Long-term infection assays of 2N and mixed 2N–N cultures of E.
www.pnas.org?cgi?doi?10.1073?pnas.0807707105Frada et al.
spores have been described in protists, such as the slime mold
Dictyostelium discoideum, as well as in several prokaryotes (36).
An alternative explanation could be that N cells are produced
regularly by meiosis in 2N cultures, but remain at very low
concentrations such that they are effectively undetectable by
microscopy or flow cytometry. In our mixed 2N–N culture
experiment (Fig. 3D), diploid cells clearly out-competed haploid
cells to the point that the growth dynamics of 2N populations
for the potential emergence of a haploid population, and viral
infection would indirectly promote succession of life cycle stages
through elimination of the more competitive diploid phase.
Ecological Implications. The visible worldwide ecological success
of the diploid stage of E. huxleyi has stimulated interest in the
underlying physiologic mechanisms that allow these calcifying
cells to form extensive blooms under certain conditions (4). Two
of the more remarkable capacities of E. huxleyi 2N cells are their
exhibit photoinhibition of photosynthesis, even at very high light
intensities (ref. 18 and references therein). These and other data
help explain how the diploid E. huxleyi comes to dominate its
phytoplankton competitors. However, in light of the growing
body of evidence indicating the devastating impact of the highly
infective EhVs on these blooms, the evolutionary advantages of
this ecological strategy are not clear. Why has investment by E.
huxleyi 2N of more resources into defense mechanisms at the
expense of growth not been selected? Why has reduction of
infectivity of EhVs to confer a clear selective advantage for
blooming in their host clones not been selected?
The ability of haploid cells to escape viral infection can explain
why the selective balance is tilted so far toward optimization of
growth rates and infectivity in E. huxleyi 2N cells and EhVs,
respectively. By increasing massively in density as a result of
successive mitoses, more cells of a given 2N clone are likely to
undergo meiosis, potentially as a direct response to viral infec-
tion. If newly formed N cells were susceptible to viral infection
they would be rapidly decimated, because viral density is highest
during bloom demise (5, 11), and because motile cells are
theoretically more likely to encounter viral particles (37).
Blooming would then have no selective advantage compared
with a strategy of maintaining low background 2N cell concen-
trations. On the other hand, transformation into a haploid
phenotype invisible to the virus provides an escape mechanism
that ensures that the genes of an individual (or clone) are passed
on to the next generation. It can be argued, therefore, that this
phenomenon dictates positive selection for rapid growth and
meiosis in the diploid host while imposing little negative selec-
tion pressure for high infectivity of the virus. It also leads to the
prediction that E. huxleyi N cells do not simply act as gametes by
mating at the first opportunity, as this would produce 2N cells
susceptible to viral attack in an environment where viruses are
still present in high concentrations. Rather, the noncalcifying
haploid phase probably plays a prominent ecological role, di-
viding and migrating with the consequence of temporally and
spatially displacing an eventual inoculum of novel 2N cells.
Evolutionary Significance: Red Queen or Cheshire Cat? Originally
proposed by Van Valen (38), the metaphor of an evolutionary
‘‘arms race’’ has been widely used for describing various biotic
interactions and termed Red Queen (RQ) dynamics in reference
to the Red Queen’s race in Lewis Carroll’s Through the Looking
Glass, in which the Red Queen states ‘‘it takes all of the running
you can do, to keep in the same place’’ (39). The idea that genetic
race between parasites and hosts was developed later by several
authors (e.g., refs. 40–42). Our results indicate that viral infec-
tion of the diploid stage of E. huxleyi promotes sexual cycling,
either directly by inducing meiosis and/or indirectly by removing
the more competitive 2N cells. A classical RQ interpretation
would be that this sexual cycling leads to increased diversity in
the following diploid generation, and that among this diversity
certain genotypes would be more resistant to viral attack, and
therefore positively selected. The fact that EhVs typically infect
a limited range of diploid E. huxleyi strains in culture (9) may
support this interpretation.
However, the limited data available from natural populations
do not indicate a constant and rapid turnover of coevolving E.
huxleyi and EhV genotypes in the oceans. In our experiments, an
EhV strain isolated from the North Atlantic was capable of
infecting host strains originating from the Mediterranean Sea
(RCC1249, RCC1213) and from the Tasman Sea off New
Zealand (RCC1216). Rapid RQ dynamics would presumably
have led to localized, genetically distinct subpopulations of host
and virus, at least in terms of resistance and recognition genes,
respectively. Furthermore, the GPA gene, encoding a highly
polymorphic protein with calcium-binding motifs and used as a
genotype marker in E. huxleyi, did not reveal variation of the
dominant genotypes over multiannual mesocosm experiments in
Norwegian fjords (11), suggesting the existence of an efficient
strategy for survival of these genotypes between blooms.
The persistence of E. huxleyi strains over multiple years in the
North Atlantic, the lack of biogeographic structuring in E.
huxleyi–EhV infectivity patterns, and in general the maintenance
of elevated growth rate and infectivity in E. huxleyi and EhV,
respectively, argue against the existence of a classical, highly
dynamic RQ equilibrium in this host–virus system. The invisi-
bility of the host haploid stage to the virus circumvents, or at
least drastically slows, the arms race. The period of respite
experienced during one life cycle phase means that resources of
the phase susceptible to viral attack can be focused on interac-
tions with direct ecological competitors rather than on devel-
oping new arms against the virus. In keeping with the RQ
metaphor taken from Lewis Carroll, we liken this theory to the
strategy used by the Cheshire Cat in Alice’s Adventures in
Wonderland (43) of making its body invisible to make the
sentence ‘‘off with his head’’ pronounced by the Queen of Hearts
impossible to execute.
RQ and Cheshire Cat (CC) mechanisms, both of which act
around a central eukaryotic process, sexual reproduction, should
not be considered mutually exclusive. CC dynamics, which rely
to some extent on separation of the sexual processes of meiosis
and fusion in time and/or space, release the host from short-term
in other directions. Evolution of genes conferring resistance to
viral attack in the host and the counteractive evolution of new
arms by the virus likely still occur, but over much longer time
scales than classically inferred. E. huxleyi is a relatively young
species (or species complex), having evolved from the Gephy-
rocapsids some 270,000 years ago (44), and thus represents an
interesting model for assessment of the pace of host–virus
coevolution and the interaction between the Cat and the Queen.
CC dynamics could, of course, also apply to predator–prey
interactions. Cases of phenotypic changes linked to predation
pressure have been documented in protists (45); however, to our
knowledge there are, as yet, no reports of phenotypic switches
associated with ploidy changes under these conditions.
Concluding Remarks. Each milliliter of seawater is known to
contain millions of viral particles, and the growing evidence of
an enormous diversity of eukaryotic viruses (46) and parasites
(47) suggests that each eukaryote species, and potentially each
clonal strain in the oceans, has its own pathogen(s). Our data
reveal a fundamental mechanism that may correlate the massive
diversity of oceanic pathogens and the maintenance of dimor-
Frada et al.
October 14, 2008 ?
vol. 105 ?
no. 41 ?
relationships involves more than mere intellectual curiosity on
the coevolution of sex and viruses. In fact, eukaryotic marine
protists are responsible for nearly half of global primary pro-
ductivity and control most of the flux of matter between the
atmosphere and the lithosphere (48). The fate of oceanic
primary production, whether sedimented to the ocean floor or
remineralized in surface waters, depends fundamentally on the
physical properties of the eukaryotic cells themselves, which
change dramatically as they differentiate over their life cycle. In
System dynamics is obviously radically different: the diploid
stage is responsible for a significant amount of planetary car-
bonate production and is thus heavily implicated in global
climate regulation (4), whereas the haploid stage does not calcify
and is probably mostly remineralized in surface waters. Under-
related phenotypic differentiation in oceanic protists will signif-
icantly advance assessment and prediction of their impact on
Strains and Culture Conditions. The E. huxleyi strains used in this study,
Sea, Spain), and RCC1213 (origin: Mediterranean Sea, Italy) from the Roscoff
Culture Collection, France (http://www.sb-roscoff.fr/Phyto/RCC), were origi-
nally initiated by micropipette isolation of a single diploid (coccolith-bearing)
cell. After diploid-to-haploid life cycle transitions of a few cells in the original
cultures, pure cultures of each phase were established from the mixed-phase
cultures by single-cell micropipette isolation. Culture purity was verified by
light microscopy before each experiment. Experiments were conducted in
triplicate in K/2 (minus Si, minus Tris) medium at 18°C, 12:12 (light:dark) cycle
and 85 ?E?m?2?s?1irradiance. The viral strains (EhV84, EhV86, EhV88, EhV163,
EhVv1, and EhVv2; ref. 6) were maintained at 4°C in K/2 medium and filtered
through a 0.2-?m filter (Minisart; Sartorius) before utilization.
Flow Cytometry and Photosynthetic Activity Analyses. Enumeration of E.
huxleyi cells and viruses was performed with a FACSCalibur flow cytometer
(Becton Dickinson) equipped with an air-cooled laser providing 15 mW at 488
nm and with the standard filter setup. Haploid and diploid E. huxleyi cells in
and side-scatter signatures (Fig. S4). Virus enumeration was performed ac-
used for sorting E. huxleyi haploid and diploid cells. The photosynthetic
during a saturating light pulse (0.6 s, 470 nm, 1,700 ?mol?m?2?s?1) with a
PHYTO-PAM (Mess und Regaltechnik) using fresh samples incubated for 10
min in the dark before analyses. The quantum efficiency of photosystem II
(Fv/Fm) was calculated by Fv/Fm ? (Fm ? F0)/Fm, where F0 is the minimum
during the saturating light pulse.
Infection Assays. The first experiment was designed to assess the response of
the haploid and diploid phases of all E. huxleyi strains to infection with each
of the viral strains. Exponentially growing cultures (200 ml; ?2 ? 106cells per
ml) of E. huxleyi strains were infected independently with each virus strain at
a virus-to-cell ratio (MOI) of 0.2. Enumeration of algae and viruses and
measurement of photosynthetic activity were performed daily. Controls
without addition of viruses were performed in parallel. The E. huxleyi
RCC1216 N and 2N strains and the EhV201 viral strain were chosen for
The effect of addition of various viral concentrations on the haploid phase
of E. huxleyi was then tested. Cultures of the haploid stage used in the
previous infection experiment (including controls) were diluted 200 times in
200 ml of fresh K/2 (minus Si, minus Tris) medium and incubated in conditions
previously described. After attaining ?2 ? 106cells per ml, duplicates were
inoculated with mois of 0.05, 1, and a control (no virus addition). Control
cultures, which had never been in contact with viruses, were inoculated with
MOIs of 0.5, 5, and a control. Samples for enumeration of algae and viruses
were collected six and three times, respectively, at irregular intervals over a
Long-Term Infection Assays. To mimic a bloom situation based on the obser-
vations of Castberg et al. (20) and Jacquet et al. (21), 200-ml exponentially
growing diploid cultures were inoculated with haploid cells in exponential
growth at a 2N/N cell ratio of 100 (?1 ? 1062N cells per ml and 1 ? 104N cells
per ml), and infected with viruses at an MOI of 0.2. Noninfected diploid
cultures with and without addition of haploid cells were used as controls.
Populations of N and 2N cells of E. huxleyi and viruses were monitored for 50
days. In the initial stages of infection, cells from the samples were sorted by
flow cytometry and cultured in 2 ml of K/2 (minus Si, minus Tris) medium.
major capsid protein (MCP) gene (?300 kb; ref. 9) from infected haploid and
diploid cells after multiple washes. Samples from N and 2N cultures were col-
lected 2 days after viral addition. Free-floating viral particles were washed away
with fresh medium by multiple filtrations of the cells on polyethersulfone mem-
from the cells on the filters (50). PCR amplifications with the primer pair MCP-F
and MCP-R were performed according to Schroeder et al. (9). A pure extract of
EhV201 DNA was used as a positive control.
1.5 h in 4% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2) with 0.25 M
sucrose. Cells were then washed three times in 0.1 M sodium cacodylate
containing decreasing concentrations of sucrose and were postfixed in 2%
osmium tetroxide in 0.1 M sodium cacodylate for 1.5 h. After washing in
distilled water, cells were dehydrated in a graded ethanol series and embed-
ded in Epon resin. Sections were double stained with uranyl acetate followed
by Reynold lead citrate. Observations were carried out on a JEOL JEM 1011
ACKNOWLEDGMENTS. We thank the Plancton Oce ´anique group at the Sta-
tion Biologique de Roscoff, especially D. Marie, F. Le Gall, A. Pagarete, P. Von
Dassow, and E. M. Bendif, for assistance with flow cytometry and algal
to M.F. by the Fundac ¸a ˜o Para a Cie ˆncia e para a Tecnologia, Portugal, and an
Actions The ´matiques et Incitatives sur Programme (ATIP) grant awarded to
C.d.V. by the Centre National de la Recherche Scientifique, France. It is part of
the pluridisciplinary project BOOM (Biodiversity of Open Ocean Microcalcifi-
ers) funded by the French Agence Nationale de la Recherche, Grant ANR-05-
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