RESEARCH ARTICLE
Light is a key factor in triggering sexual reproduction in the
pennate diatom Haslea ostrearia
Jean-Luc Mouget1, Romain Gastineau1, Olga Davidovich2, Pierre Gaudin3 & Nickolai A. Davidovich2
1
Laboratoire de Physiologie et Biochimie Végétales, EA 2160, Mer, Molécules, Santé, Université du Maine, Le Mans, France; 2Karadag Natural Reserve of
the National Academy of Sciences, p/o Kurortnoe, Feodosiya, Ukraine; and 3Ecophysiologie et Métabolisme des Microalgues, EA 2160, Mer, Molécules,
Santé, Université de Nantes, Nantes, France
Correspondence: Jean-Luc Mouget,
Laboratoire de Physiologie et Biochimie
Végétales, EA 2160, Mer, Molécules, Santé,
Université du Maine, Avenue Olivier
Messiaen, 72085 Le Mans CEDEX 9, France.
Tel.: 133 2 43 83 32 42; fax :133 2 43 83 37
95; e-mail: [email protected]
Received 19 October 2008; revised 6 March
2009; accepted 16 April 2009.
Final version published online 22 May 2009.
DOI:10.1111/j.1574-6941.2009.00700.x
MICROBIOLOGY ECOLOGY
Editor: Riks Laanbroek
Keywords
auxosporulation; diatoms; light control;
reproduction frequency; sexualization.
Abstract
Sexual reproduction is an obligatory phase in the life cycle of most diatoms, as cell
size decreases with successive vegetative divisions and the maximal cell size is only
restored by a specialized cell, the auxospore, which follows zygote formation as a
result of sexual reproduction. While in pennate diatoms the induction of sexual
reproduction depends primarily on cell–cell interactions, the importance of
different external factors for the induction of sexual reproduction is less well
known. Here, we investigated the effects of light on sexualization in the marine
benthic pennate diatom Haslea ostrearia (Gaillon) R. Simonsen. Compatible
clones were crossed and exposed to different combinations of light levels, qualities,
and photoperiods. Light was found to be a key factor for sexualization, and to a
certain extent, to control auxosporulation in H. ostrearia. The light conditions
most favorable for sexual reproduction were low irradiances (o50 mmol
photons m2 s1) and short photoperiods (6–10 h), conditions that prevail during
winter, and to a lesser extent, the higher irradiances and longer photoperiods that
correspond to the spring and fall, when blooms of this organism form in the
natural environment. Auxospore formation was very rare in continuous light, and
maximum in presence of red radiation, while it was never observed in darkness or
in radiation other than red.
Introduction
Among features distinguishing diatoms from other algae,
one of the most important is the modality of mitotic
division, which results in two daughter cells, one of which
has the same size as the mother cell, and the other is smaller
(Drebes, 1977). While the mean cell size of a population
slowly decreases in time as a result of repeated vegetative
divisions, the species-specific maximum cell size is usually
restored by a phase of sexual reproduction, when the zygotes
turn into specialized cells, known as auxospores (Chepurnov
et al., 2004). The vegetative cells must have decreased in size
to below a certain threshold before the sexual cycle is
triggered, and auxospores are produced (Geitler, 1932). The
upper threshold for sexual induction is usually close to 50%
of the initial maximum cell size, but values ranging from
30% to 75% have been reported (Davidovich, 2001). The
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maximum cell size for the species corresponds to that of the
initial cells, the new cells that are formed inside the
auxospore envelopes after the zygote/auxospore expansion
phase that follows fertilization (e.g. Drebes, 1977).
Diatoms are generally divided into two major groups on
the basis of the symmetry of the frustule and their mode
of sexual reproduction: centric forms and pennate forms
(Chepurnov et al., 2004). Centric diatoms display radial
symmetry and are oogamous, whereas pennate diatoms
display bilateral symmetry and are aplanogamous (i.e.
do not release flagellate gametes). This ‘gametangiogamy’
(Chepurnov et al., 2004) can be aniso- or isogamous, with
the copulating gametes displaying or not displaying morphological or behavioral differences. In pennate diatoms,
sexual reproduction seems to depend primarily on cell-tocell interactions between compatible cells (reviewed in
Chepurnov et al., 2004), although external factors (light,
FEMS Microbiol Ecol 69 (2009) 194–201
195
Light induction of sexual reproduction in diatoms
temperature, and medium composition) could have some
influence (Drebes, 1977). In particular, among the environmental factors that may induce sexual reproduction in
diatoms, the effects of light (particularly intensity and
photoperiod) have received some attention (reviewed by
Drebes, 1977; Davidovich, 2002; Chepurnov et al., 2004). It
is generally accepted that there is an optimum speciesspecific light regime for the stimulation of auxospore
formation.
The marine pennate diatom Haslea ostrearia (Gaillon)
R. Simonsen, a member of the Naviculaceae, is a tychopelagic
microalga distributed worldwide (Robert, 1983). This diatom regularly colonizes oyster ponds of the French Atlantic
coast, and it is famous because it synthesizes a blue pigment
‘marennine,’ responsible for the greening of oyster gills, a
phenomenon that gives an added value to the bivalves.
Auxosporulation phases in H. ostrearia [as Navicula ostrearia (Gaillon) Bory] have been identified possibly using
monoclonal cultures, thus illustrating homothallic reproduction in this species (Neuville & Daste, 1975, 1979). More
recently, 12 clones of H. ostrearia have been shown to be
heterothallic, with a type-IB2a pattern of reproduction
according to Geitler’s (1932) system, and sexual reproduction has been shown to be dependent on sexual compatibility between clones (Davidovich et al., 2009). In the
present study, we investigated the effects of light (intensity,
photoperiod, daily dose, and quality) on the induction of
heterothallic sexual reproduction in H. ostrearia.
Materials and methods
Origin and characteristics of clones
Four nonaxenic clones of H. ostrearia from the Nantes
Culture Collection (NCC), namely NCC 136, NCC 141,
NCC 158, and NCC 234, were used in the experiments
described in this paper. They were isolated by micropipette
from various samples taken from greening oyster ponds near
the Bay of Bourgneuf (France) – 46159 0 1900 N, 2107 0 5900 W for
NCC 136 and NCC 141 (sampling date, November 2, 2003)
and 46153 0 3300 N, 2114 0 1400 W for NCC 234 (sampling date,
November 30, 2007). NCC 158 resulted from a previous
crossing of NCC 141 and another clone, NCC 148.16, which
itself resulted from spontaneous, homothallic reproduction
in a culture of NCC 148, collected in spring 2000 from a
pond in Bouin (France, 46159 0 5100 N; 2101 0 5000 W). These
clones were selected based on sexual compatibility among
them (Davidovich et al., 2009), and at the beginning of the
experiments, they differed in mean cell size: 54 1 mm for
NCC 136, 46 2 mm for NCC 141, 52 1 mm for NCC 158,
and 63 1 mm for NCC 234. The decrease in size during the
2-month period corresponding to the experiments dealing
with light level and photoperiod did not exceed 3%.
FEMS Microbiol Ecol 69 (2009) 194–201
Culture conditions
Stock cultures were grown in 250-mL Erlenmeyer flasks
containing 150 mL of an artificial seawater medium (Harrison et al., 1980), at low temperature (10 1 1C) and low
irradiance (20 mmol photons m2 s1, with a 6/18 h light/
dark photoperiod). The original medium was complemented by adding Fe–NH4–citrate [1.37 mM final concentration
(f.c.)], CuSO4 5H2O (0.04 mM f.c.), folic acid (0.18 nM
f.c.), nicotinic acid (0.0325 mM f.c.), thymine (0.95 mM f.c.),
Ca-D-pantothenate (8.39 nM f.c.), and inositol (1.11 mM
f.c.) (de Brouwer et al., 2002). Crossing experiments were
conducted in a temperature-controlled room at 16 1 1C,
and cells were acclimated for at least 1 week before being
crossed.
Light conditions
Unless otherwise specified, clonal cultures were grown at
20 mmol photons m2 s1, with a 14/10 h light/dark cycle
regime during the acclimation period, conditions previously
demonstrated to allow active growth without light stress
(Mouget et al., 1999). The irradiance during the various
crossing experiments ranged from c. 3 to 150 mmol m2 s1,
and the photoperiod from continuous light to a 4/20 h light/
dark cycle (details regarding values of irradiance level and
photoperiod are specified for each set of experiments).
Illumination was provided by Philips TLD 36W/965 fluorescent tubes (white light, WL), the emission spectrum of
which, measured with a TriOS Ramses radiospectrometer, is
given in Fig. 4a. For experiments dealing with the effects of
light quality on H. ostrearia auxosporulation, sexually compatible clones were grown at 60 mmol photons m2 s1, with
a 14/10 h light/dark cycle. Clones were crossed and exposed
to 28 mmol m2 s1 irradiance level, at different wavebands:
blue light (BL), green light (GL), or red light (RL) (Lee
filters, Evas, France), with a WL control. The transmittance
spectra of the filters, measured with a Spekol 1100 spectrophotometer, are given in Fig. 4b. Irradiance was measured
using a Li-Cor LI-189 quantum meter with a Q21284
quantum sensor.
Crossing experiments
Crossing was performed by mixing parental cultures (f.c.,
2000–4000 cells mL1) that were maintained in the exponential growth phase. The mixed cultures were checked daily
for a week for signs of auxosporulation and heterothallic
reproduction. At intervals, control monoclonal cultures
were exposed to the same light conditions to check for
possible signs of homothallic reproduction, according to
Neuville & Daste (1975, 1979). Cultures were examined
under a light microscope, an Olympus CH-2, or a Nikon
TS100 inverted microscope. The reproduction frequency
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196
(as a percentage) was defined as the sum of sexualized cells
[paired gametangia containing gametes (each gametangium
counting for 1), zygotes, auxospores], or initial cells
observed in a microscope field at a given time in a culture,
relative to the total number of cells. In the various experiments conducted over the 2-month period, the reproduction frequency varied from o 1% to 4 20%. To compare
results between different series of experiments, the data were
expressed as a function of the maximum reproduction
frequency obtained for each experiment (100%), and
plotted as relative reproduction frequencies. Unless otherwise specified, experiments were run in triplicate, and for
each replicate, up to 40 microscope fields were observed to
count the cells and identify the sexual stage reached. Images
used to measure cell size (apical length) were taken with a
digital camera Sony SSC-C350P color CCD camera (Mouget
et al., 1999). Differences in reproduction frequency as a
function of light treatments were tested by ANOVA and post
hoc tests comparing the means (SUPERANOVA software package
for Macintosh, Abacus Concepts).
First crossing experiments were carried out to describe
the effects of irradiance level and photoperiod on sexualization. Crossing between parental NCC 136 and NCC 141
clones was performed in 5-cm-diameter Petri dishes (Bibby
Sterilin, Staffs, UK). Changes in the reproduction frequency
with irradiance levels were studied on cultures of the
parental clones acclimated to 20 mmol photons m2 s1,
crossed, and then exposed to different irradiances ranging
from c. 3 to 150 mmol photons m2 s1. The influence of the
photoperiod on H. ostrearia fecundity was studied on the
same parental clones grown at 20 mmol photons m2 s1, and
exposed to this irradiance, but with different photoperiods
(4–16 h of light per 24 h) after crossing. The effect of the
photoperiod for a given daily light dose was also tested by
comparing the reproduction frequency of the NCC 136 and
NCC 141 parental clones exposed to different combinations
of light levels (20–120 mmol photons m2 ms1) and photoperiods (4 h to continuous light), resulting in the same daily
light dose (1.73 mol photons day1).
The effects of continuous lighting were studied on cells of
the NCC 136 and NCC 141 clones that were first acclimated
to continuous light (20 mmol photons m2 ms1) for 1 week,
then crossed and placed in darkness for 12 h, before being
exposed to low-light conditions (6.25 mmol photons m2 s1,
8/16 h light/dark cycles). This protocol was based on the
light treatment previously used to trigger spermatogenesis
in the centric diatom Thalassiosira weissflogii (Armbrust
et al., 1990).
For experiments dealing with the effects of light quality on
H. ostrearia auxosporulation, sexually compatible clones were
crossed in sterile, 24-well microplates covered with different
Lee filters. Each well contained 2 mL of fresh medium, and
the initial cell concentration was 2000 cells mL1. The culture
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J.-L. Mouget et al.
microplates were wrapped in Parafilm ‘M’ to avoid dessication. This series of experiments took place 1 year later than
the previous ones run with NCC 136 and NCC 141, and
involved a different pair of sexually compatible clones,
namely NCC 158 and NCC 234, which displayed the same
sexual types as NCC 136 and NCC 141, respectively. Parental
cells were grown at 60 mmol photons m2 s1, with a 14/10 h
light/dark cycle. After crossing, cells were exposed to low
light (28 mmol photons m2 s1) and a short photoperiod
(6/18 h light/dark cycles), and signs of sexual reproduction
(gametangia, zygotes, auxospores, and initial cells) were
observed after about 1 week of growth.
Results
Optimum irradiance levels for sexual
reproduction in H. ostrearia
The effect of light in triggering sexual reproduction in
H. ostrearia was first assessed by measuring the reproduction
frequency as a function of the irradiance level. Changes in
the reproduction frequency with irradiance levels were
studied on cultures of the NCC 136 and NCC 141 clones
acclimated to 20 mmol photons m2 s1, crossed, and then
exposed to different irradiances ranging from c. 3 to
100 mmol photons m2 s1. When auxosporulation occurred,
first sexual (gametes, zygotes, and auxospores) and then
initial cells generally appeared 2–3 days after mating.
Compatible cells of the two clones first paired, then meiosis
took place, and gametes were formed during the next day.
If the conditions encountered were not favorable for auxosporulation, usually no sexual stages could be detected, even
after a week of vegetative growth, whereas sex was successful
in almost all replicates when conditions were favorable. A
higher reproduction frequency was observed at irradiance
levels close to or lower than growth irradiance (Fig. 1a). No
reproductive cells were observed in darkness, or in any of the
monoclonal cultures (intraclonal reproduction) exposed to
the same light conditions.
As shown in Fig. 1b, similar results were obtained in the
four series of experiments (each corresponding to a different
symbol) carried out to investigate the impact of an optimum
irradiance level on H. ostrearia fecundity. These experiments
were performed with various combinations of irradiances
(from 3 to 150 mmol photons m2 s1, depending on the
series) and photoperiods (4–16 h light day1). In these various experiments carried out over the 2-month period, the
reproductive frequency ranged from 3.4% to 4 20%. To
compare the results, the data were expressed as a function of
the maximum reproduction frequency obtained for each
experiment (referred to as 100%), and plotted as relative
reproduction frequencies (Fig. 1b). In all series of experiments, whatever the photoperiod, the highest reproduction
FEMS Microbiol Ecol 69 (2009) 194–201
197
6
(a)
4
2
0
0
25
50
75
100
125
150
Reproduction frequency (%)
Reproduction frequency (%)
Light induction of sexual reproduction in diatoms
12
(a)
10
8
6
4
2
0
0
4
8
12
16
Photoperiod (h)
20
24
4
8
12
16
Photoperiod (h)
20
24
100
(b)
80
60
40
20
0
0
25
50
75
100
125
150
Irradiance (µmol photons m–2 s–1)
Fig. 1. Reproduction frequency (a) or relative reproduction frequency
(b) as a function of irradiance levels after crosses of the Haslea ostrearia
clones NCC 136 NCC 141. In (b), symbols correspond to four different
series of experiments, differing in the combination of irradiances and
photoperiod. Values shown in (a) are means SE (n = 3).
frequency was always observed when the clones were exposed to the lowest irradiances (6–40 mmol photons m2 s1)
after crossing.
Effects of photoperiod and light dose
The influence of the photoperiod on H. ostrearia fecundity
was first studied on NCC 136 and NCC 141 clones grown at
20 mmol photons m2 s1, and exposed to the same irradiance, but with three different photoperiods (4, 8 or 14 h of
light per 24 h) after mating. The number of reproductive
cells observed after mating was significantly higher
(P o 0.05) when cells were grown over an 8/16 h photoperiod (Fig. 2a).
In five separate experiments dealing with the effect of
different photoperiods (ranging from darkness to continuous light, and all using the same irradiance, 20 mmol
photons m2 s1), the reproduction frequency ranged from
1.2% to 10.3%. The results were expressed as relative
reproduction frequencies, and are pooled in Fig. 2b (each
symbol corresponds to a different series of experiments).
Higher reproduction frequencies were observed with photoperiods ranging from 6 to 10 h light a day. Sexual reproduction never occurred in complete darkness, and auxospore
formation rarely occurred in continuous light.
FEMS Microbiol Ecol 69 (2009) 194–201
Relative reproduction frequency (%)
Relative reproduction frequency (%)
Irradiance (µmol photons m–2 s–1)
(b)
100
80
60
40
20
0
0
Fig. 2. Reproduction frequency (a) or relative reproduction frequency
(b) as a function of the photoperiod (4 h to continuous light, or darkness)
after crossing Haslea ostrearia clones NCC 136 NCC 141. In (b),
symbols correspond to five series of experiments, differing in the
combination of photoperiods. Values shown in (a) are means SE
(n = 3).
The effect of the photoperiod for a given daily light dose
was then tested by comparing the reproduction frequency of
the NCC 136 and NCC 141 clones exposed to different
combinations of light levels (20–120 mmol photons m2 s1)
and photoperiods (4 h to continuous light), resulting in the
same daily light dose (1.73 mol photons day1). Reproductive frequency was significantly higher (P o 0.05) when the
cells were exposed to 60 mmol photons m2 s1 for 8 h day1
(Fig. 3a). This experiment was replicated 2 weeks later (same
clones and light conditions). The reproduction frequencies
observed were approximately halved, and so the relative
reproduction frequencies for these two series of experiments
were pooled and plotted as a function of the light treatment
(Fig. 3b). For a given daily light dose, the highest reproduction frequencies were observed in cultures exposed to an 8-h
photoperiod.
Effects of continuous lighting
In this series of experiments, cells of the NCC 136 and NCC
141 clones were first acclimated to continuous light
(20 mmol photons m2 s1) for 1 week. The clones were then
crossed (f.c., 2000 cells mL1), placed in darkness for 12 h,
and then exposed to low-light conditions (6.25 mmol
photons m2 s1, 8/16 h light/dark cycles). In two separate
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198
J.-L. Mouget et al.
(a)
(a)
Irradiance (relative units)
Reproduction frequency (%)
1.0
0.8
0.6
0.4
0.2
4 × 120
8 × 60
16 × 30
750
80 (b)
Transmittance (%)
80
60
40
20
60
RL
GL
40
BL
20
0
350
8 × 60
16 × 30
24 × 20
4 × 120
Light treatment (h × µmol photons m–2 s–1)
experiments, reproduction frequency values (means SE)
of 32.5 1.7% (n = 15) and 40.3 2.9% (n = 8) were
observed.
Effect of light quality
This series of experiments took place 1 year later than the
previous ones run with NCC 136 and NCC 141, and
involved a different pair of sexually compatible clones,
namely NCC 158 and NCC 234, which displayed the same
sexual types as NCC 136 and NCC 141, respectively. Parental
cells were grown at 60 mmol photons m2 s1, with a 14/10 h
light/dark cycle. After crossing, cells were exposed to low
light (28 mmol photons m2 s1) and a short photoperiod
(6/18 h light/dark cycles), and signs of sexual reproduction
(gametangia, zygotes, auxospores, and initial cells) were
observed after about 1 week of growth. Under RL, the
reproduction frequency was 8.5 1.3% (n = 3), which was
not statistically different from the control in WL (10.5 1.8%, n = 3), but under BL or GL, auxosporulation never
occurred (Fig. 4c).
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Reproduction frequency (%)
Relative reproduction frequency (%)
550
650
Wavelength (nm)
(b)
Fig. 3. Reproduction frequency (a) or relative reproduction frequency
(b) as a function of the photoperiod (4, 8, 16 h or continuous light) after
crossing Haslea ostrearia clones NCC 136 NCC 141, for a given daily
light dose (1.73 mol photons day1). Values shown in (a) are means SE
(n = 3).
c
450
24 × 20
Light treatment (h × µmol photons m–2 s–1)
0
1
0
350
0.0
100
2
14
450
550
650
Wavelength (nm)
750
(c)
12
10
8
6
4
2
0
WL
BL
GL
Light quality
RL
Fig. 4. Reproduction frequency as a function of the quality of light after
crossing Haslea ostrearia clones NCC 158 NCC 234. (a) Spectral
distribution (in relative units) of the WL control. (b) Transmittance spectra
of Lee filters used to produce BL, GL, and RL. (c) Reproduction frequency
as a function of the spectral quality of the light: WL, BL, GL, and RL.
Values shown in (c) are means SE (n = 3).
Discussion
Previous studies of sexual reproduction in H. ostrearia
had demonstrated that this species was homothallic, as
auxospores were observed in monoclonal cultures (Neuville
& Daste, 1975, 1979). More recently, a study of the breeding
system of H. ostrearia revealed that heterothally also occurs
in this species, and besides the cell size threshold, sexual
compatibility between clones (mating types) is important in
determining interclonal reproduction (Davidovich et al.,
2009). With regard to the external factors that control sexual
FEMS Microbiol Ecol 69 (2009) 194–201
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Light induction of sexual reproduction in diatoms
reproduction, auxosporulation in H. ostrearia was previously studied with cultures exposed to low light levels
(c. 24 mmol photons m2 s1), and to different photoperiods,
ranging from complete darkness to continuous illumination
(Neuville & Daste, 1975, 1979). For these authors, auxosporulation, which was supposedly intraclonal, only occurred with 6 h of light a day. The present study confirms
this finding, and provides further information about light
conditions favoring auxosporulation in this species. From
our findings, it can be concluded that as long as compatible
clones of the appropriate size are used, low light levels and
short photoperiods promote sexual reproduction in the
pennate diatom H. ostrearia. The light regime that was most
successful for inducing sexual reproduction (irradiance
o50 mmol photons m2 s1, and 6–10 h photoperiod) corresponds to the light conditions prevailing during winter near
the Bay of Bourgneuf. These conditions were experienced by
the different clones collected from this area, which were used
for these experiments. On the other hand, slightly higher
irradiances (up to 100 mmol photons m2 s1) and longer
photoperiods (up to 12 h), which can be experienced in the
natural environment in spring and fall when blooms of
H. ostrearia occasionally form near the Bay of Bourgneuf
(Robert, 1983), are still favorable for auxosporulation,
although the reproductive frequency observed was lower.
Unfortunately, no data are yet available for H. ostrearia
regarding the detection of sexual stages and the occurrence
of sexual reproduction in natural environments. This would
allow us to define the optimum natural window for sexual
reproduction in this species. In another diatom species,
Pseudo-nitzschia multiseries (Hasle) Hasle, it has been shown
that maximum reproductive success is achieved in response
to light/dark cycles similar to those that occur at the
latitudes of the Canadian Atlantic coast in late fall to early
winter, when this marine diatom form blooms (Hiltz et al.,
2000). A short daylength has also been shown to promote
sexual reproduction in other pennate diatoms, such as
Rhabdonema adriaticum Kützing (Rozumek, 1968) and
Cocconeis scutellum Ehrenberg var. ornata Grunow (Mizuno
& Okuda, 1985). Nevertheless, in other species, gametogenesis and auxospore formation are not markedly affected by
changes in light/dark cycles (e.g. Davidovich & Chepurnov,
1993; Davidovich, 1994, 2002), and some are even stimulated by a long daylength (Davidovich & Chepurnov, 1993).
An appropriate light regime can induce gametogenesis,
and zygote or auxospore formation in H. ostrearia, but light
can also completely suppress sexual reproduction. This is
particularly true for high irradiances and long photoperiods,
as well as continuous light or darkness. Intriguingly, even
though sexualization in H. ostrearia was inhibited by continuous illumination, when exposure to this condition was
followed by 12 h darkness and a subsequent transfer to light
conditions favorable for auxosporulation (short photoperiod
FEMS Microbiol Ecol 69 (2009) 194–201
and low irradiance), it actually increased the percentage of
cells at sexual stages. The significance of this phenomenon is
still unclear, and further experiments are needed to investigate the possible importance of continuous light in synchronizing the cell cycle during the phase preceding sexualization, thus increasing the subsequent reproduction
fecundity in H. ostrearia. The influence of continuous light
on sexualization seems to be species specific. Continuous
light has been shown to suppress gametogenesis and sexual
reproduction in the centric Chaetoceros curvisetus Cleve
(Roshchin, 1976; Furnas, 1985), but to promote auxospore
formation in Cyclotella meneghiniana Kützing (Schultz &
Trainor, 1968), Coscinodiscus granii Gough (Roshchin,
1972), and Coscinodiscus janischii A. Schmidt (Roshchin,
1976). In the centric diatom T. weissflogii Grunow grown in
continuous light (100 mmol photons m2 s1), interrupting
growth by a 12-h period of darkness allowed the cells to
subsequently undergo spermatogenesis (Vaulot &
Chisholm, 1987). In fact, the 12-h period of darkness has
been shown not to be necessary for spermatogenesis as long
as the irradiance is subsaturating (o 100 mmol photons
m2 s1), and continuous light inhibited the sexualization
of T. weissflogii at saturating irradiances (4 100 mmol
photons m2 s1), but not at subsaturating irradiances
(Armbrust et al., 1990). In heterothallic species such as H.
ostrearia, the light regime seems to influence the early stages
of the sexual reproduction process, before the meiotic cycle.
At least in the few species tested, a shift from mitosis to
meiosis can only occur during the early G1 phase of the cell
cycle (Armbrust et al., 1990; Davidovich, 1998), but the
initial sexual stages (during which parental cells exchange
information and recognize one another) occur during the
preceding mitotic cycle, when the parental cells form pairs.
In the mixture of two sexually compatible clones of H.
ostrearia, pairing was a relatively lengthy process, because it
took place c. 2–3 days after crossing, and gametogenesis
usually started on the cell cycle following the one during
which pairing had occurred. In cultures acclimated to
continuous light, daughter cells arising as a result of mitosis
and cytokinesis had no ‘information’ about any particular
light regime. This suggests that the stimulation of sexual
reproduction observed in the mixture of cultures after
acclimation to continuous light may have resulted from the
synchronization of the cell cycle before sexualization was
triggered by the 12-h darkness.
Another factor is light quality, although its effects on
sexual reproduction in diatoms have received less attention
in the literature than those of other external factors such as
the photoperiod or light intensity (e.g. Drebes, 1977;
Chepurnov et al., 2004, and references therein). The only
data available about the influence of different wavelengths
on auxosporulation were obtained fortuitously in Chaetoceros didymus, a centric diatom in which white light in
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contrast to red light, blue light, and to a lesser extent green
light, enhanced the formation of ‘big cells,’ which were
probably auxospores (Baatz, 1941). This finding is in
accordance with the wavelength dependence of gamete
release observed in the brown alga Silvetia compressa, which
is a heterokont chromophytic alga like the diatoms (Pearson
et al., 2004). Previous studies dealing with light quality and
H. ostrearia have focused on growth, pigment composition,
chromatic adaptation, and marennine production (Tremblin et al., 2000; Mouget et al., 2004, 2005), but not on
sexualization. The findings reported in this paper demonstrate that light quality is another factor that controls sexual
reproduction in H. ostrearia, because exposure to wavelengths in the red part of the spectrum (c. 620–700 nm)
seemed to be necessary for auxosporulation to occur.
Phytochromes are photoreceptors that allow light perception in the red/far-red region of the visible spectrum in
many organisms, including purple bacteria, cyanobacteria,
algae, fungi, and plants (e.g. Smith, 2000; Montgomery &
Lagarias, 2002; Lamparter, 2004; Rockwell et al., 2006).
These biliprotein photoreceptors are light-regulated enzymes, which change their conformation when stimulated
by alternating red and far-red wavelengths. Phytochromes
allow organisms to detect changes in light quality and
quantity, as well as in photoperiod length. In plants, they
control many processes, such as germination or flower
induction (Smith, 2000). In the ascomycete Aspergillus
nidulans, phytochrome is required for asexual sporulation
(Mooney & Yager, 1990), and is implicated in sexual development (Blumenstein et al., 2005). In algae, phytochromes
are known to be involved in the regulation of cell division in
Euglena gracilis (Bolige & Goto, 2007) and in chloroplast
movement in the green algae Mesotaenium caldariorum (Wu
& Lagarias, 1997) and Mougeotia spp. (e.g. Gabrys et al.,
1997). The marine diatom T. weissflogii can perceive red and
far-red light, because expression of the fucoxanthin–chlorophyll a/c-binding protein genes is photoregulated by a
phytochrome-like sensing system (Leblanc et al., 1999).
Furthermore, genes encoding for a putative homolog of
phytochrome have been identified in Thalassiosira pseudonana (Armbrust et al., 2004). Nevertheless, the expression of
these genes is considered unpredictable, because of the low
fluences of red/far-red light underwater (Leblanc et al.,
1999), although phytochromes could be of some importance for the perception of light microenvironments,
including fluorescence emission from neighboring cells
(Ragni & Ribera d’Alcalà, 2004). However, the oyster ponds
from which H. ostrearia clones were isolated, known as
‘claires,’ are shallow and characterized by low turbidity and
transparent water. They thus constitute aquatic environments
with relatively high levels of red/far-red light in which this
ratio can change on a daily basis (Chambers & Spence, 1984).
If it can be demonstrated in situ that H. ostrearia possesses this
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
J.-L. Mouget et al.
capacity to sense red/far-red light, and to use this information
in achieving reproduction, this could explain why year on year
H. ostrearia regenerate big cells, thus maintaining populations
that are able to colonize these ponds.
Hence, different species of diatoms display different
strategies with regard to the light/dark regimes that can
induce sexual reproduction. Photoperiod, light intensity,
and light quality can all act as exogenous triggers that
control sexualization in pennate diatoms, such as H. ostrearia,
together with internal induction cues, such as decreased cell
size and sexual compatibility between the partners.
Acknowledgements
We would like to thank Fariza Hassani and Elisabeth Pradier
for laboratory assistance. We gratefully acknowledge receiving
financial help from EGIDE (France) to provide funds for
travel and other expenses, and we thank Université du Maine
for providing facilities for one of us (N.A.D.). We also thank
Steve Bates for helpful suggestions, and two anonymous
reviewers for critical comments on the manuscript.
References
Armbrust VE, Chisholm SW & Olson RJ (1990) Role of light and
the cell cycle on the induction of spermatogenesis in a centric
diatom. J Phycol 26: 470–478.
Armbrust VE, Berges JA, Bowler C et al. (2004) The genome of
the diatom Thalassiosira pseudonana: ecology, evolution, and
metabolism. Science 306: 79–86.
Baatz I (1941) Die Bedeutung der Lichtqualität für Wachstum
und Stoffproduktionplanktonischer Meeres Diatomeen.
Planta 31: 726–766.
Blumenstein A, Vienken K, Tasler R, Purschwitz J, Veith D,
Frankenberg-Dinkel N & Fischer R (2005) The Aspergillus
nidulans phytochrome FphA represses sexual development in
red light. Curr Biol 15: 1833–1838.
Bolige A & Goto K (2007) High irradiance responses involving
photoreversible multiple photoreceptors as related to
photoperiodic induction of cell division in Euglena. J Photoch
Photobio B 86: 109–120.
Chambers PA & Spence DHN (1984) Diurnal changes in the ratio
of underwater red to far red light in relation to aquatic plant
photoperiodism. J Ecol 72: 495–503.
Chepurnov VA, Mann DG, Sabbe K & Vyverman W (2004)
Experimental studies on sexual reproduction in diatoms. Int
Rev Cytol 237: 91–154.
Davidovich NA (1994) Factors controlling the size of initial cells
in diatoms. Russ J Plant Physiol 41: 220–224.
Davidovich NA (1998) Transition to sexual reproduction and
control of initial cell size in Nitzschia lanceolata. Diatom Res
13: 29–38.
Davidovich NA (2001) Species specific sizes and size range of
sexual reproduction in diatoms. Proceedings of the 16th
FEMS Microbiol Ecol 69 (2009) 194–201
201
Light induction of sexual reproduction in diatoms
International Diatom Symposium (Economou-Amilli A, ed),
pp. 191–196. University of Athens, Athens.
Davidovich NA (2002) Fotoregulyatsiya polovogo vosproizvedeniya
u Bacillariophyta (Obzor). Algologiya 12: 259–272.
Davidovich NA & Chepurnov VA (1993) Intensivnost
auksosporoobrazovaniya u dvuh vidov Bacillariophyta v
zavisimosti ot osveshchennosti i prodolzhitelnosti
fotoperioda. Algologiya 3: 34–41.
Davidovich NA, Mouget J-L & Gaudin P (2009) Heterothallism
in the pennate diatom Haslea ostrearia. Eur J Phycol 44:
251–261.
de Brouwer JFC, Wolfstein K & Stal LJ (2002) Physical
characterization and diel dynamics of different fractions of
extracellular polysaccharides in an axenic culture of a benthic
diatom. Eur J Phycol 37: 37–44.
Drebes G (1977) Sexuality. The Biology of Diatoms, Botanical
Monographs, Vol. 13 (Werner D, ed), pp. 250–283. Blackwell
Scientific Publications, London.
Furnas MJ (1985) Dial synchronization of sperm formation in
diatom Chaetoceros curvisetum Cleve. J Phycol 21: 667–671.
Gabrys H, Walczack T & Malec P (1997) Interaction between
phytochrome and the blue light photoreceptor system in
Mougeotia: temperature dependance. J Photoch Photobio B
38: 35–39.
Geitler L (1932) Der Formwechsel der pennaten Diatomeen
(Kieselalgen). Arch Protistenk 78: 1–226.
Harrison PJ, Waters RE & Taylor FJR (1980) A broad spectrum
artificial seawater medium for coastal and open ocean
phytoplankton. J Phycol 16: 28–35.
Hiltz M, Bates SS & Kaczmarska I (2000) Effect of light: dark
cycles and cell apical length on the sexual reproduction
of the pennate diatom Pseudo-nitzschia multiseries
(Bacillariophyceae) in culture. Phycologia 39: 59–66.
Lamparter TL (2004) Evolution of cyanobacterial and plant
phytochromes. FEBS Lett 573: 1–5.
Leblanc C, Falciatore A, Watanabe M & Bowler C (1999) Semiquantitative RT-PCR analysis of photoregulated gene
expression in marine diatoms. Plant Mol Biol 40: 1031–1044.
Mizuno M & Okuda K (1985) Seasonal change in the distribution
of cell size of Cocconeis scutellum var. ornata
(Bacillariophyceae) in relation to growth and sexual
reproduction. J Phycol 21: 547–553.
Montgomery BL & Lagarias JC (2002) Phytochrome ancestry:
sensors of bilins and light. Trends Plant Sci 7: 357–366.
Mooney JL & Yager LN (1990) Light is required for conidiation in
Aspergillus nidulans. Genes Dev 4: 1473–1482.
Mouget J-L, Tremblin G, Morant-Manceau A, Morançais M &
Robert J-M (1999) Long-term photoacclimation of Haslea
ostrearia (Bacillariophyta): effect of irradiance on growth rates,
pigment content and photosynthesis. Eur J Phycol 34: 109–115.
Mouget J-L, Rosa P & Tremblin G (2004) Acclimatation of Haslea
ostrearia to light of different spectral qualities – confirmation
FEMS Microbiol Ecol 69 (2009) 194–201
of ‘chromatic adaptation’ in diatoms. J Photoch Photobio B 75:
1–11.
Mouget J-L, Rosa P, Vachoux C & Tremblin G (2005)
Enhancement of marennine production by blue light in the
diatom Haslea ostrearia. J Appl Phycol 17: 437–445.
Neuville D & Daste P (1975) Observations préliminaires
concernant l’auxosporulation chez la diatomée Navicula
ostrearia (Gaillon) Bory en culture in vitro. CR Acad Sci Paris
Sér D 281: 1753–1756.
Neuville D & Daste P (1979) Observations concernant les phases
de l’auxosporulation chez la diatomée Navicula ostrearia
(Gaillon) Bory en culture in vitro. CR Acad Sci Paris Sér D 288:
1496–1498.
Pearson GA, Serrão EA, Dring M & Schmid R (2004) Blue- and
green-light signals for gamete release in the brown alga, Silvetia
compressa. Oecologia 138: 193–201.
Ragni M & Ribera d’Alcalà M (2004) Light as an information
carrier underwater. J Plankton Res 26: 433–443.
Robert J-M (1983) Fertilité des claires ostréicoles et verdissement:
utilisation de l’azote par les diatomées dominantes. PhD
Thesis, University of Nantes, Nantes.
Rockwell NC, Su Y-S & Lagarias JC (2006) Phytochrome
structure and signaling mechanisms. Annu Rev Plant Biol 57:
837–858.
Roshchin AM (1972) Vliyanie uslovij osveshcheniya na
obrazovanie auksospor i skorost deleniya kletok Coscinodiscus
granii Gough. Fiziologiya Rastenij 19: 180–185.
Roshchin AM (1976) Vliyanie uslovij osveshcheniya na
vegetativnoe razmnozhenie kletok i polovoe vosppoizvedenie
dvuh vidov tsentricheskih diatomovyh vodoposlej. Fiziologiya
Rastenij 23: 715–719.
Rozumek KE (1968) Der Einfluss der Umweltfaktoren Licht und
Temperatur auf die Ausbildung der Sexualstadlen bei der
pennaten Diatomee Rhabdonema adriaticum Kütz. Beitr Biol
Pfl 44: 365–388.
Schultz MF & Trainor FR (1968) Production of male gametes and
auxospores in the centric diatoms Cylotella meneghiniana and
C. cryptica. J Phycol 4: 85–88.
Smith H (2000) Phytochromes and light signal perception by
plants – an emerging synthesis. Nature 407: 585–591.
Tremblin G, Cannuel R, Mouget J-L, Rech M & Robert J-M
(2000) Change in light quality due to a blue-green
pigment, marennine, released in oyster-ponds: effect on
growth and photosynthesis in two diatoms, Haslea
ostrearia and Skeletonema costatum. J Appl Phycol 12:
557–566.
Vaulot D & Chisholm SW (1987) Flow cytometric analysis of
spermatogenesis in the diatom Thalassiosira weissflogii
(Bacillariophyceae). J Phycol 23: 132–137.
Wu S-H & Lagarias JC (1997) The phytochrome photoreceptor in
the green alga Mesotaenium caldariorum: implication for a
conserved mechanism of phytochrome action. Plant Cell Env
20: 691–699.
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c