A marine diatom-derived aldehyde induces apoptosis in copepod and sea urchin embryos
1 Stazione Zoologica `Anton Dohrn' Villa Comunale 1 - 80121 Napoli,
Italy
2 Istituto Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche -
83100 Avellino, Italy
* Author for correspondence (e-mail: romano{at}szn.it)
Accepted 2 July 2003
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Summary |
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Key words: apoptosis, unsaturated aldehyde, copepod, sea urchin, embryo, caspase-3, diatom
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Introduction |
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The production of aldehydes is induced by damage of diatom cells, as would
occur during grazing by copepods (Pohnert,
2000). Pohnert showed that this mechanism of cell defence is
initiated by phospholipases, with a drastic increase in the amount of free
polyunsatured eicosanoids in the first minutes after wounding. Using
fluorescent probes, he suggested that the main enzyme activity responsible for
initiation of the aldehyde-generating lipase-lipoxygenase-hydroperoxide lyase
cascade is phospholipase A2 (PLA)
(Pohnert, 2002
).
In terrestrial environments, the production of aldehydes and other
oxylipins in plants has often been associated with a wound-activated mechanism
(Matsui et al., 2000;
Rosahl, 1996
), involving the
sequential action of the above-mentioned enzymes: a lipase, lipoxygenase (LOX)
and hydroperoxide lyase (HPL) (Blée,
1998
). The aldehydes thus generated show pheromonal, bactericidal
and fungicidal activities, providing in many cases a chemical defence against
pathogens and herbivorous insects (Pohnert
and Boland, 2002
). It has also been suggested that in copepods
such compounds are an activated chemical defence by diatoms to deter future
generations of potential grazers.
Here, we examine for the first time the effects of the diatom-derived
aldehyde decadienal (DD) on the apoptotic machinery of copepod and sea urchin
embryos and compare the biological activity of this unsaturated fatty aldehyde
with the saturated aldehyde decanal. Apoptosis, or programmed cell death
(PCD), is the result of complex signal transduction pathways leading to
gene-mediated cell death. PCD is an evolutionarily conserved process, present
in both the animal and plant kingdom. Apoptotic events induce morphological
and biochemical alterations including cell shrinkage, disintegration through
blebbing and activation of specific caspases that lead to enzymatic breakdown
of DNA (Lockshin et al.,
1998). PCD plays an essential role in physiological processes such
as differentiation (Jacobson et al.,
1997
) and immune system regulation
(Krammer, 2000
;
Nagata, 1997
). Extensive
apoptosis is active in the female germline of many species, ranging from worms
to humans. Indeed, the functional lifespan of the female gonads is defined by
the size and rate of depletion of oocytes enclosed within follicles in the
ovaries at birth. The physiological goal for germline apoptosis resides in the
removal of defective cells unable to develop into fertile eggs, and to provide
essential nutrients to the surviving oocytes (reviewed in
Buszczak and Cooley, 2000
;
Morita and Tilly, 1999
). Thus,
any factor that disrupts the normal production of female gametes is a
potential threat to reproductive performance. Potential hazards to gonadal
function might be derived from environmental sources, and particularly affect
females since, unlike males, females are born with an irreplaceable number of
germ cells in their ovaries at the time of birth. For example, exposure of
women to potentially damaging agents, such as anti-cancer drugs, industrial
chemicals or even cigarette smoke, can have dramatic and irreparable effects
on the ovary by accelerating the natural process of germ cell depletion
(Tilly, 1998
).
The results presented here demonstrate that the diatom-derived aldehyde DD triggers an apoptotic mechanism in copepod and sea urchin embryos. These results are discussed in the context of laboratory findings on the toxic effects of diatoms on potential grazers such as copepods and sea urchins.
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Materials and methods |
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A second group of C. helgolandicus females were incubated in 100
ml crystallizers containing the control algae Prorocentrum minimum
(PRO), a dinoflagellate that does not impair copepod embryogenesis
(Turner et al., 2001). Embryos
produced by females fed PRO for 24 h were collected at the 8- to 32-blastomere
stage and fixed as described above, or incubated for 1 h in 5 µg
ml-1 of DD or in 5 µg ml-1 decanal. DD and decanal
solutions were prepared by diluting commercial compounds (Sigma-Aldrich,
Milan, Italy) in methanol to obtain 2 mg ml-1 stock solutions.
Copepod embryos were also incubated for 1 h in 0.5% methanol to exclude
unspecific effects of this solvent.
Copepod embryos used for DNA fragmentation experiments were obtained from females fed PRO for 24 h, and incubated in DD as described above.
Sea urchin embryo collection
Sea urchins Paracentrotus lividus (Lamarck) were collected by
SCUBA diving in the Gulf of Naples and transported to the laboratory. Soon
after their arrival, living organisms were injected with 0.5 mol
l-1 KCl to induce gamete ejection. Spawned eggs were allowed to
settle and were then washed three times with FSW and diluted to a final
concentration of 3000 eggs ml-1. Concentrated sperm were collected
and diluted immediately prior to fertilization in FSW. Fertilization occurred
in FSW for enzymatic bioassays and in FSW with 1 mmol l-1 ATA
(3-amino-1,2,4-triazole; Sigma-Aldrich) for immuno-fluorescence staining
(Buttino et al., 1999). 90 min
after fertilization, when the third mitotic division was completed, embryos
were incubated in 5 ml tissue culture wells containing 2, 5 and 10 µg
ml-1 (final concentration) of DD or decanal, prepared as described
above. A different group was used as controls. To test the effect of the
solvent, another group of embryos was incubated in 0.5% methanol, which was
the highest concentration of solvent used in the incubation experiments. The
percentage of embryos presenting membrane blebbing was determined every 30 min
for each concentration of DD, decanal and methanol.
Fluorescence labelling and confocal microscopy
C. helgolandicus embryos, incubated in DD, decanal or methanol,
were washed three times in FSW before fixation in 2-4% paraformaldehyde
overnight. Fixed embryos were then rinsed several times in PBS and immediately
stained with TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP Nick
End Labelling; Roche Diagnostics GmbH, Mannheim, Germany), or stored at
4°C in PBS containing 0.02% NaN3, until fluorescence
labelling.
Before TUNEL staining, copepod embryos were incubated for 24 h in 250 µl of 1 U ml-1 chitinase enzyme (EC3.2.1.14; Sigma-Aldrich) dissolved in 50 mmol l-1 citrate buffer, pH 6, at 25°C, to permeabilize the chitinous wall. After rinsing several times in PBS, embryos were incubated for 2 h in 0.1% Triton X-100 at room temperature, rinsed in PBS containing 1% BSA, and further incubated for 90 min in TUNEL solution at 37°C. To obtain TUNEL-positive samples, embryos were incubated for 10 min in 50 mmol l-1 Tris-HCl, pH 7.5, 10 mmol l-1 MgCl2, 0.1% dithiothreitol, containing 250 µg ml-1 DNase I (grade II from bovine pancreas; Boheringer GmbH, Mannheim, Germany) at room temperature. Negative controls were obtained by incubating embryos in label solution only, as recommended by the manufacturers of the TUNEL kit.
8-blastomere sea urchin embryos, incubated for 30, 60, 90 and 120 min in DD or in decanal, for 30 and 120 min in 0.5% methanol and controls, were gently forced through a Pasteur pipette to remove the fertilization envelope, rinsed three times in FSW, and fixed for 1 h in 4% paraformaldehyde dissolved in PBS and 0.2 mol l-1 NaCl, at room temperature. Fixed sea urchin embryos were washed several times in PBS, pH 7.4, to remove paraformaldehyde and then incubated for 1 h in asolution of 0.1% Triton X-100 and 0.1% sodium citrate, at 4°C. After washing in PBS containing 1% bovine albumin serum (BSA, Sigma-Aldrich) samples were incubated at 37°C for 90 min in TUNEL solution in a humidified chamber, in the dark. Control embryos were fixed and stained as described above. Before staining with TUNEL, a group of embryos that were not incubated in DD were used as positive and negative controls. Positive and negative controls were obtained as described above for copepod embryos.
Whole-mount sea urchin and copepod embryos were observed with an inverted
Zeiss LSM-410 confocal laser scanning microscope equipped with a 40x
water immersion objective (NA 1.2). Each image was acquired with an Argon 488
nm wavelength () laser to detect TUNEL fluorescence (green), and with
a 633 nm
laser to visualize samples in transmitted light. Images were
reconstructed three-dimensionally using the Zeiss software.
DNA extraction and fragmentation
DNA was extracted from C. helgolandicus embryos incubated for 1, 3
and 6 h with 5 µg ml-1 of DD, and their untreated controls,
after initial digestion with chitinase as follows. Embryos stored at -80°C
were thawed and resuspended in 25 µl of 50 mmol l-1 citrate
buffer, pH 6.0, before addition of an equal volume of chitinase dissolved in
the same buffer to a final concentration of 10 U ml-1. Incubation
was continued for 16 h at 25°C, followed by addition of a second amount of
enzyme (10 U ml-1) and incubation for a further 2 h. Subsequently,
genomic DNA was extracted using a commercially available kit (Nuclespin
nucleic acid purification kit), following the manufacturer's instructions
(Clontech, Palo Alto, CA, USA). The total amount of DNA recovered from 300-800
embryos was 1-4 µg. Amplification of the 16S rRNA gene by polymerase chain
reaction (PCR) was carried out as a positive control for DNA extraction, as
reported by Bucklin et al.
(1995). Using 100-200 ng of
template, and the 16SAR (5'-CGCCTGTTTAACAAAAACAT-3') and 16SBR
primers (5'-CGGTTTGAACTCAGATCACGT-3'), PCR was performed on a MJ
(Waltham, MA, USA) apparatus (Bucklin et
al., 1995
).
DNA fragmentation was measured directly on recovered DNA by loading samples
of extracted DNA (approx. 1 µg) onto 2% agarose gels, and analysis by
ethidium bromide staining. Alternatively, DNA samples were subjected to LM
(ligation mediated)-PCR, a technique designed for the detection of nucleosomal
ladders in apoptotic cells (Staley et al.,
1997). A commercially available kit (Clontech) was used to
visualise ladders that were undetectable by other methods. Briefly, fragmented
DNA was ligated with adaptor primers and these were amplified using PCR and a
24-mer primer. The resulting ladder was visualized on an agarose gel stained
with ethidium bromide.
Assay for caspase-like activity
Approximately 3000 sea urchin embryos were incubated for 30, 60, 90 and 120
min in 5 or 10 µg ml-1 DD, rinsed three times in FSW,
transferred to a 0.5 ml tube and centrifuged for 3 min at 1700
g. The pelleted embryos were stored at -80°C until the
enzymatic assay was performed. Copepod embryos were collected 1, 3, 6 and 12 h
after incubation with 5 µg ml-1 DD, and prepared using the same
protocol as sea urchin embryos. Frozen embryos were resuspended in lysis
buffer (50 mmol l-1 Tris-Cl, pH 7.6, 150 mmol l-1 NaCl,
5 mmol l-1 EDTA, 1% Nonidet NP-40, 0.5 mmol l-1
dithiothreitol, 10% glycerol, 100 µg ml-1 phenylmethanesulfonyl
fluoride, plus a cocktail of protease inhibitors: `Complete'; Boehringer) and
sonicated twice for 20 s at 20% of the maximum potency with a Sonfier 250
(Branson Ultrasonic Corporation, Danbury, CT, USA). The FlorAce Apopain Assay
Kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect
caspase-3-like activity. The kit contains a fluorescent substrate, the
acetylated peptide Ac-DEVD-AFC
(carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin), which
releases the fluorescent AFC moiety after enzymatic hydrolysis. Samples (40
µl) were incubated with 55 µmol l-1 Ac-DEVD-AFC substrate at
37°C for 2 h. Apopain included in the kit was used as a positive control
for caspase-3 activity. The caspase-3-specific inhibitor Z-DEVD-FMK was
employed to determine and quantify the non-specific caspase-3 activity in the
extracts, following the manufacturer's instructions (Bio-Rad Laboratories).
AFC fluorescence was detected using a Perkin-Elmer spectrophotometer LS50B at
395 nm excitation and 540 nm emission wavelengths. Protein concentrations were
determined using the method of Bradford
(1976). To detect other
caspase-like enzymes in the homogenate samples, a caspase-family fluorimetric
substrate set (Biovision, purchased from Alexis Corporation, Lausen,
Switzerland) was used under the same conditions. The set contained substrates
for caspases 1, 2, 5, 6, 8 and 9.
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Results |
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Two approaches were employed to verify the ability of DD to induce
apoptosis in copepods. (1) In vivo experiments. C.
helgolandicus embryos were obtained from females fed the diatom THA for
10-15 days, the time necessary to induce about 35% hatching failure
(Chaudron et al., 1996). (2)
In vitro experiments. Newly spawned embryos from females fed the
control diet PRO were directly exposed to 5 µg ml-1 DD. In
in vivo experiments, immunofluorescence assays revealed that
30±12.3% (mean ± S.D., N=200) of the embryos
were positively stained with TUNEL (Fig.
1). Fig. 1A shows a
TUNEL-positive embryo in which apoptotic nuclei appear green. Chromatin is
condensed without any apparent apoptotic bodies characteristic of late
apoptosis; however, distribution of the nuclei is asymmetrical and, in
transmitted light, the embryo appears abnormal with an irregular shape
(Fig. 1B). It is known that
C. helgolandicus females fed the non-diatom algae PRO produce almost
100% viable embryos (Poulet et al.,
1994
), but induction of apoptosis was observed in
61.7±22.5% (mean ± S.D.) of PRO-fed embryos incubated
for 60 min with 5 µg ml-1 DD
(Fig. 1C,D).
Fig. 1E,F shows a control
embryo produced by C. helgolandicus females fed PRO in which TUNEL
staining is negative with nuclei appearing dark and symmetrically distributed
(Fig. 1E). The green
fluorescent background was obtained by amplifying the brightness to highlight
unstained nuclei. Positive controls, incubated with DNase, have fluorescent
nuclei, like the DD-incubated embryos (Fig.
1G,H). After 1 h of incubation, decanal induced apoptosis in
23.7±19.4% (mean ± S.D., N=100) embryos.
After incubation in methanol the percentage of TUNEL-positive embryos was
14.0±9.9% (mean ± S.D., N=100), similar to
that observed in untreated embryos (data not shown).
|
Since treatment of C. helgolandicus embryos with DD revealed an
apoptogenic phenotype, we tried to detect DNA fragmentation in embryos that
had been treated for 1-6 h with 5 µg ml-1 of DD. Initially, DNA
recovery was very low, probably due to the external chitinous wall of the
embryos, but this problem was overcome by solubilisation with chitinase before
beginning the DNA extraction protocol, allowing us to obtain enough DNA for
further analysis. As a positive control, we amplified the 16S rRNA gene using
specific primers, and obtained a single specific fragment of approximately 460
bp (Fig. 2A) similar to that
reported by other authors (Bucklin et al.,
1995; Lindeque et al.,
1999
). Subsequently, these DNA samples were analysed for DNA
fragmentation. Fig. 2B shows an
example DNA ladder obtained from copepod embryos that had been treated with
DD. For each length of treatment, the same amount of DNA isolated from
untreated and DD treated embryos was loaded on agarose gels.
Table 1 summarizes the results
obtained from copepod embryos incubated for different times with DD.
Incubation for 1 h in the presence of 5 µg ml-1 of DD was
sufficient to induce clear, detectable DNA fragmentation into oligonucleosomal
DNA fragments compared to untreated embryos (no detectable fragmentation).
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The effects of DD were tested on sea urchin embryos at the 8-blastomere-cell stage and the results compared with those obtained for copepod embryos. Progression through apoptosis was observed by morphological cell changes, chromatin condensation and degradation, and by activation of caspases. Changes in cell morphology, such as cell shrinkage and membrane blebbing, were analysed by light microscopy after incubation at three different DD concentrations (Fig. 3). Aconcentration of 2 µg ml-1 induced blebbing in >20% of embryos 120 min after incubation and in 50% of embryos after 150 min. After 240 min, >90% of embryos showed vesciculated membranes. A concentration of 5 µg ml-1 of DD induced blebbing in 50% of embryos after almost 100 min, and 100% after 120 min incubation. All embryos incubated in 10 µg ml-1 DD showed plasma membrane blebbing 45 min after incubation. At all concentrations tested, mitotic divisions were arrested and embryos remained blocked at the 8-blastomere stage. In contrast, cell division in sea urchin embryos incubated in decanal was arrested only at concentrations >10 µg ml-1 and blebbing was never observed. Methanol incubation had no effect on cell division (data not shown).
|
TUNEL-positivity was detected using laser confocal microscopy. Fig. 4A shows an untreated sea urchin embryo at the 16-blastomere stage, fixed 3 h after fertilization. Green fluorescence, due to TUNEL labelling, was not evident, indicating the absence of DNA fragmentation. Fig. 4B shows the same embryo in transmitted light with symmetrically dividing cells. Fig. 4C,D shows the effect of 90 min incubation with 5 µg ml-1 DD: the embryo was blocked at the 8-blastomere stage; nuclei appeared green when observed for TUNEL fluorescence, indicating they had undergone apoptosis. Although the transmitted light image showed an apparently normal embryo with 8 symmetrical blastomeres (Fig. 4D), the fluorescent image clearly showed that nuclei had been subjected to DNA fragmentation, identified by single strand breaks. After 120 min incubation with 5 µg ml-1 DD, embryos appeared morphologically abnormal, with small, condensed fragments of green fluorescent chromatin (TUNEL) (Fig. 4E). Parallel transmitted light images revealed a very abnormal embryo (Fig. 4F).
|
Fig. 5 shows that the number of apoptotic embryos increased with time of incubation in 5 µg ml-1 DD. In fact, more than 40% of the embryos were positive for TUNEL after 1 hincubation in DD; the percentage increased to about 80% after 90 min of incubation. In control embryos, a physiological apoptotic process occurred in less than 20% of the embryos (N=200). The same results were obtained when embryos were incubated in methanol. The percentage of TUNEL-positive embryos incubated in 5 µg ml-1 decanal is similar to the controls until 90 min of incubation, after which it increased to 43.3±2.26% (mean ± S.D.).
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As a biochemical marker for apoptotic processes in sea urchin embryos treated with DD, we tested the ability of DD to activate caspase-like enzymes using several commercially available kits developed for vertebrate caspases. None of the caspases 1, 2, 4, 6, 8 and 9 were active in our experiments (G. Romano, unpublished data). We detected caspase-3-like activity only in sea urchin embryos after 60 min of exposure to 5 µg ml-1 of DD (Fig. 6), with maximum activity after incubation for 120 min. After this time, embryos appeared degenerated (Fig. 4). When the assay was performed in the presence of a caspase-3 specific inhibitor (ZVAD-FMK), release of the AFC moiety fell to zero, indicating that the proteolytic activity measured was attributable specifically to a sea urchin caspase-3-like enzyme. Treatment of the embryos with a higher concentration of DD (10 µg ml-1) induced an earlier and more pronounced activation of caspase-3 activity, suggesting a dose-dependent effect between DD and enzyme activation. Caspase-3-like activity was also tested on copepod embryos but we were unable to detect any activity (data not shown).
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Discussion |
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DD was previously reported to induce apoptosis in mammalian tumour cells
(Miralto et al., 1999). Here
we demonstrate that DD also induces apoptosis in copepod and sea urchin
embryos at micromolar concentrations. In copepods, DNA fragmentation was
detected both in embryos spawned by females fed the diatom THA, and in embryos
spawned by females fed the control diet PRO and then incubated directly in DD.
This is the first time that DNA fragmentation has been detected in copepods
using two independent but complementary techniques, such as laddering and
TUNEL bioassays. Similarly to the observation in copepods, DD induced TUNEL
positivity and blebbing in 8- to 16-blastomeres of sea urchin embryos. Mitotic
divisions were arrested at all DD concentrations tested, whereas apoptogenic
phenotype appearance was time- and dose-dependent. Interestingly, in both
systems, the appearance of the apoptotic phenotype followed the same kinetics,
peaking after 90-120 min after exposure to DD, which suggests possible
similarities between the two apoptogenic pathways.
Programmed cell death (PCD) is a process that is generally classified into
two main categories, depending on whether a class of proteolytic enzymes known
as caspases are activated or not. In the so-called caspase-dependent cell
death, activation of these enzymes is absolutely required to induce late and
irreversible apoptotic processes, such as DNA fragmentation and cell blebbing
(Krammer, 2000;
Lockshin et al., 1998
). By
contrast, in the caspase-independent PCD, caspase activity is not required
(Mathiasen and Jaattela,
2002
). In the present study, we did not detect caspase activity in
copepod embryos treated with DD using either enzymatic assays or
immunoblotting as evidence of proteolytic activation of caspases (data not
reported), which suggests that the PCD was caspase-independent. By contrast,
we could detect caspase-3 activity in sea urchin embryos treated with DD using
a commercially available kit designed for the mammalian version of caspase-3.
We clearly measured increased caspase-3 activity in sea urchin embryos 60 min
after addition of DD. Caspase-3 activity was tested for two reasons: because
it is conserved in animals ranging from worms (Caenorhabditis
elegans) to humans, and because caspase-3 is a downstream member of the
caspase cascade, so its enzymatic activity is enhanced compared to most
upstream caspases, such as caspase-8. At the present time, we cannot exclude
the possibility that the assay employed was not sensitive enough for copepods,
or that the substrate used for caspase-3 activity was not specific for the
copepod enzyme. Future work will help clarify this aspect.
Activation of caspases in sea urchins has also been reported for embryos
exposed to staurosporine (Voronina and
Wessel, 2001). These authors observed differences in apoptotic
response between oocytes, eggs and early sea urchin embryos, suggesting that
activation of apoptotic pathways may differ depending on the developmental
stage. Although we did not characterised sea urchin caspase-3 activity in
detail, we can speculate that it is probably structurally different from its
vertebrate homologs, since the Km for the peptide used as
a substrate (Ac-DEVD-AFC) appeared relatively higher than that of the human
enzyme used as a positive control (G. L. Russo, unpublished data). We also
tried to detect other caspase-like activity in sea urchins using substrates
available for mammalian caspases 1, 2, 5, 6, 8 and 9, without any success (G.
Romano, unpublished data). The time course of caspase-3 activation correlated
perfectly with the appearance of apoptotic nuclei determined by TUNEL. This is
unusual in a caspase-dependent PCD process, where caspase-3 activation
normally precedes DNA fragmentation and blebbing. Other studies have reported
that caspases may be activated even if not required for the progression of
apoptosis (Johnson et al.,
1999
), and DNA fragmentation may be caused by mechanisms other
than caspase-3 activation of DNase
(Hamilton et al., 1998
). Hence
increased caspase-3 activity may be an epiphenomenon, not the cause, of late
events such as DNA laddering and cell blebbing. The correspondence of these
two events, caspase-3 activation and blebbing, in sea urchin embryos may
indicate the presence of a caspase-independent PCD. This observation
strengthens the hypothesis that DD also induces a caspase-independent
apoptosis in copepod embryos, where, in fact, caspase activity was not
found.
Recently Pohnert and Boland
(1996) and Pohnert et al.
(2002
) reported the presence
of an aldehydic compound with similar reactive unsaturation and biological
activity to DD, the oxoacid
12-oxo-5-cis-8-cis-10-trans dodecatrienoic acid, in
benthic diatoms, and showed that this molecule blocked cell division in sea
urchin embryos. Hence sea urchins, which actively feed on benthic diatoms, are
potentially exposed to the effects of unsaturated reactive compounds, as are
copepods that have already been shown to graze on pelagic diatoms containing
DD and other
,ß-unsaturated aldehydes
(Ianora et al., 2003
and
references therein). The concentration of diatom cells used in our in
vivo experiments are of the same order of magnitude reached in bloom
conditions at sea (Miralto et al.,
2003
), suggesting that diatom blooms can potentially impact the
reproductive fitness of grazers, with important consequences at the population
level. Clearly more investigations are needed to clarify the molecular targets
of diatom-derived
,ß-unsaturated aldehydes, to better understand
their antiproliferative activity.
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Acknowledgments |
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