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First published online April 18, 2008
Journal of Experimental Biology 211, 1426-1433 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015859
Aldehyde-encapsulating liposomes impair marine grazer survivorship
1 Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy
2 Dipartimento di Chimica Farmaceutica e Tossicologica, Università degli
Studi di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
* Author for correspondence (e-mail: buttino{at}szn.it)
Accepted 2 March 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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7 µm) as a
delivery system for the oxylipin DD, prepared in the same size range as
copepod food and containing known amounts of DD. The aim of this work was to
relate the ingestion of DD to the reproductive failure of the copepods
Temora stylifera and Calanus helgolandicus. Liposomes were
very stable over time and after 10 days of feeding, liposomes encapsulating DD
reduced egg hatching success and female survival with a concomitant appearance
of apoptosis in both copepod embryos and female tissues. Concentrations of DD
inducing blockage were one order of magnitude lower that those used in
classical feeding experiments demonstrating that liposomes are a useful tool
to quantitatively analyze the impact of toxins on copepods.
Key words: copepod, diatom, decadienal, reproduction, egg viability, apoptosis
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Pohnert, 2005) with possible
consequences on the transfer of energy through the marine food chain to top
carnivores (Miralto et al.,
1999; Ianora et al.,
2004). This insidious mechanism, which does not deter the
herbivore from feeding but impairs its recruitment, will restrain the cohort
size of the next generation. The compounds responsible for reduced hatching
success and abnormal larval development in these small crustaceans are several
oxylipins, products of the enzymatic oxidation of fatty acids, which include
polyunsaturated aldehydes (PUAs) as well as fatty acid hydroxides, epoxy
alcohols, hydroperoxides and reactive oxygen species (ROS)
(Fontana et al., 2007). These
compounds are not constitutively present in the cells but are only produced
when the cell is damaged as would occur during grazing
(Pohnert, 2000). Owing to the
teratogenic nature of diatom oxylipins, the mechanism of chemical defense in
diatoms functions through induction of apoptosis which can occur in maturing
oocytes (Poulet et al., 2007)
or during embryo development (Romano et
al., 2003) and in newly hatched nauplii
(Ianora et al., 2004;
Poulet et al., 2003). The
toxicity of oxylipins, and more specifically of PUAs, has been demonstrated in
classical feeding experiments using diatoms as copepod food
(Ianora et al., 2003;
Poulet et al., 2007) or in
in vitro tests by incubating embryos and adults in known
concentrations of pure molecules dissolved into seawater
(Ianora et al., 1999;
Romano et al., 2003;
Adolph et al., 2004;
Caldwell et al., 2005;
Taylor et al., 2007).
Recently, Ianora et al. used the dinoflagellate Prorocentrum
minimum, which does not produce oxylipins, as a live carrier of
2-trans,4-trans-decadienal (henceforth called decadienal or
DD), which has been widely used as a model aldehyde to study the deleterious
effects of these compounds on marine invertebrates
(Ianora et al., 2004). The
daily ingestion rate of DD by females of the copepod Calanus
helgolandicus was indirectly calculated from the filtration rate of
P. minimum, considering the amount of DD adsorbed onto the algal
cells. However, since the interaction between PUAs and live carriers is
unknown, the question as to how much and for how long ingestion of oxylipins
affects copepod reproduction remains a critical point to understanding the
functional role of such compounds in the marine system.
Until now, the lack of an efficient method to deliver realistic amounts of
these compounds into the copepod body has represented a major limitation to a
deeper investigation of the toxicological impact of oxylipins in copepods.
Recently, several authors have discussed the need to develop inert carriers
delivering known concentrations of oxylipins into copepods, and able to mimic
their release as occurs during diatom feeding under natural conditions
(Caldwell et al., 2004;
Paffenhofer et al., 2005), in
order to better understand the impact of these compounds on copepod population
control at sea. Although the use of liposomes as a delivery system for drugs
and chemicals in zooplankton and in aquaculture is not new
(Hontoria et al., 1994;
Ozkizilcik and Chu, 1994;
Koven et al., 1999;
Touraki et al., 1995), this
technique has only recently been proposed in marine copepods
(Buttino et al., 2006). Buttino
and co-workers demonstrated that giant liposomes of about 7 µm diameter,
which corresponds to the same dimensional range as phytoplankton cells
ingested by copepods, were actively ingested by the copepod Temora
stylifera. Liposome uptake and palatability was confirmed using liposomes
containing a fluorescent marker (fluorescein isothiocyanate–dextran),
and 3H-labelled liposomes were used to calculate the rate of
liposome ingestion. These authors also showed that when administered with the
dinoflagellate algae P. minimum, liposomes were actively ingested
over a 48 h period, and copepods grazed twice as much as with a diet of
liposomes alone. Moreover, a diet of liposomes had no supplementary effects on
copepod egg production and egg viability, making them a good candidate as a
delivery system for chemicals in copepods.
In the present work, we use giant liposomes as a delivery system for the PUA 2-trans,4-trans decadienal (DD), to study the effect of this molecule on egg production, egg hatching success, faecal pellet production and adult survival in the copepods Temora stylifera and Calanus helgolandicus. Giant liposomes containing known quantities of DD were prepared and fully characterized. Blank or DD-encapsulating liposomes and the control diet P. minimum were supplied for 10 days to both copepod species, which represent target species used in numerous aldehyde–copepod reproduction studies. In addition, the induction of apoptosis in embryos and females were also analyzed using specific fluorescent markers. The aim of this work was to relate the ingestion of DD, via a liposome-based delivery system, to the reproductive failure of copepods in order to better understand the ecological relevance of diatom-derived oxylipins for copepod recruitment at sea.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Liposome preparation
Liposomes were prepared by a modified hand-shaking method
(Bangham et al., 1965).
Briefly, a lipid mixture containing 110 mg SPC and 40 mg cholesterol in 5 ml
of a chloroform–methanol solvent mixture (2:1 v/v) was introduced into a
250 ml round-bottomed flask. The solvent was removed in a rotary evaporator
(Laborota 4010 Digital, Heidolph, Schwabach, Germany) until formation of a
lipid film on the wall of the flask. To prepare blank liposomes, the lipid
film was hydrated with 5 ml of 0.22 µm-filtered seawater (FSW). The
resulting suspension was gently mixed in the presence of 0.5 g glass beads
until the lipid layer was removed from the glass wall. The flask was then
attached to the evaporator again, rotated at room temperature for about 30
min, and left at room temperature for 2 h. DD-encapsulating liposomes (LipoDD)
were prepared by adding 500 µl of an ethanol solution containing 0.4 mg
ml–1 DD to the organic solution containing lipids. After
preparation, LipoDD was washed twice by centrifugation at 4470
g for 30 min at 4°C to remove un-encapsulated DD. All
liposome formulations were stored at 4°C under nitrogen.
Liposome size analysis and lipid dosage
The mean diameter and size distribution of liposomes were analyzed by laser
light scattering (Coulter LS, 100Q, Beckman Coulter, Miami, USA) on a
dispersion of liposomes in FSW. Particle size was expressed as mean volume
diameter ± standard deviation (s.d.) calculated on three different
batches (N=3). The amount of lipids present in the liposome
suspension after preparation was determined using the Stewart assay
(Stewart, 1980). This test
allows the quantification of SPC concentration in the suspension; total lipid
content was calculated by assuming the same SPC:cholesterol ratio before and
after preparation. Briefly, 0.1 ml of liposome suspension (approximately at a
concentration of 0.1 mg ml–1) was added to 1.9 ml of an
aqueous 0.1 mol l–1 ammonium ferrothiocyanate solution in a
test tube. The resulting suspension was mixed with 2 ml of chloroform for 15 s
using a vortex, and then centrifuged for 5 min at 894 g The
upper layer was recovered and analyzed at 485 nm by an UV-vis
spectrophotometer (Shimadzu, Milano, Italy; model 1204), by comparison with a
standard curve.
Dosage of 2-trans,4-trans decadienal
To quantify DD in water and liposomes, the aldehyde was previously
transformed into the corresponding hydrazone by a reaction with
2,4-dinitrophenihydrazine (DNPH). DNPH solution was prepared as follows: 50 mg
DNPH were dissolved in 0.5 ml of sulfuric acid and 4.5 ml of ethanol. DD was
quantified by mixing 100 µl of the solution containing DD with 100 µl of
the DNPH solution; the resulting solution was made up to 10 ml with ethanol.
DD analysis was carried out by high-performance liquid chromatography (HPLC)
using a Luna C18 (250x4.6 mm, 5 µm) column (Phenomenex, Klwid,
Torrance, CA, USA), an HPLC LC-10AD pump (Shimadzu), a 7725i injection valve
(Rheodyne, Rohnert Park, CA, USA) and a SPV-10A UV-Vis detector (Shimadzu) set
at 360 nm. The system was controlled by a SCL-10A VP System Controller
(Shimadzu) connected to a computer. Chromatograms were acquired and analysed
by a Class VP Client/Server 7.2.1 program (Shimadzu). The analysis was
performed with a mobile phase acetonitrile:water 15:85 (v/v) in isocratic
conditions at a flow rate of 1 ml min–1.
To determine the amount of DD loaded into liposomes, 100 µl of LipoDD was transferred to a 1.5 ml Eppendorf vial and centrifuged at 9615 g for 15 min (Mikro 20, Hettich, Town, Tuttlingen, Germany). The pellet and supernatant were separated and analyzed to determine DD content in liposomes and in the suspending medium, respectively. Briefly, 50 µl of supernatant was mixed with 50 µl of DNPH solution and, after vigorous mixing by vortexing, diluted to 5 ml with ethanol; the resulting solution was analyzed by HPLC. The pellet obtained by centrifugation was washed and re-suspended in FSW to a volume of 100 µl; the suspension was mixed with 100 µl of DNPH and, after complete liposome dissolution, diluted to 1 ml with ethanol. The samples were then centrifuged at 9615 g for 15 min at 4°C, the supernatant was withdrawn, diluted 1:10 with ethanol and analyzed by HPLC. Results were expressed as DD actually loaded (µg DD mg–1 total lipids) and encapsulation efficiency calculated as the ratio between DD actually loaded and DD theoretically loaded x100. For each liposome formulation, DD actually loaded was determined soon after the preparation (t=0) and at 1, 3, 7, 9, 12 and 15 days, in order to follow the amount of DD administered during copepod feeding experiments.
Copepod experiments
Zooplankton were collected in the Gulf of Naples from March to November
2006 using a 200 µm mesh plankton net, and immediately transported to the
laboratory in an insulated box. Mature males and females of the copepod
Temora stylifera Dana were isolated under a Leica stereomicroscope
and one male and 1 female were incubated in 100 ml crystallizing dishes
containing 60 ml of 50 µm filtered seawater. After 24 h of acclimatization,
couples were transferred to new crystallizing dishes containing 60 ml of 0.22
µm filtered seawater and the dinoflagellate Prorocentrum minimum
Pavillard (Sciller) at a final concentration of 8000 cells
ml–1. This microalga was used as the copepod food source in
our liposome experiments since it does not produce aldehydes or other
oxylipins (Fontana et al.,
2007).
A group of twenty T. stylifera couples were incubated with P. minimum alone, at the above mentioned concentrations, and used as the control group (hereafter referred to as Pro). In order to verify any toxic effect of liposome formulation per se, we first tested blank liposomes at different lipid concentrations on the feeding activity and survival of T. stylifera females (Table 1). Three groups of T. stylifera couples (N=10) were incubated as for the Pro group and a specific volume of liposome suspension was added to reach final lipid concentrations of 7.5 µg ml–1 (Lipo A), 4 µg ml–1 (Lipo B) and 2.0 µg ml–1 (Lipo C; Table 1). In order to test the biological activity of DD on T. stylifera reproduction, couples (N=10) were incubated with DD-encapsulating liposomes at the lipid concentration used for Lipo C (the lipid concentration that was found not to affect survival of T. stylifera) which resulted in a DD concentration of 2.9±0.23 ng ml–1 (hereafter referred to as LipoDD).
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In another set of experiments, wild females of the copepod Calanus helgolandicus Claus were sorted from the zooplankton collected in the North Adriatic Sea during April 2006 and March 2007. About 30 C. helgolandicus females were incubated in 1 l bottles filled with 50 µm-filtered seawater and transported within 48 h to the Stazione Zoologica in Naples in an insulated box. C. helgolandicus females (N=10) were individually incubated in 100 ml crystallizing dishes containing 60 ml of 50 µm-filtered seawater. Males were not sorted since this species does not require remating for the continued production of viable eggs as in the case of T. stylifera. After 24 h of acclimatization, females were transferred to new 100 ml crystallizing dishes containing 60 ml of 0.22 µm-filtered seawater and P. minimum at a final concentration of 8000 cells ml–1 (control treatment, hereafter referred to as Pro). Another group of C. helgolandicus females (N=10) was incubated as for the Pro group with a liposome suspension at 6.3 µg ml–1 final lipid concentration (hereafter referred to as Lipo Cal; Table 1). A last group of C. helgolandicus females (N=10) was incubated with DD-encapsulating liposomes at the same lipid concentration as Lipo Cal (lipid concentration that was found not to affect survival of C. helgolandicus) which resulted in a DD concentration in the medium of 3.6±0.3 ng ml–1 (hereafter referred to as LipoDD Cal; Table 1).
All groups of copepods were incubated in a temperature-controlled chamber
at 20°C and 12 h:12 h light:dark cycle, for 10 days. Each day, T.
stylifera couples and C. helgolandicus females were transferred
to new crystallizing dishes containing fresh medium. Eggs and faecal pellets
were counted under a Zeiss inverted microscope; eggs were left to hatch for
another 48 h and percentage egg viability was calculated as described
elsewhere (Ianora et al.,
1995). Survival of T. stylifera and C.
helgolandicus females under different food conditions was also
assessed.
Fluorescent staining
Embryos produced by C. helgolandicus females fed for 8 days with
LipoDD were collected, rinsed in FSW and incubated for 30 min in 1 i.u.
ml–1 chitinase as reported elsewhere
(Buttino et al., 2004) to
permeabilize the chitinous wall. After rinsing in FSW embryos were incubated
for 30 min with Annexin V-FITC (Alexis Biochemicals, Bingham, Nottingham, UK)
at a concentration of 250 µl ml–1 FSW, rinsed again in FSW
and observed with the Zeiss inverted fluorescence microscope using 10x
and 20x objectives. Green fluorescence reveals apoptosis. Annexin V-FITC
is, in fact, a vital fluorescent probe able to bind the phosphatidylserine
that is externalized on the plasma membrane surface during early phases of
apoptotic cells (Aubry et al.,
1999). Annexin V was therefore used for measuring apoptosis in the
early stages of cellular cytotoxicity.
To verify whether DD-encapsulating liposomes also induced apoptosis in
adults, three T. stylifera females fed for 6 and 10 days on Pro, Lipo
D and LipoDD and three C. helgolandicus females fed for 9 days on
Pro, Lipo Cal and LipoDD Cal were fixed in 4% paraformaldehyde in FSW for 24 h
at room temperature. Before TUNEL analysis (terminal
deoxy-nucleotidyl-transferase-mediated dUTP nick end labelling; Roche
Diagnostics GmbH, Mannheim, Germany), copepods were frozen and thawed three
times in liquid nitrogen to fracture the carapace. The cephalosome of C.
helgolandicus females was cut to facilitate penetration of the dye.
Samples were then treated as reported by Ianora et al.
(Ianora et al., 2004) and
observed using a Zeiss LSM META-510 confocal laser scanning microscope (CLSM)
with a 488 nm wavelength argon laser and 10x or 25x water
immersion objectives.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Fig. 2B shows the percentage survival of females incubated with Lipo A and B and the control Pro. Treatment with Lipo A strongly reduced T. stylifera female survival during the experiment: survival dropped to 45% after 7 days and all females died on day 9. Less than 30% of females survived with Lipo B at the end of the experiment whereas females fed Pro had the highest survival rate over 10 days (90.7±10.6). Since Lipo A and B affected female survival, we used a lower liposome formulation with a concentration of 2 µg ml–1 lipids (Lipo C) as a carrier for decadienal.
Fig. 3 shows egg and faecal pellet production rates and egg hatching success of T. stylifera females fed blank liposomes (Lipo C), DD-encapsulating liposomes (LipoDD) and the control food Pro. All three groups had the highest egg production rates at the beginning of the experiment (day 1) with a mean of 48.4 eggs female–1 day–1 (Fig. 3A). Egg production decreased on day 2 to the end of the experiment in each treatment, more dramatically in the Lipo C group, with an egg production rate of <1 egg female–1 day–1 compared to 10 eggs female–1 day–1 produced by females fed Pro and LipoDD on day 10. On average, T. stylifera females fed Pro, LipoDD and Lipo C produced 17.1 eggs female–1 day–1, 16.9 eggs female–1 day–1, and 9.2 eggs female–1 day–1, respectively, during the experiment. These values were not statistically different from each other (one-way ANOVA, F2,27=1.08; P>0.05).
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The average number of faecal pellets produced by T. stylifera couples fed LipoDD was 65.5 pellets couple–1 day–1 similar to those of females fed Pro (54.0 pellets couple–1 day–1; Fig. 3C). Higher pellet production was initially recorded for couples fed Lipo C during the first 6 days of the experiment, with a mean of 97.2, but by day 7, the value resembled those of the Pro and LipoDD groups. On average, the Lipo C group produced significantly more pellets than the Pro and LipoDD groups (70.5 pellets couple–1 day–1, one-way variance ANOVA, F2,27=3.8; P<0.05; Tukey's post-hoc test P<0.05).
Fig. 3D shows the percentage survival of T. stylifera females after 10 days of feeding with Pro, Lipo C and LipoDD. The percentage survival was very high for both Pro and Lipo C diets during the whole experiment, with a mean of 92.4% and 95%, respectively. By contrast, the survivorship of females fed LipoDD declined steadily throughout the experiment, with only 47% live females by day 10. The average percentage survival was 76.0% in the LipoDD group, which was statistically lower than the Lipo C and Pro groups (one-way ANOVA, F2,27=6.1; P<0.01; Tukey's post-hoc test P<0.05).
Egg production rate, hatching success and faecal pellet production of C. helgolandicus females fed Lipo Cal, LipoDD Cal and Pro are reported in Fig. 4. The daily pattern with a LipoDD diet was very stable during the experiment and similar to that of Lipo Cal and Pro groups, with an average of 8.9 eggs female–1 day–1, 8.9 eggs female–1 day–1 and 11.7 eggs female–1 day–1, respectively (Fig. 4A). Hatching success for the three treatments remained high until day 7 after which hatching success for females fed LipoDD Cal was reduced to <50% and fell to 0% by the end of the experiment (Fig. 4B). By contrast, Pro and Lipo Cal diets did not reduce hatching success, which was on average 76.7% and 71.6%, respectively. These values were statistically higher than those recorded for females fed LipoDD Cal (49.8%; one-way ANOVA, F2,27=9.2; P<0.001; Tukey's post-hoc test P<0.01).
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Survival of C. helgolandicus Pro and Lipo Cal groups slightly decreased over the experiment, but remained above 70% on day 10 (Fig. 4D); only 45% of the females in the LipoDD Cal group were alive by the end of the experiment. However, the average percentage female survival was not statistically different from the other groups (one-way ANOVA, F2,27=2.08; P>0.05).
To verify if DD-encapsulating liposomes induced apoptosis in embryos, C. helgolandicus embryos produced by females fed LipoDD Cal for 8 days were stained with annexin V-FITC. Most of these embryos fluoresced green indicating that an apoptotic process had started (Fig. 5A). By contrast, embryos in the control Lipo Cal group appeared dark (Fig. 5B), as for the control group fed Pro (not shown).
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To verify if liposomes encapsulating DD induced apoptosis also in adults, C. helgolandicus females fed for 9 days on LipoDD Cal were stained with TUNEL. Fig. 6A shows the whole prosome strongly fluorescent, indicating the occurrence of apoptosis in these body tissues. At the highest magnification, the oocyte cavities in the ovary show small green fluorescent spots (Fig. 6B). By contrast, C. helgolandicus females fed Lipo Cal (Fig. 6C) and Pro (image not shown) were not fluorescent. An induction of apoptosis was also observed in T. stylifera females fed for 6 days on the LipoDD suspension and stained with TUNEL (Fig. 7). Confocal laser scanning sections revealed that one of the two gonads was positively stained during the apoptotic process (Fig. 7A,B). Control females fed Lipo C did not show any fluorescence except for an external autofluorescence of the chitinous wall (Fig. 7C). The same was true for females of the Pro group (image not shown). T. stylifera females fed LipoDD suspension for 10 days were stained green throughout the entire body, including muscles, suggesting that all organs underwent an apoptotic process in these females (Fig. 8A,B).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). These
results suggested that liposomes could be a very useful tool to deliver
bioactive molecules into the gastrointestinal tract of aquatic organisms. Here
we prepared giant liposomes encapsulating DD to unequivocally clarify the role
of polyunsaturated diatom-derived aldehydes in copepod reproduction.
Experiments in which copepods had been incubated in the presence of DD had
already been carried out (Miralto et al.,
1999; Ianora et al.,
2004). However, in these studies the relationship between the
observed effect and the amount of DD ingested by copepods was probably
overestimated due to the high volatility of this aldehyde in the incubation
medium (Miralto et al., 1999).
Moreover, DD may have been transformed prior to delivery when live carriers
were used (Ianora et al.,
2004). By contrast, liposomes are inert carriers and able to
entrap molecules with different chemical-physical characteristics. In
particular, hydrophilic molecules can be entrapped into the aqueous internal
cavity, whereas lipophilic compounds can enter into the lipid bilayer. This is
what occurs with DD, a very lipophilic molecule that can be dissolved in the
organic solution containing the lipid mix, before liposome preparation. We
found that DD loading into liposomes did not significantly change during the
time frame considered, probably due to a hydrophobic interaction between DD
and the lipids. Therefore, we performed long-term incubation experiments with
DD-encapsulating liposomes and tested the effects of these on the reproduction
of two copepod species. Blank liposomes were also tested to evaluate lipid
toxicity on copepods, with faecal pellet production used as a proxy for
copepod ingestion.
Interestingly, our results show that the production of faecal pellets
increased when T. stylifera females were fed a mixed diet of
liposomes and Pro with respect to the single diet Pro. Previous results
(Buttino et al., 2006)
demonstrated that copepod ingestion rate, calculated using radiolabelled
cholesterol, was double when liposomes where supplied together with Pro. An
increase in the number of faecal pellets was also recorded during the
experiments using lipo DD. However, whereas the number of pellets produced by
T. stylifera was significantly higher with a diet of blank liposomes
than with LipoDD, for C. helgolandicus the opposite was true. Much
more LipoDD was ingested by C. helgolandicus than liposomes without
DD. These results suggest different behaviors for the two copepod species,
with one presumably more attracted by the `flavor' of decadienal than the
other. Odorous compounds such as the aldehyde decatrienal were liberated into
water after cell disruption of some benthic diatoms, acting as a repellent to
pelagic freshwater crustaceans
(Jüttner, 2005). Such
repellence consisted of reduced swimming movements and grazing activity. In
our experiments, a similar repellent reaction was not evident, at least in one
of the two species.
An increase in liposomes ingested was not matched by increased egg
production rates for either copepod species, confirming that liposomes had no
supplementary effect on egg production as also reported by Buttino et al.
(Buttino et al., 2006). Our
results showed that when liposomes were supplied at lipid concentrations
ranging from 7.5 to 4.0 µg lipids ml–1 (Lipo A and B) most
T. stylifera females were dead a few days later and no adults
survived more than 8 days with a Lipo B formulation. This effect was not
attributed to lipid toxicity but rather to the obstruction of copepod
mouthparts by highly concentrated liposome particles that were observed in
some females (I.B., personal observations). This is why we used the lowest
lipid concentration to incubate DD into liposomes for this species. By
contrast, C. helgolandicus did not show a reduction in adult survival
at the liposome formulation used. Different responses among the two species
suggest that blank liposome incubations must be assessed for each copepod
species before using them in toxicological experiments.
Until now, the toxicity of PUAs in an aqueous medium has only been tested
by exposing organisms in vitro to dissolved PUAs in water or by using
PUA-producing diatoms as food. Our results indicate that the concentration
affecting hatching success in both copepod species was one order of magnitude
lower than those recorded in previous incubation experiments [e.g. 1 µg
ml–1 in incubation experiments
(Miralto et al., 1999;
Ceballos and Ianora, 2003;
Taylor et al., 2007)].
Concentrations blocking hatching success in our experiments were much lower in
both T. stylifera (97 ng DD) and C. helgolandicus (121 ng
DD) considering filtration rates of 0.14 ml h–1 per copepod
(Buttino et al., 2006) and
incubation concentrations of 2.9 ng ml–1 and 3.6 ng
ml–1 DD, respectively, for 10 days. Adolph et al.
(Adolph et al., 2004)
calculated similar values for feeding experiments where 30 ng
ml–1 potential oxylipins equivalent concentrations reduced
hatching success to 30% after 5 days of feeding on a diatom diet.
Another interesting finding here is that when the percentage of egg
viability for C. helgolandicus was below 25% and adult survival was
less than 50% (Fig. 4B,D),
gonad tissues of some females appeared positively stained by TUNEL
(Fig. 6A), suggesting that they
were undergoing apoptosis. The apoptotic region corresponded to oocyte 3-stage
producers (OS3) (Poulet et al.,
2007). Follicle chambers with apoptotic body-like spots were
similar to structures found in histological sections by these authors
(Fig. 6B). Poulet and
co-workers reported a concomitant arrest of OS3 maturation, characterized by
cell fragmentation and by the presence of apoptotic bodies, when C.
helgolandicus females were fed some diatom diets that reduced both egg
production and hatching. Our results also indicate a concomitance between a
reduction in egg hatching success and induction of apoptosis even if egg
production was similar to the control. Because TUNEL detects early apoptotic
events, it is probable that eggs are released but they die later, and the
observed effect indicates a reduction in hatching success rather than
fecundity. Similarly, a concomitance between TUNEL positivity and a reduction
in hatching success (
50%) occurred for T. stylifera females fed
LipoDD for 6 days (Fig. 3B,
Fig. 7A,B).
Moreover, our results also show, for the first time, that DD affected adult
survivorship. Ceballos and Ianora (Ceballos
and Ianora, 2003) reported a similar reduction in adult survival
when T. stylifera was fed the diatom Skeletonema costatum
for more than 10 days. Presumably this decrease in adult survival was due to
DD and not to poor food quality. Females of both species appeared strongly
positive for apoptosis coincidentally with reduced survival (
50%;
Fig. 8A,B,
Fig. 3D). Hence the mechanism
of chemical defense in diatoms not only functions by reducing grazing effects
of subsequent generations of copepods, as hitherto believed, but also targets
the direct predator. These compounds are of lower acute toxicity to adult
predators compared to other feeding deterrents such as dinoflagellate toxins
even though they eventually induce death if ingested for a sufficient length
of time, and lead to post-digestive reduction in fecundity or depressed
viability of the offspring. Grazing pressure is thus reduced, allowing diatom
blooms to persist when grazing pressure would otherwise have caused them to
crash.
An important application of our work is the possibility of delivering a
known quantity of toxin and being able to calculate the efficiency of
adsorption and longevity of the toxins once encapsulated. Liposomes have been
extensively used in the pharmaceutical and chemical industries for the past 30
years and in aquaculture to deliver food supplements in the diet [Buttino et
al. (Buttino et al., 2006) and
references therein]. However, until recently liposomes >6 µm were
unstable over multiday experiments (Ravet
et al., 2003) and were only used for short-term incubations. Here
we were able to produce giant (
7 µm diameter) DD-encapsulating
liposomes that were very stable over time allowing us to carry out long-term
incubations necessary to test the effects of PUAs on copepod reproductive
fitness. Further studies using liposomes are in progress to test synergistic
or antagonistic effects of different chemicals and varied nutritional content
of copepods, providing a tool to qualitatively and quantitatively analyze the
impact of toxins and nutrient supplements on copepod grazers.
LIST OF ABBREVIATIONS
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Adolph, S., Bach, S., Blondel, M., Cueff, A., Moreau, M.,
Pohnert, G., Poulet, S. A., Wichard, T. and Zuccaro, A.
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