Download fate of chlorophyll and carotenoid pigments

Journal of Plankton Research Vol.22 no.2 pp.305–319, 2000
Grazing experiments with two freshwater zooplankters:
fate of chlorophyll and carotenoid pigments
E.Pandolfini1, I.Thys2, B.Leporcq1 and J.-P.Descy1,3
1Laboratory of Freshwater Ecology, Department of Biology, FUNDP, B-5000
Namur, Belgium and 2CREBS, Centre de Recherches Public—Centre
Universitaire (CRP/CU), 162a, av. de la Faïencerie, L-1511 Luxembourg
3To
whom correspondence should be addressed
Abstract. In order to evaluate the validity of the gut pigment method to assess grazing and diet in two
freshwater zooplankters, experiments were carried out to check chlorophyll a and xanthophyll conservation during feeding. For both animals, two sets of experiments were conducted by incubating
animals in the laboratory, either isolated from a reservoir (the calanoid copepod, Eudiaptomus
gracilis) or cultured under high-food conditions (the cladoceran, Daphnia galeata). For both animals,
gut pigments and clearance rates on different types of algae were determined from the same incubations. Chlorophyll a and derivatives, as well as major algal carotenoids, were analysed by High
Performance Liquid Chromatography (HPLC). In copepods, the pigment profiles from the gut
extracts reflected the diet of the animals poorly. The animal extracts contained almost exclusively
alloxanthin (or an alloxanthin-like pigment) in large amounts, whereas the other pigments were lost
in high proportions (>70% for lutein and fucoxanthin; 57 and 78% for a-phorbins). The cladocerans
fed on the main types of algae abundant in the suspensions, with a preference, however, for small
cells. Although the main xanthophylls from these algae were detected in the Daphnia extracts, some
destruction of lutein and fucoxanthin may have occurred (18.7 and 30%). The loss rate for alloxanthin seemed more variable (0 and 68%), possibly depending on food concentration. As for the transformation of a-phorbins, E.gracilis and D.galeata behaved quite differently. The HPLC profiles of
copepod extracts always showed a very small chlorophyll a peak, along with phaeophytin a and
pyrophaeophytin a. Those from the cladoceran exhibited a large phaeophorbide a peak, along with
some chlorophyll a and phaeophytin a. In fact, D.galeata did not destroy a-phorbins under our experimental conditions but converted chlorophyll a mainly into phaeophorbide. From a comparison of our
results with data from other studies, it seems that in these two zooplankters, use of gut pigment data
for quantitative grazing assessment should be considered with caution.
Introduction
The detailed analysis of algal pigments by High Performance Liquid Chromatography (HPLC) has increasingly been used in marine and freshwater ecology,
for instance, in quantitative studies of phytoplankton composition and in detailed
investigations of particulate fluxes to the sediment [review in (Millie et al., 1993)].
In particular, the ‘gut pigment method’, based on measurements of chlorophyll a
and derivatives in zooplankton extracts, has been used widely for quantitative
assessment of grazing by marine zooplankton. Indeed, chlorophyll a and derivatives found in animals’ guts were thought to provide reliable estimates of the algal
biomass ingested by applying excretion rates determined experimentally to
pigment amount per animal [e.g. (Mackas and Bohrer, 1976; Bautista and Harris,
1992; Landry et al., 1994)]. These techniques also seemed promising to study
zooplankton grazing selectivity through the detection of carotenoid pigments
specific to various algae classes in zooplankton extracts.
However, there has been increasing evidence in the last decade that
a-phorbins (i.e. chlorophyll a and phaeopigments) are extensively destroyed
© Oxford University Press 2000
305
E.Pandolfini et al.
during gut passage in marine copepods [(e.g. (Conover et al., 1986; Lopez et al.,
1988; Penry and Frost, 1991; Mayzaud and Razouls, 1992; Head and Harris,
1992, 1994, 1996)], to such an extent that most authors agree on the need for
re-evaluating the ‘gut pigment method’ (Drits, 1997). Equations have been
proposed to correct for the destruction occurring at various stages of the
feeding process in copepods (Stevens and Head, 1998) or in other marine herbivores (Bochdansky et al., 1998). Early studies on algal xanthophylls in marine
zooplankton (Kleppel and Pieper, 1984; Kleppel et al., 1988, 1991; Nelson, 1989)
suggested that carotenoids were more stable than chlorophylls in marine mesozooplankton guts, while degraded forms were detected in the faeces of macrozooplankton. It was therefore suggested that algal xanthophylls were well
conserved in small planktonic herbivores. Subsequently, studies of selective
grazing by freshwater zooplankton have been carried out, assuming complete
carotenoid conservation in copepods, cladocerans and small zooplankton
(Quiblier et al., 1994; Quiblier-Lloberas et al., 1996). However, views on the fate
of algal carotenoids after zooplankton feeding have changed considerably. In
marine copepods, Head and Harris suggested that fucoxanthin and 199hexanoyloxyfucoxanthin from diatoms and prymnesiophytes were extensively
destroyed during gut passage (Head and Harris, 1992, 1994). Similar
conclusions have been drawn from experimental studies on marine and freshwater calanoids; Kleppel showed substantial loss of fucoxanthin in Acartia californiensis and Calanus pacificus at high and low food levels (Kleppel, 1998),
while Descy et al. found evidence of differential destruction of primary algal
xanthophylls in the freshwater species Diaptomus minutus (Descy et al., 1999).
Furthermore, Strom et al. have shown that both chlorophylls and carotenoids
may be destroyed during feeding by microzooplankton, and that the extent of
the degradation seems inversely related to protozoan size (Strom et al., 1998).
Thus, the emerging picture from these few studies on the fate of carotenoids in
zooplankton is totally different from what it was a few years ago, and the reliability of xanthophylls as markers of grazing by zooplankton may be questioned
as much as for chlorophylls.
Few experimental studies have been conducted on the fate of carotenoids in
freshwater zooplankton to date. If the gut pigment technique should be applied
with some caution in calanoid copepods, even less is known about how herbivorous cladocerans process algal pigments. Quiblier et al. conducted experiments on
D.magna fed with mixtures of algae and observed a good correspondence
between the carotenoid profiles in the animals and their food (Quiblier et al.,
1994). Chlorophyll a was converted essentially to phaeophorbides and, probably,
to ‘colourless’ products. However, when Daphnia was fed with lake water dominated by cyanobacteria, chlorophyll a was not degraded. Similar observations, i.e.
decrease of chlorophyll a and increase of phaeophorbides a in feeding suspensions, were made by Fundel et al. who conducted long-term experiments with
small and large Daphnia from Lake Constance (initial chlorophyll a range:
1.2–16.3 µg l–1) (Fundel et al., 1998). A good relationship between the phaeophorbide a/chlorophyll a ratio and Daphnia density was observed at low grazer
density, but this ratio tended to decrease at high cladoceran density, suggesting
306
Pigments in freshwater zooplankton
chlorophyll a destruction rather than conversion to phaeopigment at low food
levels.
This study presents results from feeding experiments with two common freshwater zooplankters, the calanoid copepod Eudiaptomus gracilis and the herbivorous cladoceran Daphnia galeata. We aimed to investigate the fate of algal
chlorophylls and carotenoids after zooplankton feeding, and to determine classspecific clearance rates from the decrease of the amounts of specific carotenoids
in the feeding suspensions (for copepods) or using algal counts in the control and
feeding suspensions (for cladocerans). In particular, using actual clearance of
algal groups from the suspension as a reference, we tried to assess the validity of
gut pigment analysis for measuring grazing rates and for investigating the diet of
these two species.
Method
Study lake and experimental material and design
Both species were isolated from the lake of Esch-sur-Sûre, a reservoir located in
the northern part of the Grand Duchy of Luxembourg. The lake is dimictic and
has been considered as meso-eutrophic (annual mean chlorophyll a concentration in the euphotic zone: 5.0–8.8 µg l–1; annual maximum chlorophyll a
concentration: 16.3–34.9 µg l–1; annual mean Secchi depth 4.2–4.7 m; annual
minimum Secchi depth: 1.8–2.5m). The reservoir is the main drinking water
supply for the country. Zooplankton dynamics have been followed for several
years: in the summer zooplankton, Eudiaptomus gracilis and Daphnia galeata
were often dominant species (Dohet and Hoffmann, 1995). In 1998, when this
study was carried out, phytoplankton presented the following variations. After a
spring dominance by Chrysophytes and diatoms, a clear-water phase developed
in late May and June with mainly Cryptophytes remaining in the phytoplankton;
this phase was followed by a return of Chrysophytes in July and by the development of various cyanobacteria, which became dominant in late summer. Chlorophytes were present throughout the growing season and dinoflagellates and
Euglenophytes appeared sporadically. Chlorophyll a levels, measured by HPLC
(see below) in epilimnetic phytoplankton <100 µm, were between 2.5 and
30 µg l–1.
The copepods used in the feeding experiments were picked, using Pasteur
pipettes under a dissecting microscope, from a zooplankton sample collected in
the lake with a closing plankton net (53 µm mesh size). On average, 50–70 adult
copepods (males and egg-bearing females in equivalent numbers, as far as possible) were placed in PVC cylinders with a 150 µm Nitex screen fitted to the
bottom and suspended in 250 ml beakers filled with lake water sieved through a
63 µm screen in an incubator set at the lake temperature. The animals were
allowed to acclimate, in the dark, at least overnight. The next day, the PVC cylinders containing the animals were transferred into filtered lake water for 1–3 h,
and then placed in fresh lake water for 13–18 h. Afterwards, the cylinders were
immersed for 2 min in soda water; the anaesthetized animals were collected under
the dissecting microscope, counted, and placed on a 25 mm GF/C filter, which
307
E.Pandolfini et al.
was placed in a glass scintillation vial and frozen in liquid nitrogen. Meanwhile,
the feeding suspension was fractionated on a 63 µm screen in order to separate
faecal pellets from the non-ingested algae. Both fractions were collected by filtration under a mild vacuum on 47 mm GF/C filters. Other details of the experimental protocol, including extraction in 90% acetone (HPLC grade), can be
found elsewhere (Descy et al., 1999). Two experiments were carried out using
phytoplankton of different composition, and each experiment consisted of six sets
of experimental beakers with or without animals (controls), incubated under the
same conditions.
Daphnia galeata adults (mean length 800 µm) were available from cultures run
at CRP/CU, where the cladocerans were fed with a culture of the unicellular
Chlorophyte Kirchneriella subcapitata Korš. at saturating concentration. For the
feeding experiments, 50 animals were selected and allowed to acclimate in an artificial algal assemblage made of river water sieved on a 63 µm Nitex screen and
supplemented with a culture of Cryptomonas ozolini Skuja, in order to obtain a
suspension containing Chlorophytes, diatoms and Cryptophytes. All experiments
were run in 250 ml suspensions and followed a similar design as for the copepods,
except that the PVC cylinders containing the cladocerans were designed in such
a way that the animals remained immersed throughout the transfer steps. Also,
the final feeding suspensions were not fractionated as cladocerans do not produce
coherent faecal pellets. Again, acetone extracts of the animals were obtained
following the procedure of Descy et al. (Descy et al., 1999). Two experiments were
carried out, at low and high algal biomass, and five replicates of controls and incubations with 50 animals were run through each experiment. In addition, 50 ml
from the filtered feeding suspension were eluted through Waters Sep Pak
cartridges to allow detection of possible pigments in the filtrate from passage of
subcellular material through the GF/C filters.
As the experiments with both species were run at higher animal densities than
in natural conditions, they may a priori not reflect feeding behaviour at lower
densities. However, the purpose of these incubations was to test whether gut
pigments were indicative of the algae ingested, and to evaluate possible pigment
loss. Working at lower animal densities would have required the adoption of
longer incubation times which would have led to increased risk of pigment
degradation due to bacterial activity. Nevertheless, crowded conditions may
have affected pigment loss, particularly in the case of cladocerans, as discussed
below. Similarly, all incubations were run in the dark to avoid photochemical
degradation of pigments, and we did not investigate variations of feeding activity possibly related to diel variations and photoperiod. We did, however, check
for the absence of significant variation of pigment concentration in algal
suspensions by analysing control suspensions at the beginning and at the end of
the incubations. Given that the incubations were run in the dark and that the
water was fairly rich in inorganic N and P, it was assumed that the results were
not affected by algal growth from nutrient recycling in the suspensions containing the animals.
308
Pigments in freshwater zooplankton
Grazing rates and pigment loss
Eudiaptomus gracilis grazing rate was calculated from the difference between a
given pigment concentration in the control and the final suspension (after
removal of the faecal pellets), assuming that the decrease resulted from copepod
feeding. Clearance rate (µl copepod–1 h–1) was calculated according to Adrian
(Adrian, 1991). Pigment loss related to consumption by copepods was calculated
as the difference between the amount of pigment ingested and the amount of
pigment retrieved (animals + faecal pellets), expressed as a percentage of
ingested pigment.
Daphnia galeata clearance rates were determined by counting algae units (cells
or colonies), using an inverted microscope, in three control and feeding suspensions. Most taxa were identified at the species level but clearance rates were
determined by summing algal numbers by class. The proportion of marker
pigment ingested was calculated from the percentage of cells of the corresponding algal class cleared by cladoceran grazing. Estimates of pigment loss were
based on the same formula as for copepods, except that the amounts of pigments
found in the filtrate of the feeding suspension were added to the pigments
retrieved in the particulate fraction.
For both animals, ingestion rates were determined by multiplying the clearance
rate by the algal concentration, expressed as µg C l–1. Carbon biomass was
obtained from chlorophyll a biomass using a conversion factor of 35; this value
takes into account the average C:chlorophyll a ratio for the river phytoplankton
used here (Descy and Gosselain, 1994) and the ratio found for the Cryptomonas
culture. Chlorophyll a biomass of each algal class was estimated using the
CHEMTAX program (Mackey et al., 1996). Class-specific grazing rates of copepods were determined in a similar way, using carotenoids for estimating the
biomass of the dominant algae in the feeding suspensions, i.e. fucoxanthin for
diatoms and Chrysophytes, lutein for Chlorophytes, zeaxanthin for Cyanophytes
and alloxanthin for Cryptophytes. Further details on carotenoid distribution
across algal classes, along with their amounts relative to chlorophyll a in various
categories of planktonic algae, can be found elsewhere [e.g. (Mackey et al., 1996)].
HPLC analysis
The HPLC system used comprised a Waters autosampler, a Waters 996 PDA
detector, and a Waters 470 fluorescence detector set up for optimal detection of
phaeopigments (lexc 412 nm; lem 674 nm). The separation was achieved using a
25 cm Waters Nova-Pak C18 column and the ternary gradient of Wright et al.
(Wright et al., 1991), slightly modified to allow elution of xanthophylls over a
larger range of retention times. The standard procedure consisted of the injection
of 50–100 µl of extract run through a 30 min gradient. Calibration was achieved
using three or four point calibration curves established with a mixed standard of
fucoxanthin, lutein, zeaxanthin, chlorophyll a, chlorophyll b, echinenone and bcarotene, as well as a separate phaeophytin a standard. A separate standard was
also used for alloxanthin. Standard solutions of fucoxanthin, alloxanthin and
309
E.Pandolfini et al.
chlorophyll a were purchased from VKI, Denmark, chlorophyll b from Sigma, and
lutein, zeaxanthin, echinenone and b-carotene were donated by HoffmannLaroche, Switzerland. Carotenoids not present in the mixed standard were quantified against fucoxanthin using as relative response the ratio of the specific
absorbance coefficients at 440 nm in methanol. Similarly, phaeophorbides a and
phaeophytins a were quantified against the phaeophytin a standard, assuming an
equivalent fluorescence yield.
Results
Typical absorbance and fluorescence chromatograms obtained from HPLC
analysis of animal samples after incubation are shown in Figure 1. Fucoxanthinol, a transformation product of fucoxanthin (Repeta and Gagosian, 1982), was
found in some Daphnia extracts. As it was irregularly detected, it could not be
taken into account in the fucoxanthin mass balance calculations made for estimating pigment loss.
Experiments with Eudiaptomus gracilis
The pigment profiles of the algal suspensions used in the two experiments (Figure
2, controls) indicate that they differed substantially, both in biomass and composition. In the first profile, Cryptophytes dominated the phytoplankton (with probably as much as 75% of the chlorophyll a biomass), and were accompanied by
some Chrysophytes and cyanobacteria. In the second, Chrysophytes were dominant (with ~66% of the chlorophyll a biomass), accompanied by Cryptophytes
and Chlorophytes. Clearance rates of the copepods on all algae, based on chlorophyll a removal (Figure 3A), reached 408 µl copepod–1 h–1 in the first experiment
and was about four times lower in the second (92 µl copepod–1 h–1). This difference can probably be related to the contrasting biomass range; copepods of the
first set of incubations started feeding at 3.6 µg chlorophyll a l–1 and reduced the
algal concentration by a factor of 3. They were most likely feeding at their
maximum filtration rate. By contrast, in the second experiment, the copepods
were exposed to relatively high biomass throughout the incubation, possibly
leading to a reduced filtration rate. Eudiaptomus gracilis selected food items very
differently from among the algal groups present. In the first case (Figure 3A),
they cleared mainly Cryptophytes, ingested some Chrysophytes and diatoms and,
to a much lesser extent, Chlorophytes and cyanobacteria. In the second case, they
Fig. 1. Typical absorbance and fluorescence chromatograms obtained from HPLC analysis of animal
samples after incubation; the x-axis is retention time in minutes. (A) Absorbance chromatogram, at
436 nm, of a Daphnia galeata extract (50 animals); (B) fluorescence chromatogram of the same
extract; (C) absorbance chromatogram, at 436 nm, of Eudiaptomus gracilis (70 animals); (D) fluorescence chromatogram of a faecal pellets sample of E.gracilis. Identified pigments are as follows: 1.
fucoxanthinol; 2. fucoxanthin; 3. neoxanthin; 4. violaxanthin; 5. phaeophorbide a; 6. diadinoxanthin;
7. astaxanthin; 8. alloxanthin (spectrum shown in the box in C); 9. lutein; 10. zeaxanthin; 11. canthaxanthin; 12. phaeophorbide?; 13. chlorophyll b; 14. chlorophyll a; 15. echinenone; 16. phaeophytin a;
17. bb-carotene; 18. pyrophaeophytin a.
310
Pigments in freshwater zooplankton
311
E.Pandolfini et al.
Fig. 2. Results of two feeding experiments with the freshwater copepod Eudiaptomus gracilis, isolated
from Esch-sur-Sûre Lake, incubated in lake water. Pigment amounts (pmoles) measured in the
control algal suspensions and in the different fractions (final algal suspensions, faecal pellets, animals),
at the end of a feeding experiment with adult copepods. Estimates of the percentage of pigment lost
indicated in the diagram.
Fig. 3. Results of two feeding experiments with the freshwater copepod Eudiaptomus gracilis, isolated
from Esch-sur-Sûre Lake, incubated in lake water. (A) Clearance rates calculated from pigment
removal from the feeding suspensions; fucoxanthin is a marker for Chrysophytes and diatoms, alloxanthin for Cryptophytes, lutein for Chlorophytes, zeaxanthin for cyanobacteria, and chlorophyll a for
total algal biomass. (B) Pigments detected in copepod extracts at the end of the incubations. (C)
Pigments detected in the faecal pellets at the end of the incubations.
312
Pigments in freshwater zooplankton
313
E.Pandolfini et al.
consumed exclusively Chrysophytes. In fact, calculations of ingestion rates show
that they ingested the same amount of food on both occasions (~80 ng C
copepod–1 h–1) by feeding on the most abundant prey.
Pigments found in copepod guts (Figure 1; Figure 3B) were only partly consistent with the feeding behaviour described above. Indeed, a major peak of alloxanthin was detected in the animal extracts after both experiments, although
Cryptophytes were not consumed in the second set of incubations (no decrease
of alloxanthin in the final suspension; see Figure 2). By contrast, pigments in the
faecal pellets (Figure 3C) seemed to reflect more closely the preferential ingestion of Cryptophytes and Chrysophytes in the first and the second experiment,
respectively. However, as shown by the pigment budget and by the estimation of
pigment loss, the destruction of xanthophylls was variable; 95% of the fucoxanthin ingested was lost in the second experiment, and close to 70% of lutein
and zeaxanthin in the first. Surprisingly, an excess of alloxanthin was retrieved
from the three fractions collected at the end of the incubations, especially in the
second experiment where no Cryptophytes were consumed. The bulk of this
pigment was present in the copepod extracts rather than in the faecal pellet fraction. Therefore, the presence of an animal pigment with a retention time close to
alloxanthin was suspected, but analysis of the absorption spectrum showed a
perfect match with that of alloxanthin (Figure 1C).
Experiments with Daphnia galeata
Daphnia galeata cleared the total algal biomass at similar filtration rates (Figure
4A) in both experiments (560 and 470 µl cladoceran–1 h–1, c.v. ~30% for both
values), although the incubations were run at relatively low (9.2 µg chlorophyll a
l–1) and high (35.8 µg chlorophyll a l–1) food levels. The low food level was actually close to the incipient limiting level known for the species [0.7 mg C l–1 (Stuchlik, 1991)] so that the clearance rate was not accelerated to any great extent. The
ingestion rate was lower at low food (160 ng C cladoceran–1 h–1) compared with
high food levels (765 ng C cladoceran–1 h–1). According to class-specific clearance
rates estimated from the decrease in algal numbers (Figure 4A), all main groups
of algae were consumed, with some variation. For instance, Chlorophytes in the
first experiment, in which they comprised various small taxa mostly in the edible
range for cladocerans, were preferred, while the reduced grazing rate on green
algae in the second experiment may be explained by the dominance of colonies
of Actinastrum, Dictyosphaerium and Scenedesmus, which were probably much
less edible for Daphnia. Similarly, the higher clearance of Cryptophytes in the
second experiment was probably related to the high abundance of small
Chroomonas, as their concentrations were proportionately more reduced by
cladoceran grazing than those of the larger Cryptomonas ozoloni.
The pigments found in cladoceran guts generally reflected the composition of
their diet (Figure 1; Figure 4B), i.e. the main xanthophylls of the ingested algae
were detected by HPLC and were present in proportions that corresponded well
with the group-specific clearance rates (Figure 4A). Phaeophorbide a was the
dominant form of a phorbin in the cladocerans and its concentration also
314
Pigments in freshwater zooplankton
Fig. 4. Results of two feeding experiments with the cladoceran Daphnia galeata, cultured at high food
level, incubated in a mixed algal suspension. (A) Clearance rates calculated using cell counts in control
and feeding suspensions. (B) Pigments detected in cladoceran extracts at the end of the incubation;
fucoxanthin is a marker for Chrysophytes and diatoms, alloxanthin for Cryptophytes, lutein for
Chlorophytes, zeaxanthin for cyanobacteria, and chlorophyll a for total algal biomass.
increased in the feeding suspensions. Chlorophyll a concentrations decreased in
those suspensions, whereas phaeophytin a concentrations were similar in controls
and feeding incubations.
Estimates of pigment loss rates, based on calculations of the amounts of algae
ingested from the microscope counts in the control and final suspensions, showed
relatively low and variable degrees of pigment disappearance (Figure 5). When
diatoms and green algae were ingested, loss rates for fucoxanthin and lutein were
~30 and 18.7%, respectively. The loss rates for alloxanthin differed greatly between
experiments, with 68% loss in the first experiment and no significant loss in the
second. Ingestion rates for Cryptophytes, however, were higher in the first experiment. The estimates of losses, in the case of cladocerans, were calculated taking
315
E.Pandolfini et al.
into account pigment retrieved from the ‘soluble’ fraction, which represented
between 1% (fucoxanthin) and 2.5% (alloxanthin) of the total pigment at the end
of the incubations. There was some variability associated with pigment loss estimates as they were estimated on the basis of cell counts in control and final suspensions, which have a limited accuracy (coefficient of variation 20–30%). In addition,
it is worth emphasizing that total a phorbins, after grazing by D.galeata, did not
change significantly, indicating that chlorophyll a was converted to phaeophorbide
a with 100% efficiency, without significant destruction.
Fig. 5. Results of two feeding experiments with the cladoceran Daphnia galeata, cultured at high food
level, incubated in an artificial algal suspension. Pigment amounts (pmoles) measured in the control
and final algal suspensions, at the end of a feeding experiment with adult cladocerans, and amount of
ingested pigment estimated from algae removal. Estimates of the percentage of pigment lost indicated
in the diagram.
316
Pigments in freshwater zooplankton
Discussion
The main conclusion of this experimental study is that pigment processing during
gut passage appears to be different in Eudiaptomus gracilis and Daphnia galeata,
and that it may vary for the same pigment in different species of ingested algae.
Recent research on the fate of chlorophyll a and carotenoids points to substantial pigment destruction during feeding by marine and freshwater calanoid copepods, although literature specific to xanthophylls is more sparse (Head and
Harris, 1992, 1994; Kleppel, 1998; Descy et al., 1999). These papers have reported
extensive destruction of fucoxanthin during consumption of diatoms, which may
vary with season and food concentration (Kleppel, 1998), with 0 recovery after
copepod grazing at low food levels. For chlorophyll a, most studies now agree on
variable degrees of transformation to products undetectable by HPLC and gut
fluorescence (Drits, 1997; Stevens and Head, 1998; Tirelli and Mayzaud, 1998).
These findings, together with possible variations in gut evacuation rates, are
serious restrictions to quantitative estimates of grazing rates by the gut fluorescence technique. Chlorophyll and carotenoid destruction also occurs as a result
of microzooplankton grazing (Strom et al., 1998) and seems to be inversely
related to protozoan size. The results reported here for the freshwater copepod
E.gracilis, and those for another species, Diaptomus minutus (Descy et al., 1999)
point to similar and extensive pigment alterations as a result of feeding. Therefore, pigments in animal guts are not a reliable indication of copepod diet and
should not be used to assess grazing quantitatively. An additional problem
reported here is the presence of alloxanthin in animals, even when they have not
fed on Cryptophytes. A similar intriguing observation was made with D.minutus
starved for 8 h in filtered water, and on copepods which had not fed on Cryptophytes (Descy et al., 1999). The most likely explanation is that the pigment
systematically detected in these copepods is an alloxanthin-like animal pigment,
which perhaps has a photo-protective role. It should be noted that in another
study (Descy et al., 1999), D.minutus contained large amounts of the animal
carotenoid astaxanthin, which, in this study, was observed only at low concentrations in E.gracilis.
Our results with D.galeata are consistent with those reported in two other
studies, one by Quiblier et al. (Quiblier et al., 1994) who observed relatively high
levels of carotenoid conservation in D.magna along with chlorophyll a degradation, and one by Fundel et al. (Fundel et al., 1998) who reported conversion of
chlorophyll a to phaeophorbide a except at high grazer density, where a phorbin
destruction occurred. This indicates that with regard to other grazers, food concentration may have an effect on pigment conservation in freshwater cladocerans. In
our experiments, a phorbins were not destroyed significantly, and phaeophorbides
a present in the animals increased in concentration in feeding suspensions. It is
worth mentioning, however, that the Daphnia used in these incubations came from
laboratory cultures, where they had been fed at high algal concentrations, and that
the incubations also were not run at very low food levels. A similar chlorophyll a
transformation pattern, with dominance of phaeophorbide a among the degradation products, was found in another freshwater cladoceran, Bosmina longirostris
317
E.Pandolfini et al.
(Descy, unpublished). Another factor which may have affected the fate of
pigments in our experiments is the experimental design itself. The cladocerans,
which were at higher density than under natural conditions, may have re-ingested
algae during the incubation as faeces accumulated in the feeding suspensions
(whereas faecal pellets were settled out through the Nitex screen in the copepod
experiments). Pigment re-ingestion was similarly identified as a potential cause of
further pigment degradation in experiments with microzooplankton (Strom et al.,
1998).
To summarize, whereas algal carotenoids found in the freshwater copepod
E.gracilis were not reliable for assessing their diet, those found in the cladoceran
D.galeata reflected qualitatively the type of algae ingested. In both species,
pigment amounts in the guts were not directly related to the group-specific clearance rates and therefore should not be used for estimating quantitatively the
class-specific ingestion rates. Moderate to extensive destruction of these pigments
occurred, as shown by our calculations of loss rates and by the presence of fucoxanthinol in some Daphnia extracts. Therefore, caution is recommended in using
gut pigments as indicators of cladoceran diet.
Acknowledgements
We are grateful to David Courtois for his help in the experiments with Daphnia
galeata. Part of this study was carried out with financial support of the project
MODEL of the CRP-CU. E.P. benefited from a research fellowship awarded by
the university La Sapienza, Roma, Italy.
References
Adrian,R. (1991) Filtering and feeding rates of cyclopoids feeding on phytoplankton. Hydrobiologia,
210, 217–223.
Bautista,B. and Harris,R.P. (1992) Copepod gut contents, ingestion rates and grazing impact on
phytoplankton in relation to size structure of zooplankton and phytoplankton during a spring
bloom. Mar. Ecol. Progr. Ser., 82, 41–50.
Bochdansky,A.B., Deibel,D. and Hatfield,E.A. (1998) Chlorophyll a conversion and gut passage time
for the pelagic tunicate Oikopleura vanhoeffenii (Appendicularia). J. Plankton Res., 20, 2179–2197.
Conover,R.J., Durvasula,R., Roy,S. and Wang,R. (1986) Probable loss of chlorophyll-derived
pigments during passage through the gut of zooplankton, and some of the consequences. Limnol.
Oceanogr., 31, 878–887.
Descy,J.-P. and Gosselain,V. (1994) Development and ecological importance of phytoplankton in a
large lowland river (River Meuse, Belgium). Hydrobiologia, 289, 139–155.
Descy,J.-P., Frost,T.M. and Hurley,J.P. (1999) Assessment of grazing by the freshwater copepod
Diaptomus minutus using carotenoid pigments: a caution. J. Plankton Res., 21, 127–146.
Dohet,A. and Hoffmann,L. (1995) Seasonal succession and spatial distribution of the zooplankton
community in the reservoir of Esch-sur-Sûre (Luxembourg). Belg. J. Zool., 125, 109–123.
Drits,A.V. (1997) On the possibility of the application of gut fluorescence method for studies of
herbivorous zooplankton feeding. Okeanlogiya, 37, 753–760.
Fundel,B., Stich,H.-B., Schmid,H. and Maier,G. (1998) Can phaeopigments be used as markers for
Daphnia grazing in Lake Constance? J. Plankton Res., 20, 1449–1462.
Head,E.J.H. and Harris,L.R. (1992) Chlorophyll and carotenoid transformation and destruction by
Calanus spp. grazing on diatoms. Mar. Ecol. Prog. Ser., 86, 229–238.
Head,E.J.H. and Harris,L.R. (1994) Feeding selectivity by copepod grazing on natural mixtures of
phytoplankton determined by HPLC analysis of pigments. Mar. Ecol. Prog. Ser., 110, 74–83.
Head,E.J.H. and Harris,L.R. (1996) Chlorophyll destruction by Calanus spp. grazing on
318
Pigments in freshwater zooplankton
phytoplankton: kinetics, effects of ingestion rate and feeding history, and a mechanistic interpretation. Mar. Ecol. Prog. Ser., 135, 223–235.
Kleppel,G.S. (1998) The fate of the carotenoid pigment fucoxanthin during passage through the
copepod gut: pigment recovery as a function of copepod species, season and food concentration. J.
Plankton Res., 20, 2017–2028.
Kleppel,G.S. and Pieper,R.E. (1984) Phytoplankton pigments in the gut contents of planktonic
copepods from coastal waters off southern California. Mar. Biol., 78, 193–198.
Kleppel,G.S., Frazel,D., Pieper,R.E. and Holliday,D.V. (1988) Natural diets of zooplankton off
southern California. Mar. Ecol. Prog. Ser., 49, 231–241.
Kleppel,G.S., Holliday,D.V. and Pieper,R.E. (1991) Trophic interactions between copepods and
microplankton: a question about the role of diatoms. Limnol. Oceanogr., 36, 172–178.
Landry,M.R., Peterson,W.K. and Fagerness,V.L. (1994) Mesozooplankton grazing in the Southern
California Bight. I. Population abundances and gut pigment contents. Mar. Ecol. Prog. Ser., 115,
55–71.
Lopez,M.D.G., Huntley,M.E. and Sykes,P.F. (1988) Pigment destruction by Calanus pacificus: impact
on the estimation of water column fluxes. J. Plankton Res., 10, 715–734.
Mackas,D. and Bohrer,R. (1976) Fluorescence analysis of phytoplankton gut contents and an investigation of diel feeding patterns. J. Exp. Mar. Biol. Ecol., 25, 77–85.
Mackey,M., Mackey,D.J., Higgins,H.W. and Wright,S.W. (1996) CHEMTAX—a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton. Mar. Ecol. Progr. Ser., 144, 265–283.
Mayzaud,P. and Razouls,S. (1992) Degradation of gut pigment during feeding by a subantarctic
copepod: importance of feeding history and digestive acclimation. Limnol. Oceanogr., 37, 393–404.
Millie,D.F., Paerl,H.W. and Hurley,J.P. (1993) Microalgal pigment assessment using high performance
liquid chromatography: a synopsis of organismal and ecological applications. Can. J. Fish. Aquat.
Sci., 50, 2513–2527.
Nelson,J.R. (1989) Phytoplankton pigments in macrozooplankton feces: variability in carotenoids
alterations. Mar. Ecol. Prog. Ser., 52, 129–144.
Penry,D.L. and Frost,B.W. (1991) Chlorophyll a degradation by Calanus pacificus: Dependence on
ingestion rate and digestive acclimation to food resources. Limnol. Oceanogr., 36, 147–159.
Quiblier,C., Bourdier,G., Amblard,C. and Pépin,D. (1994) Separation of phytoplanktonic pigments
by HPLC for the study of phyto-zooplankton trophic relationships. Aquat. Sci., 56, 29–39.
Quiblier-Lloberas,C., Bourdier,G., Amblard,C. and Pépin,D. (1996) A qualitative study of zooplankton grazing in an oligo-mesotrophic lake using phytoplanktonic pigments as organic markers.
Limnol. Oceanogr., 41, 1767–1779.
Stevens,C.J. and Head,E.J.H. (1998) A model of chlorophyll a destruction by Calanus spp. and implications for the estimation of ingestion rates using the gut fluorescence method. Mar. Ecol. Prog.
Ser., 171, 187–198.
Repeta,D.J. and Gagosian,R.B. (1982) Carotenoid transformations in coastal marine waters. Nature,
295, 51–54.
Strom,S.L., Morello,T.A. and Bright,K.J. (1998) Protozoan size influences algal pigment degradation
during grazing. Mar. Ecol. Prog. Ser., 164, 189–197.
Stuchlik,E. (1991) Feeding behaviour and morphology of filtering combs of Daphnia galeata. Hydrobiologia, 225, 155–156.
Tirelli,V. and Mayzaud,P. (1998) Gut pigment destruction by the copepod Acartia clausi. J. Plankton
Res., 20, 1953–1962.
Wright,S.W., Jeffrey,S.W., Mantoura,R.F.C., Llewellyn,C.A., Bjornland,T., Repeta,D. and
Welschmeyer,N. (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids
in marine phytoplankton. Mar. Ecol. Progr. Ser., 77, 183–196.
Received on March 31, 1999; accepted on August 25, 1999
319