incorporating life histories and diet quality in stable isotope interpretations of crustacean...

Incorporating life histories and diet quality in stableisotope interpretations of crustacean zooplankton

MARC VENTURA* , † AND JORDI CATALAN*

*Limnology group (CSIC-UB), Centre for Advanced Studies of Blanes (CEAB), Spanish Research Council (CSIC), Girona,

Catalonia, Spain†National Environmental Research Institute, University of Aarhus, Silkeborg, Denmark

SUMMARY

1. Stable isotope studies have been extremely useful for improving general food web

descriptions due to their ability to simultaneously summarize complex trophic networks

and track the energy flow through them. However, when considering trophic relationships

involving only two or few species, application of general isotopic interpretations based on

average fractionation values may easily lead to misleading conclusions. In these cases a

more accurate consideration of the current processes involved in the isotopic fractionation

should be considered.

2. We investigated the trophic relationships of the crustacean zooplankton assemblage in

an alpine lake (Lake Redon, Pyrenees) by means of stable isotopes of carbon and nitrogen

and applied information on their life history and biochemical composition in the

interpretation.

3. The three species occurring in the lake had distinct isotopic signatures: the two copepod

species (the cyclopoid Cyclops abyssorum and the calanoid Diaptomus cyaneus) had higher

nitrogen isotopic composition than the cladoceran (Daphnia pulicaria), indicative of a higher

trophic position of the two copepods. Most intra-specific isotopic variability was

associated with growth, while the effect of metabolic turnover was negligible. The effects

of changes in the proportion of lipids was restricted to the adults of the two copepods.

4. Daphnia Juveniles showed ontogenetic shifts in their carbon, and nitrogen isotopic

composition. Cyclops copepodites only showed changes in carbon isotopic composition.

These isotopic shifts with changes in size were the result of: (i) the prevalence of

growth over metabolic turnover as the main factor for isotopic variability and (ii) feeding,

during the growth period, on isotopically depleted food in the case of Daphnia, and on

isotopically enriched food in the case of Cyclops.

5. The carbon isotopic variation in Cyclops juveniles could be explained by fitting an

isotopic growth model that considered that they fed entirely on Daphnia. However this was

not the case for nitrogen isotopic variability. Cyclops nitrogen isotopic composition

variation and the Cyclops to Daphnia nitrogen isotopic enrichment were closely correlated

to the quantity of Daphnia protein and to the dissimilarity in the essential amino acid

composition between the two species, which can be interpreted as an indication of

consumer nitrogen limitation.

Keywords: amino acids, carbon isotopes, fractionation, nitrogen isotopes, predator–prey

Correspondence: Marc Ventura, Limnology group (CSIC-UB), Centre for Advanced Studies of Blanes (CEAB), Spanish Research

Council (CSIC), Acces a la Cala Sant Francesc, 14. 17300-Blanes, Girona, Catalonia, Spain. E-mail: [email protected]

Freshwater Biology (2008) 53, 1453–1469 doi:10.1111/j.1365-2427.2008.01976.x

� 2008 The Authors, Journal compilation � 2008 Blackwell Publishing Ltd 1453

Introduction

Traditional food-web diagrams have been criticised as

being subjective constructs that are strongly biased by

our inability to observe the relevant taxonomic, spatial

and temporal variations in trophic interactions (Paine,

1988). The use of stable isotopes has improved food-

web descriptions due to their ability to simulta-

neously capture complex interactions and track

energy flow through ecological communities

(Peterson & Fry, 1987). The ratio of stable isotopes of

nitrogen (d15N) has been used to estimate trophic

positions because the d15N of a consumer is typically

enriched by 3–4& relative to its diet (Vander Zanden

& Rasmussen, 2001; Post, 2002; McCutchan et al.,

2003; Vanderklift & Ponsard, 2003). In contrast, the

ratio of carbon isotopes (d13C) changes only little as

carbon flows through food webs (Vander Zanden &

Rasmussen, 2001; Post, 2002; McCutchan et al., 2003;

Vanderklift & Ponsard, 2003).

Several assumptions are usually made when apply-

ing stable isotopes to field studies. These are based

on experimental studies and have been questioned

(Gannes, O’Brien & Martınez del Rio, 1997; Post, 2002).

For instance, the assumption of constancy in nitrogen

fractionation has frequently proved invalid (e.g. Webb,

Hedges & Simpson, 1998; Adams & Sterner, 2000;

Gaye-Siessegger et al., 2004), and the application of

constant average nitrogen fractionation values to

establish trophic positions should be applied with

caution and usually only when dealing with broad and

complex food webs with multiple trophic links and

many species (Post, 2002). In studies of trophic

relationships involving only two or few species,

potential factors of variability in isotopic fractionation

should be taken into account, including aspects of basic

biochemical composition, diet quality and life history.

The relative proportions of the main biochemical

compounds affect the isotopic composition of an

organism due to the distinct isotopic compositions of

proteins, lipids and chitin (Macko et al., 1990; Kling,

Fry & O’Brien, 1992). Changes in food quality can lead

to high nitrogen fractionation, equivalent to changes of

up to two trophic levels under the assumption of

constant 3–4& enrichment (Webb et al., 1998; Adams

& Sterner, 2000; Oelbermann & Scheu, 2002; Gaye-

Siessegger et al., 2004). The lack of a sufficient source of

amino acids in a given diet forces a higher nitro-

gen turnover in consumers and, therefore, a higher

isotopic discrimination (Hobson & Clark, 1992; Gan-

nes, Martınez del Rio & Koch, 1998). This deficiency

can either be due to the lack of total protein (Fantle

et al., 1999; Adams & Sterner, 2000; Gaye-Siessegger

et al., 2004) or to an amino acid imbalance between the

consumer and its diet (McClelland & Montoya, 2002).

Finally, depending on the isotopic turnover of each

organism, its isotopic signature will be related to

variable sources used throughout the life of the

organism (O’Reilly et al., 2002). Experimental studies

have shown that isotope turnover times may be linked

to metabolism, either related to maintenance or

growth (Hesslein, Hasllard & Ramlal, 1993; Ponsard

& Averbuch, 1999). The relative importance of both

isotopic incorporation routes differ between homeo-

therms and cold-water poikilotherms. For homeo-

therms Ponsard & Averbuch (1999) concluded that

turnover time was very quick due to a much higher

importance of maintenance metabolism over growth

incorporation. However, for poikilotherms living in

cold environments, growth has been shown to be of

much greater importance than maintenance metabo-

lism and the isotopic composition of poikilotherms

will therefore be a summary of their growing period

(Hesslein et al., 1993; Grey, 2000; Herzka & Holt, 2000).

Among freshwater zooplankton, copepods are

known for their trophic shift from the naupliar stage

to copepodites and adults, increasing their prey size

and switching feeding mode from grazing to hunting

(Dussart & Defaure, 1995; Santer, 1998). Copepod

predation on cladocerans can be a result of direct

predation of adults on small bodied Daphnia (Gliwicz

& Lampert, 1994; Gliwicz & Umana, 1994) or of

copepodite predation on the eggs by directly entering

into egg pouches of large bodied Daphnia species

(Gliwicz & Stibor, 1993; Hanazato & Dodson, 1995).

This latter predation mode is very relevant in alpine

lakes (Gliwicz & Boavida, 1996) and becomes a

mechanism leading to life cycle synchronization

(Gliwicz, Slusarczyk & Slusarczyk, 2001). Since cope-

pods have a relatively higher nitrogen and lower

phosphorus content than cladocerans (Ventura, 2006),

they are susceptible to nitrogen limitation. Therefore,

the study of copepods feeding on cladocerans is an

excellent case study for determining the extent to

which the isotopic composition of natural communi-

ties is affected by food quality changes.

Lake Redon, is an oligotrophic lake with

average microbial biomass ratios of 10 : 2 : 2 : 1 for

1454 M. Ventura and J. Catalan

� 2008 The Authors, Journal compilation � 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1453–1469

phytoplankton : bacteria : heterotrophic nanoflage-

lates : ciliates during the ice-free period (Felip et al.,

1999). The lake zooplankton assemblage has only

three crustacean species, one cladoceran (Daphnia

pulicaria Forbes), one cyclopoid copepod (Cyclops

abyssorum Sars) and the calanoid copepod Diaptomus

cyaneus Gurney. Rotifers are usually less abundant

than crustaceans (the ratio between Daphnia and

rotifer biomass is approximately 10 : 1 during the

ice-free period; Camarero et al., 1999). Daphnia and

Cyclops are present in the plankton all year round,

while Diaptomus appears only during summer

(Ventura & Catalan, 2005). The feeding habits of the

first two species are well described, while those of the

calanoid copepod are unknown. D. pulicaria is a

generalist primary consumer (grazer), and C. abysso-

rum has been described as a secondary consumer

(carnivore) preying on Daphnia whenever it can

(Fryer, 1957; Schindler, 1971; Vandenbosch & Santer,

1993). It has been shown, however, that C. abyssorum

may survive on a strictly algal diet (Whitehouse &

Lewis, 1973; Hopp & Maier, 2005). Studies using

stable isotopes have shown C. abyssorum to exhibit a

more substantially enriched nitrogen isotopic ratio

compared to other copepods, such as Cyclops vicinus

Uljanin (Santer, Sommerwerk & Grey, 2006). In the

case of Lake Redon, as in other alpine lakes (Gliwicz &

Boavida, 1996; Gliwicz et al., 2001), it is to be expected

that the main diet of Cyclops will consist of Daphnia

due to the lower availability of other prey items.

The specific aims of this study were (i) to describe

the seasonal isotopic composition of the three species

and their trophic relationships; (ii) to estimate the

relative influence of lipids and chitin in the isotopic

variability of the three species; (iii) to determine

whether the isotopic composition of Cyclops could be

explained based on the isotopic composition of Daph-

nia and (iv) to examine whether the variability of the

isotopic distance between Cyclops and Daphnia, espe-

cially for nitrogen isotope, changed depending on the

protein quantity or quality of its putative food.

Methods

Study site

Lake Redon (formerly Lake Redo) is an oligotrophic

glacial cirque lake located at 2240 m.a.s.l. in the central

Pyrenees (42�38¢N, 0�46¢E). It is relatively large (24 ha)

and one of the deepest lakes (73 m) in the Pyrenees. It is

dimictic, covered by ice during half of the year, usually

from mid-December until late May or the beginning of

June (Fig. 1). The three crustacean zooplankton species

studied had contrasting life histories although all

produced a single cohort per year. A complete descrip-

tion of their life cycles during the study period can be

found in Ventura & Catalan (2005), but the most notable

features are as follows. Cyclops adults survived below

the ice cover and reproduced before the ice cover

melted. The appearance of nauplii (copepods have six

02468

101214

Tem

pera

ture

(o

)C

1999

paD

painh

uracil

aim oib

%(a

)ss

0

20

40

60

80

100ovigerous femalesnon-ovigerous femalesjuveniles

1998

Cyc

lrossyba

spoum

%(moib

)ssa

0

20

40

60

80

100

NaupliiCopepodites CI, CII, CIIICopepodites CIV, CVAdults

rolhC

ollyhp

(a

µL

g–1

)

0.0

0.5

1.0

1.5

2.0

2.5

12 1 2 3 4 5 6 7 8 9 10 11 12

hpaD

upain

racilai

tulcrep

sggE

hc

0

2

4

6

Fig. 1 Seasonal changes in water temperature, chlorophyll a, the

relative contribution of Cyclops abyssorum small copepodites

(I–III), large copepodites (IV and V) and adults of the total C.

abyssorum biomass, the average ± SD of the number of eggs per

clutch, and the relative contribution of Daphnia pulicaria

ovigerous females and juveniles to the total D. pulcaria biomass.

Daphnia juveniles were those not reaching sexual maturity,

which in this year corresponded to a size lower than 2 mm. For

temperature and chlorophyll a, filled circles are the water

column average values; dotted lines follow maximum and

minimum values recorded on each sampling occasion. The black

filled bar shows the ice-covered period.

Trophic relationships in alpine crustacean zooplankton 1455


naupliar, and five copepodite stages before adults

develop) coincided with the spring production maxi-

mum. Copepodites CI–CIII, the stages described to be

sufficiently small to enter a Daphnia egg pouch (Gliwicz

& Stibor, 1993; Gliwicz & Lampert, 1994; Gliwicz &

Boavida, 1996), dominated Cyclops biomass during July

and August (Fig. 1). Most Daphnia adult females also

survived below the ice cover, postponing reproduction

until the ice-free period (Fig. 1). Over-wintering

females (termed ‘first cohort females’ in our study)

were therefore clearly distinguishable from the juve-

niles born during the ice free period (termed ‘second

cohort females’). Due to their long life span these first

generation Daphnia females were all longer than

2.25 mm (Ventura & Catalan, 2005), the minimum size

allowing copepodites to enter their egg pouch (Gliwicz

& Lampert, 1994). The main reproductive period in

which most females had eggs occurred after the spring

overturn from June to August, with a peak in July when

all females carried eggs. In contrast, the appearance of

Daphnia juveniles did not occur until September (Fig. 1)

after the CIII copepodites had become CIV, suggesting

that this delay could have been due to copepodite

predation. A second indication of copepodite predation

on Daphnia eggs was the increased variability in the

number of eggs per female (Gliwicz & Lampert, 1994),

which was higher during the 2 months when the small

copepodites were present in the lake (Fig. 1). Further-

more, Daphnia females were discovered with copepo-

dites within their egg pouches in the July and August

samples. From September onwards, due to the larger

size of copepodites, predation on Daphnia was probably

restricted to direct predation on smaller Daphnia juve-

niles, which were found until November (Ventura &

Catalan, 2005). Coinciding with the end of the autumn

overturn and the start of the ice cover, few adult males

appeared and sporadic ephippia were produced.

Diaptomus was the only strictly diapausing species,

completing its life cycle within 3 months. In Lake

Redon, Diaptomus emergence also coincided with the

spring production maximum, and during their pres-

ence in the plankton of the lake they dominated the

zooplankton biomass (Ventura & Catalan, 2005).

Sample collection and preparation

The lake was sampled on 14 dates from December

1998, just after the lake was completely ice-covered,

until December 1999 when it was ice-covered again.

Samples were collected at the deepest point of the lake

either by drilling through the ice or from a platform

anchored all summer at the same spot. Zooplankton

samples were collected by vertical hauls from 65 m to

the surface with a 200 lm net. Sampled individuals

were kept alive and transported cold (4 �C) until they

were frozen ()20 �C) in the laboratory within a few

hours of collection. After thawing the samples, from

10 to several hundreds of individuals, depending on

the weight of each species and stage, were quickly

sorted under a dissecting microscope and were either

placed into pre-weighted tin capsules for stable

isotope analysis or in Teflon capsules for analysis of

total amino acids, chitin and lipids. At least three

combined sample replicates were analysed for each

species and stage for each sampling date, with the

exception of a few cases where sample material did

not suffice. Individuals were kept cold (<4 �C) during

the sorting process which lasted a few hours for all

samples. Dry weight (DW) was determined for all

samples after drying at 60 �C for 24 h and weighing

on a high precision microbalance (Ohaus Analytical

Plus, AP250D-0; Ohaus corporation, Florham park,

NJ, U.S.A.). Body lengths were measured in each

sample under an Olympus inverted microscope

(Daphnia from the upper edge of the head to the base

of tail spine; copepods from the anterior end of the

cephalothorax to the posterior end of the furca).

Chemical analyses

Dried samples for stable isotope analysis were packed

into tin capsules with vanadium pentaoxide as cata-

lyser to assure complete combustion. Samples were

analysed in a Delta C Finningan MAT mass spec-

trometer (Thermo Fisher Scientific, Inc., Waltham,

MA, U.S.A.) coupled online with a Carlo Erba CHNS

elemental analyzer (CE Instruments, Wigan, U.K.)

via a Finnigan conflo 2 interface. Specific stan-

dards were used for calibrating the isotopic signal:

sucrose (IAEA CH6), polyethilene (IAEA CH7) and

graphite (IAEA-USGS 24) for carbon, and ammonium

sulfate (IAEA-USGS 25, IAEA-N1 and IAEA-N2) and

potassium nitrate (IAEA-NO3) for nitrogen (Gonfian-

tini, 1978). Complete batches of all standards were run

at the beginning and at the end of each analytical

session, and IAEA CH6 and CH7, and IAEA-N1 and

IAEA-NO3 were run for every 12 samples to control

for linearity. Special care was taken with respect to



weighing the samples and the standards to ensure

similar amplitudes. Results are reported using atmo-

spheric nitrogen and PeeDee belemnite carbonate as

references. Reproducibility was better than 0.1& and

0.3& for d13C and d15N respectively.

Protein content was measured as the sum of

individual amino acids. The amino acid analysis also

allowed for the combined determination of chitin,

since N-acetylglucosamine, the molecular constituent

of chitin, is an amino-sugar that after hydrolysis

appears in the aminogram as glucosamine. Total

amino acid samples were vacuum sealed and hydro-

lysed with HCl 6N at 112 �C for 16 h. An internal

norleucine standard was included in every sample

prior to hydrolysis to increase reproducibility. The

analysis was performed on a Biochrom20 (Amersham-

Pharmacia Biotech, Roosendaal, the Netherlands)

ion-exchange amino acid auto analyser following the

ninhidrine method of Spackman, Stein & Moore

(1958). A standard solution of 20 amino acids and

glucosamine was run for every 10 samples.

Total lipids were quantified gravimetrically.

Between 0.3 and 0.6 mg of dried zooplankton were

placed in a dichlormethane : methanol (2 : 1, v ⁄v)

solution (Folch, Lees & Sloane Stanley, 1957) and

sonicated for 30 min in an ultra-sound bath for lipid

extraction. The non-lipidic remains were then col-

lected on pre-weighted GF ⁄F Whatman filters, which

were dried and re-weighted. Lipid content was

determined by weight difference.

Biochemical composition influence on stable isotope

composition

To determine the possible sources of isotope variation,

we analysed the concentration of lipids and chitin for

each species. Lipids are known to have a more

depleted signature than whole-body carbon (DeNiro

& Epstein, 1977), while chitin has a very depleted

signature compared to whole-body nitrogen (Macko

et al., 1990). Isotopic ratios of the lipid and chitin free

fractions were calculated following:

d13CLF ¼ d13CSA�ðMLP � fLPÞ and

d15NCTF ¼ d15NSA�ðMCT � fCTÞ

where LF stands for lipid free fraction, SA refers to the

isotopic composition of the sample, MLP and MCT are

the mass fractions of lipids and chitin, and ƒLP and ƒCT

are the isotopic fractionation in lipid and chitin

compartments respectively. In this study we assumed

the fractionation in zooplankton lipid inhabiting a

similar temperature regime (ƒLP = )3; Kling et al.,

1992) and a fractionation in chitin of ƒCT = )9 (Macko

et al., 1990).

Dissimilarity index

Average Euclidean distance (Djk) was used to estimate

the dissimilarity between the amino acid composi-

tions of Cyclops and Daphnia.

Dij ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiXn

i¼1

Xij � Xik

� �2

s

where Xij and Xik are the percentages of the amino

acid i of Daphnia (j) or Cyclops (k), and n is the number

of amino acids considered. The index was calculated

with all the amino acids or separately for the essential

amino acids (lysine, phenylalanine + tyrosine,

leucine, valine, threonine, isoleucine, histidine, cyste-

ine + methionine) and non-essential amino acids

(glutamic acid, aspartic acid, alanine, arginine, gly-

cine, proline and serine).

Results

Intra-specific and gender differences

The three planktonic crustacean species inhabiting

Lake Redon differed in their mean stable isotope

composition of C and N (Table 1), the differences

being significant in one-way repeated measures-

ANOVAANOVA tests for d13C (F2,77 = 21.5, P < 0.001) and

d15N (F2,77 = 727.9, P < 0.001). To check for differences

between species pairs we conducted a Tukey’s post hoc

test. While the d13C signature of Cyclops was signif-

icantly different from Daphnia (P = 0.012) and Diapto-

mus (P = 0.001), there were non-significant differences

between Daphnia and Diaptomus (P = 0.212). The d15N

signature differed for all three species (P < 0.001).

Daphnia males only appeared occasionally and then

in very low densities, while copepod males and

females occurred in similar numbers when present

in the lake. We analysed males and females of the two

copepod species to determine whether there were

gender differences in isotopic signatures. Cyclops

males appeared only in January 1999, while Cyclops



females inhabited the lake from December 1998 to July

1999. As for Diaptomus, both sexes were present

during the same period. To avoid potential seasonal

differences in the diet of females, we compared the

isotopic composition of males and females of both

species when co-existing. Males and females of the

two species showed the same d15N values (Table 1),

one-way ANOVAANOVA showing no significant gender dif-

ferences either in Diaptomus (F1,10 = 1.99, P = 0.189) or

in Cyclops (F1,4 = 0.54, P = 0.502). Diaptomus presented

the same carbon isotopic values for both sexes

(F1,10 = 0.28, P = 0.611). However, there were gender

differences in the d13C values of Cyclops (F1,4 = 80.63,

P < 0.001), females being slightly more depleted

(Table 1).

Seasonal variability and trophic position as inferred

from stable isotope data

The plot of d13C values throughout the seasonal cycle

(Fig. 2a) showed that the adults of both long-lived

species (Daphnia and Cyclops) during ice-cover and

thawing periods had a much lower variability than

their respective offspring during the ice-free period.

Table 1 Mean and SD of the stable isotopic composition of the pelagic zooplankton species of Lake Redon

Species Sex or stage d13C SD d15N SD n Time period

Daphnia pulicaria 1st cohort females )31.8 0.9 )2.98 0.82 20 12 ⁄ 98–8 ⁄ 99

2nd cohort females )27.9 2.9 )2.99 0.47 12 8 ⁄ 99–12 ⁄ 99

Diaptomus cyaneus Females )31.8 2.4 )0.77 0.51 6 8 ⁄ 99–9 ⁄ 99

Males )31.2 2.0 )0.43 0.28 6 8 ⁄ 99–9 ⁄ 99

Nauplii )32.9 )5.32 1 6 ⁄ 99

Cyclops abyssorum Females )28.6 0.6 0.69 0.27 16 12 ⁄ 98–7 ⁄ 99

Males )27.1 0.1 0.83 0.13 3 1 ⁄ 99

Copepodites )29.5 1.6 )0.22 0.29 17 8 ⁄ 99–12 ⁄ 99

Number of samples analysed (n) and the occurrence period of distinct stages and genders for each species are also indicated.

D.c. n a uplii

δ 3 1

) ‰

(

C

–36

–34

–32

–30

–28

–26

–24

–22

δ 5 1

) ‰

(

N

–7

–6

–5

–4

–3

–2

–1

0

1

2

D.c. na uplii

(a)

(b)

1 2 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

1998 1999

D. p . 1st. coh o rt f e m a les D. p . 2nd . coho r t f e m a les C. a . females C. a . copep odites D. c . a dults

Fig. 2 Seasonal changes in the average

± SE of (a) carbon isotopic composition

and (b) nitrogen isotopic composition of

the planktonic crustacean species of Lake

Redon, Daphnia pulicaria (D. p.), Cyclops

abyssorum (C. a.) and Diaptomus cyaneus

(D. c.). The duration of ice cover is

indicated by the black line at the top of the

figure.



The primary consumer Daphnia exhibited the highest

d13C variation. The adults of the calanoid copepod

Diaptomus changed their d13C from July to August

matching the d13C values of Daphnia. Diaptomus

nauplii also had an isotopic carbon signature similar

to that of Daphnia during their month of co-existence.

The seasonal changes in d15N of the different

species (Fig. 2b) showed a lower variability than

d13C, fluctuations in Daphnia being larger than in

Cyclops. The d15N values of Diaptomus adults were

similar to those of Cyclops copepodites and those of

Diaptomus nauplii were close to those of Daphnia

during the period they co-existed in the lake.

To describe the trophic positions of the two cope-

pod species of Lake Redon, we used the Daphnia

signature as a reference, since due to its unselective

feeding mode it constitutes a good estimate for the

base of the food chain (Vander Zanden & Rasmussen,

1999; Post, 2002; Matthews & Mazumder, 2003). The

average difference in isotopic nitrogen ratios (Dd15N)

between Cyclops and Daphnia was 3.5& (5.3–2.2&),

and 2.7& between Diaptomus and Daphnia (3.0–2.4&)

(Fig. 2b). Thus, Cyclops adults were at the top of the

zooplankton food chain and could potentially be

feeding on Daphnia. Diaptomus adults were almost

one trophic level above Daphnia, but were slightly

more depleted than Cyclops.

The Dd13C between Cyclops and Daphnia was 1.4&

on average but varied widely ()5.3& to +4.5&)

(Fig. 2a). The Dd13C between Diaptomus and Daphnia

was 0.3&. Carbon fractionation between consecutive

trophic levels is small (c. 0.4&: Post, 2002; Vanderklift

& Ponsard, 2003; McCutchan et al., 2003); therefore,

according to the Dd13C results, one would estimate

that Diaptomus could actually be feeding on Daphnia,

whereas Cyclops was not, contradicting what was

indicated by Dd15N.

Influence of biochemical composition on stable isotope

signatures

After removing the lipid isotopic influence, the average

d13C enrichment was +1.4& for Cyclops, +1.5& for

Daphnia and +0.8& for Diaptomus. The lipid-free

fraction had an overall similar seasonal variability to

the bulk isotopic values in Daphnia and Cyclops (Fig. 3a)

and, as a consequence, the major seasonal isotopic

fluctuations of these two species were not attributable

to lipid changes. In contrast, part of the isotopic

variability of Cyclops females and adults of Diaptomus

could be attributed to changes in lipid content. Cyclops

females started reproducing in April, which implied a

progressive decrease in their lipid content (Table 2).

The d13C of reproducing females was significantly

δ Δ 3 1

) ‰

(

C

–8

–4

0

4

8

sam p le lipid f r ee f r action

δ 5 1

) ‰

(

N

–9

–6

–3

0

3

C. a . sam p le C. a . chitin f r ee f r action D. p. sam p le D. p. ch itin f r ee f r action

δ Δ

5 1

‰

( N

) –6

–3

sam p le chitin f r ee f r action

199 9

δ 3 1

) ‰

(

C

–32

–28

–24

–20

D . p . sam p le D. p . lipid f r ee f r actio n

C. a. sa m p l e C. a. lipid f r ee f r action

(a) (b)

(c) (d)

12 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

1 998 19 99

12 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

199 8

Fig. 3 Seasonal changes in (a) the total body and lipid free carbon isotopic composition of Daphnia pulicaria (D. p.) and Cyclops

abyssorum (C. a.) and of (b) the total body and chitin free nitrogen isotopic composition of the same species. The isotopic distance (D)

between both species is also shown for (c) total body and lipid free fraction for carbon and (d) total body and chitin free fraction for

nitrogen.



higher than that of non-reproducing females (Table 2),

but this difference disappeared after correcting for the

lipid lost during the reproductive period (Table 2). The

total lipid content of Diaptomus adults also decreased

from 45.5% of DW to only 5.5% from July to August

due to reproductive investment (Table 2). This

decrease explained 30% of the isotopic change. Non-

reproducing Daphnia females had higher d13C than

reproducing females in both the bulk and lipid-

corrected d13C (Table 2) and, therefore, carbon isotopic

differences between the two cohorts were not attribut-

able to the large difference in lipid content between

them. Lipid effects on Dd13C were almost negligible

between Daphnia and Cyclops (Fig. 3c) and between

Daphnia and Diaptomus (data not shown).

For all species, subtraction of the chitin fraction

resulted in a slight increase (0.3–0.5&) in d15N

(Table 2). The chitin composition was constant for

the three species, and correction for chitin changes

therefore had only a small effect on the seasonal

variability of the nitrogen isotope (Fig. 3b; Diaptomus

data not shown). There were non-significant differ-

ences between non-reproducing and reproducing

females of Cyclops and adults of Diaptomus. In

contrast, Daphnia first cohort non-reproducing females

had a higher d15N than reproducing females in both

bulk and chitin free composition (Table 2). Similarly,

Dd15N values among species remained unaltered for

the three species when corrected for chitin content

(Fig. 3d).

Ontogenetic isotopic shifts

The isotopic composition of Cyclops copepodites

increased in d13C throughout summer (Fig. 2), coin-

ciding with a progressive increase in the DW of the

different stages found in each consecutive month

(Fig. 4). By contrast, Daphnia second cohort females

did not exhibit a clear temporal trend in isotopic

composition. Their d13C increased from August to

September, after which it decreased progressively

until December (Fig. 2). Similarly, their d 15N

increased from August to October and then decreased

progressively until December. This pattern resembled

the development of average size (length and DW),

since Daphnia second cohort females had the highest

proportion of youngest individuals in October,

increasing their average weight until December

(Fig. 4). Expressing isotopic composition in terms of

length showed that Daphnia second cohort females

became more depleted in d13C and d15N with growth,

and the good correlation between both isotopes and

Table 2 Isotopic composition (average ± SE) of non-reproducing and reproducing first cohort females of Daphnia pulicaria,

females of Cyclops abyssorum and adults of Diaptomus cyaneus

Species

Carbon Nitrogen

Non-reproductive Reproductive d.f. F P-value Non-reproductive Reproductive d.f. F P-value

Daphnia pulicaria

Bulk d13C ⁄d15N (&) )31.6 ± 0.23 )32.9 ± 0.17 1,18 8.47 0.009 )2.8 ± 0.23 )3.9 ± 0.74 1,18 74.0 <0.001

Lipid free d13C ⁄ chitin

free d15N (&)

)29.9 ± 0.31 )32.5 ± 0.16 1,18 32.7 <0.001 )2.6 ± 0.23 )3.6 ± 0.75 1,18 26.4 <0.001

Lipids ⁄ chitin (% DW) 55.8 ± 4.26 14.1 ± 0.50 2.0 ± 0.10 3.4 ± 0.12

Diaptomus cyaneus

Bulk d13C ⁄d15N (&) )33.5 ± 0.27 )29.5 ± 0.16 1,11 166.2 <0.001 )0.4 ± 0.17 )0.8 ± 0.17 1,11 1.6 0.238


free d15N (&)

)32.1 ± 0.26 )29.4 ± 0.14 1,11 87.7 <0.001 0.0 ± 0.17 )0.2 ± 0.18 1,11 0.4 0.556

Lipids ⁄ chitin (% DW) 45.5 ± 0.22 5.5 ± 1.57 4.6 ± 0.16 6.3 ± 0.11

Cyclops abissorum

Bulk d13C ⁄ d15N (&) )29.0 ± 0.05 )28.3 ± 0.21 1,14 9.0 0.01 0.8 ± 0.03 0.7 ± 0.05 1,13 3.5 0.084


free d15N (&)

)27.4 ± 0.08 )27.2 ± 0.20 1,14 2.1 0.169 1.1 ± 0.04 1.1 ± 0.06 1,13 0.3 0.593

Lipids ⁄ chitin (% DW) 53.4 ± 1.74 34.7 ± 1.20 3.7 ± 0.10 4.4 ± 0.17

Both the bulk and the biochemical compound-free composition (either removing the effects of variable lipid composition for carbon, or

chitin for nitrogen) are provided. The average ± SE compositions of both compounds (lipids below d13C and chitin below d15N) used

for the calculations are also provided. Differences between non-reproductive and reproductive individuals were tested using one-way

A N O V AA N O V A. d.f., degrees of freedom; F, F-test; P, probability of significance; DW, dry weight.



length (r2 = 0.61, P = 0.001 and r2 = 0.5, P = 0.005 for

d13C and d15N respectively) (Fig. 5) indicated that

isotope enrichment was related to ontogenic develop-

ment. Furthermore, the two isotopes were positively

correlated with each other (r2 = 0.35, P = 0.026), indi-

cating that the causative mechanism might be the

same. Also the carbon isotopic composition of Cyclops

copepodites exhibited an ontogenetic pattern but, in

this case, enrichment in d13C increased with growth.

In contrast, it showed no d15N relation with growth

other than the increase in d15N from copepodites

CII–CIII (Fig. 5).

Modelling Cyclops isotopic composition

The high correlation between the d13C values of

Cyclops juveniles and their DW indicates that growth

was the main factor for isotopic change, with tissue

turnover being negligible. In order to better evaluate

this hypothesis, we used the turnover equation of Fry

& Arnold (1982) to fit the d13C data:

dt¼ dfþðdi�dfÞðWt

WiÞc

where df and di are the final and initial isotopic

composition of Cyclops, Wt is the DW at each growth

stage (t), and Wi is the initial weight. The value c, the

exponent of metabolic decay, is indicative of the

relative contribution of growth and tissue turnover

(Fry & Arnold, 1982). At c = )1, tissue replacement is

absent or not detectable and the equation becomes a

simple mass balance model in which dt is a function of

growth alone. For values of c less than )1, both

growth and tissue turnover contribute to the isotopic

shift. The results of fitting the curve by nonlinear

curve fitting and restricting c to less than or equal

to )1 confirmed that the best model was c = )1

(r2 = 0.964, P = 0.013) and accordingly that tissue

turnover was negligible.

The isotopic compositions of the juveniles of Cyclops

and second cohort females of Daphnia were not

significantly correlated (P = 0.397 and P = 0.977 for

d13C and d15N respectively; Fig. 6a,b). However, this

lack of correlation does not imply that Cyclops could

not have been feeding on Daphnia, since it does not

take into account the changes in the growth rate of

Cyclops. Therefore, in order to determine whether the

isotopic composition of Cyclops might be explained by

the assumption that it was mainly feeding on Daphnia,

we employed a food-based isotopic shift model based

on the growth rate of copepodites, assuming the

influence of tissue turnover to be negligible (see

above). The model simply adds ingested carbon and

nitrogen from food (Daphnia in this case) proportion-

ally to the average population growth rate at

every time step, and allows for food isotopic variation

from step to step (Parker, Anderson & Lawrence,

1989):

dPredt¼ dPredt�1Wt�1=WtþðdfoodþfaÞð1�Wt�1=WtÞ

where dpred is the carbon or nitrogen isotopic compo-

sition of the predator, W is the DW of the individual,

10

20

30

40

50

60

1.2

1.4

1.6

1.8

2.0

Cyclops abyssorum(copepodites)

yrD

(thgiew

µnI

gd–1

)

1

2

3

4

CII

CIII

CIII > CIV

CIV > CV

CV > CIV

CV > CIV

Daphnia pulicaria(second generation females)

yrD

µ(thgiew

dnIg

–1)

Length

()

mm

8 9 10 11 121999

8 9 10 11 121999

7

(a)

(b)

Fig. 4 Changes in the average (circles) and SE (vertical error

bars) of the population average length and dry weight of (a)

Daphnia pulicaria second cohort females and (b) dry weight of

Cyclops abyssorum copepodites. The stage dominating each

month is shown. When more than two stages were present their

order of importance in the total sample abundance is indicated

by >.



dfood is the average isotopic composition of food

between time t and t ) 1, and ƒa is the trophic

fractionation (3.4& for nitrogen and 0.5& for carbon).

The model successfully predicted the d13C of

Cyclops copepodites, explaining 95% of the measured

isotopic change (P < 0.001) (Fig. 6c). Contrary to d13C,

the fitted model for d15N was not significant

(P = 0.168) (Fig. 6d), and the modelled isotopic

change only accounted for 41% of the measured

change. This discrepancy between modelling carbon

and nitrogen isotopic changes suggested that an

additional fractionation mechanism prevailed in the

case of nitrogen.

Two main causes of changes in nitrogen isotopic

fractionation between consumer and food have been

described: insufficient protein availability in the food

and poor quality of food (i.e. proteins with different

amino acid composition between the consumer and

food). Measurement of the protein content showed

that Cyclops had higher protein content than Daphnia

throughout the study period (average DW% ± SE

was 43.6 ± 1.6 and 27.0 ± 1.1 for Cyclops and Daphnia

respectively). In addition, the relationship between

Daphnia protein content and Cyclops–Daphnia Dd15N

showed a negative correlation (r2 = 0.65, P = 0.053;

Fig. 7a). Therefore, if Cyclops were feeding on Daphnia,

protein limitation could be expected. To evaluate the

potential additional relevance of amino acid compo-

sition, we related the Dd15N between Cyclops and

Daphnia to the dissimilarity in their amino acid

composition. We calculated the amino acid average

dissimilarity between the two species either using all

amino acids (Fig. 7b), only essential amino acids

(Fig. 7c) or only non-essential amino acids (Fig. 7d).

There was a highly significant positive correlation

between Dd15N and amino acid dissimilarity based on

δ 3 1

) ‰

(

C

–36

–32

–28

–24

Length (m m )

0 . 8 1 . 2 1 . 6 2 . 0

δ 5 1

( N

‰

)

–4.2

–3.6

–3.0

–2.4

–1.8

Daphnia pulicaria

Daphnia pulicaria

r 2 = 0.61, P = 0.00 1

r 2 = 0. 5, P = 0.00 5

δ 3 1

( C

‰

)

–32

–30

–28

Dry weight (µg i n d –1 )

1 2 3 4

δ 5 1

( N

‰

)

–1

0

1

Cyclops abyssorum

Cyclops abyssorum

r2 = 0.99, P < 0.0001

(a) (c)

(b) (d)

C I I

C II I

C I I I > CI V

C IV >CV

C V > C I V

C V > CIV

Fig. 5 Ontogenetic changes in Daphnia pulicaria (a) carbon and (b) nitrogen isotope composition in relation to length and in Cyclops

abyssorum of (c) carbon and (d) nitrogen isotope composition in relation to dry weight. Values for Cyclops are monthly population

averages ± SE corresponding to different copepodite developmental stages. The stage dominating each month is shown. When more

than two stages were present, their order of importance in total sample abundance is indicated by >.



total and essential amino acids, but not based on to

non-essential amino acid dissimilarity. The months

with the highest Dd15N had also the highest amino

acid disimilarity between Cyclops and Daphnia. In

conclusion, both protein quality and quantity

appeared as relevant factors for explaining the

Dd15N of Cyclops and Daphnia.

As mentioned above, the changes in nitrogen

isotopic composition of Cyclops copepodites were not

related to growth or to the changes in the isotopic

composition of Daphnia. Therefore, due to the rele-

vance of the essential amino acids and protein

quantity on the Dd15N, we expected that either one

or both of these parameters would explain part of the

variance unaccounted for by the food-based isotopic

shift model. We fitted a linear regression model with

the d15N of Cyclops copepodites as the dependent

variable and d15N of Cyclops copepodites predicted by

the food based isotopic shift model, the essential

amino acid dissimilarity between the Cyclops copepo-

dites and Daphnia, and the protein concentration of

Daphnia, as independent variables. We used a step-

wise procedure to fit the best minimal model. The

variables selected were the essential amino acid

dissimilarity (Deaa) and the protein content (Pc) of

Daphnia (d15NCyclops = 0.445 Deaa ) 0.018 Pc ) 0.466,

r2 = 0.854, P = 0.055). The selection of the two food

quality variables shows that metabolic processes are

r 2 = 0. 99, P < 0.001

Pr edi c ted Cycl ops abyssorum δ 13 C (‰ )

–33 –32 –31 –30 –29 –28 –27

M

d e r u s a e C

y c l

m

u r o s s y b a s p o

δ 3 1

) ‰

(

C

r 2 = 0. 41, P = 0.17

P r ed icted C y clops abyssorum δ 15 N (‰ )

–0.8 –0.6 –0.4 –0.2 0.0 0.2 0.4

d e

r u

s a

e M

C

y m

u

r o

s s

y b

a s

p o

l c

δ51

(N

‰)

D a phni a pul ic aria δ 13 C (‰)

–32 –30 –28 –26 –24

C y c

l m

u r o s s y b a

s p o δ

3 1 )

‰

( C

–33

–32

–31

–30

–29

–28

–27

–33

–32

–31

–30

–29

–28

–27

Daph nia p u li cari a δ 15 N ( ‰ )

–3.6 –3.4 –3.2 –3.0 –2.8 –2.6 –2.4

l c

y C

u

r o

s s

y b

a s

p o

m δ 5

1 (

N

‰ )

–0.8

–0.6

–0.4

–0.2

0.0

0.2

0.4

–0.8

–0.6

–0.4

–0.2

0.0

0.2

0.4

P = 0. 397 P = 0.977

(a) (b)

(c) (d)

Fig. 6 Upper panels: comparison between the measured isotopic composition of (a) carbon and (b) nitrogen of growing juveniles of

Daphnia pulicaria and copepodites of Cyclops abyssorum in Lake Redon. Lower panels: comparison between the measured Cyclops

abyssorum copepodites values for (c) carbon and (d) nitrogen isotopic composition and that modelled by an isotopic enrichment model

assuming Cyclops abyssorum feeding on Daphnia pulicaria.



more relevant in explaining the changes in the d15N of

Cyclops copepodites than is the isotopic composition

of Daphnia itself (represented in this case by the food-

based isotopic shift model).

Discussion

Relevance of life history incorporation into stable isotope

interpretations

In this study we have found that growth is a relevant

factor for explaining the isotopic composition changes

of both carbon and nitrogen isotopes in Daphnia and

for carbon isotopes in Cyclops. Several experimental

studies have demonstrated that for poikilotherms

inhabiting low temperature habitats such as alpine

lakes, growth has a greater importance than mainte-

nance metabolism in determining the isotopic

composition in different animal groups such as fishes

(Hesslein et al., 1993; Herzka & Holt, 2000), zooplank-

ton (Grey, 2000; Tamelander et al., 2006) and shrimps

(Fry & Arnold, 1982). Our field observations for

crustacean zooplankton are consistent with this

experimental evidence and thus may be of general

relevance for many other low temperature poikilo-

therms and the interpretation of their isotopic ratios.

For example, based on the average Dd13C between

Cyclops and Daphnia (1.4&), one would predict that

Cyclops was either not predating on Daphnia or that

the fractionation factor for carbon was well above the

average. However, by incorporating the life history

information in the food-based isotopic shift model, we

showed that the isotopic composition of Cyclops can

be predicted based on a pure diet of Daphnia (or any

other food with the same isotopic composition of

Daphnia) and a common fractionation factor of 0.4&.

3. 0

3. 5

4. 0

4. 5

Dap

hnia

pro

tein

con

tent

(%

dry

wei

ght)

20

25

30

35

Δδ 15 N Cyclops-D a phnia (‰)

2 3 4

aD-spolcy

Cnhp

iama

indi

ssim

ilari

tydica

o

2.0

2.5

3.0

Δδ 15 N Cyclops-D ap hnia (‰)

2 3 4

dissimilarity

polc

yC

hpa

D-s

ain

maic

ao n

id

dissimilarity

polc

yC

hpa

D-s

ain

madi

caon

i

1. 5

2. 0

2. 5

3. 0

to tal amin o acids r 2 = 0. 85 8, P = 0.008

essen t ial am ino acids r 2 = 0.93 1, P = 0.0009

n on-essential am ino aci d s r 2 = 0.46 1, P = 0.138

pro t ein con t ent r 2 = 0 . 65, P = 0.053

(a) (b)

(c) (d)

Fig. 7 Comparison between (a) the Cyclops–Daphnia nitrogen isotopic dissimilarity and the protein content of Daphnia pulicaria,

(b) the total amino acid average Euclidean distance between the two species, (c) the essential amino acid average Euclidean

distance, (d) the non-essential amino acid average Euclidean distance.



mv

Cross-Out

mv

Replacement Text

enrichment or fractionation

Ontogenetic isotopic changes

Within zooplankton several ecological factors may

cause intra-specific isotopic variation independently

of food isotopic change, such as ontogenetic diet

changes or gender differences in food utilization. For

example, many copepods increase the size of their

prey from early naupliar stages to copepodites and

adults (Dussart & Defaure, 1995), and thus an isotopic

shift during ontogeny can be expected. In this study,

Diaptomus nauplii in June had a similar isotopic

signature to that of Daphnia (Table 1 and Fig. 2),

which agrees with the fact they both graze on small

planktonic organisms. In July, the dominant Diapto-

mus stage was adult and no nauplii were found. In

July, Diaptomus adults had the same isotopic compo-

sition as Cyclops copepodite CII, and both were 2.65&

in d15N above the Daphnia of July. Assuming that

nauplii in July would have had a signature similiar to

that of Daphnia, we predict that both Diaptomus and

Cyclops copepodite CII were almost one trophic level

above, and that in the case of Cyclops the shift from

primary consumer to secondary consumer occurs

during the first copepodite stage. This is probably

related to the major structural change that copepods

face from nauplius to copepodite CI. This result

agrees with what has been found in experimental

(Gliwicz & Stibor, 1993; Hanazato & Dodson, 1995)

and field studies (Gliwicz & Boavida, 1996; Gliwicz

et al., 2001). The progressive ontogenetic isotopic

enrichment observed for Cyclops copepodites was

therefore not due to a trophic shift but resulted from

the conjunction of two aspects: (i) predominance of

growth as the explanatory factor of body isotopic

change (reduced or negligible metabolic turnover)

and (ii) feeding on isotopically isotopic enriched food

during the main growth periods. This enriched food

was actually Daphnia, which was isotopically enriched

compared to Cyclops during the period in which

Cyclops were predominantly growing (Fig 2a), and

showed that copepodite predation can be an ecolog-

ically relevant but generally neglected cause of Daph-

nia population growth reduction.

Similarly to Cyclops d13C, changes in C and N

isotopes in Daphnia were related to changes in size.

There was a prevalence of growth effect combined

with feeding on isotopically depleted food during the

periods of growth. Other recent studies have found

similar Daphnia size-based isotopic variation in both

d13C (Matthews & Mazumder, 2006) and d15N

(Matthews & Mazumder, 2007). In the case of d13C,

these authors showed that the size-based isotope

variations in Daphnia were related to differential size-

based habitat selection in vertical gradients in the d13C

of particulate organic matter. In particular, they

showed that the epilimnion was enriched compared

to the hypolimnion, which corresponded with a

predominantly allochthonous origin of the epilimnetic

carbon and a predominantly autochthonous origin of

the hypolimnetic carbon. In our study site, the d13C of

the dominant vegetation of the Lake Redon catchment

was )24.9& and that of epilimnetic seston )25.4 ±

0.2& (Catalan et al., 2004), a value very similar to

those of the smaller stages of Daphnia ()24.1&),

whereas the hypolimnetic seston of Lake Redon had

an average isotopic composition of )28.5 ± 0.6&

below the thermocline (Catalan et al., 2004). Since

seston can be found to be enriched by about 3& with

respect to zooplankton in low phosphorous lakes

(del Giorgio & France, 1996), zooplankton in Lake

Redon feeding below the thermocline could have a

d13C of )31.5&, a value similar to those found for the

largest Daphnia in our study. Some species of Daphnia

(including D. pulicaria, the species inhabiting

Lake Redon) increase their filter size with ontogeny,

and increase the mean size of captured particles

(Lampert, 1974; Brendelberger, 1991). Ontogenetic

isotopic changes in these species are therefore to be

expected if the different size particles have distinct

isotopic compositions. A possible explanation for the

observed isotopic changes could be a diet change with

increasing phytoplankton contribution. In contrast to

d13C, the sestonic d15N was similar (c. )2&) between

epilimnion and hypolimnion in Lake Redon (Catalan

et al., 2004) and thus it could not explain the size-

based isotopic change in Daphnia. A similar pattern

has been described by Matthews & Mazumder (2007),

who found that seston had lower variability than

Daphnia. The origin of such variability is still an open

question.

Changes in adult isotopic composition

Changes with reproduction differed among the three

study species. Cyclops had the same d15N or d13C

when corrected for changes in lipid and chitin

content. Diaptomus adults also had the same d15N

but their d13C decreased in reproducing adults. This



decrease was only partially explained by a strong

decrease in lipid content. The lack of complete

explanation could be due to the use of an erroneous

fractionation factor for lipids. The value used in this

study ()3&) was chosen since it was obtained for

zooplankton living in similar conditions (Kling et al.,

1992). However, a more recent study focussing on

different zooplankton groups has found values rang-

ing from )4.4& to )8.2& (Smyntek et al., 2007), which

could explain almost all the difference in the isotopic

composition change observed in Diaptomus with

reproduction.

Contrasting with the two copepods, reproducing

Daphnia first cohort females increased their d13C and

d15N compared with over-wintering females. Changes

in biochemical composition were not responsible for

this change, since lipid correction increased the

difference in d13C from 1.3& to 2.6&, while chitin

correction had no effect on d15N. Similar results have

been found experimentally by Grey (2000) and

Tamelander et al. (2006). Their studies demonstrated

that adult copepods (the freshwater copepod Cyclops

sp. in the first study and the marine Calanus glacialis in

the second) did not equilibrate their body isotopic

composition with the composition of the experimental

food. Grey (2000) also found that Daphnia rapidly

equilibrated its body content with the signature of

experimental food and concluded that this was very

likely related to differences in the moulting charac-

teristics of copepods and cladocerans. While cope-

pods stop moulting once they are adults, cladocerans

continue moulting with the production of each egg

clutch (Tessier et al., 1983). Daphnia females of Lake

Redon did not start reproduction until May (Ventura

& Catalan, 2005). Reproducing Daphnia females (June

and July) were therefore more depleted in d13C or

d15N than non-reproducing females. Furthermore,

non-reproducing females had a more constant isoto-

pic composition (Fig. 2). Therefore, it is very likely

that only reproducing Daphnia females reflect the

variability occurring in their resources. In contrast,

non-reproducing Daphnia females and the isotopic

composition of Cyclops adults did not necessarily

reflect the composition of their diet, but rather a

‘summary’ of diet through their ontogeny.

Copepods show a sexual size dimorphism, which is

more pronounced in cyclopoid than in calanoid

copepods (Gilbert & Williamson, 1983). Two main

hypotheses have been suggested to explain the

adaptive evolution of this dimorphism: sexual selec-

tion and ecological causation (Shine, 1989). If the

second hypothesis is true, one would expect that

trophic gender differences would be more pro-

nounced in cyclopoid copepods, which should be

reflected in differences in their isotopic composition.

The almost identical isotopic composition of males

and females of both copepod species suggests that

there is no difference in the feeding of males and

females, at least, until their last stage. In fact, copepod

males and females have an almost identical morphol-

ogy during their ontogeny, and it is not until they

moult from copepodite V to adults that their mor-

phology distinctively differs. The question of whether

males and females feed on similar resources in the last

stage remains a subject for future studies.

Variability in Cyclops d15N enrichment

Cyclops d15N variability was not related to growth, but

to the protein concentration of Daphnia and the

essential amino acid dissimilarity between the two

species. Experimental studies have also reported a

lower protein content of the diet (e.g. in crabs by

Fantle et al., 1999; Daphnia by Adams & Sterner, 2000

and fish by Gaye-Siessegger et al., 2004) or an amino

acid imbalance between the consumer and its diet

(e.g. in birds by Hobson & Clark, 1992) that substan-

tially increases the tissue-diet isotopic fractionation.

The proposed mechanism for an increase in tissue-

diet fractionation with lower protein content is related

to a lower growth rate of animals and a progressive

increase in d15N of the animal through isotopic

selective excretion of ammonia (e.g. in crabs Fantle

et al., 1999 and fish Trueman, Mcgill & Guyard, 2005).

Unlike the effects of gross protein availability, the

effects of a changing amino acid composition on the

tissue-diet fractionation are much less studied. Some

of the amino acids are known to be essential for

animals because they are unable to synthesize the

carbon skeleton of the amino acids. This explains the

preservation of the d13C signature of the essential

amino acids from the diet, while the non-essential

amino acids undergo intense turnover and thus

fractionation (Fantle et al., 1999). This explanation for

d13C cannot be applied to d15N, since amino acids

derived from protein breakdown are deaminated to

keto acids, a portion of which is reaminated and

reincorporated into body tissue. Each transamination



results in nitrogen isotope discrimination, with

excreted nitrogen being depleted in 15N and the

retained amino acids being correspondingly enriched

(Scrimgeour et al., 1995). The specific mechanisms

driving changes in the d15N of individual amino acid

are not well described. A comparison of the individ-

ual amino acid d15N of a marine rotifer and those with

the d15N values of its food showed that only seven of

the 13 amino acids analysed had a clear d15N

fractionation (McClelland & Montoya, 2002), half of

them being essential. Therefore, the physiological

mechanisms behind the relationship between essential

amino acid dissimilarity and the Cyclops–Daphnia

Dd15N found in this study require further investiga-

tion. However, a possible explanation may be an

increased amino acid metabolism in diets deficient in

essential amino acids, including remobilization of

stored amino acids; the same not being true in the case

of high deficiency in non-essential amino acids.

Finally, the lack of correlation between the Cyclops

d15N and growth and its positive correlation with the

protein and amino acid dissimilarity between Daphnia

and Cyclops may be interpreted as evidence of

nitrogen or amino acid limitation of Cyclops growth.

In a parallel study, we found that Cyclops contained

almost half of the phosphorus content that was

present in Daphnia during the whole seasonal cycle

(Ventura & Catalan, 2005). Therefore, it seems

unlikely that copepods would suffer from phospho-

rous limitation when feeding on Daphnia. This is

probably true for most carnivorous cyclopoid cope-

pods feeding on cladocerans, since all cyclopoids have

a lower phosphorus content than cladocerans

(Ventura, 2006). Similarly, Daphnia had a higher

phosphorus content than seston (Ventura & Catalan,

2005), suggesting that its growth might be limited by

phosphorous availability, which would also explain

why both d13C and d15N showed a similar relationship

with Daphnia growth (Fig. 5a,b).

In conclusion, our results show that in field studies

dealing with a community of a small number of

species, stable isotope variation can only be used to

study diet changes and nutrient limitations when life

histories are properly considered.

Acknowledgments

We thank LL. Camarero, B. Claramunt, A. Miro, P.

Renom, T. Buchaca and G. Cots for their assistance

with the field work, and R. Franco for laboratory

assistance. The total amino acid analysis and the

stable isotope C and N analyses were performed at the

Serveis Cientıfico Tecnics at the University of Barce-

lona, whose assistance is kindly acknowledged. A.M.

Poulsen assisted with linguistic ⁄editorial corrections.

We thank T. Buchaca, T. Larsen, the editor and

anonymous referees for very helpful comments on

the manuscript. The research was partially supported

by the European Commission projects MOLAR

(ENV4-CT95-0007) and EUROLIMPACS (GOCE-

CT-2003-505540) and the Spanish MEC project

TRAZAS (CGL2004-02989). MV was partially sup-

ported by a Marie Curie post-doctoral grant (MEIF-

CT-2005-010554) and a Juan de la Cierva contract.

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(Manuscript accepted 23 January 2008)



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