effects of copper on energy metabolism and larval development in the midge chironomus riparius

Effects of copper on energy metabolism and larval development

in the midge Chironomus riparius

MARIA J. SERVIA,1,2,3 ALEXANDRE R.R. PERY,2 MICHELINE HEYDORFF,1 JEANNE GARRIC2

AND LAURENT LAGADIC1,*1UMR 985 Inra-Agrocampus Ecobiologie et Qualite des Hydrosystemes Continentaux,

Equipe Ecotoxicologie et Qualite des Milieux aquatiques, 65 rue de Saint Brieuc, F-35042 Rennes,Cedex, France

2Laboratoire d’Ecotoxicologie, Cemagref, 3bis quai Chauveau, F-69336 Lyon, Cedex 9, France3Field Station of Hydrobiology ‘‘Encoro do Con’’, Castro de Agudin – Cea, E-36617 Vilagarcia de

Arousa, Pontevedra, Spain

Accepted 15 December 2005 / Published online 24 March 2006

Abstract. When spiked in sediments, copper is known to reduce growth of Chironomus riparius larvae andthe production of eggs by adult females. The aim of this work was to better understand the origin of thesephenomena by studying the effects of copper using developmental and energetic biomarkers, such aschanges in larval weight and age and changes in the levels of sugars and lipids. Four-day-old C. ripariuslarvae were exposed to nominal concentrations of copper of 0, 6.5, 12.5, 25 and 50 mg/kg of dry sediment(silica) in 0.6 l beakers. They were fed ad libitum and exposures were stopped at 7 and 9 days after thebeginning of the tests. The larvae were weighed, sexed and aged. For each sex, the larvae belonging to thephases the most frequently found in the beakers were selected for dissection and measurement of energyreserves. The increase in the concentration of copper resulted in an increasing delay in larval growth in bothsexes. Desynchronized development was observed, as shown by the increase in the number of individualsthat remained in the third instar or early phases of the fourth instar, as well as by a reduction in age ofmales. Concerning energy reserves, the levels of sugars (glycogen, trehalose and glucose) in the dissectedlarvae remained almost constant among levels of exposure. In contrast, at the highest copper concentration(50 mg/kg), triglyceride levels suffered a slight reduction whereas the level of free glycerol significantlyincreased. It is concluded that selection of C. riparius larvae for both sex and age improves the relevance ofsome energy-yielding substrates as indicators of adverse physiological effects of copper.

Keywords: midge larvae; copper; growth; energy-yielding substrates; sugars; lipids

Introduction

The cost of the metabolic processes needed forcombating a toxicant might modify the pattern of

physiological energy allocation of an organism(Calow, 1991; Forbes and Forbes, 1994). There-fore, classical endpoints used in ecotoxicity testssuch as survival, growth or reproduction, mayultimately reflect changes in energy metabolism ofthe individuals. This has already been shown in anumber of aquatic animals exposed to copper,

*To whom correspondence should be addressed:

Tel.: +33-223-485-237; Fax: +33-223-485-440;

E-mail: [email protected]

Ecotoxicology, 15, 229–240, 2006

� 2006 Springer Science+Business Media, Inc. Printed in The U.S.A.

DOI: 10.1007/s10646-005-0054-0

which is known to affect a range of endpoints suchas oxygen consumption, blood variables, andenergy efficiency (US EPA, 2003), with possibleconsequences on individual performances. Sucheffects have been extensively investigated in fish(DeBoeck et al., 1997a, b; Handy et al., 1999;Smith et al., 2001; Campbell et al., 2002; Ali et al.,2003), amphibians (Mounaji et al., 2003; Carattinoet al., 2004; Redick and La Point, 2004), andmolluscs (Widdows and Johnson, 1988; Belangeret al., 1990; Cheung and Wong, 1998; Elfwing andTedengren, 2002). Effects of copper on the ener-getic costs associated with individual performanceshave also been studied in arthropods. In terrestrialenvironments, insects have received the bulk of thefocus (Michaud and Grant, 2003; Lapointe et al.,2004; Nursita et al., 2005). In aquatic ecosystems,most of the studies have been performed in cla-doceran crustaceans (Wong, 1993; Khangarot andRathore, 2003), in which population-level effectsof copper are well documented (reviewed in Starkand Banks, 2003). Moreover, De Coen and Jans-sen (2003a, b) established a link between severalenergy-related biomarkers and population char-acteristics of Daphnia magna. Extrapolation ofthese relationships to other organisms would rep-resent an invaluable contribution to the knowledgeof the ecotoxicological relevance of energetic bio-markers.

The life cycle of C. riparius (Diptera, Chiro-nomidae) can be fully described by energy-basedmodeling, as shown by Pery et al. (2002). With arelevant understanding of the energetic mecha-nisms for feeding, reproduction and growth, theauthors could predict successfully the growth,emergence and reproduction patterns for differentstarting densities and different amounts of dailyfeeding from 0.1 to 0.6 mg/larva/day (this latterdiet corresponded to ad libitum conditions). As aconsequence, it can be expected that any effect of achemical on physiological energetics would pro-duce effects on the life cycle. This is precisely thecase with copper. Pery et al. (2003b) showed thatthe effects of copper on C. riparius larval growthwere due to an increase of the energetic costs ofgrowth rather than a decrease of feeding. Reduc-tion in individual length occurred followingexposure to copper-spiked silicate with concen-trations above 6 mg/kg, and effects on survivalwere observed for concentrations above 40 mg/kg.

Moreover, Ducrot et al. (2004) showed thatincreasing exposure to copper from 0 to 50 mg/kgresulted in a decrease in the number of eggs per eggmass produced by females.

In the present study, the effects of exposure tocopper (Cu)-spiked sediment on sugar and lipidreserves were investigated in C. riparius larvae. Inaddition, developmental effects were studied usinglarval weight, and by determining the larval instarand phase in the fourth instar, in order to establisha link between energetic and developmentalassessment endpoints.

Materials and methods

Chemicals

Glycogen (type VIII from Crepidula fornicata),a-D(+)-Glucose, anthrone, trehalase (a-a-Treha-lose glucohydrolase from porcine kidney; EC3.2.1.28) and copper (cupric sulfate anhydrous,CuSO4) were purchased from Sigma ChemicalCompany Europe (Saint Quentin Fallavier,France). D(+)-Trehalose was obtained from AcrosOrganics (Noisy Le Grand, France), and glucosereagent kit (Nr 991-85108) containing GOD-PAPand mutarotase was generously provided by WakoChemicals GmbH (Neuss, Germany). All otherchemicals used were of analytical grade.

Experimental design

Due to the small size of the larvae, two separateexperiments were needed in order to obtain enoughmaterial for measuring sugars (Experiment 1) andlipids (Experiment 2). In both cases, the experi-mental conditions were the same as in Pery et al.(2002, 2003b).

Sediment spiking – Artificial sediments (silicasand with particle size distribution: 90% between50 and 200 lm, and 10% under 50 lm) were pre-conditioned and spiked with copper as described inPery et al. (2003b). The copper stock spikingsolution was obtained by dissolving 10 g CuSO4 in1 l ultra-pure water. To test the efficiency of thespiking, copper concentration was measured in thespiked sediment of Experiment 1 at days 0 and 7.Copper was quantified using atomic absorptionphotometry with an Analyst Perkin Elmer

230 Servia et al.

photometer. Prior to analysis, sediment sampleswere dried, milled and mineralized (with 2.5 mlnitric acid and 7.5 ml hydrochloric acid per 500 mgsediment during 12 h at room temperature, thenduring 2 h in a boiling water bath).

Larvae exposure – Midge (C. riparius) larvae,from our laboratory culture, were exposed tosediment-spiked copper as described in Pery et al.(2003b). At the beginning of the test, 20 four-days-old C. riparius larvae were put into each beaker,and three replicates were allocated to each treat-ment. Four-days-old larvae were used preferablybecause copper is known to be much more toxicfor younger larvae (Pery et al., 2003a). The larvaewere fed with 0.6 mg of powdered Tetramin�

(Tetrawerke, Melle, Germany) fish food per larvaper day (ad libitum conditions).

Previous work on larval growth modeling (Peryet al., 2002) showed that C. riparius larvae grownin the same laboratory conditions present an initialperiod of somatic growth (from hatching to10 days after hatching) and then a period of ga-metal investment (from 10 days after hatching tillpupation), where larvae increase in weight but notin length, and accumulate reserves for reproduc-tion. As a consequence, exposures were stopped atdays 7 and 9 after the beginning of the test (i.e. 11and 13 days after hatching). These two periods ofexposure were chosen due to the different rates ofdevelopment of male and female larvae (Peryet al., 2002, 2003b), and because detection ofdepletion in energetic reserves by those timesmight reveal impairment in the reproductive out-put. For each copper concentration and samplingdate, the larvae from three replicated beakers wereused.

Copper concentration was measured in larvaeexposed during Experiment 1. Ten larvae weretaken randomly in each replicate for nominal con-centrations of 12.5 and 25 mg/kg so that the resultscould be compared with those obtained by Peryet al. (2003b). Prior to analysis, the larvae weremaintained for 10 min at room temperature in a100 ml solution of 3�10)3 M EDTA to removecopper from the surface of the organisms. Theywere then dried in an incubator, weighed individ-ually, and mineralized using 1 ml nitric acid per10 mg organism during 1 h at room temperature,

followed by a slight heating (ca. 60 �C) during 1 h.Ultra-pure water (1 ml) and nitric acid (0.5 ml)were then added before heating at 60 �C during halfan hour. This procedure was repeated until com-plete mineralization. Copper concentration wasmeasured using atomic absorption photometry, asfor sediment samples.

Endpoints

Growth and development stage – The developmentof the imaginal discs and wet weight were usedhere as growth endpoints. The state of develop-ment of the imaginal discs can be followed allthroughout the fourth larval instar (Wulker andGotz, 1968; Ineichen et al., 1983; Hahn andSchulz, 2002). As the levels of energetic com-pounds greatly vary throughout the fourth larvalinstar (M.J. Servia, personal observation), the useof imaginal disc development can be used to knowwhether the larvae have reached a certain age withthe same amount of reserves, even if copper in-creases the time to attain that age. In addition,observation of the imaginal discs allows determi-nation of larval sex, which can be used for inde-pendent analysis of the effects of copper ondevelopment and energetic biomarkers for malesand females.

Levels of energy-yielding substrates – Only lar-vae belonging to the development phases the mostfrequently found in the beakers (66% of the re-trieved individuals) were selected for dissection,discarding the extreme cases (i.e. undevelopedlarvae) that appeared at the highest copper con-centrations. The larvae were bled to collect he-molymph, and eviscerated to eliminate the gutwith its content. The levels of energy-yieldingcompounds (sugars and lipids) were measured inboth the hemolymph and eviscerated body. Forsugars, glycogen concentration was measured inthe eviscerated body, and the levels of glucose andtrehalose were followed in the hemolymph,whereas for lipids, the amounts of glycerol andtriglycerides were measured in both tissues. Ener-getic compounds were measured spectrophoto-metrically, according to Van Handel (1965) andMasumura et al. (2000) for sugars, and to Weberet al. (2003) for glycerides.

Copper-induced Changes in Energetics in Chironomus riparius Larvae 231

Statistical analysis

Chi-square tests were used to check for differencesin mortality both among replicates and amonglevels of exposure. Data on instar developmentwere analyzed using GLM techniques (Crawley,1993), using sex, copper exposure and interactionbetween both factors as explanatory variables. Foreach duration of exposure, data from Experiments1 and 2 were grouped before analysis. Thresholdvalue for significant differences between concen-trations was fixed at 0.0125 (Bonferroni correctionfor multiple comparisons). GLM analysis wereperformed using R 1.8.1. software package.

Larval weights and the levels of sugars andlipids (glycerol and triglycerides) were comparedusing two-way ANOVA with ‘‘Phase’’ and ‘‘Cop-per concentration’’ as the explanatory factors foreach sex in each experiment. In those cases whereeither ‘‘Copper concentration’’ or the interactionbetween the two factors were significant, one-wayANOVA (factor: ‘‘Copper concentration’’) fol-lowed by a Tukey HSD post-hoc test was per-formed for each phase, in order to detectdifferences among levels of exposure. For lipids, inthose cases where only two replicated measure-ments were performed due to the low amount oftissue and/or hemolymph available, statistical sig-nificance of the trends was also assessed by fittingthe data with a linear description, then testing ifthe slope of the description curve was significantlydifferent from 0 (Spiegel, 1975).

All the analyses were performed separately forthe two durations of exposure (7 and 9 days), ex-cept for testing the differences in phase and inweight for the different groups of larvae.

Results

Copper concentration in sediment and larvae

In Experiment 1, measurements of the levels ofcopper in the sediments showed a spiking efficiencyof 97±4% for all the concentrations with threesamples per concentration. For C. riparius larvae,body copper residues were 218±25 and402±53 mg/kg dry weight (n=3) for nominalconcentrations of 12.5 and 25 mg/kg, respectively.These results are in accordance with those

obtained in the experiment performed by Pery et al.(2003b) where the internal copper concentration inC. riparius larvae were 199±40 and 430±58 mg/kg for exposure concentrations of 12.5 and 25 mg/kg, respectively. The spiking efficiency and internalcopper concentration in the larvae were not mea-sured in Experiment 2, but it was assumed they didnot greatly vary since the spiking procedure andexposure conditions were identical to those used inExperiment 1 and in Pery et al. (2003b).

Larval development

Average larval survival in the controls was79.58±2.99% in Experiment 1 (n = 3) and92.92±2.18% in Experiment 2 (n=3). No signifi-cant differences in mortality were found among thecontrol replicates (v2, p>0.05). As expected, in-crease in the concentration of copper resulted in anincrease in mortality (v2-test; v2Exp-1=151.011,p<0.001; v2Exp-2=286.532, p<0.001) (Fig. 1).

After 7 days of exposure, only sex had a sta-tistically significant effect on the development oflarvae (p<0.0001). Treatment and the interactionbetween both factors were not significant(p=0.074 and p=0.708, respectively). After 9 daysof exposure, sex and copper had a statisticallysignificant effect on the development of larvae(p<0.0001 for both factors). The interaction be-tween both factors was not significant (p=0.58).Among copper concentrations, 25 and 50 mg/kgwere statistically different from controls (p=0.002and p=0.0002, respectively). The two lower con-centrations had no statistically significant effectson larval development (Fig. 2a–d). Some larvaesuffered serious developmental arrest, and re-mained as third instars or in phase 1 of the fourthinstar, even after 9 days of exposure, as shown forthe larvae submitted to the highest copper con-centrations (Table 1).

Note that in Experiment 1, control larvae,especially males, remained slightly younger thanlarvae exposed to the lowest concentrations ofcopper (Fig. 2a and b). This might be due to aproblem of aeration in some beakers that wasdetected and solved on the first day of the exper-iment. Even if the mean development phasedecreased with increasing amount of copper in thesediment, in most cases, the weight of the larvaebelonging to the same phase and sex did not

232 Servia et al.

significantly vary among treatments. Only in somecases (females in phases 3 and 4 in Experiment 1,females in phase 4 and males in phase 8 inExperiment 2), ANOVA showed that larvae ex-posed to certain copper concentrations presentedsignificantly lower weights than larvae exposed tothe other copper levels. However, those differenceswere limited (1–2% larval dry weight) and did notshow any clear tendency.

Concentration of energy-yielding substrates

Whether they were measured in the hemolymph oreviscerated body, the levels of sugars did notgreatly vary among treatments (Fig. 3). In malelarvae, body glycogen concentration did not differamong treatments, for both 7-day and 9-dayexposures (ANOVA; p=0.243 and p=0.619,respectively) (Fig. 3a and d). In female larvae,

glycogen level significantly varied only for the 9-day exposure, where larvae exposed to 6.5 and25 mg/kg copper showed significantly higher val-ues as compared to control larvae (Tukey HSDpost-hoc test; p=0.004 and p=0.010, respectively).In the hemolymph, the levels of free glucose andtrehalose did not significantly vary among treat-ments (Fig. 3b, c, e and f).

Compared to sugars, lipid concentrations inC. riparius larvae exposed to copper showedslightly higher variations among treatments(Figs. 4 and 5). In peripherical body layers, thelevel of glycerol increased after 7 days of exposure(Fig. 4a) whereas it remained stable after 9 days ofexposure (Fig. 4c). In both male and female larvaeexposed for 7 days, the slope of the curves wassignificantly different from zero (p=0.032 andp=0.006, respectively), due to the high levels ofglycerol measured for the 50 mg/kg exposure

0

10

20

30

40

50

60

70

Contro l 6.5 12. 5 25 50

Copper concentration (mg/kg)

Larv

al m

orta

lity

(%)

0

10

20

30

40

50

60

70

Control 6.5 12.5 25 50La

rval

mor

talit

y (%

)

(a)

(b)

Figure 1. Mortality of C. riparius larvae for each copper concentration in the sediment. Data are presented as means±SE (error

bars; n=3) for Experiments 1 (a) and 2 (b).


concentration (Fig. 4a). However, ANOVA indi-cated that the differences between control andexposed larvae can be considered as significant forfemales (p=0.052) but not for males (p=0.096).After 9 days of exposure, no significant differences

were observed in body glycerol levels in male andfemale larvae (ANOVA, p=0.590 and p=0.157,respectively). The levels of triglycerides measuredin the eviscerated body decreased for the 7-dayexposure period (Fig. 4b) but not for the 9-day

Table 1. Mean percentages of third instar and young fourth instar (phase 1) C. riparius larvae at the end of each experiment after

7 and 9 days of exposure

Treatment

7-day exposure 9-day exposure

3rd instar 4th instar (phase 1) 3rd instar 4th instar (phase 1)

Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2

Control 0 0 2.50 0 0 0 0 0

(1.44)

Copper exposure (mg/kg)

6.5 0 1.67 0 1.67 0 0.83 0 0

(1.67) (0.83) (0.83)

12.5 0 2.50 0 4.17 0 0 0 0

(1.44) (2.20)

25 0 10.00 1.67 3.33 0 2.50 0 1.67

(1.44) (1.67) (2.20) (1.44) (0.83)

50 5.83 20.83 3.33 3.33 1.67 0.83 1.67 3.33

(0.83) (4.17) (1.67) (0.83) (0.83) (0.83) (0.83) (2.20)

Standard errors to the means are given in brackets.

Figure 2. Development phase of C. riparius larvae exposed during 7 and 9 days (a, b and c, d, respectively) to each copper concen-

tration in Experiments 1 (a and c) and 2 (b and d). Data are presented as means±SE (error bars; n=30) for males (black bars)

and females (white bars). For each experiment and duration of exposure, ANOVA indicated significant differences between male

and female larvae (p<0.0001). Significant differences between control and exposed larvae, for both sexes: **p=0.002;

***p=0.0002 (Tukey HSD post-hoc test).

234 Servia et al.

exposure period (Fig. 4d). Whatever the durationof exposure, no significant differences were de-tected for the slope of the curves, nor for thecomparison between control and exposed larvae.

In the hemolymph, the levels of free glycerolslightly decreased (Fig. 5a) or remained stable(Fig. 5c) when the concentration in copperincreased. The steepest slope was obtained for malelarvae after 7 days of exposure, but it was not sig-nificantly different from zero (p=0.063). ANOVAdid not detect any significant differences in hemol-ymph glycerol levels between exposed and controllarvae. Triglyceride levels in the hemolymph greatly

varied in both male and female larvae after 9 daysof exposure to copper (Fig. 5d), but differencesbetween control and exposed larvae were onlysignificant in females (ANOVA, p=0.024), due tothe highest value recorded for the 6.5 mg/kgexposure concentration (Tukey HSD post-hoc test;p=0.036).

Discussion

Copper is well known to affect the larval devel-opment of chironomids (Nebeker et al., 1984;

Figure 3. Changes in the levels of glycogen in the eviscerated body (a, d), and of glucose (b, e) and trehalose (c, f) in the hemol-

ymph in C. riparius larvae exposed during 7 and 9 days (a–c and d–f, respectively) to increasing copper concentrations in the sedi-

ment. Data are presented as means±SE (error bars; n=3) for males (d) and females (s). Significant differences between control

and exposed larvae, for both sexes: *p=0.01; **p=0.004 (Tukey HSD post-hoc test).


Kosalwat and Knight, 1987; Timmermans et al.,1992; Besser et al., 1995; Watts and Pascoe, 1996;Pery et al., 2003b; De Haas et al., 2004). InC. riparius exposed to this metal, growth reductionhave been reported for the entire larval period(Pery et al., 2003b; De Haas et al., 2004). Ourapproach, based on the determination of devel-opment phases in the fourth instar, showed that infact exposed larvae need more time to pass fromone phase to the next, as compared to controllarvae (Fig. 2). For high copper exposure con-centrations, such a growth delay combined withtoxic effects led to enhanced larval mortality(Fig. 1). Besides the delay in growth, our workshowed that copper caused a desynchronizeddevelopment in the population, which can be ob-served by the increase in the number of larvae thatremained in the third instar or phase 1 of thefourth instar, as well as by the important reductionin age of males, which was abnormally close tothat of females. The sexual difference in larval

growth rate (Fischer and Rosin, 1969; Pery et al.,2002, 2003b) usually results in protandry (i.e. thephenomenon whereby males emerge slightly beforefemales), which may assist in outbreeding and/orin enhancing mating success (Armitage, 1995).Reduction in the difference in age between malesand females due to the exposure of the larvalpopulation to copper may therefore impairreproduction.

Copper-induced changes in C. riparius larvaldevelopment reported here suggest that less energywas available for individual growth. Reduced foodintake may be one reason for the decrease inphysiological energy. Indeed, it has been shownthat C. riparius larvae suffer delays in growth whenfood is restricted (e.g., Ristola et al., 1999; Peryet al., 2002). Copper is known to induce mor-phological deformities of the mentum and man-dibles in chironomids (Janssens de Bisthovenet al., 1992, 1998; Martinez et al., 2003). There-fore, it cannot be excluded that mouthpart

0

0.2

0.4

0.6

0.8

1

Gly

cero

l (µg

/mg

tissu

e)

0

0.2

0.4

0.6

0.8

1

0

4

8

12

16

0

4

8

12

16

Trig

lyce

rides

(µg

/mg

tissu

e)


6.5 12.5 25 50Control


6.5 12.5 25 50Control


(a) (c)

(b) (d)

Figure 4. Changes in the levels of glycerol (a, c) and triglycerides (b, d) in the eviscerated body of C. riparius larvae exposed dur-

ing 7 and 9 days (a, b and c, d, respectively) to increasing copper concentrations in the sediment. Data are presented as

means±SE (error bars; n=3) for males (d) and females (s). For glycerol levels, ANOVA indicated differences between control

and exposed female larvae were at the significance threshold (p=0.052).

236 Servia et al.

abnormalities may have reduced the feeding abilityof C. riparius larvae exposed to copper in thepresent study. Another explanatory hypothesis forreduced energy availability for growth in thelarvae exposed to copper refers to biochemicalmechanisms set up to counteract effects of thetoxicant, e.g. metal-induced production of metal-lothionein-like proteins (Gillis et al., 2002; Sarkaret al., 2004) and heat-shock proteins (Karouna-Reinier and Zehr, 2003). Increased energy demandmay thus result from such an energy reallocation,so that enhanced mobilization of energy reservesmay occur. In such circumstances, the levels ofstorage and circulating forms of sugars and lipids,the two main sources of metabolic energy in in-sects, may vary according to the stress intensity.

Changes in the concentrations of sugars andlipids have been observed when insects are exposedto metals. For sugars, the most common finding isthat fat body glycogen reserves are reduced, whilelevels of sugars in the hemolymph might be either

reduced, or increased (Bischof, 1995; Ortel, 1996;Choi et al., 2001, and references therein). In ourcase, levels of sugars remained almost constantamong levels of exposure. The only significantvariations were observed in females exposed to 6.5and 25 mg/kg copper during 9 days of exposure,where abnormally high levels of glycogen weremeasured (Fig. 3d). Altogether, these results sug-gest that C. riparius larvae need to accumulate acertain amount of sugar reserves before pupation.In chironomids, larvae represent the only feedingstage. The status of sugar and lipid reserves duringthe larval stage may therefore have consequenceson critical events of the life cycle such as pupationand metamorphosis. In particular, glycogen isconsidered as the main source of energy duringmetamorphosis and non-feeding adult life stage(Hamburger et al., 1996). De Haas et al. (2004)demonstrated at low copper concentration( £50 mg/kg), the influence of food quantity wasdominant, and until copper concentration reached

0

10

20

30

40

50

60

Fre

e gl

ycer

ol (

µg/m

l)

0

10

20

30

40

50

60

-50

50

150

250

350

450

Hem

olym

ph tr

igly

cerid

es (

µg/m

l)

-50

50

150

250

350

450

n.d. n.d. n.d.

n.d.

(a) (c)

(b) (d)



6.5 12.5 25 50Control


6.5 12.5 25 50Control

*

Figure 5. Changes in the levels of free glycerol (a, c) and triglycerides (b, d) in the hemolymph of C. riparius larvae exposed during

7 and 9 days (a, b and c, d, respectively) to increasing copper concentrations in the sediment. Data are presented as means±SE

(error bars; n=3) for males (d) and females (s). n.d.: not detected for males, females, or both. For hemolymph triglyceride levels,

ANOVA indicated significant differences between control and exposed female larvae (p=0.024). Significant differences between

control and exposed female larvae: *p=0.036 (Tukey HSD post-hoc test).


a critical threshold value of 200 mg/kg, larvaewere able to compensate for effects of the toxicantwhen the amount of food available increased(maximum food supply: 0.5 mg/larva/day). This isin accordance with our results; as larvae were fedad libitum (0.6 mg/larva/day), they were able tofind enough food to compensate for the energyexpenditure devoted to counteract the effects ofcopper, so that the level of sugars remained atlevels compatible with further requirements fordevelopment.

Conversely, even if differences among the levelsof lipids between exposed and control C. ripariuslarvae were not always statistically significant, it isworthy to note that, in both 7-day and 9-dayexposures, females showed reductions of triglyce-ride levels from control to the 50 mg/kg exposureconcentration (ca. 50% and 30% reduction,respectively). These figures are in accordance withthe reduction in adult female fecundity (expressedas the number of eggs per egg mass) found byDucrot et al. (2004) after exposure of larvae tocopper, thus suggesting a possible link betweenboth endpoints. Interestingly, as female body tri-glycerides were reduced, body free glycerol in-creased when exposure concentration of copperincreased (107% and 33% for 7-day and 9-dayexposure, respectively). Glycerol plays an impor-tant role in multiple biochemical pathways, andsignificant variations in its level might be related tochanges in body triglycerides. However, careshould be taken when trying to relate directly tri-glyceride decrease and glycerol increase, sinceglycerol participates in many biosynthetic andcatabolic pathways, and it can be originated also,for example, from degradation of glycogen (Storey,1997; Van der Horst et al., 1997; Goto et al., 2001).

In summary, our study provides a first clue forthe comprehension of the energetic costs of metalexposure in C. riparius larvae and of their links toother life cycle endpoints like growth or repro-duction (Pery et al., 2003b). It also confirms thatselection of the larvae for both sex and age im-proves the relevance of energy-yielding substratesas indicators of adverse physiological effects ofcopper, females being more responsive than malesto copper exposure. However, selection of larvaeof approximately the same age also implied theelimination from the analyses of the individualsmost affected by copper exposure, such as those

that remained as third instars or in the initialphases of the fourth instar. Indeed, as all the larvaewere killed when experiments were stopped, we donot know whether those small larvae would haveshown more drastic reductions of their energeticreserves if close to pupation. Complementarystudies would help to clarify those aspects.

Acknowledgments

This research was supported by the French Min-istry of Ecology and Sustainable Development(MEDD), through the ‘‘National Programme ofEcotoxicology’’ (PNETOX). M.J. Servia wasgranted by a MEDD–INRA–CEMAGREF post-doctoral joint fellowship. The authors thank thetechnical staffs of the Experimental Unit ofAquatic Ecology and Ecotoxicology (INRA,Rennes, France) and of the Laboratory ofEcotoxicology (CEMAGREF, Lyon, France) fortechnical support. They also thank Wako Chemi-cals GmbH (Neuss, Germany) for their generousgift of the glucose reagent kit. They are gratefulto Th. Caquet for computing data statistical anal-ysis and for valuable and stimulating discussions.

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effects of copper on energy metabolism and larval development in the midge chironomus riparius

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