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Journal of Insect Physiology 52 (2006) 897–904

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Importance of sterols acquired through host feeding in synovigenicparasitoid oogenesis

Nathalie Mondya,�, Marie-France Corio-Costetb, Aurelie Bodina, Nicole Mandona,Fabrice Vanniera, Jean-Paul Mongea

aUniversite Franc-ois-Rabelais, IRBI UMR CNRS 6035, Parc Grandmont, F-37200 Tours, FrancebUMR 1065 Sante vegetale (INRA-ENITA), INRA-Bordeaux, BP 81, F-33883 Villenave d’Ornon, France

Received 9 November 2005; received in revised form 16 March 2006; accepted 20 March 2006

Abstract

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a host feeding ectoparasitoid of fourth-instar larvae or pupae of Callosobruchus

maculatus (Coleoptera; Bruchidae) infecting Vigna unguiculata seed and pods (Fabacae). Parasitoid females are synovigenic, i.e. they are

born with immature eggs and need to feed from the host in order to sustain egg production. In this study, the role of sterols obtained

through host feeding in parasitoid oogenesis are examined. Quantitative and qualitative analyses of the sterol contents in each partner of

the tritrophic interaction show that a parasitoid female’s larval sterol contents is sufficient to produce only 30% of the total number of

eggs laid throughout a female’s life cycle. In a second step, by manipulating the composition of the sterols hemolymph in the host, it is

shown that cholesterol obtained through adult nutrition plays a crucial role in the eggs viability but does not affect the egg production

quantitatively. This result has important implications for understanding both the nutrient allocation strategy in this species and the

impact of cholesterol in parasitoid reproduction.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Sterols; Steroids; Host feeding; Parasitoid; Eggs viability

1. Introduction

The nature and the pattern of nutritional resourcesallocation to reproduction have critical consequences onthe fitness of organisms (e.g. Roff, 1992; O’Brien et al.,2000; Jervis et al., 2001) and strongly influence reproduc-tive and foraging strategies (e.g. Boggs, 1992; Mangel andHeimpel, 1998; Fischer et al., 2004). Resources allocated tobreeding may be acquired from stored reserves or currentfeeding (Wheeler, 1996; Tammaru and Haukioja, 1996).Income breeder species acquire and expend resourcessimultaneously, as opposed to capital breeder species thatgather resources over a long period and then expend themin a brief reproductive phase. Nonetheless, organisms

e front matter r 2006 Elsevier Ltd. All rights reserved.

sphys.2006.03.007

ing author. Current address: UMR CNRS 5023 Ecologie

es fluviaux, Universite Claude Bernard Lyon 1, 43 Bd du

918, F-69622 Villeurbanne Cedex, France.

3 28 93; fax: +33 4 72 43 11 41.

ess: nathalie.mondy@univ-lyon1.fr (N. Mondy).

rarely are pure capital or income breeders; but rather theydraw varying degrees of nutrients from reserves andcurrent feeding (Boggs, 1997a, b; Truman and Riddiford,1999; O’Brien et al., 2004, Casas et al., 2005). Synovigenicparasitoids offer an ideal opportunity to study how insectsmanage their larval and adult resources (Flanders, 1950;Jervis et al., 2005). These parasitic wasps mature eggsduring their entire adult lifetime. Females lay their eggs inor on the bodies of their hosts and feed off them as theydevelop. The host is also the main source of nutrients forthe foraging female (Jervis and Kidd, 1986; Heimpel andCollier, 1996). Many studies have focused on the cost andbenefits of host feeding in parasitoids (e.g. Jervis and Kidd,1986; Collier, 1995; Rivero and West, 2005). Significantpercentages of sugars and proteins which serve to meet thehigh amino acid demands associated with egg productionare obtained through host feeding by the adult female(Rivero et al., 2001; Giron et al., 2002). A recent study onlifetime nutrient dynamics reveals that Eupelmus vuilleti, a

ARTICLE IN PRESSN. Mondy et al. / Journal of Insect Physiology 52 (2006) 897–904898

synovigenic ectoparasitoid, could show different nutrimentmanagement strategies for carbohydrates, proteins, andlipids (Casas et al., 2005).

Sterols are key elements in insect nutritional ecology.Indeed, unlike most plants and animals, insects lack thecapacity to synthesise de novo the steroid nucleus. Theydepend on exogenous sources of sterols for normal growth,development and reproduction (Clark and Bloch, 1959).Sterols are required as membrane components, as pre-cursors to important steroid hormones and as a signallingmolecule bound to the hedgehog group of proteinsaffecting developmental processes (review in Behmer andNes, 2003). The requirement for sterols in Hymenopteranspecies has been particularly studied in phytophagousspecies, like bees and leaf-cutting ants (e.g. Svoboda andLusby, 1986; Svoboda et al., 1986; Maurer et al., 1992;Feldlaufer et al., 1993; Feldlaufer and Hartfelder, 1997).To our knowledge, the only reports regarding the sterol ofparasitoids involved Brachymeria lasus, Pachycrepoideus

vindemiae and Microplitis demolitor (Thompson, 1981;Ritter and Johnson, 1991) and dealt with the sterolrequirement during larval growth. Because synovigenicparasitoids must feed as adults to reproduce, sterols gainedthrough host feeding may play an important role in theirreproduction. Generally, cholesterol is the major sterolfound in insects and is found accumulated in eggs (e.g.Costet et al., 1987; Behmer et al., 1999; Jouni et al., 2002).The particular importance of cholesterol to developingembryos was demonstrated by Costet et al. (1987) on newlymoulted adult female Locusta migratoria. Even if choles-terol plays a major role in growth and membraneformation, the significance of cholesterol in eggs may alsorelate to the fact that it serves as a precursor to numerousecdysteroids which regulate a number of critical processesthroughout embryonic development (Lanot et al., 1988;Behmer and Nes, 2003).

Eupelmus vuilleti (Hymenoptera; Eupelmidae), a solitaryparasitoid producing yolk-rich eggs during its imaginalstage, seems to be very appropriate to explore whatstrategy—capital or income breeder—is adopted by asynovigenic parasitoid for its acquisition of sterols. Indeed,the nutritional ecology of this species is well understood(Rivero and Casas, 1999; Rivero et al., 2001; Giron et al.,2002; Giron and Casas, 2003; Casas et al., 2005). Thefemales oviposit on the third to fourth larval instars ofCallosobruchus maculatus (Coleoptera; Bruchidae) whichdevelop within pods and seeds of Vigna unguiculata

(Fabacae). During host feeding, parasitoid females feedfrom the host by puncturing its cuticle and creating afeeding tube with secretion from their ovipositor. Thefemales, then turn and use the feeding tube to extract thehost hemolyph with their mouthparts (Giron et al., 2002).In order to advance knowledge about the significance ofsterols gained from host feeding in parasitoid reproduction,we measured quantitatively and qualitatively the sterolcomposition of the plant, the phytophagous insect and itsparasitoid. In a second step, experimental modification of

the sterol composition of the phytophagous insect’shemolymph, allowed us to test the biological significanceof sterol acquired by the parasitoid females through hostfeeding (female fecundity, egg viability, and ecdysteroidcontent in eggs).

2. Materials and methods

2.1. Breeding conditions

C. maculatus and E. vuilleti were bred in the laboratoryaccording to the methodology described by Jaloux et al.(2004). All experimental procedures were carried out in abreeding room with a temperature cycle of 35: 25 1C at 12:12 hL: D photoperiod, and a constant 70% r.h. Innutritional experiments, fourth-instar larvae of C. macula-

tus provided to female wasps had been previously extractedfrom the V. unguiculata seeds and placed inside a gelatinecapsule with holes on the top following Gauthier andMonge (1999). This system does not alter the naturalbehaviour of females or their life expectancy and facilitatesthe recording of the number and the collecting of laid eggs.

2.2. Sterol composition of the plant, the phytophagous and

the parasitoid

Sterol analyses were performed on 5 g of crushed V.

unguiculata seed from the Californian Black Eyes variety(about 25 seeds). C. maculatus analyses were performedboth on a pool of 60 fourth-instar larvae and on 30 ml ofhemolymph collected with a graduated micro-capillaryconnected to a manual pump. Analyses of sterols ofE. vuilleti were carried out on 70 males and 70 femalescollected at emergence and on a sample of 200 eggs. Allthese analyses were performed in triplicates.Each sample was freeze dried and ground in an ultra-

turrax homogenizer in the presence of dichloromethane–methanol (2:1, v:v). After the solvent evaporation, theextract was saponified using KOH (6%, w:v) in methanol.This step was omitted when steryl esters were recovered.The following steps were similar to those described byCorio-Costet et al. (1989). The unsaponifiable material wasextracted three times with hexane, and pooled extracts weredried. The residue was chromatographied on Merck HF254 TLC plates with dichloromethane as the developingsolvent. The free 4-desmethylsterols bands and the totalesterified sterols band were scraped off and each type waseluted in dichloromethane. The esterified sterols weresaponified using KOH and extracted as described above.This procedure led to the isolation of free and esterified 4-desmethyl sterols. After acetylation, samples containing aninternal standard of cholesterol were analysed by GC(Quartz capillary column, 30m� 0.25mm) coated withOV-1 (J&W Scientific, Folsom, CA).Because of the small sample number and to avoid the

overuse of significance tests, only comparisons between thecholesterol concentration of C. maculatus larvae, E. vuilleti

ARTICLE IN PRESSN. Mondy et al. / Journal of Insect Physiology 52 (2006) 897–904 899

males and females were made. A Kruskal–Wallis testfollowed by a Mann–Whitney U test for post hoccomparisons was used in a procedure adapted for verysmall sample sizes (Scherrer, 1984).

2.3. Effects of nutritional cholesterol on the parasitoid

oogenesis

2.3.1. Diet preparation

The sterol composition of C. maculatus hemolymph wasmanipulated in order to study the effects of host feeding onparasitoid oogenesis. Because of the hydrophobic characterof sterol, sterols purchased from Sigma were made solublein oil (5mg of sterol in 1ml of oil). Colza oil was chosenbecause it contained only 0.17% phytosterols, mainlysitosterol and campesterol. Together, 2 ml of an emulsion(1:1, v:v) of water/colza oil containing additional choles-terol, stigmasterol or sitosterol was injected into C.

maculatus larvae. Water/colza oil without added sterolswas used as a control. On average, C. maculatus larvaeused for injection contained 270.17 ml of hemolymph. In2 ml of each emulsion injected in C. maculatus, the quantityof sterols was 1.62 and 6.62 mg of total sterols in water/oiland in water/oil plus sterols, respectively. The total sterolsand cholesterol concentrations of the host hemolymphafter the injection are noted in Table 1. Injections wereperformed with a graduated micro-capillary connected to amanual pump under a microscope. The parasitoid wasprovided with a live treated host 1 h after injection. In apreliminary experiment, it was verified that the sting of theinjection did not modify the reproductive physiology of thewasp females.

2.3.2. Experimental set-up

Newly emerged parasitoid females were isolated for 10 hduring which time their reproductive system matured. Theywere then mated and used for experiment. In order todetermine the importance of sterols acquired through hostfeeding in the wasp’s reproduction, we used C. maculatus

larvae without injection (treatment NI), C. maculatus

injected with an emulsion of oil and water (treatment Oi)

Table 1

Total sterols and cholesterol concentrations of the C. maculatus

hemolymph (mg/ml) after the different injections

C. maculatus Total sterols Cholesterol

mg/ml of host hemolymph

NI: without injection 0.4 0.33

Oi: injection of oil/water 0.6 0.16

Ch: injection of oil/water plus

cholesterol

1.85 1.45

Si/St: injection of oil/water plus

sitosterol/stigmasterol

1.85 0.16

Before injection, C. maculatus larvae contained 2ml (70.17) of hemo-

lymph.

and C. maculatus injected with oil and water plusadditional cholesterol, stigmasterol or sitosterol (treat-ments Ch, St, Si respectively). Comparisons between (NI)and (Oi) allowed us to test the injection effect. Compar-isons between (Oi) and (Ch, Si, St) allowed us to test theeffects of a diet diluted in cholesterol compared to a dietenriched in different sterols.In all treatments, 10 female parasitoids were kept

individually in a Petri dish containing five C. maculatus

which was renewed daily over the entire lifetime of thewasp. Each day, the number of eggs laid by the female wascounted and the presence of host feeding tubes wascontrolled to check that the parasitoids had fed. All theeggs laid by the 10 females of a treatment were pooled and,simultaneously, 30 eggs were kept in controlled conditionto record their viability and 60 eggs were collected atrandom to measure their ecdysteroid titre.Means values of the duration of female life and fecundity

were compared using a parametric one-way ANOVA. Posthoc comparisons for all treatments were performed using amultiple comparison test based on student’s t statistic witha Bonferoni correction. Comparison between the hatchingrates was made using a w2 test. Computations were carriedout using SYSTAT 9/PC software (2000).

2.3.3. Ecdysteroid analyses

Because of the ecdysteroid EIA detection limit, the 60eggs collected for this analysis were pooled in batches of 20.Thus, all points of measure were repeated three times.Immunoassays (EIA) used for the determination ofecdysteroids employed a peroxidase conjugate of 20E asa tracer (De Reggi et al., 1992). The L2 polyclonalantibody (gift from Dr. Delbecque, University of Bor-deaux, France) was very sensitive to ecdysone, 2-deoxy-ecdysone and 3-dehydro- ecdysone, but ca. six times lesssensitive to 20-hydroxy-ecdysone. Eggs were homogenizedby sonication in 100% methanol and stored at �18 1C untilassayed. After centrifugation (10 000g, 10min), super-natants were dried in a Speed-Vac apparatus andsuspended in 0.1M phosphate buffer (pH 7.4). Eachsample analysed was measured at least in duplicate.Because this ligand was used for reference curves,ecdysteroid titres are expressed in ecdysone equivalents.The data were analysed with a Kruskal–Wallis H test

(a ¼ 0:05) (SYSTAT).

3. Results

3.1. Sterol composition of plants, phytophagous insects and

parasitoids

Identification and measurement of free and esterifiedsterols of the plant, the phytophagous insect and itsparasitoid are shown in Table 2. The predominant sterolin V. unguiculata seed was stigmasterol (D5 sterol), account-ing for 48.7% of the total sterols. In addition, 21.5% ofsitostanol (D0 sterol) was also detected. Other 24-alkylsterols

ARTICLE IN PRESS

Table 2

Relative percentage of major sterols (quantity of major sterols in mg/mg of dry weight) isolated from V. unguiculata seed, fourth-instar larvae and

hemolymph of C. maculatus and newly emerged females, males and eggs of E. vuilleti

Sterolsa,b V. unguiculata C. maculatus E. vuilleti

Seed Larvae Hemolymph Males Females Eggs

Cholesterol tr 83.9 (0.76) 83.4 (0.33) 80.6 (0.87) 95.6 (1.34) 78 (11.7)

7-dehydrocholesterol 0.4 (1.5) — — tr — —

Campesterol 10.2 (39.1) 2.9 (0.02) 2.9 (0.01) 2.8 (0.03) tr 3.9 (0.53)

Stigmasterol 48.7 (185.4) 6.1 (0.06) 7.2 (0.03) 4.2 (0.05) 1.3 (0.2) 11.6 (1.88)

Sitosterol 11.4 (44.7) 5.4 (0.05) 6.5 (0.03) 9.4 (0.11) 1.3 (0.2) 6.5 (0.95)

Sitostanol 21.5 (82.3) 1.1 (0.01) — tr — —

Others 7.8 (28.6) tr — 1.7 (0.2) 1.7 (0.3) —

Total sterols

mg/mg of dry weight 381.3 0.9 0.4c 1.1 1.4 15

mg/insect (SE) — 4.14 (0.07) — 0.36 (0.1) 1.23 (0.1) 66 (3.2)d

Free sterols (%) 99.5 92.9 96.1 99.9 99.9 100

Esterified sterols (%) 0.5 7.1 3.4 0.1 0.1 —

SEM do not exceed 20%.aEsterified sterols and other minor sterols were not listed to facilitate comparison.bCholesterol [5a-cholesta-5-en-3b-ol], 7-dehydrocholesterol [5a-cholesta-5,7-dien-3b-ol], campesterol [24-methyl-5a-cholesta-5-en-3b-ol], stigmasterol

[24-ethylcholesta-5,22-dien-3b-l], sitosterol [24-ethylcholesta-5-en-3b-ol], sitostanol [24-ethylcholestan-3b-ol].cExpressed in mg/ml of hemolymph.dExpressed in ng/egg.

N. Mondy et al. / Journal of Insect Physiology 52 (2006) 897–904900

such as campesterol and sitosterol (respectively, 10.2% and11.4%) represented the bulk of the remaining sterols. Tracesof cholesterol (C27 sterol) were found. Only 0.5% of thetotal sterols were esterified.

Both larvae and hemolymph of C. maculatus had asimilar sterol profile composed of mainly cholesterol(83.9% and 83.4%, respectively). The remaining sterolsconsisted of a small percentage of seed phytosterols(C24-methyl and ethyl sterols). Each accounted for1.1–7.2% of the total sterols. In addition, a small quantityof sitostanol was present in the larvae. Esterified sterolswere higher in larva than in its hemolymph (7.1% vs.3.4%) and consisted of equal quantities of cholesterol,stigmasterol and sitosterol (data not shown). On average, aC. maculatus larva contained 4.14 mg70.07 total sterolsand the concentration of total sterols in its hemolymph was0.4 mg/ml.

In both female and male E. vuilleti parasitoids,cholesterol was also the main sterol representing 95.6%and 80.6%, respectively, of the total sterols. Surprisingly,males contained more than 16% phytosterols, especiallysitosterol, whereas there were very few remaining phytos-terols in females (less than 3% of the total sterols).Differences among the cholesterol concentrations (inmg/mg of dried weight) of C. maculatus larvae, E. vuilleti

males and females were found to be statistically significant(Kruskall–Wallis test, Hc ¼ 7:2, P ¼ 0:05). Post hoccomparisons showed that differences observed bothbetween host larvae and male or female wasps, andbetween male and female wasps were statistically signifi-cant (U ¼ 0; P ¼ 0:05). Thus, even if the sterol profiles ofmales and females were reflective of the sterol contents of

their hosts, female wasps would accumulate qualitativelymore cholesterol (+15%) than male wasps.The sterol profile of parasitoid eggs was more complex

and quite different from those of males and females. Theeggs had relatively less cholesterol (78%) but morestigmasterol (11.6%). A total of 66 ng73.2 of sterols werefound per egg. In males, females and eggs, esterified sterolsrepresented less than 0.5% of the total sterols.Assuming a complete mobilisation of sterols, including

membrane sterols, by an egg-laying female, its totalsterol content would only allow for the production of18.6 eggs.

3.2. Effects of nutritional cholesterol on the parasitoid

oogenesis

3.2.1. Parasitoids fecundity

Modification of the quality of the host hemolymph had asignificant effect on the total number of eggs laid byfemales (Table 3). Indeed females reared on C. maculatus

without injection (NI) laid significantly more eggs duringtheir entire lifetime compared to females Oi, Ch, St and Si(F ¼ 25:2, df ¼ 4, po0:001). This difference between thenumbers of eggs laid by females NI compared to the otherswas due to the higher survival rate of these females(F ¼ 9:65, df ¼ 4, po0:05).Surprisingly, the total number of eggs laid by females

feeding on host with hemolymph diluted (Oi) or supple-mented (Ch, St, Si) in sterol was not significantly different.The maximum number of eggs obtained from the larvalsterol stock (18.6) is attained on day 3 for NI females andon average on day 5 for Oi, Ch, St and Si females.

ARTICLE IN PRESS

Table 3

Lifetime total number of eggs laid, adult lifetime duration and number of eggs laid per day (Mean7SE) of E. vuilleti female fed on non injected C.

maculatus (NI) or on C. maculatus with hemolymph cholesterol diluted (Oi) or with hemolymph supplemented with cholesterol (Ch), stigmasterol (St) or

sitosterol (Si)

Adult lifetime duration (days) Eggs number laid per female

During its lifetime Per days

NI: without injection 17.470.6a 11776a 6.770.2

Oi: oil/water injection 9.770.7b 59.9714b 5.571.2

Ch: cholesterol injection 9.971.3b 62.4712b 5.270.8

St: stigmasterol injection 10.871b 38.477b 3.271.7

Si: sitosterol injection 10.671.4b 58.8712b 4.970.7

Significant differences between treatments are indicated by different letters (Bonferoni method after one-way ANOVA).

0

20

40

60

80

100

Hat

ched

egg

s (%

)

(A)

0

20

40

60

80

100

0 6 10 11 12 13 14 15 16

Hat

ched

egg

s (%

)

Time (days)

(B)

1 2 3 4 5 7 8 9

Fig. 1. Percentage of hatched eggs laid per E. vuilleti female fed on

C. maculatus with hemolymph sterols modified or not: (A) non-injected

C. maculatus (NI: ~), sterols hemolymph diluted (Oi: J) or supplemented

with cholesterol (Ch: ’) and (B) with sterols hemolymph supplemented

with phytosterols: stigmasterol (St: D) and sitosterol (Si: � ).

0.0

0.5

1.0

1.5

[1-2] [3-4] [5-6] [7-8] [9-10] [11-12] [13-14] [15-16]

Time (days)

eq E

pg/

egg

Fig. 2. Means (7SE) of ecdysteroid contents (eq E pg) per egg laid by

E. vuilleti female reared with non-injected C. maculatus (NI: ~), with

sterols host hemolymph diluted (Oi: J) or with host hemolymph

supplemented with cholesterol (Ch: ’), with stigmasterol (St: D) or withsitosterol (Si: � ).

N. Mondy et al. / Journal of Insect Physiology 52 (2006) 897–904 901

3.2.2. Percentage of hatched eggs

Fig. 1 showed the hatchability of eggs laid by adultfemales fed on either non-injected or injected hosts.Comparisons between the number of hatched eggs inrelation to the five treatments showed a significantdifference (w2 ¼ 184, df ¼ 4, po0:01). During the entirelifetime of the female, the percentage viability of the eggslaid by females NI was 78.1%. This viability fluctuatedbetween 100% and 67% over 14 days. Then it decreasedsimultaneously with the drop in female survival. The eggslaid during the first 5 days by females reared on Oi hosts inwhich hemolymph sterols had been diluted were viable.Egg hatchability results obtained from females Ch clearlyshowed that the addition of cholesterol to host hemolymphincreased the egg viability compared to the eggs laid byfemales Oi (73% for the females Ch vs. 34% for the females

Oi, (w2 ¼ 149, df ¼ 2, po0:01)). The hatchability of eggsCh and NI were not significantly different (w2 ¼ 2:4,df ¼ 2, p ¼ 0:1). Parasitoids feeding on host hemolymphsupplemented with stigmasterol (St, 52%) or with sitosterol(Si, 53%) did not show any significant difference(w2 ¼ 0:06, df ¼ 2, p ¼ 0:8). The viability of eggs laid byfemales feeding on host hemolymph supplemented withthese two phytosterols was significantly lesser than theviability recorded for females Ch (St/Ch w2 ¼ 28:2, df ¼ 2,po0:01; Si/Ch w2 ¼ 25:7, df ¼ 2, po0:01).

3.2.3. Egg ecdysteroid contents

Fig. 2 shows the ecdysteroid titres of eggs (means andSE) laid by female wasps, reared on hosts injected with thedifferent sterols, compared with controls injected withcolza/oil, throughout their entire lifetimes. Whatever thetreatment administered, females invested the same quantityof ecdysteroids in their eggs for a 10-day period afteremergence (Kruskall–Wallis test, pX0:05) and a slowreduction of this investment was observed. The first eggslaid contained on average 270.3 pg equivalent ecdysone.Eggs laid on the 9th and 10th days contained0.8370.15 pg-equivalent ecdysone. This decrease of ecdys-teroids in the eggs was not apparently linked with eggviability (Fig. 2). It was noted that from the 11th day to the

ARTICLE IN PRESSN. Mondy et al. / Journal of Insect Physiology 52 (2006) 897–904902

16th day, the eggs laid by control females had a constanttitre of ecdysteroids 0.5470.02 pg-equivalent ecdysone peregg.

4. Discussion

The adaptive significance of host feeding in parasitoidshas been the subject of numerous studies but most havefocused only on the acquisition of proteins for vitellogen-esis. Our results emphasise for the first time the role ofsterols, and cholesterol in particular, acquired through hostfeeding for the production of viable eggs by a synovigenicparasitoid.

It is necessary to analyse the sterol composition of eachpartner of the tritrophic interaction in order to understandtheir nutritional strategies. In agreement with previousstudy on legume seeds (Akihisa et al., 1992), D5-phytoster-ols were in the majority (stigmaterol, sitosterol andcampesterol) in V. unguiculata seeds. But generally,sitosterol was the major sterol of seeds and stigmasterolwas less than 10% of total sterols. V. unguiculata exhibitedstigmasterol (48.7%) as the major D5 sterol and we alsofound a high content of sitostanol (21.5%). Other seeds ofVigna genera have important quantities of stigmasterol(31.6% in Vigna mungo). Esterified sterols were negligiblein these seeds.

The current finding that cholesterol constitutes over 84%of the tissue sterols in C. maculatus is consistent with thefact that these insects are able to convert dietary plantsterols to cholesterol like other phytophagous beetles(Svoboda and Lusby, 1994). This ability to perform sucha dealkylation at the 24-position appears to be character-istic of plant-eating insects (e.g. Ritter and Wientjens, 1967;Svoboda et al., 1995). Furthermore, in agreement with aprevious study on the dietary sterols of Callosobruchus

chiniensis (Ishii, 1951 in Behmer and Nes, 2003), C.

maculatus directly used sterols saturated on ring B (D0

sterol), such as sitostanol. The patterns of sterols seen inboth the hemolymph and in whole larvae were similar.

Our results showed that newly emerged E. vuilleti havesterol compositions that reflect those of their hosts, C.

maculatus. They contained cholesterol as major tissuesterol and had low levels of C28 and C29 phytosterols. Thus,they usually have adequate cholesterol available in theirdiets, and they appear to use dietary sterols with nomodification. This apparent incapacity to modify dietarysterols will have to be clarified using more controlledconditions.

Tissue sterol profiles can also differ between male andfemale parasitoids. E. vuilleti had on average 1.470.1 mg oftotal sterols/mg of dry weight for the females vs.1.170.1 mg of total sterols/mg of dry weight for the males.However, female tissues were enriched in cholesterolcompared with male tissues (+52% of total sterols). Thecholesterol/phystosterol ratio in parasitoid males (1/5) wasthe same as in their host. On the contrary, E. vuilleti

females contained a 33-fold higher quantity of cholesterol

than phytosterols. It seems that cholesterol may berequired for an essential physiological process in thefemale, probably oogenesis. For instance, Lasser et al.(1966) showed that there was 3-fold higher concentrationof cholesterol in the reproductive organs of females of acockroach, Eurycotis floridana, compared with males.Thus, E. vuilleti females may selectively absorb cholesterolfrom the host tissues, and that during the oogenesisallocation of sterols the phytosterols are preferentiallyaccumulated in eggs. Besides sterols, others lipids may beincorporated directly into the tissues of parasitoids. Indeed,several studies have suggested that most adult parasitoidsseem to be incapable of lipogenesis (Ellers, 1996; Olsonet al., 2000; Giron and Casas, 2003).Finally, it was observed that E. vuilleti did not

contain esterified sterols compared with its host. Numerousstudies have shown that insects can absorb dietarycholesterol in free and ester form. However, after absorp-tion, a small fraction of the free form is subjected tointracellular esterification (Jouni et al., 2002). The sig-nificance of esterification is unknown but it has beensuggested that it could serve as a temporary intracellularstorage mechanism (Turunen, 1985). The small quantity ofcholesterol reserves in this synovigenic parasitoid con-firmed the female’s need to acquire cholesterol during adultlife for oogenesis.Our study showed interesting results concerning the

parasitoid’s strategy of sterols acquisition. A newlyemerged E. vuilleti female contains only 1.2370.1 mg oftotal sterols whereas one egg contains 66 ng of sterols. Sothe amount of sterols sequestered in the eggs laid during 12days in optimal egg-laying condition for NI females (117on average) exceeds by 6-fold the total body store of sterolsin the newly emerged adult insect. While Casas et al. (2005)convincingly showed that females behave as capitalbreeders for lipids, in general, the current detailed analysisof one particular category of lipids clearly shows that forsterols, females are mainly income breeders. These twoclaims are compatible, however, as sterols make up only1.5% of lipids. Lipids are a diverse group of molecules andtheir heterogeneity may imply variations in their manage-ment, especially sterols which are not a source of energybut a principal component of primary metabolism (mem-brane synthesis and regulation).To understand the physiological role in reproduction of

sterols that are acquired during adult feeding, the sterolcomposition of the host hemolymph was experimentallymanipulated. We note that in all the treatments in whichfemale wasps were fed on injected larvae, there was anegative effect of the injection on both the rate of eggproduction and the total number of eggs produced,compared with the results obtained with the wasps rearedon non-injected C. maculatus. This was presumablybecause the injection diluted by 2-fold the non-sterolnutrients, in particular proteins and sugars, which are veryimportant for survival and egg production of this species(Rivero et al., 2001; Casas et al., 2005). Results obtained

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with wasps fed on injected larvae clearly showed thatcholesterol gained by host feeding did not affect fecunditybut played a crucial role in egg viability. These results arein agreement with those of Behmer and Grebenok (1998)done on the diamondback moth and Mondy and Corio-Costet (2000) on the European grapevine moth. Theyconfirmed that the mechanism driving egg productionmight be independent from that determining egg viability,which is clearly linked to dietary sterols.

We demonstrated that adult females which gained verylittle cholesterol by host feeding (Oi) laid viable eggs onlyduring the first 5 days. Maternal cholesterol invested inthese eggs may correspond to the stock of sterol that isderived from larval feeding. When females obtainedcholesterol by host feeding (Ch), egg viability was restored.Results obtained with host hemolymph supplemented inphytosterols (St and Si) were more variable. However, thetotal viability of these eggs was less, in each case, thanthe viability for eggs laid by females NI or Ch. In addition,the quantity of ecdysteroids introduced into eggs by femalesreared on an untreated host or on a host with a modifiedsterol profile was not significantly different. Hence, whateverthe composition of the adult diet, the cholesterol derivedfrom larval feeding is sufficient for biosynthesis ofecdysteroids invested in eggs. Based on these results, wecautiously suggest that for the reproduction of synovigenicparasitoids, the sterols gained by host feeding are used inmembrane synthesis rather than in ecdysteroid synthesis.

To conclude, in this study, we showed that E. vuilleti

needs sterols from both larval and adult food. Females canmature and lay only their first batch of eggs from sterolsgained during the larval stage. Then, if hosts are abundant,females can both feed and lay simultaneously. The fact thatthe host is both the oviposition site and the nutritionalsource may have favoured the evolution of incomebreeding.

Acknowledgments

We are grateful to Prof. J. Casas, Dr. B. Jaloux and Dr.D. Giron for comments on earlier drafts which improvedthe manuscript and to Dr. JP Delbecque for the initiationto the use of EIA analyses. We thank G. Hollyfield forreviewing the English of this manuscript.

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