Superparasitism limitation in an aphid parasitoid: cornicle secretion avoidance and host discrimination ability

Journal of Insect Physiology 47 (2001) 339–348www.elsevier.com/locate/jinsphys

Superparasitism limitation in an aphid parasitoid: cornicle secretionavoidance and host discrimination ability

Y. Outremana,*, A. Le Raleca, M. Plantegenesta, B. Chaubeta, J.S. Pierreb

a E.N.S.A.R., Laboratoire d’Ecologie et Sciences Phytosanitaires, 65, rue de Saint-Brieuc, 35042 Rennes Cedex, Franceb Universitede Rennes I, U.M.R. 6552, Campus Beaulieu, 35042 Rennes Cedex, France

Received 28 February 2000; accepted 29 September 2000

Abstract

Superparasitism avoidance by the endoparasitoidAphidius rhopalosiphiDe Stefani Perez on the grain aphid,Sitobion avenaewas studied. Experiments were carried out in which aphids were exposed to two consecutive attacks by parasitoids. Results showedthat superparasitism avoidance inA. rhopalosiphiwas mediated by two successive stimuli whose effectiveness depended on thetime interval between attacks. For short time intervals (,16 h), host rejections were mainly associated with the presence of driedcornicle secretion on the host’s body which was exuded during the first attack. The repellency of this secretion declined with thetime interval between attacks, becoming ineffective 2 days after the first parasitization, and allowed females to reject up to 30%of parasitized hosts. For longer time intervals ($16 h), host rejection behavior was a response of parasitoid females to internalchanges in host quality associated with parasite development. This response gradually increased with an increase in time interval,reaching no more than 60%, 96 h after initial parasitization. This host discrimination ability did not allow females to distinguishbetween hosts parasitized by themselves or by conspecifics. Consequently, these findings suggest that superparasitism is a commonevent inA. rhopalosiphiand especially on recently parasitized hosts. 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Aphidius rhopalosiphi; Superparasitism; Host discrimination; Host defensive behaviors;Sitobion avenae

1. Introduction

Parasitoid wasp females have to decide which hoststo accept for oviposition and this decision stronglydepends on the characteristics of the hosts (Visser et al.,1992). An important feature is whether the encounteredhost has already been parasitized or not (Ueno, 1994).Contrary to prey consumed by predators, attacked hostsremain in their habitat, but in a parasitized state. There-fore, these hosts can be encountered again by the sameor another parasitoid, and can be accepted again for ovi-position, resulting in superparasitism. In solitary parasit-oids, only one individual can develop in a host andsupernumerary individuals are eliminated through intra-host competition (Hubbard et al., 1987). Generally, theoldest parasite eliminates all the younger competitors

* Corresponding author. Tel.:+33-223-48-55-65; fax:+33-223-48-51-70.

E-mail address:[email protected] (Y. Outreman).

0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (00)00142-6

(Mangel, 1989). Therefore, a parasitized host is of lowerquality for a parasitoid female (Nelson and Roitberg,1995). Accordingly, even if superparasitism can beadaptive in some situations (for a review see van Alphenand Visser, 1990), most solitary wasps tend to avoid it.

Avoidance of superparasitism generally implies theability of female parasitoids to distinguish betweenunparasitized and parasitized hosts, termed host dis-crimination ability (van Lenteren, 1981). Such an abilityhas a strong selective advantage as females can avoidwasting eggs laid in lower quality hosts (Bakker et al.,1985). Many studies have reported that parasitoids candiscriminate between parasitized and unparasitized hosts(Ueno, 1994), and this ability is generally mediatedthrough host markers present externally and/or intern-ally. Some solitary parasitoids mark the host they justattacked externally with either a pheromone depositedduring oviposition (e.g., Hofsvang, 1988; Vo¨lkl andMackauer, 1990) or a physical mark left on the host body(e.g., Boldt and Ignoffo, 1972; Takasu and Hirose,1988). Internal cues for host discrimination can originate

340 Y. Outreman et al. / Journal of Insect Physiology 47 (2001) 339–348

either from some parasitoid injected substances (Vinson,1976; Hubbard et al., 1987) or from host quality changesassociated with parasitism (Cloutier et al., 1984;Hofsvang, 1988; Mackauer, 1990). The level of recog-nition of these markers can vary between parasitoidfemales. Some researchers have recently demonstratedthat female parasitoids can discriminate between hostscontaining their own eggs and those containing conspe-cifics’ eggs (van Dijken and Waage, 1987; Vo¨lkl andMackauer, 1990; Ueno, 1994; van Baaren et al., 1994).This is advantageous as it allows a female to avoid ovi-position in hosts containing their own progeny (self-superparasitism) and hence, competition between theirown offspring (Visser et al., 1992).

In contrast, occasional or total lack of host discrimi-nation has been observed in a few solitary parasitoids(Rosenheim and Mangel, 1994). In the aphid parasitoidAphidius rhopalosiphiDe Stefani Perez (Hymenoptera:Braconidae), experiments failed to demonstrate theability of females to discriminate between parasitizedand unparasitized hosts (Gardner et al., 1984). However,this result was not supported by Outreman et al. (2000),who showed that females of this species were able torecognize already parasitized hosts but that this abilityseems imperfect, leading to some already parasitizedaphids being attacked a second time.

The present study aimed to assess the level of super-parasitism avoidance inA. rhopalosiphiand to under-stand the mechanisms involved in host discrimination.The host used in the experiments was the grain aphid,Sitobion avenaeFabricius (Homoptera: Aphididae). Theability of females to distinguish between hosts parasit-ized by themselves or by conspecifics was also assessed.The mechanisms of host discrimination were approachedby analyzing the behavior of females towards aphidscontaining parasitoids at different developmental stages,thus determining if superparasitism avoidance ismediated by external or internal cues. Apart from hostdiscrimination, other factors could limit superparasitismin A. rhopalosiphi. Aphids are known to exhibit variousdefensive reactions against attacking parasitoids andsuch behavioral responses can influence oviposition to aconsiderable degree (Stary´, 1970; Michaud andMackauer, 1994). Gardner et al. (1984) showed that amechanical defensive behavior (i.e., quick motions ofbody) of the cereal aphidMetopolophium dirhodum,stimulated by a firstA. rhopalosiphiattack, induced ahigh level of superparasitism avoidance. Accordingly,the present study also aimed to assess the effect ofdefensive behaviors ofS. avenaeon the probability ofbeing parasitized once again by an attackingA. rhopalo-siphi female.

2. Materials and methods

2.1. Parasitoids and hosts

A. rhopalosiphiused for the experiments originatedfrom individuals captured in June 1996 at Rennes,France, and reared in the laboratory on a mixed-age cul-ture ofS. avenaefeeding on winter wheat,Triticum aes-tivum, cv. “Arminda”. Host aphids originated from oneparthenogenetic female collected in 1990 in the Rennesarea. Colonies of bothA. rhopalosiphiand S. avenaewere maintained in climate rooms at 20°C, 70±10%R.H., and a 16 h light:8 h dark photoperiod. For theexperiments, only second-instar larvae ofS. avenaewereused as hosts. To obtain parasitoid females, mummieswere collected and placed individually in gelatine cap-sules. Newly emerged females were enclosed in plastictubes (22×1 cm) containing moistened cotton, dropletsof honey diluted in water (dilution factor: 80% honeyand 20% water) and one male for mating. All the femalesused for the experiments were 1 day old.

2.2. Superparasitism avoidance in A. rhopalosiphi

The aims of this experiment were (i) to assess the rateof superparasitism avoidance inA. rhopalosiphion selfand conspecifically parasitized hosts, (ii) to examine theeffect of the time elapsed since the first parasitization onsuperparasitism, and (iii) to determine the mechanismsleading to superparasitism avoidance. Aphids were thenexposed to two successive attacks achieved either by thesame female or by two unrelated females at differenttime intervals. For each treatment, the level of super-parasitism avoidance was estimated by the analysis ofthe number of eggs found in aphids after these two con-secutive attacks. Then, the mechanisms involved in thesuperparasitism limitation were assessed via analysis ofthe behavior of parasitoids and aphids observed duringattacks. In particular, possible host rejection related tothe detection of an internal or external cue and theresponse of females to host defensive behaviors wereanalyzed.

Eight healthy aphids feeding on a piece of wheat leafwere successively exposed to anA. rhopalosiphifemalein a glass Petri dish (3.5 cm in diameter). Each host wasattacked once (a host was considered as being attackedwhen the female had stung and departed from it) andsubsequently isolated in a plastic tube with a wheat leaf.The parasitoid female was enclosed in a plastic tube con-taining a drop of honey syrup. Both the female and theeight attacked hosts were kept under laboratory con-ditions. After 1, 2, 4, 8, 16, 24, 48, 72 or 96 h, the eightpreviously attacked hosts were successively exposed (inthe same order as during the first phase) to a secondparasitoid female. This female had been allowed toattack once (in a glass Petri dish) a healthy host placed

341Y. Outreman et al. / Journal of Insect Physiology 47 (2001) 339–348

on a piece of wheat leaf just before the second encounterin order to reduce egg-laying pressure. For each timeinterval, the second female wasp was either the samefemale as in the first run (self-discrimination series) oranother female that had previously attacked eight otheraphids (conspecific-discrimination series). During thefirst and the second encounters, the behaviors of bothfemales and hosts were continuously recorded using avideo camera (an encounter could last between 5 and10 s) and the following behaviors were noted: antennaldrumming, number of ovipositor insertions, defensivebehaviors of hosts and host rejections. Four behaviorsleading to a host rejection inA. rhopalosiphiwere dis-tinguished: (i) antennal rejection: an antennal drummingon the host’s body could result in an immediate rejec-tion; (ii) mechanical defensive behavior of aphids: quickmotions of legs and/or body and escape reactions couldreduce the tendency of the wasp to undertake a stabbingattack; (iii) sting rejection: the female could reject thehost after ovipositor insertion. As sting rejection inA.rhopalosiphi could not be distinguished from ovi-position, such host rejection was deduced from theresults of both dissection and video analysis; (iv) corni-cle secretion avoidance: during the first attack, aphidscould exude a small drop of yellowish fluid fromcornicles. Once emitted, this secretion rapidly solidifiesin the air, remaining on the ends of the cornicles, andaphids with dried cornicle secretion on their body couldbe rejected by females at the second attack.

At the end of each experiment, aphids were isolatedas previously described. They were dissected in a dropof saline solution under a microscope and any parasitoidspresent were counted. To avoid underestimation of thenumber of ovipositions resulting from competitionbetween parasitoid individuals, the dissections were car-ried out only 3–4 h after the end of the experiment. Atthat time, only parasitoid eggs can be found and to facili-tate their detection, neutral red was added to the dissec-tion solution (previous tests showed thatA. rhopalosiphieggs are detected readily by this method). Each treat-ment was repeated 10–12 times. Experiments were car-ried out at 20±2°C and 60±10% R.H., and designed ina randomized complete block.

In order to analyze the level of superparasitism avoid-ance, the observed distributions of the number of eggsper host found at dissection were compared with anexpected distribution built under the null assumption thatA. rhopalosiphihad no host discrimination ability. Tocompute this expected distribution, an estimate of theprobability of parasitization after a single attack wasneeded. We thus carried out a separate experiment toestablish the proportion of aphids actually parasitizedafter a single attack. Eight healthy hosts were separatelyand successively exposed to a female and only one attackper aphid was allowed. Attacks were observed continu-ously with a camera connected to a video tape recorder

in order to analyze the influence of host behavioral reac-tions on attack success. The attacked aphids were dis-sected 3 days after the attack. At that time, hatching ofthe parasitoid larva had normally occurred and thus,detection of parasitoids was much easier. Because pre-liminary dissections showed no evidence of eggs beingencapsulated or destroyed, it was assumed that the num-ber of larvae recovered at dissection indicated the actualnumber of eggs deposited by the parasitoid. Twenty-fiveparasitoid females were used for this preliminary experi-ment. Of the 198 attacks observed, 184 (92.9%) resultedin successful egg laying. Among these 184 successfulattacks, 158 (79.8% of all attacks) contained one parasitelarva and 26 (13.1% of all attacks) contained two larvae.Hence, cases of superparasitism were observed, althoughonly one attack per host was allowed. This wasexplained by the behavioral analysis, which revealed thata single attack could consist of multiple stabbing, andhence resulted in successive ovipositions. Besides, 14(7.1%) attacked hosts escaped from parasitism.Behavioral analysis suggested that these unsuccessfulattacks might result from aphid defense: a parasitoidstabbing attack can elicit an aphid movement (quickmotions of legs or body) which could influence attacksuccess.

From these results, we computed the expected distri-bution of the number of eggs per host after two success-ive attacks under the null hypothesis H0 of no host dis-crimination ability. For this, we assumed that (i) twosuccessive encounters with the same host were inde-pendent (i.e., no host discrimination ability), (ii) amaximum of two eggs can be laid per attack, and (iii)all eggs laid survived until dissection. Letp0, p1 andp2

be the probabilities of a host containing 0, 1 or 2 parasit-oids after a single attack, respectively. From a separateexperiment, we got the estimatesp0=0.0707 (i.e.,14/198),p1=0.7980 (i.e., 158/198), andp2=0.1313 (i.e.,26/198). Therefore, under H0, in a sample of sizen, theexpected frequenciesE(Nj) of the number of hosts con-taining j parasitoids at dissection after two attacks arethen given by:

E(N0)5np20

E(N1)52np0p1

E(N2)5n(p2112p0p2)

E(N3)52np1p2

E(N4)5np22

E(Nj )50 for j.4

For each treatment, a G-test with Williams’ correction(Scherrer, 1984) was used to compare the observed dis-tribution of parasitoid eggs per host to the expected one.


2.3. Repellent effect of the aphid cornicle secretion

As shown above, some host rejections seemed to berelated to the presence of dried cornicle secretion on thehost’s body. Such a result suggests that this secretioncould have a repellent effect. The present experimentaimed to support this effect of the cornicle wax on theparasitoid’s behavior. For this purpose, the wasp’sbehavior towards two kinds of hosts in a series of choicetests was observed. Parasitoids were individually placedin the center of a 6-cm diameter glass Petri dish contain-ing one healthy host and one healthy host with driedcornicle secretion. Hosts were 2 cm apart. Hosts withdried cornicle wax were obtaining by mechanically sti-mulating aphids 30 min prior to the trial with an entomo-logical pin. Once emitted, secretion remained on theends of the cornicles and aphids were rapidly placed ona piece of wheat leaf for the test.

In every test, the kind of the encountered hosts wasnoted and then host acceptance and host rejection wererecorded, respectively, if a parasitoid stung the aphidwith its ovipositor or abandoned it without ovipositorinsertion. Once a female departed from the encounteredhost, it was replaced immediately with another. Experi-ments ended when a female had encountered a total of16 aphids. The two-choice test was repeated 10 times.Experiments were carried out at 20±2°C and 60±10%R.H., and designed in a randomized complete block.

For statistical analysis, the numbers of hosts of eachkind that were encountered by the wasps were comparedby a paired-samplet-test (Sokal and Rohlf, 1981).

3. Results

3.1. Superparasitism avoidance in A. rhopalosiphi

3.1.1. Dissection analysisObserved distributions of the number of parasitoid

eggs per host after two successive attacks were shownto differ significantly from the distribution expectedunder the hypothesis of no host discrimination (Fig. 1).This result did not depend on the time interval betweenthe two successive attacks or on whether the two attackshad been made by the same female (Fig. 1A) or twounrelated females (Fig. 1B). Therefore, the assumptionof independence between two successive encounterswith the same host was rejected. Closer examination ofobserved distributions showed that the number of hostswith more than one parasitoid was smaller than expectedand conversely, an excess of hosts containing a singleegg was found. This suggests that females had avoidedoviposition in already attacked hosts. However, thisavoidance was partial as some level of superparasitismwas still found in all experiments. In fact, the level of

superparasitism avoidance appeared to vary with thetime interval between the two attacks. Comparisons withthe expected distribution under the null hypothesis ofno host discrimination suggested that the levels of hostrejection (i.e., superparasitism avoidance) were rela-tively low in the early phase of parasitism, but increasedwith the time interval between successive encounters.Whatever the time elapsed since the first parasitization,no significant difference between self- and conspecific-discrimination distributions was found (Fisher’s exacttests,P.0.05).

3.1.2. Behavioral analysisThe mean percentage of host rejections observed

through behavioral analysis is presented in Fig. 2. Thetime-dependence of the level of superparasitism avoid-ance inA. rhopalosiphi, described in Section 3.1.1, isevident from the behavioral analysis. Females rejectedabout 40% of hosts that had already been attacked 1 hbefore, and this level of host rejection greatly decreasedwith increasing time interval down to about 20%, 8 hafter the first oviposition. From this lowest level, themean frequency of host rejection increased slowly,reaching about 65% when 96 h separated the two attacks.Whatever the time interval between the two attacks,A.rhopalosiphi females rejected as many self-attackedhosts as conspecific-attacked hosts (c2

8df=2.34,P=0.968).Fig. 3 presents the frequency distribution of the four

behaviors leading to host rejections inA. rhopalosiphias a function of the time duration separating the twosuccessive attacks. No significant difference betweenself- (Fig. 3A) and conspecific-discrimination distri-butions (Fig. 3B) was found at any time interval betweenencounters (Fisher’s exact tests,P.0.05). Host rejectionbehavior at short time intervals was obviously differentfrom that observed at long time intervals. In the earlyphase of parasitism, host rejection was mainly related tothe presence of dried cornicle secretion on the host body.Actually, about 40% of aphids were shown to emit corni-cle secretion during the first attack (among these hosts,only five exuded this secretion prior to being stung), andthese aphids can still have dried secretion on the endsof the cornicles at the second attack (Table 1). However,both the number of hosts bearing secretion residues atthe second attack and the repellency of this secretiondecreased with the time interval between attacks (Table1). Consequently, the effect of the cornicle wax wasmaximal shortly after the first attack, allowing up to 30%of host rejections, and then it declined with increasingtime interval, becoming ineffective 48 h after the firstparasitization (Fig. 3).

From 16 h after the first attack onward, some rejec-tions of parasitized hosts after sting behavior appeared(Fig. 3). The frequency of sting rejections increased sub-sequently with the length of the interval between attacksuntil it accounted for nearly all host rejection. This


Fig. 1. Distribution of the number of parasitic eggs found inside a grain aphid attacked twice byA. rhopalosiphifor different time intervalsbetween encounters. (A) Self-discrimination series; (B) conspecific-discrimination series. All replicates were grouped in each treatment. Doubleasterisks indicate a highly significant difference between expected and observed distributions at each time interval between encounters (G-testswith William’s correction,P,0.001). No significant difference between self- and conspecific-discrimination distributions was found at each timeinterval between encounters (Fisher’s exact test,P.0.05).

Fig. 2. Host rejection frequency (mean±s.e.) of parasitized grainaphids byA. rhopalosiphiobserved on video-recording in relation totime elapsed from first parasitization.

behavior led to the rejection of 60–65% of hosts 96 hafter initial parasitization. Finally, antennal rejectionsand mechanical defensive behavior of aphids contributedto a low level of superparasitism avoidance. Rejectionsdue to mechanical host defense accounted for about 5%of rejections, and remained independent of time interval,contrary to antennal rejections that were effective only

for short time intervals, allowing up to 6% of rejections1 h after initial parasitization (Fig. 3).

The behavioral observations have shown four differ-ent types of host rejection behavior inA. rhopalosiphi.It remained to be determined if the frequency of theserejection behaviors was sufficient to explain theobserved distributions of the number of eggs per hostfound at dissections. As shown above, these observeddistributions were significantly different from the distri-bution expected under the hypothesis of no host dis-crimination (i.e., the two successive encounters with thesame host were independent). Because parasitoidfemales rejected some hosts on the second attack, theassumption of independence between two successiveencounters was then false. The expected distribution wasthen redefined by introducingm, the probability that aparasitized host is rejected at the second attack. Thisparameter was estimated using the behavioral obser-vations (Fig. 2). Accordingly, in a sample of sizen, theexpected frequenciesE(Nj) of hosts containingj eggs atdissection after two attacks were modified as follows:

E(N0)5np20

E(N1)5n[p0p11p1p0(12m)1p1m]

E(N2)5n[p0p21p1p1(12m)1p2p0(12m)1p2m]

E(N3)5n[2p1p2(12m)]


Fig. 3. Distribution of host rejection types byA. rhopalosiphion onceattacked grain aphids observed on video-recording in relation to timeelapsed from first parasitization. (A) Self-discrimination series; (B)conspecific-discrimination series. All replicates were grouped in eachtreatment. No significant difference between self- and conspecific-dis-crimination distributions was found at each time interval betweenencounters (Fisher’s exact test,P.0.05).

E(N4)5n[p22(12m)]

E(Nj)50 for j.4

A G-test with Williams’ correction (Scherrer, 1984) wasused to compare the observed distribution of parasitoidsper host to that expected for each treatment. Observeddistributions did not differ any more from those expectedand this did not depend on the time between the twosuccessive attacks and on whether the two attacks hadbeen made by the same female or two conspecifics.Consequently, the expected distributions, taking intoaccount the observed level of rejection, considerablyimproved the prediction of the parasitoid’s behavior onits second attack over the basic expected distribution(i.e., under the hypothesis of no host discrimination).

Thus, the observed number of eggs per host found atdissections is likely related to the four different types ofhost rejection behavior described and quantified throughthe behavioral analysis.

3.2. Repellent effect of the aphid cornicle secretion

A. rhopalosiphi females showed an ovipositionrestraint regarding a host having dried cornicle secretionon its body. In the two-choice test, the presence of corni-cle wax on the healthy host’s body did indeed affect theacceptance rate, as only 37.7% of encountered healthyaphids bearing secretion were accepted for oviposition,while 100% of encountered healthy hosts withoutsecretion were accepted. At last, it is worth noting thateach kind of host was equally encountered by females(paired-samplet-tests,P.0.05), suggesting that corniclewax acted at very short range or on contact, and did notapparently affect parasitoid orientation.

4. Discussion

A. rhopalosiphiexhibited a tendency to avoid layingeggs in previously parasitized aphids. The results suggestthat this superparasitism avoidance inA. rhopalosiphiismediated by two stimuli which act independently, suc-cessively, and whose efficiency varies with the timeelapsed since initial parasitization. During the “early”phase of parasitism, females often rejected alreadyattacked hosts that bore dried cornicle secretion on theirbody. The results of the second experiment strongly sug-gest that these host rejections were elicited by the repel-lency of the cornicle secretion. This response to corniclewax would then allow parasitoids to indirectly avoidsuperparasitism. The repellency of this secretion wasmaximum shortly after the first oviposition, leadingfemales to reject up to 30% of parasitized hosts, and thenit quickly declined with increasing time interval betweenattacks, becoming ineffective 2 days after the first parasi-tization. Moreover, some antennal rejections wereobserved in the early phase of parasitism and their pres-ence coincided with the efficiency of the effect of corni-cle secretion (Fig. 3). This may indicate that this hostrejection behavior might be due to the presence of driedsecretion spread out over the aphid’s body.

For longer time intervals, a second stimulus allowingsuperparasitism avoidance appeared. Female behaviorindeed suggests that first reactions to an internal cueoccur 16 h after the first attack. Perception to that cuegradually increased with time interval between attacks,leading to the rejection of 60% of already parasitizedaphids when 96 h separated the two attacks. In additionto these stimuli, at each time interval, about 5% of pre-viously parasitized aphids were rejected by means ofmechanical defensive behavior (quick motions of legs or


Tab

le1

Fre

quen

cyof

S.

ave

na

eaph

ids

(i.e.

,th

egr

ain

aphi

d)th

atex

uded

corn

icle

secr

etio

ndu

ring

the

first

atta

ckof

A.

rho

pa

losi

ph

ifem

ales

and

beha

vior

alre

spon

ses

ofth

ese

was

psto

war

dsho

sts

that

still

had

drie

dco

rnic

lese

cret

ion

onth

eir

body

atth

ese

cond

atta

ck

Exp

erim

enta

lse

ries

Tim

ein

terv

albe

twee

nN

umbe

rof

Num

ber

ofho

sts

Hos

tsex

udin

gco

rnic

leH

osts

havi

ngdr

ied

Hos

tsre

ject

edha

ving

Rat

eof

repe

llen

cyof

two

atta

cks

(h)

repl

icat

esat

tack

edse

cret

ion

durin

gfir

stse

cret

ion

atse

cond

drie

dse

cret

ion

corn

icle

secr

etio

nat

tack

atta

ck

Sel

f-di

scrim

inat

ion

111

8835

3224

0.75

212

9332

2518

0.72

411

8829

2116

0.76

812

9235

3214

0.44

1612

9038

236

0.26

2412

9238

112

0.18

4811

8241

61

0.17

7210

7845

0–

–96

1071

270

––

Con

spec

ific-

disc

rimin

atio

n1

1188

3431

240.

772

1187

3431

160.

524

1296

3829

150.

528

1292

3936

180.

5016

1294

3419

50.

2624

1296

316

00.

0048

1295

356

10.

1772

1174

370

––

9610

7827

0–

–


body and escape reactions) on the second attack. Thisratio appeared to remain similar to that found on healthyhosts. It could then be assumed that parasitism has noeffect on these aphid defensive responses. Thus, contraryto what has been shown inM. dirhodum(Gardner et al.,1984), the mechanical defensive behavior ofS. avenaedoes not seem to limit superparasitism inA. rhopalosi-phi. This difference suggests that the host defensiveresponses against an attacking parasitoid could varybetween cereal aphid species.

The presence of cornicle secretion on a newly parasit-ized S. avenaeusually repelled wasps. The response tothis chemical cue occurs at very short range or on con-tact, and volatiles released, if any, do not apparentlyinfluence parasitoid orientation. This secretion emittedin response to predator attack (Nault et al., 1973) orparasites (Goff and Nault, 1974) has a defensive function(Nault and Phelan, 1984) and acts in two different ways.Firstly, the contained volatile (E)-β-farnesene elicits dis-persal of nearby aphids, operating as an alarm phero-mone (Bowers et al., 1972; Edwards et al., 1973). Sec-ondly, the sticky and hardening consistency of thissecretion, based on wax-like triglycerides (Callow et al.,1973), can glue organs (e.g., mouthparts, antennae, ovi-positor, etc.) of the attacking enemies (Strong, 1967).Although some studies indicated that cornicle secretioncould act as a contact kairomone in aphid parasitoids(Grasswitz and Paine, 1992; Battaglia et al., 1993, 1994;Micha and Wyss, 1996),A. rhopalosiphi respondednegatively to this secretion. This negative response couldhave some consequences onA. rhopalosiphi females’fitness since it accounts for up to 30% of previously par-asitized host rejections. However, the efficiency of corni-cle secretion decreases as the secretion dries, leading toincreasing levels of superparasitism. The activecompound(s) of cornicle secretion may be then eithervolatile or otherwise unstable when in air (Grasswitz andPaine, 1992).

To account for the repellent effect of corniclesecretion, it can be suggested that females perceive thepresence of secretion residues as a marked host that hasalready been attacked (i.e., as a host recognition cue).Since very few aphids exuded this secretion prior tobeing stung, it would seem to be highly reliable as arecognition cue of already parasitized hosts. Theresponse of wasps to cornicle secretion also appears tobe adaptive for the following reason. Cornicle wax isproduced when enemies attack aphid colonies and thiscauses potential hosts of colonies to either disperse orto enter a defensive mood. In consequence, femalesshould have difficulties in finding the dispersing aphidsor should encounter defensive hosts that cannot be easilyparasitized. Recognition of already disturbed host colon-ies through cornicle secretion could then allowA. rhopa-losiphi females to spend less time foraging on them.

The second observed behavior associated with host

rejection events was sting rejection. This behavior sug-gests a reaction to an internal cue that is directly associa-ted with the presence of a developing parasite in the host.However, this parasitized host recognition cue appearsto require several hours to become effective. No evi-dence of sting rejection was actually observed for timeintervals of less than 16 h. After this threshold delay,sting rejection increased as the time interval since thefirst attack increased, leading to decreased levels ofsuperparasitism. This gradual setting-up reached amaximum of 60% of rejection at a time interval of 96h. The relatively long delay that must elapse before anyevidence of discrimination suggests that parasitoid-injected or deposited substances are unlikely to beinvolved. To account for these dynamic changes, twomechanisms are often suggested (Cloutier et al., 1984;Chow and Mackauer, 1986; Hofsvang, 1988): (1) aninternal change in host composition associated with thedevelopment of the parasitoid, or (2) the presence of amarker gradually released by the parasite into the hosthaemolymph. Whatever the exact mechanism, theinternal cue seems not to vary betweenA. rhopalosiphifemales. An identical behavioral response of waspstowards self- and conspecific-parasitized hosts wasindeed found. This suggests that a female ofA. rhopalos-iphi cannot discriminate between aphids containing itsown eggs and those containing conspecifics’ eggs.

The observed pattern of host discrimination inA. rho-palosiphi differs from that reported in other species ofaphid parasitoids. Firstly, an increasing host rejectionwith an increasing time interval from initial parasitiz-ation has already been observed in other Aphidiinae(Cloutier et al., 1984; Chow and Mackauer, 1986;Hofsvang, 1988). However, in these species, perfect hostdiscrimination ability (i.e., 100% of parasitized hostrecognition) is reached after a delay of only 1 day. InA. rhopalosiphi, perfect host discrimination is reachedafter a much longer delay because only 60% of hostsparasitized were recognized 4 days after the initial paras-itization. This peculiar pattern of host discrimination hasa consequence on parasitoid fitness. InA. rhopalosiphi,when females lay an egg in an aphid which has alreadybeen parasitized for more than 24 h, the pay-off is nil,as the second egg laid never develops (Outreman,unpublished data). Thus, oviposition in a host parasitizedfor more than 1 day has no functional value. Secondly, insome Aphidiinae, including some of those which exhibitinternal discrimination ability after 24 h, an external cueis deposited during oviposition that permits a high rateof superparasitism avoidance on freshly parasitized hosts(Mackauer et al., 1996). The observed results during theearly phase of parasitism may indicate that no markerof recently parasitized hosts was selected in this aphidparasitoid. However, host rejections in the early phaseof parasitism elicited by the presence of dried corniclesecretion on the host’s body could partly compensate for


the apparent lack of parasitoid host external cues forsuperparasitism avoidance. Nevertheless, the observedlevel of host rejections induced by this original factorsuggests that superparasitism on freshly parasitized hostsis a common event inA. rhopalosiphi.

Host discrimination and superparasitism are two keyfeatures of parasitoid behavior, determining their forag-ing strategy and reproductive success. The present studyprovides some results on superparasitism avoidance inthe aphid parasitoidA. rhopalosiphi. Females of thisspecies were shown to be able to recognise already para-sitized aphids. This result contradicts Gardner et al.’s(1984) observations and confirms the assumptions ofOutreman et al. (2000). However, this discriminationability is time-dependent and it induces some superpara-sitism, even when the pay-off of the second egg is equalto zero. Furthermore, the repellent effect of corniclesecretion tends to limit superparasitism during the periodwhere the mechanism of host discrimination is not yetefficient. This effect could partly replace the function ofan external marker found in other Aphidiinae. In thosespecies, it could be of some interest to investigatewhether the cornicle secretion also has a repellent effect.If not, this could partly explain why an external markerhas not been selected inA. rhopalosiphi.

Finally, the results indicate that the defensive behaviorof an animal towards a natural enemy could have a func-tional value for the latter. Here, chemical defensivebehavior of the hostSitobion avenaecould indirectlyenable some avoidance of superparasitism in the parasit-oid A. rhopalosiphi.

Acknowledgements

We thank E. Wajnberg (I.N.R.A. Antibes) and X. Fau-vergue (I.N.R.A. Antibes) for their continuous encour-agement and criticisms and for their precious patience,and J.F. Arnaud (Universite´ de Rennes I) and C. Rispe(I.N.R.A. Rennes) for their critical reading of the manu-script. We are grateful to two anonymous referees fortheir useful comments and suggestions on an earlier draftof the manuscript. We thank also F. Lefevre (E.N.S.A.Rennes) for technical assistance and P. Stary´ (Instituteof Entomology, Czech Republic) for parasitoid identifi-cation. This study was supported by grants from theMinistere de l’Agriculture and from the Ministe`re del’Education, de la Recherche et de la Technologie,France.

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Superparasitism limitation in an aphid parasitoid: cornicle secretion avoidance and host discrimination ability