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Acceptability and suitability of the European Ostrinia nubilalisHbnerfor Macrocentrus cingulum Brischke from Asia and Europe

E.D.M. Campan a,b,, S. Havard a,b,c, A. Sagouis a,b, C. Plissier a,b, F.J. Muller d, C. Villemant d, Y. Savriama d,D. Gury e,f, J. Hu e,f,g, S. Ponsard e,f

a Universit de Toulouse, INP, UPS, EcoLab (Laboratoire Ecologie Fonctionnelle et Environnement), 118 route de Narbonne, 31062 Toulouse, Franceb CNRS, EcoLab, 31062 Toulouse, Francec

IAS, InVivo AgroSolutions, 83 Av. de la Grande Arme, 75116 Paris, Franced CNRS, Musum National dHistoire Naturelle, UMR 7205, CP50 Entomologie/Hymnoptres, 45 rue Buffon, 75005 Paris, Francee Universit de Toulouse, ENFA, UMR5174 EDB (Laboratoire Evolution & Diversit Biologique), 118 route de Narbonne, F-31062 Toulouse, FrancefCNRS, Universit Paul Sabatier, UMR5174 EDB, F-31062 Toulouse, Franceg State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China

h i g h l i g h t s

In Asia, the parasitoid M. cingulum

parasitizes the maize pest Ostrinia

furnacalis.

In Europe, the maize pest O. nubilalis

is not a suitable host for M. cingulum.

In the laboratory, the Asian M.

cingulumparasitizes bothOstrinia

species similarly.

The AsianM. cingulum is a potential

candidate to controlO. nubilalis in

Europe.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:

Received 24 October 2013

Accepted 21 March 2014

Available online 1 April 2014

Keywords:

ParasitoidHost acceptability

Host suitability

New association

Polyembryony

Centroid size

a b s t r a c t

We examined whether Macrocentrus cingulum(Hymenoptera: Braconidae) of Asian origin could serve as a

biological control agent of the maize pest Ostrinia nubilalis (Lepidoptera: Crambidae) in Europe.M. cingu-

lumis already present in Europe, where it does not parasitize O. nubilalisbutOstrinia scapulalis, a related

species feeding on wild dicotyledons. In contrast, M. cingulum have been imported from Europe and Asia

into North America (whereO. nubilalis had been accidentally introduced from Europe), and does parasit-

izeO. nubilalisthere. We conducted laboratory infestations to assess host acceptability (parasitoids pro-

pensity to oviposit) and suitability (parasitoids ability to develop) of European O. nubilalisfor M. cingulumof European and Asian origin, and ofOstrinia furnacalis (their original host) for Asian M. cingulum. Asian M.

cingulumparasitized EuropeanO. nubilalisas readily asO. furnacalis, and developed equally well in terms

of: % female cocoons, time to first emergence from the cocoon, total number of adult offspring, % female

offspring and adult longevity. Adult female parasitoids were significantly larger when emerging from O.

nubilalis, mixed-sex and male cocoons were significantly more and less frequent, respectively. The

acceptability ofO. nubilalis was significantly lower for European than for AsianM. cingulum, and its suit-

ability was zero. AsianM. cingulum appears a potential candidate for introduction as a biological control

http://dx.doi.org/10.1016/j.biocontrol.2014.03.013

1049-9644/2014 Elsevier Inc. All rights reserved.

Corresponding author at: Laboratoire Ecologie Fonctionnelle et Environnement,

UMR 5245 (CNRS-UPS-INPT), Btiment 4R1, Universit Paul Sabatier Toulouse III,

118, route de Narbonne, 31062 Toulouse Cedex 9, France. Fax: +33 561558901.

E-mail address: [email protected](E.D.M. Campan).

Biological Control 74 (2014) 1320

Contents lists available at ScienceDirect

Biological Control

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y b c o n

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agent of a major maize pest, European O. nubilalis, provided environmental impact studies, economic

analyses, and foreseeable interactions with other biological control agents such as the egg parasitoid

Trichogramma brassicae(Hymenoptera: Trichogrammatidae) are satisfying.

2014 Elsevier Inc. All rights reserved.

1. Introduction

Classical biological control consists in introducing natural ene-

mies into places where they do not occur naturally, to control pests

considered undesirable or too abundant (Waage et al., 1988). Tar-

gets are often exotic species accidentally introduced into a new

geographic area, and a logical place to look for potential biological

control agents is the pests area of origin. However, rather than or

in addition to seeking to re-assemble species communities that

exist elsewhere, Hokkanen and Pimentel (1984, 1989) advocate

the creation of new associations, -i.e., introducing natural ene-

mies from places where the pest is absent and where they attack

species of the same genus or family as the target pest.

Here, we examine the possibility that the larval parasitoid Mac-

rocentrus cingulum Brischke (Hymenoptera: Braconidae, also

known asM. abdominalis Fabricius (Baker et al., 1949),Macrocen-

trus grandii Goidanich or Macrocentrus gifuensis Ashmead (Van Ach-

terberg and Haeselbarth, 1983)) of Asian origin might be a

promising candidate for introduction into Europe as a biological

control agent of a major maize pest, the European corn borer

(ECB) Ostrinia nubilalis Hbner (Lepidoptera: Crambidae). At first

glance, this may seem very unlikely. Indeed,M. cingulumis already

present in Europe (Van Achterberg, 1993; Yu et al., 2005), where it

parasitizes Ostrinia scapulalis Walker sensu Frolov et al. (2007), a

related host species that feeds mostly on mugwort (Artemisia vul-

garis L.). However, despite intensive surveys (Plissi et al., 2010

and references therein;Folcher et al., 2011),M. cingulumhas never

been recorded emerging from ECB in Europe, even in sympatry

with infested O. scapulalis. Moreover,Plissi et al. (2010)detected

M. cingulum DNA in field-collected ECB in France, suggesting that

M. cingulum does oviposit in European ECB (ECBE) at least to a

certain extent , but is unable to complete development until adult

stage in this host. In Eastern Asia, where the ECB is absent, M. cin-

gulumparasitizes the maize pest Ostrinia furnacalis Gune (ACB)

(He et al., 2000). Hence, it might seem that only the ACB and O.

scapulalis, not the ECB, are suitable hosts for M. cingulum.

On the other hand, evidence from biological control attempts in

North America calls this conclusion in question. The ECB was acci-

dentally introduced there in the early 20th century (Baker et al.,

1949). Over the following decades, several parasitoids including

M. cingulumfrom Europe and Asia were released in various loca-tions (Thompson and Parker, 1928; Baker et al., 1949). Only incom-

plete information is available about which releases resulted in

long-term establishment of self-sustaining parasitoid populations.

Nonetheless,M. cingulumappears to have been the most successful

among the released species, and it can still be found nowadays par-

asitizing North American O. nubilalis (ECBNA) field populations

(Plissi et al., 2012; Sked, 2003 and included references), although

releases stopped long ago. Hence, the global result of these releases

is thatM. cingulum somehow became established in North Amer-

ica. If not by the M. cingulum introduced from Europe, American

populations must have been founded by those introduced from

Asia. Available phylogeographic analyses albeit on a very limited

sample of American individuals, all from the same location are

consistent with this hypothesis (Plissi et al., 2012), as are mostrecorded short-term success rates of the releases (Baker et al.,

1949). Similar intraspecific geographic variability in the ability to

parasitize a given host species has been documented, e.g., within

the moth parasitoids Cotesia sesamiae (Hymenopera: Braconidae)

(Ngi-Song et al., 1998) and Cotesia flavipes (Potting et al., 1997)

or the fruit fly parasitoids Asobara tabida (Hymenoptera: Braconi-

dae) (Kraaijeveld and Van Alphen, 1994) and Leptopilina boulardi

(Hymenoptera: Figitidae) (Dupas and Boscaro, 1999).

Alternatively or in addition to such polymorphism among geo-

graphic taxa ofM. cingulumfor their ability to parasitize ECB, there

might be polymorphism among geographic taxa of ECB for their

ability to prevent M. cingulum from developing to adult stage. A

population bottleneck during the colonization of North America

by ECB and/or post-colonization evolution of ECBNA populations

may have resulted in such polymorphism. A previous study

(Havard et al., 2014) did not detect any difference in encapsulation

ability between ECBNAand ECBEpopulations, but they may differ in

other aspects of immune response. Geographic intra-specific dif-

ferences in quality as a host for a given parasitoid species have also

been documented elsewhere (e.g., withinDrosophila melanogaster:

Kraaijeveld and Van Alphen, 1995).

We conducted laboratory experiments to assess the acceptabil-

ity for oviposition and suitability for parasitoid development of

ECBEas a host for Asian M. cingulum. To this effect, we compared

the propensity to oviposit and the ability to develop to adult stage

of Asian M. cingulum (MA) in its original host, the ACB, with those of

(1) MA in ECBEand (2) European M. cingulum (ME) in ECBE.

2. Materials and methods

2.1. Establishment of colonies and origin of insects

All rearings and experiments were conducted at 2123 C, with

a 16:8 Light:Dark photoperiod and 74% Relative Humidity. Adult

parasitoids had access to a piece of paper soaked in ca. 10% honey

water during rearing, mating or parasitism experiments, but had

access to only water .when offspring longevity was assessed.

We founded our MAand ACB strains with a mixture of individ-

uals from the corresponding laboratory strains maintained at the

State Key Laboratory of Biocontrol (Sun Yat-Sen University,

Guangzhou, China) and the Institute of Plant Protection (Chinese

Academy of Agricultural Sciences, Beijing, China). These strains

had been founded with individuals obtained from wild ACB feeding

on maize and collected in various places across China including but

not limited to the surroundings of Hangzhou. Our MA strain was

routinely reared on our ACB strain. Adult M. cingulum were kept

in BugDorm population cages (30 30 30 cm). Once exposed

to parasitism, individual hosts were never put back to the rearing

even if they survived until adult stage, so as to avoid selecting

for resistance against the parasitoid.

Our ECBEstrain was founded from larvae collected on maize

near Toulouse (France). The MEwe used did not come from a lab-

oratory culture, but directly from parasitized O. scapulalis larvae

collected in mugwort during their winter diapause near Amiens

(France). These diapausing larvae were kept at 5 C in the dark un-

til being transferred to diapause-breaking conditions (transparent

plastic boxes containing water-soaked paper next to the larvae toincrease humidity, and kept under the same temperature, photope-

riod and moisture conditions as rearings and experiments). ECBEand ACB larvae were reared on artificial diet (Zhou et al., 1980).

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2.2. Treatments

MA females were allowed to parasitize ACB, their natural host

(MA-ACB), or ECBE, the new host (MA-ECB), to compare their

acceptability and suitability. In addition, MEfemales were allowed

to parasitize ECBElarvae (ME-ECB) to compare the acceptability ofECBE for MA vs. ME, and to confirm that ECBE suitability for ME is

near or equal to zero, as suggested by field observations (Plissi

et al., 2010). To compare host mortality in our trials with that of

hosts not exposed to parasitism, we conducted the same trials

without parasitism for ECBE (Control-ECB) and ACB (Control-

ACB). It would have been interesting to also study ME being al-

lowed to parasitize O. scapulalis, their natural host, but this was

impossible for practical reasons: we repeatedly tried but failed to

establish a strain ofO. scapulalis.

2.3. Parasitism experiments

All M. cingulum females used in our experiments were 3 days

old. Just after emergence, adult parasitoids were placed in1811.5 7 cm plastic boxes containing several tens of same-

age males and females, resulting in a very high probability for fe-

males to be mated after three days (Parker, 1931). Females had

no contact with any host larva before the experiment, hence no

prior oviposition experience.

Maize stems were collected in late July, when plants were still

green andca. 1.8 m high. Stems were cleaned and deep-frozen at

20 C. Before being fed to the larvae, the needed amount of maize

was defrosted and cut into pieces the size of a matchstick.

For each treatment or control (MA-ACB, MA-ECB, ME-ECB, Con-

trol-ECB, Control-ACB), we conducted 16 replicates. For each repli-

cate (Fig. 1), sixty 3rd-instar host larvae were removed from

artificial diet, placed in a plastic box ( 12 cm, height 8 cm) and

fed on maize for 24 h so that they produced frass (which stimulatesparasitoid oviposition: Ding et al., 1989). Then (d0), ten 3-d-old

parasitoid females were placed in the box and left with the host

larvae, maize and frass for 24 h. On d1, surviving parasitoids were

counted and discarded, surviving larvae were counted and trans-

ferred onto artificial diet. Of 60 larvae, on average 55 survived until

d1 (minimum 45, maximum 60).

2.3.1. Host acceptability

On d4, 20 larvae from each replicate of MA-ACB, MA-ECB, ME-

ECB were deep-frozen for later molecular detection ofM. cingulum

DNA. Total DNA was extracted from the entire body of each larva

using the DNeasy Blood and Tissue kit (Qiagen, Venlo, The Nether-

lands) with a single elution in 200lL. Extracts were stored at20 C until PCR amplification. We used specific primers Mcing28f

(50-ACACTATCACGCACATTCGC) and Mcing28r (50-AACTGTGTT

ATCGACAAACTATAGAGC) (Molecular Ecology Resources Primer

Development Consortium, 2012). We conducted PCRs in 25ll con-

taining 5 ll Taq (Qiagen), 3.8 ll H2O, 1 ll DNA and 0.1 ll of each

primer at a concentration of 50 nmol/ml. The PCR included a dena-

turation at 94 C for 3 min followed by 35 cycles of 95 C for 1 min,

61 C for 45 s and 72 C for 1 min, and a final elongation at 72 C for

7 min.

PCR products were separated by electrophoresis on a 2% agarose

gel stained with ethidium bromide. All PCR amplifications included

a negative control a total DNA extract from an unparasitized ACB

or ECBElarva, depending on which host species was being assessed

, and a positive control a DNA extract from the European orAsianM. cingulum, depending on the origin of the parasitoid being

assessed. Although each treatment was replicated 16 times, only

the first 15 sets of 20 larvae were submitted to PCR, as time was

short and results sufficiently clear-cut.

We estimated initial parasitism rates for each replicate as the

proportion of hosts yielding an amplification among the 20 larvae

submitted to PCR.

2.3.2. Host suitability

M. cingulum is polyembryonic (Parker, 1931): after being laid

into a 3rd or 4th instar host larva, each egg divides into several em-

bryos. During the hosts 5th instar, the parasitoid larvae exit and

pupate together as a cocoon mass containing up to 80 individuals

(White and Andow, 2008). Henceforth, the term cocoon refers toone such cocoon mass.

All larvae except those used for PCR were left on artificial diet

until they pupated (if parasitism failed), turned into a M. cingulum

cocoon (if parasitism succeeded), or died as a host larva. Host pu-

pae were counted and destroyed. Parasitoid cocoons were isolated

in individual Petri dishes ( 5.5 cm), checked daily for adult

Fig. 1. Experimental procedure (numbers are for 1 replicate).

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emergence, and classified as all-female, all-male, mixed or

non viable if only females, only males, both males and females,

or no adult emerged, respectively. We estimated efficient parasit-

ism as the number ofM. cingulumcocoons from which at least one

adult emerged (viable cocoons) over the total number of viable

cocoons and host pupae obtained from a given replicate. Basedon previous experience, we considered that most if not all host pu-

pae would survive to adult stage, but we did not formally check it

(they were destroyed at pupal stage). We estimated post-d1 mor-

tality (i.e., mortality after the parasitization phase, Fig. 1) as 1

minus the ratio between the number of host larvae that turned into

either a pupa or a viable cocoon and the number of larvae that

were transferred onto artificial diet after d1 (subtracting the 20 lar-

vae submitted to PCR when applicable).

The number of adults of each sex emerging from each cocoon

was recorded. Time to first emergence for a given cocoon is the

time interval between d0 and the first adult emergence. Adult lon-

gevity was estimated for a subsample of 197 males and 106 fe-

males from MA-ECB and 272 males and 68 females from MA-ACB

by transferring newly emerged adults into individual Petri disheswith moist paper and checking them daily until death. The size

of the right forewing was estimated for a subsample ofM. cingulum

females (131 emerged from ECBE, 271 from ACB) by characterizing

its shape by geometric morphometrics and calculating its centroid

size. To this effect, wings were mounted on glass slides in 95% eth-

anol and photographed using a Leica x2 panachromatic objective

(magnification: 1.6) with a 5 megapixels CCD Leica DFC420 camera

fitted to a Leica Z6 APO dissecting microscope. Fifteen landmark

data were collected on each picture using tpsDig2 2.16 (Rohlf,

2006).

2.3.3. Female parasitoid survival

For each replicate, we recorded the proportion ofM. cingulum

females retrieved alive on d1 from the 10 introduced on d0, in or-der to check whether hosts had been submitted to similar parasit-

ism pressure in all treatments. Unexpectedly, this comparison

revealed that parasitoid female survival from d0 to d1 had been

significantly lower in MA-ACB than in both other treatments (see

Section4). Therefore, we used parasitoid survival as an additional

explanatory variable when applicable in our data analyses.

3. Data analysis

All analyses except those of wing size were conducted using

software R (R Core Team, 2012). We compared proportions (initial

and efficient parasitism, female parasitoid survival between d0 and

d1, proportions of all-male, all-female or mixed-sex cocoons, pro-

portion of females in the adult offspring) or counts (number of

adult offspring) using generalized linear mixed models (GLMMs)

fitted using procedure glmmPQL (library nlme), with proportions

considered drawn from a binomial and counts from a Poisson dis-

tribution. When the deviance:degrees of freedom ratio was great-

er than one, proportions or counts were considered drawn from a

quasi-binomial or quasi-Poisson distribution, respectively. Treat-

ment and Replicate were modelled as fixed and random effects,

respectively. In addition, in order to correct for a possible differ-

ence in exposure to parasitism (see Section2.3.3.), Female parasit-

oid survival between d0 and d1 was included as a second fixed

effect when the initial parasitism rate or the proportion of

mixed-sex cocoons were the dependent variable. In two cases

(comparison of efficient parasitism rates between treatments),

we had to compare positive values (in MA-ECB and MA-ACB) withvalues consistently equal to zero (in ME-ECB), a situation where

GLMM is inappropriate. In these two cases, we first compared all

three treatments using a non-parametric KruskalWallis test

followed by Dunns multiple contrasts post hoc test (Siegel and

Castellan, 1988) using procedure kruskalmc (library pgirmess,

P= 0.05) to identify significantly different pairs of treatments, then

compared only MA-ECB and MA-ACB using a GLMM to maximize

statistical power. We compared initial and efficient parasitism

using paired t-tests (one per treatment, checking that the differ-ences were normally distributed using Shapiro tests: details not

shown). Finally, we used a Cox proportional hazards regression

model fitted using procedure coxph (library survival) to examine

the influence of Treatment and Cocoon type (male, female or mixed

on time to first emergence and of Treatment and Sex on adult lon-

gevity. In all analyzes including more than one fixed effect, all

interactions were tested in a preliminary analysis and removed

from the final analysis because they were found non-significant

(P> 0.05).

The analysis of wing sizes was carried out using MorphoJ ver-

sion 1.05c (Klingenberg, 2011). Centroid size was computed from

a generalized Procrustes fit as the square root of the sum of

squared distances of all landmarks to their centroid (e.g., Dryden

and Mardia, 1998). For a subset of 20 specimens all from the samecocoon, wings were mounted and digitized twice to assess mea-

surement error due to these steps. In this subset, a one-way nested

Procrustes ANOVA of centroid size was used to test for the impor-

tance of mounting and digitizing error compared to variation

among individuals (e.g.,Klingenberg et al., 2002). As measurement

error (mounting and digitizing) appeared negligible (F= 1.39,

P = 0.138) compared to variation among individuals (F= 312.65,

P< 104), each wing was digitized only once for the complete data-

set and a simpler one-way Procrustes ANOVA was used, with Cen-

troid Size as the dependent variable, Host species as the main

effect and a Remainder term that included all measurement er-

rors. The influences of differences among cocoons or sample sizes

were eliminated by correcting for the differences in means

(Klingenberg et al., 2001).

All average values are given 1SEM.

4. Results

4.1. Exposure to parasitism

Unexpectedly, the proportion of female parasitoids that sur-

vived until the end of d0 was significantly different between

MA-ACB and MA-ECB (48.2 7.7% vs. 72.5 6.0%, respectively,

t= 2.601, P= 0.014). It did not differ significantly (t= 1.659,

P= 0.108) between MA-ECB and ME-ECB (57.5 5.8%).

4.2. Host acceptability

Average initial parasitism rates (Fig. 2) in MA-ACB and MA-ECB

were 69.3 3.9% and 71.3 5.1%, respectively, and did not differ

significantly from each other (t= 0.125, P = 0.901). In contrast,

initial parasitism was lower in ME-ECB (26.3 6.3%) than in

MA-ECB (t= 4.902, P< 104). We detected no significant influence

of female parasitoid survival on initial parasitism rate

(t= 0.772, P= 0.447).

4.3. Host suitability

In ME-ECB, efficient parasitism rate was 0%, as no parasitoid co-

coon ever formed in this treatment. KruskalWallis tests con-

ducted on all three treatments detected a significant influence of

Treament on efficient parasitism (v2 = 32.782, P< 10

7), whichwas due to both MA-ACB and MA-ECB differing significantly from

ME-ECB (P< 103), but not from each other (P> 0.05). Efficient par-

asitism rates (Fig. 2) i n MA-ACB and MA-ECB were on average


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72.7 4.0% and 74.6 6.0%, respectively, and did not differ signifi-cantly from each other (t= 0.252, P= 0.805) according to the

GLMM analysis.

There was no significant difference between initial and efficient

parasitism in MA-ACB (t= 0.403, P= 0.693) or MA-ECB (t= 1.224,

P= 0.241), but there was in ME-ECB (t= 4.480,P< 103).

Average post-d1 mortality rates (Fig. 3) in MA-ACB (29.9 3.1%)

and MA-ECB (25.5 2.8%) were not significantly different from

each other (t= 1.007, P = 0.322), but mortality was significantly

lower in ME-ECB (16.3 2.6%) than in MA-ECB (t= 2.320,

P= 0.028). Post-d1 mortality was significantly lower in Control-

ACB (11.0 1.6%) than in MA-ACB (t= 5.666,P< 104), but no sig-

nificant difference was detected between Control-ECB

(19.0 4.3%) and MA-ECB (t= 1.187, P= 0.245) or ME-ECB

(t= 0.776,P= 0.444). Finally, mortality was significantly higher in

Control-ECB than in Control-ACB (t= 2.263, P= 0.039).

Therewasno significantdifferencebetween theproportion of all-

female cocoons among viable cocoons in MA-ACB (13.0 3.5%)vs.

MA-ECB (8.6 1.7%,t= 0.884, P= 0.391). In contrast, there was a

significantly larger proportion of all-male cocoons in MA-ACB

(65.9 5.1%) than in MA-ECB (49.9 5.2%, t= 2.896, P= 0.011),

offset by a significantly smaller proportion of mixed-sex cocoons

in MA-ACB (21.0 3.3%) than in MA-ECB (41.4 4.7%, t= 3.314,

P= 0.005). As for infestation rates (see above), the proportion of

femaleparasitoids that surviveduntil d1had nosignificantinfluence

on the proportion of mixed-sex cocoons: t= 0.785, P= 0.446).

Despite these differences in the proportion of male and mixed-

sex cocoons, the proportion of females among the adult offspring

was not significantly different between MA-ACB (22.1 4.6%) and

MA-ECB (23.8 3.4%,t= 1.170, P= 0.260,Fig. 4). This was the net

result of MA-ACB cocoons tending to produce more male and fewer

mixed-sex cocoons which should decrease the proportion of fe-males but also fewer adults per cocoon, the difference tending

to be stronger for males than for females which should increase

the proportion of females (21 and 17 individuals in all-male and

all-female cocoons, respectively, and 10 females and 11 males in

mixed-sex cocoons, against 25 and 18 individuals, and 12 females

and 16 males in the corresponding categories for MA-ECB). The

average total number of adult offspring was also not significantly

different between MA-ECB (480.1 59.4) and MA-ACB

(370.6 41.3,t= 1.701,P= 0.110,Fig. 4).

We detected no effect of Treatment on Time to first emergence

(v2 = 0.230, P= 0.132), but Cocoon type (male, female or mixed)

had a significant influence (v2 = 18.654, P< 104). Time to first

emergence was very similar for all-male cocoons in MA-ECB

(27.9 0.3 days) and MA-ACB (27.3 0.2 days) and all-female co-coons in MA-ECB (27.7 0.5 days) and MA-ACB (27.3 0.2 days),

and slightly shorter in mixed-sex cocoons (MA-ECB:26.6 0.2 days,

MA-ACB: 27.2 0.2 days,Fig. 5).

We found no significant difference in adult longevities between

Sexes (v2 = 0.136, P= 0.712). The influence of Treatment was sig-

nificant (v2 = 4.828, P = 0.028), but differences were very small:

the average adult longevities of M. cingulum offspring were

3.6 0.1 days for MA-ECB and MA-ACB males, and 3.5 0.1 and

3.7 0.1 days for MA-ACB and MA-ECB females, respectively.

Female offspring size (as estimated by right forewing centroid

size) was significantly larger in MA-ECB (2.47 0.02 mm) than in

MA-ACB (2.30 0.01 mm,F= 82.29, P< 104).

5. Discussion

5.1. The ECB is not a suitable host for ME

Initial parasitism rate was significantly lower when ECBElarvae

were exposed to MEthanto MA. Efficient parasitism was zero, as no

MEcocoon ever formed in ECBE. Post-d1 mortality was not signifi-

cantly different from control mortality. Hence, our experiments

confirm that MEdoes oviposit in ECBEbut is unable to develop to

the adult stage, and does not even appear to cause any additional

mortality to parasitized host larvae. This was suspected by Plissi

et al. (2010), who detected M. cingulum DNA in field-collected ECBE,

while no adult ever emerged from such larvae. The massive para-

sitism of O. nubilalis by M. cingulum described by Thompson

and Parker (1928)in Europe was most probably actually restricted

Fig. 2. Initial and efficient parasitism rates.

Fig. 3. Post-d1 mortality of the host larvae. Fig. 4. Number of adult parasitoid offspring and percentage of females in it.


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Finally, if introduced, MA may interact with other biological

control agents attacking ECBE Nosema spp. (Microspora: Nose-

matidae), Beauveria sp. (Hypocreales: Clancipitaceae), and insects

parasitizing egg or larval stages (Thompson and Parker, 1928).

The outcome of these interactions is difficult to predict, and likely

depends on many factors such as environment, phenology and ini-tial parasitism rates. M. cingulumlongevity appears negatively af-

fected by infection by Nosema spp. in their ECBA host (Andreadis,

1980; Cossentine and Lewis, 1987), and parasitism byM. cingulum

has been found negatively correlated with the rate of infection by

Nosemaspp. when the latter is high (Andreadis, 1982), suggesting

that M. cingulumcontributes little to biological control of ECBA in

populations where the prevalence of Nosema spp. is high. In con-

trast, concerning the only natural enemy commercially released

at broad scale, the egg parasitoid Trichogramma brassicae,M. cingu-

lum might have a complementary action by parasitizing larvae

hatching from eggs that escaped T. brassicae. The latter is exten-

sively used in Europe (e.g., on 80,000 ha in France: LeRoux et al.,

2008, i.e., 20% of the total surface treated against ECB) but can be

overwhelmed when initial parasitism rates are particularly high.When this happens, usingM. cingulummight reduce pest pressure

in the subsequent generation or year, thereby restoring conditions

whereT. brassicae can again provide optimal crop protection.

5.4. Potential benefits of an introduction of MAinto Europe

As far as we know, M. cingulum is not used in inundative re-

leases in the USA or in Asia. This might be because no cost-effective

rearing method is available and/or because other means of crop

protection (pesticides or genetically modified (Bt-) maize) are con-

sidered sufficient. The situation might be different in Europe ifBt-

maize adoption rates remain low and if the trend towards limita-

tion of pesticide use intensifies. Moreover, if maize prices continue

to rise, farmers may become willing to spend more than before on

crop protection.

If inundative biological control (Waage et al., 1988) proved too

expensive, introduction might be an option. The cost would be

lower, and the North American experience shows that self-sustain-

ing M. cingulumpopulations can persist for several decades. In Chi-

na and the USA, the self-sustaining populations of M. cingulum

typically cause parasitism rates ranging between 0% and 20%,

which does not maintain ACB or ECB belowlevels where other crop

protection methods are no longer needed, but even a moderate

parasitism rate may be worth the moderate cost of a one-time

introduction.

A possibly promising attempt could be to introduce American

M. cingulum instead of or in addition to MA. If certain traits facili-

tate parasitismon ECB as opposed to ACB , these traits may have

been selected for in North American populations.

Globally speaking, the case ofM. cingulumillustrates the impor-

tance of accurate taxonomy in biological control endeavors: dis-

covering that O. scapulalis and O. nubilalis are different species

was a first step to understand why introductions of European M.

cingulumagainst ECBNA mostly failed.Plissi et al.s (2012)study

suggesting that ME populations may result from a westward

expansion of genetically diverse MA populations, during which

they lost some generalist traits, may explain why introductions

of Asian rather than European M. cingulum apparently succeeded

in North America and might succeed in Europe.

Acknowledgments

We thank Wang Zhenying for providing ACB and M. cingulumindividuals, E. Sturm et R. Calvignac for help with insect rearing,

J. Cami and R. Campan for providing maize and two anonymous

referees for comments. Funds were provided by project BIOCOS-

MAC (ANR-08-STRA-05-02) and an APC-Universit Toulouse III

grant. Laboratoire Evolution and Diversit Biologique is partly sup-

ported by TULIP (ANR-10-LABX-41).

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