Testing semiochemicals from aphid, plant and conspecific: attraction of Harmonia axyridis

Insect Science (2012) 19, 372–382, DOI 10.1111/j.1744-7917.2011.01449.x

ORIGINAL ARTICLE

Testing semiochemicals from aphid, plant and conspecific:attraction of Harmonia axyridis

Pascal D. Leroy1, Thomas Schillings1, Julien Farmakidis1, Stephanie Heuskin2, Georges Lognay2,Francois J. Verheggen1, Yves Brostaux3, Eric Haubruge1 and Frederic Francis1

Departments of 1Functional and Evolutionary Entomology, 2Analytical Chemistry and 3Applied Statistics, Computer Science and

Mathematics, University of Liege, Gembloux Agro-Bio Tech, Gembloux, Belgium

Abstract Harmonia axyridis Pallas (Coleoptera: Coccinellidae) is an invasive specieaffecting the dynamics and composition of several guilds. Nowadays, no biological con-trol method is available to reduce the populations of this harmful coccinellid. Attractantsand semiochemicals seem to be the best alternative but only few studies have testedthe impact of semiochemicals on this Asian lady beetle. In this work, through wind-tunnel experiments, semiochemicals from aphids (Z,E-nepetalactone, [E]-β-farnesene,α-pinene and β-pinene), from coccinellids ([-]-β-caryophyllene) and from the nettle Ur-tica dioica L. were evaluated as potential attractants. The nettle volatile compounds ([Z]-3-hexenol and [E]-2-hexenal) were extracted using a Clevenger Apparatus

R©and identified

by headspace gas chromatography–mass spectroscopy. In the wind-tunnel experiments,the main components of the aphid alarm pheromone as well as a component of the aphidsexual pheromone strongly attracted both sexes of the Asian lady beetle while (-)-β-caryophyllene only attracted few individuals and had no impact on the males. The nettleextract as well as the (Z)-3-hexenol oriented both males and females to the odor source.The (E)-2-hexenal was shown to have no effect on females even if this green leaf volatileattracted males. Because Z,E-nepetalactone was identified as the most efficient attrac-tant in the wind-tunnel experiments, this volatile was also tested in a potato field whereH. axyridis has been showed to respond to this semiochemical. This study highlightedthat Z,E-nepetalactone orientated the Asian lady beetle H. axyridis under natural condi-tions, indicating that this volatile compound could certainly help for an efficient biologicalcontrol approach against this invasive specie.

Key words aphids, attractant, Harmonia axyridis, plant, semiochemicals, wind-tunnel

Introduction

The Asian lady beetle, Harmonia axyridis Pallas(Coleoptera: Coccinellidae) was introduced into Europeand North America as an efficient biological control agentof different arthropod pests. Indeed, this predator presents

Correspondence: Pascal D. Leroy, Department of Functionaland Evolutionary Entomology, University of Liege GemblouxAgro-Bio Tech, Passage des Deportes 2, B-5030 Gembloux,Belgium. Tel: +32 81 62 22 81; fax: +32 81 62 23 12; email:[email protected]

all the characteristics required to be used against manypests: large body size, high voracity and high predationefficiency (Soares et al., 2001; Labrie et al., 2006), highcolonization aptitude (With et al., 2002), rapid develop-ment, high fecundity and low susceptibility to pathogensor natural enemies (Marco et al., 2002). However, forthese reasons, this exotic predator has rapidly become aninvasive specie presenting negative impacts on native coc-cinellids and affecting the dynamics and composition ofseveral guilds (Soares et al., 2008). In this sense, sev-eral recent studies have demonstrated that native ladybeetles (e.g. Adalia bipunctata L. and Coccinella septem-punctata L.) are consumed by this exotic lady beetle in

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field crops (Koch, 2003) since H. axyridis adults andlarvae both have been shown to present a higher pre-dation and foraging efficiency than indigenous species(Labrie et al., 2006; Lanzoni et al., 2004). Furthermore,H. axyridis constitutes a human nuisance by damagingfruit crops in late summer (Kovach, 2004; Koch et al.,2004) and by migrating from the field into houses whereit massively aggregates during winter and by excretinghemolymph (containing alkaloids) with an unpleasantodor that can lead to allergic reactions (Huelsman et al.,2002).

Presently and according to Kenis et al. (2008), nobiological control method is available to lower popula-tion densities in the natural environment and to limitthe impact of the lady beetle H. axyridis on nativespecies. Indeed, natural enemies including pathogens (thefungi Metarhizium anisopliae [Ginsberg et al., 2002] andBeauveria bassiana [Roy et al., 2007]), para-sitoids (Strongygaster triangulifera [Diptera: Tachinidae][Katsoyannos & Aliniazee, 1998] and Dinocampus coc-cinellae [Hymenoptera: Braconidae] [Obrycki et al.,1985]), predators (birds, ants, spiders [Dutcher et al.,1999]) and a parasitic mite (Coccipolipus hippodamiae[Acari: Podapolipidae] [Webberley et al., 2004]) do notshow sufficient potential to regulate the H. axyridisinvasion.

Nowadays, attractant and deterrent semiochemicalsseem to represent the best option to develop efficienttrapping systems against this invasive and harmful coc-cinellid. In this sense, Verheggen and colleagues (2007)have previously studied the impact of the H. axyridisvolatile aggregation pheromone (-)-β-caryophyllene andthe effect of the main aphid alarm pheromone compo-nent (E)-β-farnesene on this coccinellid. These authorsdemonstrated that both (-)-β-caryophyllene and (E)-β-farnesene attract the Asian lady beetle under laboratoryconditions. In contrast, some plant compounds, such ascamphor and menthol, have proved to be repellent toH. axyridis in field and laboratory tests (Riddick et al.,2000).

Interestingly, during field observations, Alhmedi andcolleagues (2006, 2007, 2009) noted that H. axyridis wasmainly found on Urtica dioica L., the perennial and cos-mopolitan stinging nettle well known as a food resourcefor many insects. The Asian coccinellid was particularlyshown to feed on this nettle infested with Microlophiumcarnosum Buckton, an aphid consumed by a wide rangeof beneficial insects (Perrin, 1975).

Based on all these observations, the present workhas focused on semiochemicals from different origins:(i) Z,E-nepetalactone identified as a component of theaphid sex pheromones (Dawson et al., 1987); (ii) (E)-

β-farnesene, α-pinene and β-pinene: the main compo-nents of the aphid alarm pheromone (Mondor et al.,2000; Pickett & Griffiths, 1980; Francis et al., 2005a,b);(iii) (-)-β-caryophyllene, the H. axyridis volatile aggre-gation pheromone (Brown et al., 2006); and (iv) (Z)-3-hexenol and (E)-2-hexenal, the main volatile compoundsidentified in this study from damaged nettle leaves. In afirst step, all these volatile compounds were tested throughwind-tunnel experiments with the aim to identify attrac-tants and/or repellents affecting H. axyridis foraging be-havior. In a second step, semiochemicals that had a sig-nificant impact on H. axyridis during the wind-tunnelexperiments were tested in a potato field to determinetheir efficiency under natural conditions.

The main goal of this research was to determine poten-tial chemical cues that could be used in a biological controlapproach against the invasive coccinellid H. axyridis.

Materials and methods

Insects

In a climate-controlled room (16-h light photoperiod;60% ± 5% RH; 20 ± 2◦C), host-plants – Vicia faba L.– were grown in 9 × 8 cm plastic pots containing a mix-ture of vermiculite and perlite (1/1) and were infestedwith the aphid Aphis fabae Scopoli. In the same climaticconditions, but in a different room, H. axyridis larvaewere obtained from a mass-production: adults were rearedwith sugar, pollen and water and the oviposition was in-duced during 3 h by the introduction of aphid-infestedhost-plants in rearing boxes (20 × 15 × 15 cm plasticboxes pierced with two holes and re-covered with metalscreening on each lateral side). The complete life cy-cle took place in the rearing boxes, daily supplied withaphids. Initial adult individuals were collected in fieldcrops between July and December 2009 near to Gembloux(Belgium).

Nettle volatiles extraction

Urtica dioica L. nettles were collected in Gemblouxand directly used to extract the volatile compounds. Thenettle leaves, after washing, were manually damaged inorder to simulate an aphid infestation. A whole nettle plant(damaged leaves + stems) (≈70 g) was then placed into750 mL of milli-Q water to realize the extraction with aClevenger Apparatus

R©(Filter Service, Eupen, Belgium)

during 2 h using boiling water. After 30 min of cooling,the aqueous extract was transferred into a 10 mL vial. Fivereplications were performed.

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Nettle volatile compounds identification

One hundred microliters of nettle extract were placedinto vials (20 mL) maintained at 40◦C during 30 min withagitation speed at 250 r/m. A volume of 1 mL of headspacevolatile vapor was injected into gas chromatography–massspectroscopy (GC-MS). GC-MS analyses were carried outon an Agilent 7890A GC System coupled with an Agilent5975C inert XL EI/CI mass selective detector with Triple-Axis Detector and equipped with a HP-5MS (Agilent,Diegem, Belgium) capillary column (30 m × 0.25 mminternal diameter, 0.25 μm film thickness). The oven tem-perature program was initiated at 40◦C, held for 5 minthen raised first at 8◦C/min to 220◦C, held for 5 min andraised in the second ramp at 25◦C/min to 290◦C and heldfor 5 min. Other operating conditions were as follows:carrier gas, He; with a constant flow rate of 1 mL/min;injector temperature, 270◦C; splitless mode. Mass spectrawere taken at 70 eV. Mass range was from mass-to-chargeratio (m/z) 35 to 350 amu (atomic mass unit). The nettlevolatile components were identified by comparing theirmass spectra fragmentation patterns with those stored inthe Wiley275 L computer library. Five replications wereperformed for the analyzed samples and for blanks (emptyvials).

Wind-tunnel assays

The (-)-β-caryophyllene (identified from theH. axyridis aggregation site), the Z,E-nepetalactone(a component of the aphid sex pheromones), the (E)-β-farnesene, the α-pinene and the β-pinene (themain components of the aphid alarm pheromone)as well as the (Z)-3-hexenol and (E)-2-hexenal (the2 main volatile compounds identified from nettleplants) were tested on H. axyridis. The chemical cues(-)-β-caryophyllene (97.7%), (E)-β-farnesene (83.8%)and Z,E-nepetalactone (96.7%) were produced afteressential oil fractionation (flash chromatography) bythe Department of Analytical Chemistry (University ofLiege, Gembloux Agro-Bio Tech) according to Heuskinet al. (2009, 2010) while the other semiochemicals(GC putity >97%) were purchased from Sigma-Aldrich(Steinheim, Germany). Each semiochemical was formu-lated in paraffin oil solution (10 mg/mL). Rubber septawere filled with 100 μL of the solutions and fixed on aV. faba plant stem. The aqueous nettle extract was alsotested by filling a rubber septum with 100 μL of theextraction product.

Clean plants constituted the negative controls and thepositive control consisted of plants infested with 50 A.

Fig. 1 Wind-tunnel to record Harmonia axyridis behaviors inresponse to semiochemicals. 1, Vicia faba plants + the odorsource (rubber septum); 2, lady beetles; 3, Fan; 4, wind direction.

fabae aphids. Ten H. axyridis (3–5 weeks old) were in-troduced into the wind-tunnel, 70 cm downwind from theodor source. The wind-tunnel was constructed of glass(L × W × h = 80 × 9.0 × 9.0 cm) and a fan was usedto pump the air through the tunnel and toward the insectswith a velocity of 4 cm/s (laminar flow) (Fig. 1). Temper-ature and RH were 20 ± 2 ◦C and 60%–70%, respectively.Individuals were given 1 h to respond in each set of ex-periments and their positions were observed every 20 minaccording to three defined zones: Zone 1 comprising thelure (plant + semiochemical) and Zone 3 representingthe departure point of the insects. The wind-tunnel wascleaned with hexanol and water after each experiment.Three replications per sex were performed for each testedlure in order to test 30 individuals per sex. The test datawere then analyzed by fitting a generalized linear mixedmodel on the number of individuals per zone to test theinfluence of treatments and sex of individuals on theirdistribution in the wind-tunnel. Multiple comparisons ofthe means were realized by contrast comparison to thecontrol treatment. The statistical analyses of results wereperformed using the software R 2.10.0 (R DevelopmentCore Team).

Field experiments

The field study was conducted during 3 weeks fromJuly 12 to August 1, 2010 (week 1, July 12–18;week 2, July 19–25; week 3, July 26–August 1) ina 6 ha potato field (Solanum tuberosum var. Ditta)(50◦30.565′N, 4◦51.305′E). Dispensers (rubber septa)containing 100 μL of a solution of Z,E-nepetalactonein paraffin oil (1 mg/100 μL) were prepared and installedon yellow water pan traps. Twenty of these traps were de-ployed randomly at 1 m above ground level in the potatofield. Twenty other traps with dispensers only containingparaffin oil were also deployed randomly (negative con-trol). Traps were separated by 30 m of potato plants. Thenumbers of trapped H. axyridis were noted after 1 week,distinguishing males and females. Average numbers oftrapped H. axyridis were compared by one-way analy-sis of variance (ANOVA) followed by Dunnett’s post-hoc

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test (comparison with a control) (R statistical softwarev2.10.1).

Results

Identification of the nettle volatile compounds

Headspace-GC-MS analyses of the nettle extract al-lowed the determination of two main volatile compounds:the (Z)-3-hexenol and the (E)-2-hexenal (Fig. 2), two typ-ical green leaf volatiles (GLVs) commonly emitted bydamaged or attacked plants (Arimura et al., 2009; Staudtet al., 2010).

Effects of semiochemicals from the aphid alarm and sexpheromones on H. axyridis

(E)-β-farnesene, α-pinene and β-pinene, significantlyattracted the lady beetle near the lure both for males(Fig. 3A) and females (Fig. 3B) during the wind-tunnelexperiments. Indeed, when compared to the negative con-

trol, a significantly higher number of coccinellids wereobserved near the plants associated with volatile com-pounds from the aphid alarm and sex pheromones. Morespecifically, for both sexes, α-pinene strongly attractedH. axyridis since 60.0% ± 0.5% and 61.0% ± 0.6%of individuals were observed in Zone 1, including theodorant lure, for males (z = 3.737 and P = 0.000 2, z-test) and females (z = 4.804 and P < 0.000 1, z-test).β-pinene also significantly attracted both sexes (50.0%of males and 55.0% of females) near the odor source(z = 2.727 and P = 0.006 4; z = 4.271 and P < 0.000 1, z-test for males and females, respectively). (E)-β-farnesenewas also attractive with a less marked effect (46.0% ofmales and 39.0% of females) (z = 2.275 and P = 0.022 9;z = 2.613 and P = 0.009 0, z-test for males and females,respectively)

The semiochemical Z,E-nepetalactone also signifi-cantly affected lady beetle behavior since 70.0% ± 0.3%of females (z = 5.661 and P < 0.000 1, z-test) and52.0% ± 0.6% of males (z = 2.947 and P = 0.003 2,z-test) were directed toward the odour source (Fig. 4A

Fig. 2 Chromatogram obtained by headspace gas chromatography–mass spectroscopy analyses of the nettle extract. (E)-2-hexenal(peak number 1) and (Z)-3-hexenol (peak number 2).

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Fig. 3 Harmonia axyridis attraction near the lure (in Zone 1) in response to semiochemicals from the aphid alarm pheromones (E)-β-farnesene, α-pinene and β-pinene. A, males and B, females. Average percentage of individuals recovered near the lure (Zone 1) andelsewhere in the wind-tunnel (n = 30 × 3 observations). ∗, ∗∗ and ∗∗∗ respectively indicate significant difference from the control atP < 0.05, P < 0.01 and P < 0.001.

Fig. 4 Harmonia axyridis attraction near the lure (in Zone 1) in response to the aphid sexual pheromone Z,E-nepetalactone. A, malesand B, females. Average percentage of individuals recovered near the lure (Zone 1) and elsewhere in the wind-tunnel (n = 30 × 3 ob-servations). ∗∗ and ∗∗∗ respectively indicate significant difference from the control at P < 0.01, P < 0.001.

and Fig. 4B). For each tested semiochemical, no signif-icant differences were noted between males and females(P > 0.05, z-test).

Effects of the H. axyridis volatile aggregation pheromoneon H. axyridis

In our experiments, the mean number of individuals ob-served near the lure did not significantly differ for bothsexes in response to (-)-β-caryophyllene: 2.6 ± 0.7 malesand 3.4 ± 0.9 females were located near the odor source(z = –1.262, P = 0.207, z-test). However, when com-

pared to the respective negative controls, the H. axyridisvolatile aggregation pheromone did not significantly at-tract males (z = 0.108 and P = 0.9139, z-test) (Fig. 5A)while females (34.0% ± 0.9%) significantly respondedto this semiochemical (z = 2.088 and P = 0.0367, z-test)(Fig. 5B).

Effects of the nettle extract and of the identified nettlevolatile compounds on H. axyridis

The nettle extract but also the two main GLVs identi-fied in the extraction product significantly attracted H.

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Fig. 5 Harmonia axyridis attraction near the lure (in Zone 1) in response to its aggregation pheromone (-)-β-caryophyllene. A, malesand B, females. Average percentage of individuals recovered near the lure (Zone 1) and elsewhere in the wind-tunnel (n = 30 × 3observations). ∗, ∗∗∗ and NS respectively indicate significant difference from the control at P < 0.05, P < 0.001 and no significantdifference.

Fig. 6 Harmonia axyridis attraction near the lure (in Zone 1) in response to the nettle extract and to the semiochemicals identifiedfrom the nettle extract (Z)-3-hexenol and (E)-2-hexenal. A, males and B, females. Average percentage of individuals recovered near thelure (Zone 1) and elsewhere in the wind-tunnel (n = 30 × 3 observations). ∗, ∗∗, ∗∗∗ and NS respectively indicate significant differencefrom the control at P < 0.05, P < 0.01, P < 0.001 and no significant difference.

axyridis (Fig. 6A and Fig. 6B): 61.0% ± 0.3% of females(z = 4.806 and P < 0.0001, z-test) and 48.0% ± 0.4%of males (z = 2.500 and P = 0.012 4, z-test) respondedto the nettle extract. (Z)-3-hexenol significantly attracted43.0% ± 0.5% of females (z = 3.074 and P = 0.002 1,z-test) and 46.0% ± 0.7% of males (z = 2.273 andP = 0.023 0, z-test). The second GLV, (E)-2-hexenal,presented the same attractive property for males since54.0% ± 0.3% of males (z = 3.176 and P = 0.001 4,z-test) were attracted toward the lure. Females were notattracted by this later volatile compound (z = 1.868 andP = 0.061 7, z-test). Males and females presented the

same behavior in response to (Z)-3-hexenol (z = 0.300and P = 0.764, z-test); females were more attracted bythe (E)-2-hexenal than males (z = 3.000 and P = 0.002 7,z-test).

Field experiments

Because Z,E-nepetalactone was identified as the mostefficient attractant through the wind-tunnel assays and be-cause this volatile has not been tested under natural condi-tions on H. axyridis, it was used as a potential attractant in

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Fig. 7 Average number (+SE) of Harmonia axyridis (males and females) caught per trap in a potato field in response to Z,E-nepetalactone (1 mg dose) (n = 20). ∗ and ∗∗ respectively indicate significant difference from the control at P < 0.05 and P < 0.01.NS indicates no significant difference from the control. Error bars indicate standard errors. C-, negative control.

a potato field over 3 weeks. If no significant results wereobtained in response to Z,E-nepetalactone during the firstweek of observation (tobs = −0.835 3 and P = 0.623 2;tobs = 0.835 3 and P = 0.623 2, respectively for males andfemales), H. axyridis was significantly trapped when thissemiochemical was associated to the water pan traps dur-ing the second (tobs = 2.80 9 and P = 0.024; tobs = 3.121and P = 0.013, respectively for males and females) andthe third (tobs = 2.986 and P = 0.017; tobs = 3.635 andP = 0.005, respectively for males and females) weeks ofexperiments: more males and females were observed intraps comporting dispensers of Z,E-nepetalactone in com-parison with the control traps (dispensers only filled withparaffin oil) (Fig. 7). In response to Z,E-nepetalactone,2.0 ± 0.3 males and 3.0 ± 0.7 females were caught pertrap during the second week while 5.0 ± 1.1 males and6.0 ± 1.0 females were found in the traps during the thirdweek. These average numbers of trapped individuals wereall significantly higher than those noted with the negativecontrol (P < 0.05 for males and females, one-way ANOVAfollowed by Dunnett’s post-hoc test, comparison with thenegative control).

Discussion

The current study on the invasive coccinellid H. axyridisis the first one realised in a wind-tunnel, the most ap-propriate system to determine the impact of semiochem-icals on insects, since this kind of assay enables directobservations of the upwind attraction responses undercontrolled environmental conditions (Rojas, 1999; Tasinet al., 2006). The most commonly found semiochemicalsassociated with aphids (alarm pheromone components:[E]-β-farnesene, α-pinene, β-pinene and the main sexpheromone component: Z,E-nepetalactone) were testedas potential attractants as well as the volatile aggre-gation pheromone of H. axyridis, (-)-β-caryophyllene.Furthermore, nettle extract and the two GLVs identified

from the extraction product of Urtica dioica L., ([Z]-3-hexenol and [E]-2-hexenal) were also envisaged as poten-tial kairomones for the Asian lady beetle.

Semiochemicals from aphids and volatile compoundsfrom damaged or attacked plants are largely known to actas kairomones for many predatory and parasitic insects,inducing specific behaviors like the active search for preyor egg-laying (Han & Chen, 2002; Zhu & Park, 2005;Pareja et al., 2007; Francis et al., 2004; Verheggen et al.,2008). In this sense and as shown in the present study, (E)-β-farnesene, emitted both by aphids (alarm pheromone)and plants, was demonstrated to act as an attractant for sev-eral coccinellids: Coccinella septempunctata (Al Abassiet al., 2000; Ninkovic et al., 2001; Nakamuta, 1991),Adalia bipunctata (Hemptinne et al., 2000; Francis et al.,2004), Hippodamia convergens (Acar et al., 2001; Zhuet al., 1999), Coleomegilla maculata (Zhu et al., 1999)and H. axyridis (Verheggen et al., 2007; Mondor &Roitberg, 2000; Zhu et al., 1999).

In this study, the two other semiochemicals from theaphid alarm pheromone (α-pinene and β-pinene formu-lated in paraffin oil) were also identified as attractantsfor the Asian lady beetle as well as Z,E-nepetalactone,the main aphid sex pheromone component. If Z,E-nepetalactone was studied through electroantennographicstudies on Colemegilla maculata (Zhu et al., 1999), itsattractiveness on coccinellids was never envisaged. How-ever, our results demonstrated the high potential of thislatter semiochemical since it attracted more than half ofthe tested males and 70.0% of the tested females in thewind-tunnel experiments.

Furthermore and for the first time to our knowledge,our results obtained with Z,E-nepetalactone in the potatofield confirmed that the main aphid sex pheromone com-ponent strongly attract the coccinellid H. axyridis undernatural conditions. The sex pheromone component, Z,E-nepetalactone, was also shown to be an attractant forthe adults of Chrysopa oculata (Zhu et al., 2005) and

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Chrysopa cognata (Boo et al., 1998), confirming the keyrole played by this semiochemical toward aphid naturalenemies.

Also, through the wind-tunnel assays, α-pinene wasidentified as an attractant both for males and females(60.0% of individuals were observed near the odorant lurefor both sexes). This volatile was previously identifiedto play a significant role in attracting the coccinellidsChilocorus kuwanae (Zhang et al., 2009) and H. axyridis(Xue et al., 2008).

Not just coccinellids are known to be attracted by semio-chemicals from aphids, and many studies have demon-strated the attractive properties of (E)-β-farnesene on hov-erflies (Verheggen et al., 2008; Francis et al., 2005a,b),Chrysopidae (Boo et al., 1998; Zhu et al., 2005) andAphidiidae (Glinwood et al., 1999).

Concerning the H. axyridis aggregation pheromone,(-)-β-caryophyllene, our wind-tunnel assays only demon-strated the attractive effect of this cue on females, whilemales did not respond to this volatile compound. On onehand, this result confirms those obtained by Verheggenet al. (2007) and Alhmedi et al. (2010) showing that (-)-β-caryophyllene attract this lady beetle. On the other hand,only females responded to this cue in our study, while thework of Verheggen and colleagues (2007) reported thesame attractive effect on males and females. The follow-ing hypothesies could explain the differences between theH. axyridis responses reported in the present study andthose reported in Verheggen and colleagues (2007): ourstudy was performed on another H. axyridis generation,the H. axyridis larvae were reared on two different aphidspecies (A. pisum or A. fabae), the experimental producewere not the same (four-armolfactometer or wind-tunnel)and different (-)-β-caryophyllene doses were tested (purechemical or chemical formulated in paraffin oil). Oth-erwise, an earlier study showed that (-)-β-caryophylleneacts as an attractant to adults of Chrysoperla carnea (Flintet al., 1979). This indicates that (-)-β-caryophyllene notonly affects H. axyridis behavior but also acts as a mes-senger for C. carnea, another important aphid predator.

Because the Asian coccinellid H. axyridis was shown tofrequently feed on the plant Urtica dioica L. attacked byaphids (Alhmedi et al., 2010), an extraction of the volatilecompounds from this nettle was performed. Two typicalGLVs were identified as (Z)-3-hexenol and (E)-2-hexenalemitted by plants in response to damage and/or attack.The aqueous nettle extract as well as the two GLVs for-mulated in paraffin oil were attractive to both H. axyridismales and females, confirming that semiochemicals fromplants help coccinellids to locate plants and/or prey. Inthis context, (Z)-3-hexenol was identified as a synomonefor the coccinellids Coccinella septempunctata (Han &

Chen, 2002), Coleomegilla maculate (Zhu et al., 1999),Epilachna fulvosignata (Murray et al., 1972) and Stetho-rus punctum picipes (James, 2005) while (E)-2-hexenalwas determined as a synomone for Coccinella septem-punctata (Han & Chen, 2002).

In contrast to our results, Alhmedi and colleagues(2010) did not record behavioral responses for H. axyridiswhen in presence of (E)-β-farnesene, (Z)-3-hexenol andβ-pinene. One possible explanation for this lack of re-sponse could be that only low doses (5 μg in olfactoryexperiments and 10 μg for the oviposition bioassays) weretested by these authors while insect behavioral responsesare known to vary according to volatiles concentrationsapplied (Zhu et al., 1999). Because we have chosen to testall semiochemicals at a 1 mg dose, like in other worksconducted in wind-tunnels on other insects (e.g. Coraciniet al., 2004; Knudsen et al., 2008), the next step shouldbe the use of lower doses.

The use of semiochemicals from aphids and from nettleextracts could certainly help for an efficient biologicalcontrol approach against H. axyridis. More particularly,this study highlights Z,E-nepetalactone as having highpotential for biological control, since this semiochemicalacts as an attractant for the Asian lady beetle H. axyridisin the field.

Acknowledgments

This research was funded by the Walloon Region MinistryGrant (WALEO2: SOLAPHIDRW/FUSAGX 061/6287).The funders had no role in study design, data collec-tion and analysis, decision to publish, or preparation ofthe manuscript. We sincerely thank Delphine Durieuxfor providing us with lady beetles (Department of Func-tional and Evolutionary Entomology, University of Liege,Gembloux Agro-Bio Tech) and Modeline Joseph for herhelp concerning the statistical analyses.

Disclosure

The authors declare that they have no conflicts of interest,including specific financial interests and relationships andaffiliations relevant to the subject of their manuscript.

References

Acar, E.B., Medina, J.C., Lee, M.L. and Booth, G.M. (2001)Olfactory behaviour of convergent lady beetles (Coleoptera:Coccinellidae) to alarm pheromone of green peach aphid(Hemiptera: Aphididae). Canadian Entomology, 133, 389–397.

C© 2011 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 372–382

380 P. D. Leroy et al.

Al Abassi, S.A., Birkett, M.A., Pettersson, J., Pickett, J.A.,Wadhams, L.J. and Woodcock, C.M. (2000) Response of theseven-spot ladybird to an alarm pheromone and an alarmpheromone inhibitor is mediated by paired olfactory cells.Journal of Chemical Ecology, 26, 1765–1771.

Alhmedi, A., Haubruge, E. and Francis, F. (2010) Intraguildinteractions implicating invasive species: Harmonia axyridisas a model species. Biotechnologie, Agronomie, Societe etEnvironnement, 14, 187–201.

Alhmedi, A., Haubruge, E. and Francis, F. (2009) Effect of sting-ing nettle habitats on aphidophagous predators and parasitoidsin wheat and green pea fields with special attention to the in-vader Harmonia axyridis Pallas (Coleoptera: Coccinellidae).Entomological Science, 12, 349–358.

Alhmedi, A., Haubruge, E., Bodson, B. and Francis, F. (2006)Inter- and intra-guild interactions related to aphids in nettle(Urtica dioica L.) strips closed to field crops. Communica-tions in Agricultural and Applied Biological Sciences, 71,413–423.

Alhmedi, A., Haubruge, E., Bodson, B. and Francis, F. (2007)Aphidophagous guilds on nettle (Urtica dioica) strips closeto fields of green pea, rape and wheat. Insect Science, 14,419–424.

Arimura, G.I., Matsui, K. and Takabayashi, J. (2009) Chemicaland molecular ecology of herbivore-induced plant volatiles:Proximate factors and their ultimate functions. Plant CellPhysiology, 50, 911–923.

Boo, K.S., Chung, I.B., Han, K.S., Pickett, J.A. and Wadhams,L.J. (1998) Response of the lacewing Chrysopa cognata topheromones of its aphid prey. Journal of Chemical Ecology,24, 631–639.

Brown, A.E., Riddick, E.W., Aldrich, J.R. and Holmes, W.E.(2006) Identification of (-)-β-caryophyllene as a gender-specific terpene produced by the multicolored Asian ladybeetle. Journal of Chemical Ecology, 32, 2489–2499.

Coracini, M., Bengtsson, M., Liblikas, I. and Witzgall, P. (2004)Attraction of codling moth males to apple volatiles. Ento-mologia Experimentalis et Applicata, 110, 1–10.

Dawson, G.W., Griffiths, D.C., Janes, N.F., Mudd, A., Pickett,J.A., Wadhams, L.J. and Woodcock, C.M. (1987) Identifica-tion of an aphid sex pheromone. Nature, 325, 614–616.

Dutcher, J.D., Estes, P.M. and Dutcher, M.J. (1999) Interactionsin entomology: Aphids, aphidophaga and ants in pecan or-chards. Journal of Entomological Science, 34, 40–56.

Flint, H.M., Salter, S.S. and Walters, S. (1979) Caryophyllene:An attractant for the green lacewing. Environmental Entomol-ogy, 8, 1123–1125.

Francis, F., Lognay, G. and Haubruge, E. (2004) Olfac-tory responses to aphid and host plant volatile releases:E-β-farnesene an effective kairomone for the predatorAdalia bipunctata. Journal of Chemical Ecology, 30, 741–755.

Francis, F., Martin, T., Lucas, G. and Haubruge, E. (2005a) Roleof (E)-beta-farnesene in systematic aphid prey location byEpisyrphus balteatus larvae (Diptera: Syrphidae). EuropeanJournal of Entomology, 102, 431–436.

Francis, F., Vandermoten, S., Verheggen, F., Lognay, G. andHaubruge, E. (2005b) Is the E-β-farnesene only volatile ter-penoids in aphids? Journal of Applied Entomology, 129, 6–11.

Ginsberg, H.S., Lebrun, R.A., Heyer, K. and Zhioua, E.(2002) Potential nontarget effects of Metarhizium anisopliae(Deuteromycetes) used for biological control of ticks (Acari:Ixodidae). Environmental Entomology, 31, 1191–1196.

Glinwood, R.T., Du, Y.J. and Powell, W. (1999) Responses toaphid sex pheromones by the pea aphid parasitoids Aphid-ius ervi and Aphidius eadyi. Entomologia Experimentalis etApplicata, 92, 227–232.

Han, B.Y. and Chen, Z.M. (2002) Composition of the volatilesfrom intact and mechanically pierced tea aphid–tea shootcomplexes and their attraction to natural enemies of the teaaphid. Journal of Agricultural and Food Chemistry, 50, 2571–2575.

Hemptinne, J.L., Gaudin, M., Dixon, A.F.G. and Lognay, G.(2000) Social feeding in ladybird beetles: adaptive signifi-cance and mechanism. Chemoecology, 10, 149–152.

Heuskin, S., Godin, B., Leroy, P., Capella, Q., Wathelet, J.P.,Verheggen, F., Haubruge, E. and Lognay, G. (2009) Fast gaschromatography characterization of purified semiochemicalsfrom essential oils of Matricaria chamomilla L. (Asteraceae)and Nepeta cataria L. (Lamiaceae). Journal of Chromatogra-phy A, 1216, 2768–2775.

Heuskin, S., Rozet, E., Lorge, S., Farmakidis, J., Hubert, P.,Verheggen, F., Haubruge, E., Wathelet, J.P. and Lognay, G.(2010) Validation of a fast gas chromatographic method forthe study of semiochemical slow release formulations. Jour-nal of Pharmaceutical and Biomedical Analysis, 53, 962–972.

Huelsman, M.F., Jasinski, J., Young, C. and Kovach, J. (2002)Multicolored Asian lady beetle (Harmonia axyridis) as a nui-sance pest in households in Ohio. Proceedings of the 4thInternational Conference on Urban Pests, July 7–10, 2002(eds. S.C. Jones, J. Zhai & W.H. Robinson), pp. 243–250.Charleston, SC, USA.

James, D.G. (2005) Further field evaluation of syntheticherbivore-induced plant volatiles as attractants for beneficialinsects. Journal of Chemical Ecology, 31, 481–495.

Katsoyannos, P. and Aliniazee, M.T. (1998) First record ofStrongygaster triangulifera (Loew) (Diptera: Tachinidae) asa parasitoid of Harmonia axyridis (Pallas) (Coleoptera: Coc-cinellidae) in western North America. Canadian Entomology,130, 905–906.

Kenis, M., Roy, H.E., Zindel, R. and Majerus, M.E.N. (2008)Current and potential strategies against Harmonia axyridis.BioControl, 53, 235–252.

C© 2011 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 372–382

Attraction of Harmonia axyridis 381

Knudsen, G.K., Bengtsson, M., Kobro, S., Jaastad, G., Hofsvang,T. and Witzgall, P. (2008) Discrepancy in laboratory and fieldattraction of apple fruit moth Argyresthia conjugella to hostplant volatiles. Physiological Entomology, 33, 1–6.

Koch, R.L. (2003) The multicoloured Asian lady beetle, Harmo-nia axyridis: a review of its biology, uses in biological controland non-target impacts. Journal of Insect Science, 3, 1–16.

Koch, R.L., Burkness, E.C., Burkness, S.J. and Hutchison,W.D. (2004) Phytophagous preferences of the multicoloredAsian lady beetle (Coleoptera: Coccinellidae) to autumnripening fruit. Journal of Economic Entomology, 97, 539–544.

Kovach, J. (2004) Impact of multicolored Asian lady beetles as apest of fruit and people. American Entomology, 50, 159–161.

Labrie, G., Lucas, E. and Coderre, D. (2006) Can developmen-tal and behavioral characteristics of the multicolored Asianlady beetle Harmonia axyridis explain its invasive success?Biological Invasions, 8, 743–754.

Lanzoni, A., Accinelli, G., Bazzocchi, G.G. and Burgio, G.(2004) Biological traits and life table of the exotic Harmoniaaxyridis compared with Hippodamia variegata and Adaliabipunctata (Col. Coccinellidae). Journal of Applied Entomol-ogy, 128, 298–306.

Marco, D.A., Paez, S.A. and Cannas, S.A. (2002) Species inva-siveness in biological invasions: a modeling approach. Bio-logical Invasions, 4, 193–205.

Mondor, E. and Roitberg, B. (2000) Has the attraction of preda-tory coccinellids to cornicle droplets constrained aphid alarmsignaling behavior? Journal of Insect Behavior, 3, 321–329.

Mondor, E.B., Baird, D.C., Slessor, K.N. and Roitberg, B.D.(2000) Ontogeny of alarm pheromone secretion in pea aphids.Acyrthosiphon pisum. Journal of Chemical Ecology, 26,2857–2882.

Murray, R.D.H., Martin, A. and Stride, G.O. (1972) Identifica-tion of the volatile phagostimulants in Solanum campylacan-thum for Epilachna fulvosignata. Journal of Insect Physiol-ogy, 18, 2369–2373.

Nakamuta, K. (1991) Aphid alarm pheromone component, (E)-beta-farnesene, and local search by a predatory ladybeetle, Coccinella septempunctata Bruckii mulsant(Coleoptera: Coccinellidae). Applied Entomology andZoology, 26, 1–7.

Ninkovic, V., Al Abassi, S. and Pettersson, J. (2001) The in-fluence of aphid-induced plant volatiles on ladybird beetlesearching behavior. Biological Control, 21, 191–195.

Obrycki, J.J., Tauber, M.J. and Tauber, C.A. (1985) Perili-tus coccinellae (Hymenoptera: Braconidae): parasitizationand development in relation to host-stage attacked. An-nals of the Entomological Society of America, 78, 852–854.

Pareja, M., Moraes, M.C.B., Clark, S.J., Birkett, M.A. andPowell, W. (2007) Response of the aphid parasitoid Aphid-

ius funebris to volatiles from undamaged and aphid-infestedCentaurea nigra. Journal of Chemical Ecology, 33, 695–710.

Perrin, R.M. (1975) The role of the perennial stinging nettle,Urtica dioica, as a reservoir of beneficial natural enemies.Annals of Applied Biology, 81, 289–297.

Pickett, J.A. and Griffith, D.C. (1980) Composition of aphidalarm pheromones. Journal of Chemical Ecology, 6, 349–360.

Riddick, E.W., Aldrich, J.R., De Milo, A. and Davis, J.C. (2000)Potential for modifying the behavior of the multicolored Asianlady beetle (Coleoptera: Coccinellidae) with plant-derivednatural products. Annals of the Entomological Society ofAmerica, 93, 1314–1321.

Rojas, J.C. (1999) Electrophysiological and behavioural re-sponses of the cabbage moth to plant volatiles. Journal ofChemical Ecology, 25, 1867–1883.

Roy, H.E., Brown, P.M.J., Rothery, P., Ware, R.L. andMajerus, M.E.N. (2007) Interactions between the fungalpathogen Beauveria bassiana and three species of coccinellid:Harmonia axyridis, Coccinella septempunctata and Adaliabipunctata. BioControl, 53, 265–276.

Soares, A.O., Borges, I., Borges, P.A.V., Labrie, G. and Lucas,E. (2008) Harmonia axyridis: What will stop the invader?BioControl, 53, 127–145.

Soares, A.O., Coderre, D. and Schanderl, H. (2001) Influence ofphenotype on fitness parameters of Harmonia axyridis Pallas(Coleoptera: Coccinellidae). European Journal of Entomol-ogy, 98, 287–293.

Staudt, M., Jackson, B., El-Aouni, H., Buatois, B., Lacroze, J.P.,Poessel, J.L., Sauge, M.H. and Niinemets, U. (2010) Volatileorganic compound emissions induced by the aphid Myzuspersicae differ among resistant and susceptible peach cultivarsand a wild relative. Tree Physiology, 30, 1320–1334.

Tasin, M., Backman, A.C. and Bengtsson, M. (2006) Wind tun-nel attraction of grapevine moth females, Lobesia botrana, tonatural and artificial grape odour. Chemoecology, 16, 87–92.

Verheggen, F.J., Fagel, Q., Heuskin, S., Lognay, G., Francis, F.and Haubruge, E. (2007) Electrophysiological and behavioralresponses of the multicolored Asian lady beetle, Harmoniaaxyridis Pallas, to sesquiterpene semiochemicals. Journal ofChemical Ecology, 33, 2148–2155.

Verheggen, F.J., Arnaud, L., Bartram, S., Gohy, M. andHaubruge, E. (2008) Aphid and plant secondary metabolitesinduce oviposition in an aphidophagous hoverfly. Journal ofChemical Ecology, 34, 301–307.

Webberley, K.M., Hurst, G.D.D., Husband, R.W., Schulenburg,J.H.G.V.D, Sloggett, J.J., Isham, V., Buzcko, J. and Majerus,M.E.N. (2004) Host reproduction and a sexually transmit-ted disease: causes and consequences of Coccipolipus hippo-damiae distribution on coccinellid beetles. Journal of AnimalEcology, 73, 1195–1200.

C© 2011 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 372–382

382 P. D. Leroy et al.

With, K.A., Pavuk, D.M., Worchuck, J.L., Oates, R.K. andFisher, J.L. (2002) Threshold effects of landscape structureon biological control in agroecosystems. Ecological Applica-tions, 12, 52–65.

Xue, J., He, J., and Xie, Y. (2008) Attractive effect of plantvolatiles on Harmonia axyridis (Pallas). Chinese Journal ofApplied & Environmental Biology, 4, 494–498.

Zhang, Y., Xie, Y., Xue, J., Peng, G. and Wang, X. (2009) Effectof volatile emissions, especially α-pinene, from Persimmontrees infested by Japanese wax scales or treated with methyljasmonate on recruitment of ladybeetle predators. Environ-mental Entomology, 38, 1439–1445.

Zhu, J., Cosse, A.A., Obrycki, J.J., Boo, K.S. and Baker, T.C.(1999) Olfactory reactions of the twelve-spotted lady beetle,

Coleomegilla maculata and the green lacewing, Chrysoperlacarnea to semiochemicals released from their prey and hostplant: electroantennogram and behavioural responses. Jour-nal of Chemical Ecology, 25, 1165–1177.

Zhu, J., Obrycki, J.J., Ochieng, S.A., Baker, T.C., Pickett, J.A.and Smiley, D. (2005) Attraction of two lacewing species tovolatiles produced by host plants and aphid prey. Naturwis-senschaften, 92, 277–281.

Zhu, J.W. and Park, K.C. (2005) Methyl salicylate, a soybeanaphid-induced plant volatile attractive to the predator Coc-cinella septempunctata. Journal of Chemical Ecology, 31,1733–1746.

Accepted April 8, 2011

C© 2011 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 19, 372–382