Research Article VOCs-Mediated Location of Olive Fly

Research ArticleVOCs-Mediated Location of Olive Fly Larvae by the BraconidParasitoid Psyttalia concolor A MultivariateComparison among VOC Bouquets from Three Olive Cultivars

Giulia Giunti1 Giovanni Benelli1 Giuseppe Conte1 Marcello Mele12 Giovanni Caruso1

Riccardo Gucci12 Guido Flamini3 and Angelo Canale12

1Department of Agriculture Food and Environment University of Pisa Via del Borghetto 80 56124 Pisa Italy2Interdepartmental Research Center Nutrafood ldquoNutraceuticals and Food for Healthrdquo University of Pisa Pisa Italy3Department of Pharmacy University of Pisa Via Bonanno 6 56126 Pisa Italy

Correspondence should be addressed to Giovanni Benelli benelligiovannigmailcom

Received 1 October 2015 Revised 11 January 2016 Accepted 13 January 2016

Academic Editor Heather Simpson

Copyright copy 2016 Giulia Giunti et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Herbivorous activity induces plant indirect defenses as the emission of herbivorous-induced plant volatiles (HIPVs) which couldbe used by parasitoids for host location Psyttalia concolor is a larval pupal endoparasitoid attacking a number of tephritid fliesincluding B oleae In this research we investigated the olfactory cues routing host location behavior of P concolor towards B oleaelarvae infesting three different olive cultivars VOCs from infested and healthy fruits were identified using GC-MS analyses Intwo-choice behavioral assays P concolor females preferred infested olive cues which also evoked ovipositional probing by femalewasps GC-MS analysis showed qualitative and quantitative differences among volatiles emitted by infested and healthy olivesVolatile emissions were peculiar for each cultivar analyzed Two putative HIPVs were detected in infested fruits regardless ofthe cultivar the monoterpene (E)-120573-ocimene and the sesquiterpene (E-E)-120572-farnesene Our study adds basic knowledge to thebehavioral ecology of P concolor From an applied point of view the field application of the above-mentioned VOCs may help toenhance effectiveness of biological control programs and parasitoid mass-rearing techniques

1 Introduction

The olive tree (Olea europea) is an economically importantcrop in the Mediterranean basin holding about 98 ofworldrsquos olive groves [1] In the last decades olive crop was alsowidespread in novel regions such as China Brazil and SouthAfrica increasing olive production up to 204 million tonsin 2013 one of the highest production levels ever recordedOn the other hand the olive crop spread has determineddiffusion of the most devastating insect pest of olives theolive fruit fly Bactrocera oleae (Rossi) (Diptera Tephritidae)Its diffusion occurred in the Mediterranean regions for over2000 years and more recently in California olive grows [2]B oleae is a monophagous pest feeding exclusively on Oleaspecies Olive fruit fly females lay an egg under the fruitsurface thus the larvae develop inside olive fruits until they

open an exit hole before pupate On table olive groves theoviposition puncture leads to a serious reduction of cropvalue while exit holes and pulp degradation can determinea quality and quantity loss of olive oil production B oleaeinfestation can reduce oil yield [3 4] alter several qualityparameters (eg acidity peroxide value UV absorbance)[1 5ndash7] and even negatively impact chemical compositionwhich determine oil taste and flavor [1 6ndash10] Volatilesprofiles are known to be influenced by abiotic factors [11]but also B oleae infestation could induce critical changes ofvolatile emissions [12]

Herbivorous feeding activity is known to induce a varietyof biochemical changes in plants It is well known that plantsrespond to herbivoresrsquo presence activating their defensesystem [13] but they can also trigger indirect defenses asthe emission of herbivorous-induced plant volatiles (HIPVs

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7827615 10 pageshttpdxdoiorg10115520167827615

2 BioMed Research International

hereafter) [14 15] The role of kairomones on parasitoidhost location has been widely investigated [16ndash18] and it hasbeen demonstrated that many plants rely on volatile signalsinduced by phytophagous feeding to attract their naturalenemies [14 19 20] Moreover despite the evidence aboutthe influence of B oleae infestation on the quality and thequantity of volatile compounds emitted in olive oils [7 8]no information is available to assess the presence of HIPVsproduced by infested olive fruits However differential emis-sions have been already proved for Tephritidae-infested andhealthy fruits [21ndash24] highlighting the production of severalHIPVs able to evoke electrophysiological and behavioralresponses in parasitoid wasps [23 24]

Psyttalia concolor (Szepligeti) (Hymenoptera Braconi-dae) is a koinobiont larval pupal endoparasitoid able toparasitize at least fourteen tephritids on different wild andorcultivated plants including B oleae and Ceratitis capitata(Wiedemann) the Mediterranean fruit fly [25] P concolorfemales rely on a number of stimuli to successfully locate theirhost Indeed female wasps are able to distinguish betweeninfested and healthy fruit preferring the first one even ifjust olfactory cues are provided [23] In addition it wasdemonstrated that apple and peach fruits infested by Ccapitata larvae emitted peculiar volatiles recognized by Pconcolor wasps and able to attract selectively mated females[23] HIPVs from apple and peach fruits are also able toattract and prolong the time spent performing searchingbehavior in P concolor virgin males probably raising theirchances to locate receptive females nearby host microhab-itat [26] Furthermore even synthetic blends reproducinginfested peaches or apples were found able to be attractive forP concolor mated females and virgin males [23 26]

In this research we investigated the importance of olfac-tory cues used by P concolor females to locate their hostmicrohabitat We hypothesize that the HIPVs from B oleae-infested olive fruits may play a pivotal role in affecting Pconcolor host location as described for the same parasitoid ona different tephritid host [23] Olive fruits from three differentcultivars were tested to determine parasitoid attractivenessand volatile organic compounds (VOCs) emissions cv Fran-toio and cv Leccino (traditionally cultivated in Italy) andcv Arbequina (typical of Spanish olive groves) Firstly weevaluated femalesrsquo preferences among healthy and infestedfruits in two-choice bioassay providing both visual andolfactory cues or olfactory stimuli alone in order to evalu-ate the magnitude of volatiles attractiveness Subsequentlyvolatiles emitted by healthy and infested olive fruits wereSPME-sampled and analyzed by gas chromatography-massspectrometry (GC-MS) to estimate differentially emissionsattributable to herbivoresrsquo activity and to indicate possibleHIPVs

2 Material and Methods

21 Parasitoid Rearing P concolor wasps were reared asdescribed by Canale and Benelli [25] Insects were main-tained in Pisa Laboratory under controlled conditions (22∘Cplusmn 1 50ndash60 relative humidity and natural photoperiod)

during their entire life Adult parasitoids were allowed toemerge in transparent Plexiglas tubes (diameter 40 cm length50 cm) into which 1500 adults were introduced (male femalesex ratio 03ndash05) To obtain pupae from which the adultemerged a nylon mesh bag containing around 700 thirdinstar C capitata larvae was posed into a cage and exposedto P concolor wasps for 20 minutes Parasitized pupae wereplaced into smaller Plexiglas cages (diameter 20 cm height30 cm) and there P concolor adults were allowed to emerge ata density of 50 specimens per cage (males females sex ratio03) Insects were stored at laboratory conditions [22 plusmn 1∘C50plusmn5 relative humidity and 16 8 (L D) photoperiod] for 7days after the parasitoidsrsquo emergence to allow mating beforetesting Adult insects were fed on a semisolid diet (honeymixed with pollen) and with water ad libitum

22 Plant Material Olive fruits from three different cultivars(Frantoio Leccino and Arbequina) were used for behav-ioral assay and GC-MS analysis Olives were collected onSeptember 15 2014 in Tuscan olive groves [Frantoio andLeccino fromTorrita di Siena (43∘151015840498610158401015840N 11∘781015840965810158401015840E)and Arbequina from Rapolano Terme (43∘271015840709710158401015840N11∘601015840709810158401015840E)] from 5ndash3-year olive trees Healthy or infestedolives from each cultivar were collected manually stored intoglass jars (diameter 10 cm length 20 cm) and transferred tolaboratory conditions within 3 hours The fruits were firstlydivided according to the maturation index (MI) whereby theskin and flesh colors were scored to a 0 to 7 scale [27] andolives with MI from 2 to 7 were discharged Among infestedolives we selected the ones attacked second or early thirdinstar larvae with no exit holes on the olive surface Healthyfruits were selected avoiding crushed and naturally damagedones

Before being tested fruits were stored at laboratory condi-tion for 2ndash5 days All olives used for both behavioral and GC-MS tests were subsequently dissected to check the presenceor the absence of B oleae larvae inside the fruits

23 Effect of Olfactory Cues from Infested Olives on ParasitoidAttractiveness Bioassays were conducted using the still airarena described by Benelli et al [28] A Plexiglas unit (150 times150 times 30mm) was covered on the top with a removable glasspanel to create the arenaTheunit presents a circular chamber(diameter 40mm) in the center to release the specimenand two other identical chambers connected with linearpaths (length 20mm width 10mm) where the stimuli wereallocated

To assess if infested olives are attractive for P concolorin a first experiment mated females were allowed to chooseamong three healthy or infested olive fruits of each cultivar Inaddition to investigate the role of olfactory stimuli in leadingparasitoid host location a second experiment was designedAs in the first experiment three infested or healthy fruits wereplaced into the test chambers but a piece of filter paper wasposed ahead of the fruits to avoid visual contact with theparasitoid female released in the central chamber

A replicate starts when a wasp was gently transferred tothe released chamber and observed for 8min A wasp was

BioMed Research International 3

considered to have to choose a cue when it remains in thesame chamber for at least 20 s actively searching for a hostand the replicate was considered complete when the wasp leftthe chamber Wasps that show no choice after 7min were notconsidered With each new wasp the arena was rotated 90∘and the relative position of cues was randomized After eachassay the arena was cleaned washing firstly with warm waterthen rinsed in a water bath with mild soap subsequentlywashed with hot water and eventually cleaned with distilledwater [29] 30 mated females were tested for each treatmentFor each bioassay the (i) latency time (time elapsing from thestart of the replicate and the effective choice) (ii) femalersquos firstchoice (iii) time spent on the chosen chamber (iv) numberof antennal drumming series (performed in close proximityof the stimulus) and (v) number of oviposition attempt(performed on the fruits or on the filter paper surface) wererecorded

For each choice-test a likelihood chi-square test withYates correction (with 120572 = 005) was used to comparethe proportion of parasitoids choosing a given cue [30]The other measured variables were analyzed in JMP 7 byusing a general linear model with one fixed factor (ie thetreatment)

24 Effect of B oleae Infestation onVOCs Production Supelco(Bellefonte PA USA) SPME devices coated with poly-dimethylsiloxane (PDMS 100 120583m) were used to sample theheadspace of three olive fruits (healthy or infested by B oleae)inserted into a 30mL glass vial and allowed to equilibratefor 30min SPME sampling was performed using the samenew fibre preconditioned according to the manufacturerinstructions for all the analyses Sampling was accomplishedin an air-conditioned room (25 plusmn 1∘C) to guarantee astable temperature After the equilibration time the fibrewas exposed to the headspace for 30min Once samplingwas finished the fibre was withdrawn into the needle andtransferred to the injection port of the GC-MS system Allthe SPME sampling and desorption conditions were identicalfor all the samples Furthermore blanks were performedbefore each first SPME extraction and randomly repeatedduring each series For each cultivar three replicates (eithercontaining three olives) for both infested and healthy fruitswere provided Quantitative comparisons of relative peaksareas were performed between the same chemicals in thedifferent samples

Gas chromatographyelectron impact mass spectroscopy(GC-EIMS) analyses were performed with a Varian CP-3800gas chromatograph equipped with a DB-5 capillary column(30m times 025mm coating thickness = 025 120583m) and a VarianSaturn 2000 ion trap mass detector (emission current 10microamps count threshold 1 countmultiplier offset 0 voltsscan time 100 second prescan ionization time 100microsec-onds scan mass range 20ndash300119898119911 ionization mode EI)The following analytical conditions were used injector andtransfer line temperature at 250 and 240∘C respectively oventemperature programmed from 60 to 240∘C at 3∘Cminminus1carrier gas helium at 1mLminminus1 splitless injection Iden-tification of the constituents was based on comparison of

the retention times (RT) with those of pure compoundscomparing their linear retention indices (LRI) relative to theseries of n-hydrocarbons and on computer matching againstcommercial (NIST 98 and ADAMS) and homemade librarymass spectra built from pure substances and components ofknown oils and MS literature data [31ndash35]

For each compound and chemical class the area integra-tion report was transformed into log values before statis-tical analysis The normal distribution of data was checkedusing Shapiro-Wilk test To evaluate differences in volatileemissions between infested and healthy fruits of the threecultivars the variance was analyzed with JMP 7 by using ageneral linear model with one fixed factor (ie fruit healthstatus) In addition a general linear model with two factorshealth status and cultivar was performed 119910

119895= 120583 + 119868

119895+ 119862119895+

(119867119895times 119862119895) + 119890119895 in which 119910

119895is the observation 120583 the overall

mean 119868119895the fruit infestation status (119895 = 1-2) 119862

119895the cultivar

(119895 = 1ndash3) 119868119895times119862119895the interaction infestation status times cultivar

and 119890119895the residual error

Principal Component Analysis (PCA) was achieved onnormalized values of each VOC to derive different variables(principal components) that summarize the original dataPCA analysis was performed using JMP software PCAcalculated linear combination of the original data extractingeigenvalues and eigenvectors of a correlation matrix ofvolatilesrsquo areas and highlighted principal components theorthogonal and linear combination of the original variablesTwo-dimensional score plots were created to determine ifvolatiles from different olive fruit cultivar or with differentinfestation degree could be clustered into classes Then aMultifactorial Analysis (MFA) was performed to assess com-mon factors explaining volatilesrsquo variability using amaximumlikelihood estimation procedure and aVARIMAXorthogonalrotation technique by JMP Scores of common factors werecalculated as described byMacciotta et al [36] Furthermorefactors scoreswere analyzed using a general linearmodelwithinfestation status and cultivars as fixed factors to enlightenthe relationship between a common factor and the varioustreatments Discriminant analysis also performed usingJMP software used different volatiles which can be highlycorrelated to a given fixed variable (ie infestation status)as a set of independent variables A step-wise method wasused to select a set of independent variables with 1198772 gt 01The ratio (Wilksrsquos lambda) between the generalized within-category dispersion and the total dispersion was considered[37]

3 Results

31 Effect of Olfactory Cues from Infested Olives on ParasitoidAttractiveness P concolor mated females showed significantpreferences for infested fruits over healthy ones when bothvisual and olfactory cues were provided (Arbequina 1205942 =108333 df = 1 119875 = 00010 Frantoio 1205942 = 85667df = 1 119875 = 00034 Leccino 1205942 = 65667 df = 1 119875 =00104) (Figure 1) No significant differences were recordedfor latency times and times spent on the chosen chamber(Table 1) while wasps which had preferred infested fruits of


Table 1 Choice time spent by Psyttalia concolor females during searching behavior on healthy and Bactrocera oleae-infested olives in two-choice bioassay in still air arena

Cultivar TreatmentInfested olives Healthy olives

119865 119875 valueChoice timeMean plusmn SE (s) Replicates Choice time

Mean plusmn SE (s) Replicates

Arbequina Visual + olfactory 268 plusmn 30 25 255 plusmn 70 5 00327 08579ns

Olfactory 336 plusmn 25 24 208 plusmn 64 6 47747 00374lowast

Frantoio Visual + olfactory 268 plusmn 28 23 218 plusmn 43 7 07723 03870ns

Olfactory 158 plusmn 26 22 125 plusmn 48 8 04192 05227ns

Leccino Visual + olfactory 346 plusmn 29 22 308 plusmn 45 8 04642 05013ns


Within a row the asterisk indicates a significant difference (119875 lt 005)ns not significantSE standard error

Table 2 Number of antennal drumming series performed by Psyttalia concolor females during searching behavior on healthy and Bactroceraoleae-infested olives in two-choice bioassay in still air arena


119865 119875 valueDrumming seriesMean plusmn SE (N) Replicates Drumming series

Mean plusmn SE (N) Replicates



Frantoio Visual + olfactory 79 plusmn 13 23 16 plusmn 08 7 6833 00142lowast


Leccino Visual + olfactory 98 plusmn 16 22 14 plusmn 05 8 96881 00042lowast



Arbequina (visual + olfactory)

Arbequina (olfactory)

Frantoio (visual + olfactory)

Frantoio (olfactory)

Leccino (visual + olfactory)

Leccino (olfactory)

Infested

Infested

Infested

Infested

Infested

Infested

lowast

lowast

lowast

lowast

lowast

lowast

Number of Psyttalia concolor females

Healthy

Healthy

Healthy

Healthy

Healthy

Healthy

Figure 1 Attractiveness of Bactrocera oleae-infested fruits towardsPsyttalia concolormated females effect of visual and olfactory Two-choice bioassays were conducted in a still air arena with olive fruitsinfested or not by olive fruit fly larvae providing visual and olfactorystimuli associated or only olfactory cuesThirty wasps were tested ineach bioassay For each test asterisks indicate significant differencesin the number of wasps choosing different cue (1205942 test with Yatescorrection 119875 lt 005)

Frantoio andLeccino varieties performed a greater number ofdrumming series on infested fruits (Table 2) No ovipositionattempts were noted when females visited healthy olives

conversely to oviposition and probing behaviors recorded ininfested fruits of all varieties

Infested olives were positively located and chosen evenin absence of visual stimuli when fruits were hidden byfilter paper Indeed P concolor females preferred to prospectchambers containing infested fruits over healthy ones (Arbe-quina 1205942 = 133667 df = 1 119875 = 00003 Frantoio 1205942 =65667 df = 1 119875 = 00104 Leccino 1205942 = 85667 df = 1119875 = 00034) (Figure 1) No significant differences for latencytimes were found but P concolor females spent longer timesin the chamber with Arbequina infested fruits than withhealthy Arbequina olives (Table 1) Indeed when Arbequinaor Leccino infested fruit odor was preferred by tested waspsfemales accomplished a higher number of drumming series(Table 2) No oviposition attempts were recorded for waspschoosing healthy fruit chamber while interestingly somewasps were noted to perform probing behavior in the filterpaper or in the glass walls of the chamber containing infestedolives

32 Effect of B oleae Infestation on VOCs Production of OliveFruits from Different Cultivar GC-MS analysis identifiedover 100 different volatile compounds Differential emissionsattributable to herbivore activity were found for all cultivarsIn detail we found 6 compounds significantly increased


in infested olives and 6 volatiles were exclusively producedby infested fruits of Arbequina variety (SupplementaryTable S1 see Supplementary Material available online athttpdxdoiorg10115520167827615) In Frantoio 3 com-pounds were exclusive and 3 increased and one decreased ininfested olives (Supplementary Table S2) while in Leccinowe found 4 compounds increasing and 4 exclusively presentin infested fruits (Supplementary Table S3) In detail thethree cultivars present 2 common VOCs prevalently pro-duced by infested olive fruits (E)-120573-ocimene and (E-E)-120572-farnesene Among chemical classes monoterpenes hydro-carbons increased in all cultivars (Arbequina 119865 = 80698df = 1 119875 = 00468 Frantoio 119865 = 354752 df = 1 119875 =00040 Leccino 119865 = 143467 df = 1 119875 = 00193) IndeedArbequina infested fruit showed different emissions of ke-tones (119865 = 1038718 df = 1 119875 lt 00001) and sesquiterpeneshydrocarbons (119865 = 241958 df = 1 119875 = 00079) whileFrantoio increased monoterpenes oxygenated (119865 = 175960df = 1 119875 = 00138) and aromatic hydrocarbons (119865 =505679 df = 1 119875 = 00021) From two factors general linearmodel 36 compounds and 6 chemical classes were noted tobe significant for at least one factor (Supplementary Table S4)

Furthermore PCA followed by discriminant analysisallowed a more precise partition of cultivar and infestationeffects on volatile emission from fruitsThe Kaiser coefficientwas around 100 since no correlations existed between themajorities of the compounds Six principal components wereanalyzed explaining 66730 of variation (Table 3) Figure 2shows PCA results and two-dimensional score plots werecreated to highlight different clusters relative to different olivefruit cultivar anddifferent infestation status (Figure 3) Eigen-vectors of single VOCs are provided in Supplementary TableS5 and rotated factor patterns in Supplementary Table S6The rotated factors with an eigenvector of at least plusmn05 weremarked in bold and considered for the following analysis Atwo-way general linear model was provided to understandwhich sources of variation had a significant effect on the sixanalyzed factors as reported by Supplementary Table S7 Onthis basis we labeled the six factors as Factor 1 ldquoInfestationrdquoFactor 2 ldquocv Frantoiordquo Factor 3 ldquoItalian Varietiesrdquo Factor4 ldquoInfestation cv Leccinordquo Factor 5 ldquocv Arbequinardquo andFactor 6 ldquocv Leccinordquo Discriminant analysis was providedfor one source of the variations (ie infestation status)Wilksrsquo Lambda test showed a 119875 value lt 00001 and nomisclassified variables were recorded Step-wise methodemphasized 11 variables highly correlated to infestation status(Table 4) Two VOCs (6-methyl-3-methylene-5-hepten-2-one and 2611-trimethyldodecane) resulted positively cor-related with Canonical 1 representing compounds typicallyassociated with healthy olives while the other 9 compoundswere expression of infested status (Figure 3)

4 Discussion

Olfactory stimuli from host-infested fruits are known to beessential during host location behavior for many braconidsincluding species attacking larval stages [24 38ndash43] For Pconcolor the presence of chemical compounds was demon-strated produced by C capitata-infested apples and peaches

Table 3 Principal component identified after Principal ComponentAnalysis (PCA) of volatile emissions from three olive cultivarsBolded components were analyzed using a General Linear Modelto determine source of variation

Principalcomponent Eigenvalue Percentage Cumulative

percentage1 195168 20986 209862 136287 14654 356403 108109 11625 472654 65221 7013 542785 62379 6707 609856 53423 5744 667307 50994 5483 722138 43666 4695 769089 39679 4267 8117510 35437 3810 8498511 33083 3557 8854212 25223 2712 9125513 21784 2342 9359714 20431 2197 9579415 17363 1867 9766116 11942 1284 9894517 09813 1055 100000

Table 4 Volatiles identified after discriminant analysis Positivecorrelations with Canonical1 indicate volatiles representative ofhealthy fruits while negative correlations compounds are expressiveof infested olives

Compound Correlation withCanonical1

6-Methyl-3-methylene-5-hepten-2-one 015583672Dihydromyrcenol minus0316570777Terpinolene minus0446911371Methyl carvacrol minus0759893869Linalool acetate minus04454356142611-Trimethyldodecane 0421648972Cyclosativene minus0355430074(EE)-120572-Farnesene minus0535648427Liguloxide minus01179655011-Hexadecene minus021094794trans-Methyl dihydrojasmonate minus0279958519

able to attract both mated females and virgin males [23 26]The evidence that olfactory cues from infested fruits evokebehavioral responses from mated P concolor females and ofthe presence of compounds that were produced exclusively orin higher amount by infested olives supports our hypothesisthat VOCs could act as short-range attractant playing a keyrole during host-seeking also in this tritrophic system Pconcolor were attracted preferentially by infested olives bothwhen visual stimuli were provided or not suggesting thatthe presence of feeding larvae inside the fruit is crucial for


Heptadecane

DodecaneLinalool acetate

Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal

Butylated hydroxyanisole

Butylated hydroxytoluene

2611-Trimethyl dodecane1-Hexadecene

Methyl carvacrol

Liguloxide

6-Methyl tridecane

Undecanol1-Octanol-2-butyl

Isobornyl acetate

Epi-longipinanol

Lilial

Epoxy alloaromadendrene

trans-Methyl dihydrojasmonate

58-Diethyl dodecane

2610-Trimethyl tetradecanetridecane

17-Octadien-3one 2methyl-6-methylene

DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene

120573-caryophyllene

120572-Cedrene

(E)-120573-farnesene

120572-BisabololCis-120572-bergamotene

minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)

minus10 00 05 10minus05

Component 1 (21)

trans-120572-Bergamotene

(Z)-120573-Ocimene

(Z)-120573-Farnesene

(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)

Figure 2 Principal Component Analysis (PCA) of volatile profiles from infested and healthy fruits of three different olive cultivars (a) PCAloading plot showing volatile correlations with the first and second principal component (b) PCA score plot highlighting cluster of volatilesattributable to cultivar or infestation status e Arbequina infested fruits I Arbequina healthy fruits ◼ Frantoio infested fruits ◻ Frantoiohealthy fruitsX Leccino infested fruits loz Leccino healthy fruits

host location Indeed oviposition behavior was performedfrom females just when they chose infested fruit stimuliInterestingly ovipositor probing responses were performedalso by P concolor females which did not come directly

in contact with olive fruits but only sensing infested oliveodorsThis behavior already described forP concolor femalesattracted by some syntheticHIPVs [23] is uncommon amongparasitic wasps since usually they need an integration of


120140160180200220240260280

Cano

nica

l 2

0minus100 minus50minus200 minus150minus250

Canonical 1

TreatmentHealthyInfested

Figure 3 Canonical plot from discriminant analysis showingcompounds highly correlatedwith Canonical 1 variable representingthe infestation status in olive cultivars

visual and olfactory stimuli to perform a complete hostlocation sequence [21] Moreover P concolor females showedprobing behaviors on the chamber glass surface in presenceof volatile emitted by all the three cultivars inducing alsolonger active searching activities with particular reference toantennal drumming

To determinewhatever change in volatile emissions couldexplain parasitoid behavior B oleae-infested and healthyolive fruits were analyzed Among over 100VOCs identifiedby SPME and GC-MS techniques only two volatiles werefound to increase in infested olives in all the three cultivarsa monoterpene (E)-120573-ocimene and a sesquiterpene (E-E)-120572-farnesene which are already known as constituent ofthe odors of olive oils and processed table olives [11 44ndash46] However since (E)-120573-ocimene is attractive to severalbraconid species with special reference to Aphidius species[47ndash49] and (E-E)-120572-farnesenewhich has been demonstratedto attract Opius dissitus Muesebeck wasps [50] these twocompounds can be considered as putative kairomones for Pconcolor Although we observed mainly quantitative changesin volatile emissions among infested andhealthy olives whichis not uncommon even in similar tritrophic systems [23 5152] indeed several plants react to herbivore damages byproducing blends of metabolites with changes in number orin their proportions [21 24]

Moreover PCA analysis has highlighted that VOCs emis-sions are peculiar for each cultivar and chemicals which weredifferentially emitted after herbivore infestation changeddepending on the olive varieties Indeed after multifactorialanalysis we could describe the variability due to baselinehealthy cultivar emissions using three different factors (Fac-tor 2 Factor 5 and Factor 6) explaining each one VOCemission of a specific cultivar In addition Italian cultivars(cv Frantoio and cv Leccino) showed common volatilesexplained by Factor 3 which were never produced by theSpanish one (cv Arbequina) On the other hand infestationstatus could be explained for all the three cultivars by a com-mon factor (Factor 1) but we identified also some exclusive

compounds which were emitted only by Leccino olives underB oleae infestation (Factor 4) Moreover Arbequina varietyshowed to emit differentially the larger number of VOCsBeside (E)-120573-ocimene and (E-E)-120572-farnesene Arbequinainfested olives increased the emission of other 4 compounds(methyl carvacrol n-tridecane trans-120572-bergamotene andcis-120573-farnesene) and produced specifically 6 compounds[(Z)-120573-ocimene 2-methyl-6-methylene-17-octadien-3-onecyclosativene 1-undecanol cis-120572-bergamotene and 35-di-tert-butylpyrocatechol] Most of them are known to becommon floral compounds but interestingly some of themare recognized pheromones for several hymenoptera species[53ndash55] while (Z)-120573-ocimene is known to be an attractantfor the braconid Diachasmimorpha longicaudata (Ashmead)[24] When attacked by B oleae Leccino cultivar similar toArbequina increased the production of the floral compound2-methyl-6-methylene-17-octadien-3-one In addition Lec-cino infested olives increased the emission of limoneneand exclusively produced 4 monoterpenes (isocineole 120574terpinene dihydromyrcenol and terpinolene) the majorityof which are HIPVs produced by mango fruits positivelytested by Carrasco et al [24] on D longicaudata waspsFrantoio cultivar seems to be the less odorant varieties sinceit produced only 5 compounds when herbivory attack suc-ceeds As described for Arbequina Frantoio infested olivesemitted more (E)-120573-ocimene and methyl carvacrol but theyspecifically generated [besides (E-E)-120572-farnesene] dihydroc-itronellol and heptylcyclohexane which to the best of ourknowledge were never investigated for their attractivenesstoward insects Interestingly infested Frantoio olives showedto decrease the production of 2611-trimethyldodecane apeculiar VOC never identified on olives or olive oils Con-versely the majority of the other identified VOCs are com-mon volatiles emitted by olive oils [11 44 56ndash59] processedtable olives [45 46] leaves [44 60] and olive fruits regardlessof their infestation status [61]

Hence the cultivar seems to be the higher source ofvariation for VOC emissions in olive trees For this reason wecannot exclude that other VOCs produced specifically by onecultivar or increased in not all varieties may act as attractanttoward P concolor wasps Indeed it was demonstrated thatalso healthy fruits can produce volatiles attractive for parasiticwasps [24] and generalist parasitoid as P concolor could beable to perceive cues from non-infested plant to locate hostmicrohabitat Thus short-range volatiles produced by theplants or as excretion of the feeding larvae andor vibrationalstimuli from the hosts could be useful to the successfullocalization [62]

Overall since HIPVs are known to act as kairomones forseveral parasitic wasps [24 63 64] further researches areneeded to assess the activity of the highlighted compoundson parasitoid behavior Indeed knowledge about tritrophicsystem communications has potential implications also onbiological control programs [65] Synthetic kairomones havebeen already tested in field conditions for parasitoid attrac-tion [64ndash66] but beside field applications HIPVs may alsobe employed to enhance mass-rearing techniques


5 Conclusions

Our results support the hypothesis that chemical cues pro-duced by olive fruits under B oleae attack route the hostlocation behavior of P concolor females acting as short-range kairomones Olfactory cues seem to have a key rolein host-seeking behavior for two olive cultivars traditionallycultivated in Italy (cv Frantoio and cv Leccino) and oneSpanish variety (cv Arbequina) Indeed behavioral assayshave shown that P concolor mated females can perceivethe presence of host larvae inside a fruit when visual andolfactory stimuli were provided but also when visual per-ception was forbidden In addition females choosing fruitsinfested by B oleae performed longer searching activitieswith particular references to antennal drumming seriescompleted on the fruit or in close proximity and olfactorycues from infested olives evoked ovipositor probing behavioreven in absence of direct contact between the parasitoid andthe fruits SPME and GC-MS analysis have also supportedthe presence of volatiles attributable to herbivore activitywhich can be indicated as putative HIPVs In detail wefound 12 volatiles increasing or exclusively emitted by infestedArbequina olives 5 in Frantoio and 8 in Leccino onesInterestingly the three cultivars showed 2 common VOCsproduced as response of B oleae infestation (E)-120573-ocimeneand (E-E)-120572-farneseneMoreover PCA andMFAhighlightedthat the cultivar is a higher source of variation for VOCemissions in olive trees

SinceHIPVs are recognized as kairomones of a number ofparasitic wasps [23 24 63 64] further studies are necessaryto ensure the behavioral activity of these investigated volatilestoward P concolor parasitoids Synthetic kairomones maybe useful to improve biological control programs but eventechniques for parasitoid mass-rearing Moreover even if theefficacy of synthetic kairomonal molecules has been alreadyproved in field conditions [54ndash66] the role of HIPVs on theforaging behavior of beneficial arthropods in agroecosystemsneeds to be investigated deeper to enable their safe commer-cial applications [19]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This studywas partially funded byUniversity of Pisa Progettodi Ricerca di Ateneo PRA 2015 0012The authors are gratefulto H Simpson and the two anonymous reviewers for improv-ing an earlier version of this paper The authors would like tothank Pierluigi Cioni for discussion about the interpretationof mass spectra

References

[1] F Mraicha M Ksantini O Zouch M Ayadi S Sayadi andM Bouaziz ldquoEffect of olive fruit fly infestation on the quality

of olive oil from Chemlali cultivar during ripeningrdquo Food andChemical Toxicology vol 48 no 11 pp 3235ndash3241 2010

[2] K M Daane and M W Johnson ldquoOlive fruit fly managing anancient pest in modern timesrdquo Annual Review of Entomologyvol 55 pp 151ndash169 2010

[3] S E Michelakis P Neuenschwander and R Cavalloro ldquoEsti-mates of the crop losses caused byDacus oleae (Gmel) (DipteraTephritidae) in Crete Greecerdquo in Proceedings of the CECIOBCInternational Symposium on Fruit Flies of Economic Importancepp 603ndash611 AA Balkema Publishers Athens Greece Novem-ber 1982

[4] PNeuenschwander and SMichelakis ldquoThe infestation ofDacusoleae (Gmel) (Diptera Tephritidae) at harvest time and itsinfluence on yield and quality of olive oil in Creterdquo Zeitschriftfur Angewandte Entomologie vol 86 no 1ndash4 pp 420ndash433 1978

[5] R Gucci G Caruso A Canale et al ldquoQualitative changes ofolive oils obtained from fruits damaged by Bactrocera oleae(Rossi)rdquo HortScience vol 47 no 2 pp 301ndash306 2012

[6] J A Pereira M R Alves S Casal and M B P P OliveiraldquoEffect of olive fruit fly infestation on the quality of olive oil fromcultivars Cobrancosa Madural and Verdeal TransmontanardquoItalian Journal of Food Science vol 16 no 3 pp 355ndash365 2004

[7] A Tamendjari F Angerosa and M M Bellal ldquoInfluence ofBactrocera oleae infestation on olive oil quality during ripeningof Chemlal olivesrdquo Italian Journal of Food Science vol 16 no 3pp 343ndash354 2004

[8] F Angerosa L D Giacinto andM Solinas ldquoInfluence ofDacusoleae infestation on flavor of oils extracted from attacked olivefruits by HPLC and HRGC analyses of volatile compoundsrdquoGrasas y Aceites vol 43 no 3 pp 134ndash142 1992

[9] R Aparicio M T Morales and D L Garcıa-GonzalezldquoTowards new analyses of aroma and volatiles to understandsensory perception of olive oilrdquo European Journal of LipidScience and Technology vol 114 no 10 pp 1114ndash1125 2012

[10] A M Gomez-Caravaca L Cerretani A Bendini et al ldquoEffectsof fly attack (Bactrocera oleae) on the phenolic profile andselected chemical parameters of olive oilrdquo Journal of Agriculturaland Food Chemistry vol 56 no 12 pp 4577ndash4583 2008

[11] G Benelli G Caruso G Giunti et al ldquoChanges in olive oilvolatile organic compounds induced by water status and lightenvironment in canopies of Olea europaea L treesrdquo Journal ofthe Science of Food and Agriculture vol 95 no 12 pp 2473ndash2481 2015

[12] R Malheiro S Casal S C Cunha P Baptista and J A PereiraldquoOlive volatiles fromPortuguese cultivars CobrancosaMaduraland Verdeal Transmontana role in oviposition preference ofBactrocera oleae (Rossi) (Diptera Tephritidae)rdquo PLoS ONE vol10 no 5 Article ID e0125070 2015

[13] GAGonzalez-AguilarM E Tiznado-Hernandez R Zavaleta-Gatica and M A Martınez-Tellez ldquoMethyl jasmonate treat-ments reduce chilling injury and activate the defense responseof guava fruitsrdquo Biochemical and Biophysical Research Commu-nications vol 313 no 3 pp 694ndash701 2004

[14] J D Hare ldquoEcological role of volatiles produced by plants inresponse to damage by herbivorous insectsrdquo Annual Review ofEntomology vol 56 pp 161ndash180 2011

[15] R Gols J M Bullock M Dicke T Bukovinszky and JA Harvey ldquoSmelling the wood from the trees non-linearparasitoid responses to volatile attractants produced bywild andcultivated cabbagerdquo Journal of Chemical Ecology vol 37 no 8pp 795ndash807 2011


[16] M Dicke and I T Baldwin ldquoThe evolutionary context forherbivore-induced plant volatiles beyond the lsquocry for helprsquordquoTrends in Plant Science vol 15 no 3 pp 167ndash175 2010

[17] E Hatano G Kunert J P Michaud and W W WeisserldquoChemical cues mediating aphid location by natural enemiesrdquoEuropean Journal of Entomology vol 105 no 5 pp 797ndash8062008

[18] I Kaplan ldquoTrophic complexity and the adaptive value ofdamage-induced plant volatilesrdquo PLoS Biology vol 10 no 11Article ID e1001437 2012

[19] I Kaplan ldquoAttracting carnivorous arthropods with plantvolatiles the future of biocontrol or playing with firerdquo Biologi-cal Control vol 60 no 2 pp 77ndash89 2012

[20] MDicke andMW Sabelis ldquoHowplants obtain predatorymitesas bodyguardsrdquoNetherlands Journal of Zoology vol 38 no 2 pp148ndash165 1987

[21] M L Henneman E G Dyreson J Takabayashi and R ARaguso ldquoResponse to walnut olfactory and visual cues by theparasitic waspDiachasmimorpha juglandisrdquo Journal of ChemicalEcology vol 28 no 11 pp 2221ndash2224 2002

[22] P E Kendra A L Roda W S Montgomery et al ldquoGas chro-matography for detection of citrus infestation by fruit fly larvae(Diptera Tephritidae)rdquo Postharvest Biology and Technology vol59 no 2 pp 143ndash149 2011

[23] G Benelli S Revadi A Carpita et al ldquoBehavioral and electro-physiological responses of the parasitic wasp Psyttalia concolor(Szepligeti) (Hymenoptera Braconidae) to Ceratitis capitata-induced fruit volatilesrdquoBiological Control vol 64 no 2 pp 116ndash124 2013

[24] M Carrasco P Montoya L Cruz-Lopez and J C RojasldquoResponse of the fruit fly parasitoid Diachasmimorpha longi-caudata (Hymenoptera Braconidae) to mango fruit volatilesrdquoEnvironmental Entomology vol 34 no 3 pp 576ndash583 2005

[25] A Canale and G Benelli ldquoImpact of mass-rearing on the hostseeking behaviour and parasitismby the fruit fly parasitoidPsyt-talia concolor (Szepligeti) (Hymenoptera Braconidae)rdquo Journalof Pest Science vol 85 no 1 pp 65ndash74 2012

[26] G Benelli and A Canale ldquoDo tephritid-induced fruit volatilesattract males of the fruit flies parasitoid Psyttalia concolor(Szepligeti) (Hymenoptera Braconidae)rdquo Chemoecology vol23 no 3 pp 191ndash199 2013

[27] G Beltran M Uceda M Hermoso and L Frias ldquoMaduracionrdquoin El Cultivo del Olivo D Barranco R Fernandez-Escobar andL Rallo Eds pp 159ndash183 Mundi-Prensa Madrid Spain 2007

[28] G Benelli C Stefanini G Giunti S Geri R H Messingand A Canale ldquoAssociative learning for danger avoidancenullifies innate positive chemotaxis to host olfactory stimuli in aparasitic wasprdquoNaturwissenschaften vol 101 no 9 pp 753ndash7572014

[29] A Carpita A Canale A Raffaelli A Saba G Benelli andA Raspi ldquo(Z)-9-tricosene identified in rectal gland extracts ofBactrocera oleaemales first evidence of amale-produced femaleattractant in olive fruit flyrdquo Naturwissenschaften vol 99 no 1pp 77ndash81 2012

[30] R C Sprinthall Basic Statistical Analysis Allyn amp BaconBoston Mass USA 2003

[31] YMasadaAnalysis of Essential Oils byGasChromatography andMass Spectrometry John Wiley amp Sons 1976

[32] W Jennings Qualitative Analysis of Flavor and FragranceVolatiles by Glass Capillary Gas Chromatography AcademicPress New York NY USA 1980

[33] N W Davies ldquoGas chromatographic retention indices ofmonoterpenes and sesquiterpenes on methyl silicon and Car-bowax 20M phasesrdquo Journal of Chromatography A vol 503 pp1ndash24 1990

[34] R P Adams Identification of Essential Oil Components by GasChromatographyMass Spectrometry Allured Publishing CarolStream Ill USA 1995

[35] E Stenhagen S Abrahamsson and F W McLafferty Registryof Mass Spectral Data John Wiley amp Sons Hoboken NJ USA1974

[36] N P P Macciotta D Vicario C Di Mauro and A Cappio-Borlino ldquoA multivariate approach to modeling shapes of indi-vidual lactation curves in cattlerdquo Journal of Dairy Science vol87 no 4 pp 1092ndash1098 2004

[37] R I Jenrich ldquoStepwise discriminant analysisrdquo in StatisticalMethods for Digital Computers K Enslein A Ralston and HWilf Eds pp 76ndash95 John Wiley amp Sons New York NY USA1960

[38] R H Messing L M Klungness E B Jang and K A Nishi-jima ldquoResponse of the melon fly parasitoid Pysttalia fletcheri(Hymenoptera Braconidae) to host-habitat stimulirdquo Journal ofInsect Behavior vol 9 no 6 pp 933ndash945 1996

[39] A Eben B Benrey J Sivinski and M Aluja ldquoHost speciesand host plant effects on preference and performance ofDiachasmimorpha longicaudata (Hymenoptera Braconidae)rdquoEnvironmental Entomology vol 29 no 1 pp 87ndash94 2000

[40] L L Stelinski L J Gut A V Pierzchala and J R MillerldquoField observations quantifying attraction of four tortricidmoths to high-dosage pheromone dispensers in untreated andpheromone-treated orchardsrdquo Entomologia Experimentalis etApplicata vol 113 no 3 pp 187ndash196 2004

[41] J W P Silva J M S Bento and R A Zucchi ldquoOlfac-tory response of three parasitoid species (Hymenoptera Bra-conidae) to volatiles of guavas infested or not with fruit flylarvae (Diptera Tephritidae)rdquo Biological Control vol 41 no 3pp 304ndash311 2007

[42] M M Ero C J Neale E Hamacek T Peek and A R ClarkeldquoPreference and performance of Diachasmimorpha kraussii(Fullaway) (Hymenoptera Braconidae) on five commercialfruit speciesrdquo Journal of Applied Entomology vol 135 no 3 pp214ndash224 2011

[43] D F Segura M M Viscarret S M Ovruski and J L CladeraldquoResponse of the fruit fly parasitoid Diachasmimorpha long-icaudata to host and host-habitat volatile cuesrdquo EntomologiaExperimentalis et Applicata vol 143 no 2 pp 164ndash176 2012

[44] G Flamini P L Cioni and I Morelli ldquoVolatiles from leavesfruits and virgin oil from Olea europaea cv Olivastra Seg-gianese from Italyrdquo Journal of Agricultural and Food Chemistryvol 51 no 5 pp 1382ndash1386 2003

[45] S Dabbou M Issaoui F Brahmi et al ldquoChanges in volatilecompounds during processing of Tunisian-style table olivesrdquoJournal of the American Oil Chemistsrsquo Society vol 89 no 2 pp347ndash354 2012

[46] A Sansone-Land G R Takeoka and C F Shoemaker ldquoVolatileconstituents of commercial imported and domestic black-ripetable olives (Olea europaea)rdquo Food Chemistry vol 149 pp 285ndash295 2014

[47] D U Yongjun G M Poppy W Powell J A Pickett L J Wad-hams and C M Woodcock ldquoIdentification of semiochemicalsreleased during aphid feeding that attract parasitoid Aphidiuservirdquo Journal of Chemical Ecology vol 24 no 8 pp 1355ndash13681998


[48] C M De Moraes M C Mescher and J H TumlinsonldquoCaterpillar-induced nocturnal plant volatiles repel conspecificfemalesrdquo Nature vol 410 no 6828 pp 577ndash580 2001

[49] M E F Hoballah C Tamo and T C J Turlings ldquoDifferentialattractiveness of induced odors emitted by eight maize varietiesfor the parasitoid Cotesia marginiventris is quality or quantityimportantrdquo Journal of Chemical Ecology vol 28 no 5 pp 951ndash968 2002

[50] J-N Wei and L Kang ldquoElectrophysiological and behavioralresponses of a parasitic wasp to plant volatiles induced by twoleaf miner speciesrdquo Chemical Senses vol 31 no 5 pp 467ndash4772006

[51] A Hern and S Dorn ldquoInduction of volatile emissions fromripening apple fruits infested with Cydia pomonella and theattraction of adult femalesrdquo Entomologia Experimentalis etApplicata vol 102 no 2 pp 145ndash151 2002

[52] A Hern and S Dorn ldquoInduced emissions of apple fruit volatilesby the codling moth changing patterns with different timeperiods after infestation and different larval instarsrdquo Phyto-chemistry vol 57 no 3 pp 409ndash416 2001

[53] F Francis S Vandermoten F Verheggen G Lognay and EHaubruge ldquoIs the (E)-120573-farnesene only volatile terpenoid inaphidsrdquo Journal of Applied Entomology vol 129 no 1 pp 6ndash11 2005

[54] A Lenoir P DrsquoEttorre and C Errard ldquoChemical ecology andsocial parasitism in antsrdquoAnnual Review of Entomology vol 46pp 573ndash599 2001

[55] F C Abdalla and C da Cruz-Landim ldquoDufour glands inthe hymenopterans (Apidae Formicidae Vespidae) a reviewrdquoBrazilian Journal of Biology vol 61 no 1 pp 95ndash106 2001

[56] B Baccouri S B Temime E Campeol P L Cioni D DaoudandM Zarrouk ldquoApplication of solid-phasemicroextraction tothe analysis of volatile compounds in virgin olive oils from fivenew cultivarsrdquoFoodChemistry vol 102 no 3 pp 850ndash856 2007

[57] J-F Cavalli X Fernandez L Lizzani-Cuvelier and A-MLoiseau ldquoCharacterization of volatile compounds of French andSpanish virgin olive oils by HS-SPME identification of quality-freshness markersrdquo Food Chemistry vol 88 no 1 pp 151ndash1572004

[58] Y Ouni G Flamini M Issaoui et al ldquoVolatile compounds andcompositional quality of virgin olive oil from Oueslati varietyinfluence of geographical originrdquo Food Chemistry vol 124 no4 pp 1770ndash1776 2011

[59] S Vichi L Pizzale L S Conte S Buxaderas and E Lopez-Tamames ldquoSolid-phasemicroextraction in the analysis of virginolive oil volatile fraction characterization of virgin olive oilsfrom two distinct geographical areas of northern Italyrdquo Journalof Agricultural and Food Chemistry vol 51 no 22 pp 6572ndash6577 2003

[60] E Campeol G Flamini S Chericoni S Catalano and RCremonini ldquoVolatile compounds from three cultivars of Oleaeuropaea from Italyrdquo Journal of Agricultural and Food Chem-istry vol 49 no 11 pp 5409ndash5411 2001

[61] R Malheiro S Casal S C Cunha P Baptista and J APereira ldquoMadural and verdeal transmontana role in ovipositionpreference of Bactrocera oleae (Rossi)(Diptera Tephritidae)rdquoPloS ONE vol 10 no 5 Article ID e0125070 2015

[62] A Canale ldquoPsyttalia concolor (Hymenoptera Braconidae) roleof host movement and host substrate in ovipositor-probingbehaviourrdquo Bulletin of Insectology vol 56 no 2 pp 211ndash2132003

[63] H K M Dweck G P Svensson E A Gunduz and O Ander-brant ldquoKairomonal response of the parasitoid Bracon hebetorSay to themale-produced sex pheromone of its host the greaterWaxmothGalleriamellonella (L)rdquo Journal of Chemical Ecologyvol 36 no 2 pp 171ndash178 2010

[64] H Yu Y Zhang K Wu W G Xi and Y G Yu ldquoField-testingof synthetic herbivore-induced plant volatiles as attractants forbeneficial insectsrdquo Environmental Entomology vol 37 no 6 pp1410ndash1415 2008

[65] M Uefune Y Choh J Abe K Shiojiri K Sano and JTakabayashi ldquoApplication of synthetic herbivore-induced plantvolatiles causes increased parasitism of herbivores in the fieldrdquoJournal of Applied Entomology vol 136 no 8 pp 561ndash567 2012

[66] S Colazza A Fucarino E Peri G Salerno E Conti and FBin ldquoInsect oviposition induces volatile emission in herbaceousplants that attracts egg parasitoidsrdquo Journal of ExperimentalBiology vol 207 no 1 pp 47ndash53 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


hereafter) [14 15] The role of kairomones on parasitoidhost location has been widely investigated [16ndash18] and it hasbeen demonstrated that many plants rely on volatile signalsinduced by phytophagous feeding to attract their naturalenemies [14 19 20] Moreover despite the evidence aboutthe influence of B oleae infestation on the quality and thequantity of volatile compounds emitted in olive oils [7 8]no information is available to assess the presence of HIPVsproduced by infested olive fruits However differential emis-sions have been already proved for Tephritidae-infested andhealthy fruits [21ndash24] highlighting the production of severalHIPVs able to evoke electrophysiological and behavioralresponses in parasitoid wasps [23 24]

Psyttalia concolor (Szepligeti) (Hymenoptera Braconi-dae) is a koinobiont larval pupal endoparasitoid able toparasitize at least fourteen tephritids on different wild andorcultivated plants including B oleae and Ceratitis capitata(Wiedemann) the Mediterranean fruit fly [25] P concolorfemales rely on a number of stimuli to successfully locate theirhost Indeed female wasps are able to distinguish betweeninfested and healthy fruit preferring the first one even ifjust olfactory cues are provided [23] In addition it wasdemonstrated that apple and peach fruits infested by Ccapitata larvae emitted peculiar volatiles recognized by Pconcolor wasps and able to attract selectively mated females[23] HIPVs from apple and peach fruits are also able toattract and prolong the time spent performing searchingbehavior in P concolor virgin males probably raising theirchances to locate receptive females nearby host microhab-itat [26] Furthermore even synthetic blends reproducinginfested peaches or apples were found able to be attractive forP concolor mated females and virgin males [23 26]

In this research we investigated the importance of olfac-tory cues used by P concolor females to locate their hostmicrohabitat We hypothesize that the HIPVs from B oleae-infested olive fruits may play a pivotal role in affecting Pconcolor host location as described for the same parasitoid ona different tephritid host [23] Olive fruits from three differentcultivars were tested to determine parasitoid attractivenessand volatile organic compounds (VOCs) emissions cv Fran-toio and cv Leccino (traditionally cultivated in Italy) andcv Arbequina (typical of Spanish olive groves) Firstly weevaluated femalesrsquo preferences among healthy and infestedfruits in two-choice bioassay providing both visual andolfactory cues or olfactory stimuli alone in order to evalu-ate the magnitude of volatiles attractiveness Subsequentlyvolatiles emitted by healthy and infested olive fruits wereSPME-sampled and analyzed by gas chromatography-massspectrometry (GC-MS) to estimate differentially emissionsattributable to herbivoresrsquo activity and to indicate possibleHIPVs

2 Material and Methods

21 Parasitoid Rearing P concolor wasps were reared asdescribed by Canale and Benelli [25] Insects were main-tained in Pisa Laboratory under controlled conditions (22∘Cplusmn 1 50ndash60 relative humidity and natural photoperiod)

during their entire life Adult parasitoids were allowed toemerge in transparent Plexiglas tubes (diameter 40 cm length50 cm) into which 1500 adults were introduced (male femalesex ratio 03ndash05) To obtain pupae from which the adultemerged a nylon mesh bag containing around 700 thirdinstar C capitata larvae was posed into a cage and exposedto P concolor wasps for 20 minutes Parasitized pupae wereplaced into smaller Plexiglas cages (diameter 20 cm height30 cm) and there P concolor adults were allowed to emerge ata density of 50 specimens per cage (males females sex ratio03) Insects were stored at laboratory conditions [22 plusmn 1∘C50plusmn5 relative humidity and 16 8 (L D) photoperiod] for 7days after the parasitoidsrsquo emergence to allow mating beforetesting Adult insects were fed on a semisolid diet (honeymixed with pollen) and with water ad libitum

22 Plant Material Olive fruits from three different cultivars(Frantoio Leccino and Arbequina) were used for behav-ioral assay and GC-MS analysis Olives were collected onSeptember 15 2014 in Tuscan olive groves [Frantoio andLeccino fromTorrita di Siena (43∘151015840498610158401015840N 11∘781015840965810158401015840E)and Arbequina from Rapolano Terme (43∘271015840709710158401015840N11∘601015840709810158401015840E)] from 5ndash3-year olive trees Healthy or infestedolives from each cultivar were collected manually stored intoglass jars (diameter 10 cm length 20 cm) and transferred tolaboratory conditions within 3 hours The fruits were firstlydivided according to the maturation index (MI) whereby theskin and flesh colors were scored to a 0 to 7 scale [27] andolives with MI from 2 to 7 were discharged Among infestedolives we selected the ones attacked second or early thirdinstar larvae with no exit holes on the olive surface Healthyfruits were selected avoiding crushed and naturally damagedones

Before being tested fruits were stored at laboratory condi-tion for 2ndash5 days All olives used for both behavioral and GC-MS tests were subsequently dissected to check the presenceor the absence of B oleae larvae inside the fruits

23 Effect of Olfactory Cues from Infested Olives on ParasitoidAttractiveness Bioassays were conducted using the still airarena described by Benelli et al [28] A Plexiglas unit (150 times150 times 30mm) was covered on the top with a removable glasspanel to create the arenaTheunit presents a circular chamber(diameter 40mm) in the center to release the specimenand two other identical chambers connected with linearpaths (length 20mm width 10mm) where the stimuli wereallocated

To assess if infested olives are attractive for P concolorin a first experiment mated females were allowed to chooseamong three healthy or infested olive fruits of each cultivar Inaddition to investigate the role of olfactory stimuli in leadingparasitoid host location a second experiment was designedAs in the first experiment three infested or healthy fruits wereplaced into the test chambers but a piece of filter paper wasposed ahead of the fruits to avoid visual contact with theparasitoid female released in the central chamber

A replicate starts when a wasp was gently transferred tothe released chamber and observed for 8min A wasp was








119895= 120583 + 119868

119895+ 119862119895+

(119867119895times 119862119895) + 119890119895 in which 119910



119895the cultivar




3 Results






























Leccino (olfactory)

Infested

Infested

Infested

Infested

Infested

Infested

lowast

lowast

lowast

lowast

lowast

lowast


Healthy

Healthy

Healthy

Healthy

Healthy

Healthy









4 Discussion




percentage1 195168 20986 209862 136287 14654 356403 108109 11625 472654 65221 7013 542785 62379 6707 609856 53423 5744 667307 50994 5483 722138 43666 4695 769089 39679 4267 8117510 35437 3810 8498511 33083 3557 8854212 25223 2712 9125513 21784 2342 9359714 20431 2197 9579415 17363 1867 9766116 11942 1284 9894517 09813 1055 100000






Heptadecane


Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal




Methyl carvacrol

Liguloxide

6-Methyl tridecane


Isobornyl acetate

Epi-longipinanol

Lilial



58-Diethyl dodecane



DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene


120572-Cedrene



minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)


Component 1 (21)


(Z)-120573-Ocimene


(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)





120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








119895= 120583 + 119868

119895+ 119862119895+

(119867119895times 119862119895) + 119890119895 in which 119910



119895the cultivar




3 Results






























Leccino (olfactory)

Infested

Infested

Infested

Infested

Infested

Infested

lowast

lowast

lowast

lowast

lowast

lowast


Healthy

Healthy

Healthy

Healthy

Healthy

Healthy









4 Discussion




percentage1 195168 20986 209862 136287 14654 356403 108109 11625 472654 65221 7013 542785 62379 6707 609856 53423 5744 667307 50994 5483 722138 43666 4695 769089 39679 4267 8117510 35437 3810 8498511 33083 3557 8854212 25223 2712 9125513 21784 2342 9359714 20431 2197 9579415 17363 1867 9766116 11942 1284 9894517 09813 1055 100000






Heptadecane


Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal




Methyl carvacrol

Liguloxide

6-Methyl tridecane


Isobornyl acetate

Epi-longipinanol

Lilial



58-Diethyl dodecane



DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene


120572-Cedrene



minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)


Component 1 (21)


(Z)-120573-Ocimene


(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)





120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Leccino (olfactory)

Infested

Infested

Infested

Infested

Infested

Infested

lowast

lowast

lowast

lowast

lowast

lowast


Healthy

Healthy

Healthy

Healthy

Healthy

Healthy









4 Discussion




percentage1 195168 20986 209862 136287 14654 356403 108109 11625 472654 65221 7013 542785 62379 6707 609856 53423 5744 667307 50994 5483 722138 43666 4695 769089 39679 4267 8117510 35437 3810 8498511 33083 3557 8854212 25223 2712 9125513 21784 2342 9359714 20431 2197 9579415 17363 1867 9766116 11942 1284 9894517 09813 1055 100000






Heptadecane


Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal




Methyl carvacrol

Liguloxide

6-Methyl tridecane


Isobornyl acetate

Epi-longipinanol

Lilial



58-Diethyl dodecane



DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene


120572-Cedrene



minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)


Component 1 (21)


(Z)-120573-Ocimene


(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)





120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 Discussion




percentage1 195168 20986 209862 136287 14654 356403 108109 11625 472654 65221 7013 542785 62379 6707 609856 53423 5744 667307 50994 5483 722138 43666 4695 769089 39679 4267 8117510 35437 3810 8498511 33083 3557 8854212 25223 2712 9125513 21784 2342 9359714 20431 2197 9579415 17363 1867 9766116 11942 1284 9894517 09813 1055 100000






Heptadecane


Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal




Methyl carvacrol

Liguloxide

6-Methyl tridecane


Isobornyl acetate

Epi-longipinanol

Lilial



58-Diethyl dodecane



DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene


120572-Cedrene



minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)


Component 1 (21)


(Z)-120573-Ocimene


(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)





120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Heptadecane


Cyclosativene

Undecane

Valencene

7-Epi-sesquithujene

Dodecanal




Methyl carvacrol

Liguloxide

6-Methyl tridecane


Isobornyl acetate

Epi-longipinanol

Lilial



58-Diethyl dodecane



DecanolLongifolene

(Z)-2-Tridecene

Dihydro myrcenol

Nonanal

Neo menthol

120572-Copaene


120572-Cedrene



minus10

minus05

00

05

10

Com

pone

nt 2

(14

7)


Component 1 (21)


(Z)-120573-Ocimene


(a)

0 5 10minus5minus10

Component 1 (21)

minus10

5

0

5

10

Com

pone

nt 2

(14

7)

(b)





120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


120140160180200220240260280

Cano

nica

l 2


Canonical 1










5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 Conclusions





Acknowledgments


References













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article VOCs-Mediated Location of Olive Fly