Establishing abiotic and biotic factors necessary for reliable male pheromone production and ...

20 Pages·2014·0.38 MB·English

1
Establishing abiotic and biotic factors necessary
for reliable male pheromone production
and attraction to pheromones by female
plum curculios Conotrachelus nenuphar
(Coleoptera: Curculionidae)
VirginiaHock1,GéraldChouinard,ÉricLucas,DanielCormier,TracyLeskey,
StarkerWright,AijunZhang,AndréPichette
Abstract—Theplumcurculio(PC),ConotrachelusnenupharHerbst(Coleoptera:Curculionidae),is
akeypestofstoneandpomefruitinNorthAmerica.Thoughgrandisoicacid(GA)wasidentifiedasa
male-producedaggregationpheromoneforthisspecies,othercomponentslikelyexist,ashavebeen
identified for various curculionids. To better determine these components, an understanding of the
conditionsnecessaryforoptimumpheromoneproductionandattractionisneeded,thisisessentialfor
theimprovementofmonitoringtechniquesandtoachievebetterbiologicalcontrol.Thegoalofthis
studywastodeterminethebioticandabioticfactorsinfluencingboththeresponsetopheromonesand
pheromone production. Tests were conducted in a dual-choice still-air vertical olfactometer using
live male PCs as odour sources and live females as responders, to determine which physiological
factors (age, number of males, mating status) influenced female response to males. Head-space
collections of GA production under various conditions (airflow rate and frequency, collection zone
strata, variation of humidity, temperature, and presence of a harbourage) were also done, as were
electroantennograms (EAG) using synthetic pheromone mixtures. Results revealed that for both
strains, the odour of two virgin mature males elicited significantly greater and more consistent
attraction from mature virgin females than other ages and numbers of males when compared with
the control. Head-space collections indicate that male PC have increased production of GA under
high humidity in the presence of fruit, indicating that these conditions are necessary for optimal
pheromone production and collection. EAG studies revealed significant responses to GrandLures I,
II, III/IV and to the positive enantiomer of GA, and the amplitude of the signal varied with
concentration. Our data identify the optimal physiological state and conditions at which
pheromone collections should be performed, and what physiological life stages respond to these
stimuli. These results have implications for optimising monitoring tools for this serious crop pest.
This species has a northern univoltine strain and a southern multivoltine strain, both of which
were examined in this study.
Received14March2013.Accepted9October2013.
V.Hock,1LaboratoiredeProductionFruitièreIntégrée,Institutderechercheetdedéveloppementenagroenvironne-
ment(IRDA),335,chemindesVingt-CinqEst,Saint-Bruno-de-Montarville,Québec,CanadaJ3V0G7;andLaboratoire
de lutte biologique, Département des sciences biologiques, Université du Québec à Montréal (UQAM), Case postale
8888,succursaleCentre-ville,Montréal,Québec,CanadaH3C3P
G.Chouinard,D.Cormier,LaboratoiredeProductionFruitièreIntégrée,Institutderechercheetdedéveloppement
enagroenvironnement(IRDA),335,chemindesVingt-CinqEst,Saint-Bruno-de-Montarville,Québec,CanadaJ3V0G7
É. Lucas, Laboratoire de lutte biologique, Département des sciences biologiques, Université du Québec à Montréal
(UQAM),Casepostale8888,succursaleCentre-ville,Montréal,Québec,CanadaH3C3P
T. Leskey, S. Wright, United States Department of Agriculture, Agricultural Research Service – Appalachian Fruit
ResearchStation,2217WiltshireRoad,Kearneysville,WestVirginia25430,UnitedStatesofAmerica
A. Zhang, Invasive Insect Biocontrol and Behaviour Laboratory, United States Department of Agriculture (USDA),
Agricultural Research Service – Plant Science Institute, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350,
UnitedStatesofAmerica
A.Pichette,UniversitéduQuébecàChicoutimi,555,boul.del'Université,Chicoutimi,Québec,CanadaG7H2B1
1Correspondingauthor:(e-mail:[email protected])
Subjecteditor:VéroniqueMartel
doi:10.4039/tce.2014.1
Can.Entomol.00:1–20(2014) ©2014EntomologicalSocietyofCanada
2 Can.Entomol. Vol.00,2014
Résumé—Lecharançondelaprune,ConotrachelusnenupharHerbst(Coleoptera:Curculionidae),
est un ravageur important des fruits à noyau et à pépins en Amérique du Nord. Bien que l'acide
grandisoïque ait été identifié comme une phéromone d'agrégation produite par les mâles de cette
espèce,ilestprobablequed'autrescomposéssecondairesexistentcommec’estlecaspourd'autres
espèces de curculionidés. Afin d’identifier ces possibles composés additionnels, une bonne
compréhension des conditions nécessaires à l’émission et à l’attraction aux phéromones est
nécessaire, en vue de l'amélioration des techniques de dépistage et de lutte. Le but de cette étude
était de mesurer l’effet de facteurs biotiques et abiotiques qui influencent à la fois la réponse aux
phéromonesetlaproductiondephéromones.Destestsontétéeffectuésdansunolfactomètrevertical
(sans courant d'air) à deux voies avec des mâles vivants comme émetteurs de la phéromone
d'agrégation et avec des femelles comme réceptrices des odeurs afin de déterminer les facteurs
physiologiques (âge, quantité de phéromone, statut d’accouplement) qui influencent la réponse des
femellesauxmâles.Descollectesdephéromonesontétéfaitessousdiversesconditions(débitd'airet
fréquencedelacollection,humidité,température,stratesetabris,etc.)demêmequedesétudesmenées
sur l'électroantennogramme (EAG) enutilisant des mélanges dephéromones synthétiques. Pour les
deuxsouches,l'odeurdedeuxmâlesviergesmaturesasuscitéuneréponseplusforteetplusconstante
de la part des femelles vierges matures que des autres âges et nombres de males. Les collectes de
phéromones ont révélé que le charançon de la prune produit de plus grandes quantités d’acide
grandisoïqueenconditionsdefortehumiditéetenprésencedefruit.TouteslesodeursdeGrandLures
testées (I, II, III/IV) et l'énantiomère positif de l'acide grandisoïque ont provoqué des réponses
significativesdescharançonsparEAG.L’amplitudedusignaldel’EAGaégalementvariéselonla
concentrationdesodeurstestées.Cesrésultatsprécisentlesconditionsphysiologiquesducharançonde
laprunepourlesquellesl’attractionauxphéromonesestlaplusimportante,etdansquellesconditions
ils produisent la plus grande quantité de phéromone. Ils aident aussi à identifier les conditions
optimales dans lesquelles les collections de phéromones doivent être effectuées afin d'analyser la
phéromone d'agrégation et d’optimiser les outils de lutte contre ce ravageur majeur des cultures. Il
existedeuxsouchesdecetteespèce,unesoucheunivoltinedunord,etunesouchemultivoltinequise
trouveplusausud.Lesdeuxsouchesontétéétudiéesdanscetteétude.
Introduction the pepper weevil (Anthonomus eugenii Cano;
Coleoptera: Curculionidae) (Eller et al. 1994), and
Theplumcurculio(PC)Conotrachelusnenuphar the strawberry blossom weevil (Anthonomus rubi
Herbst (Coleoptera: Curculionidae) is a major pest Herbst;Coleoptera:Curculionidae)(Innocenzietal.
ofpomeandstonefruitsinNorthAmerica,capable 2001), which produce multiple component aggre-
of damaging 90% of fruit at harvest (Vincent and gation pheromones. Although onlyone component
Roy 1992). There are two strains of this insect, a (thepositiveenantiomerofGA)hasbeenfoundthus
univoltinenorthernoneandamultivoltinesouthern far (Eller and Bartelt 1996), it is likely that such a
one(Racetteetal.1992),whicharereproductively closespeciesalsoproducesablendofcompounds.
incompatible (Padula and Smith 1971; Zhang and While advancements in the control of PC
Pfeiffer2008;Zhangetal.2010). have been made (Piñero et al. 2011) the use of
To locate fruit and mates for feeding and repro- pheromones in monitoring PC still requires
ductivepurposesPCuseolfactorycues(Butkewich improvement. Analysis of behavioural and physio-
etal.1987;ButkewichandProkopy1993;Ellerand logical factors influencing pheromone production
Bartelt1996;Leskeyetal.2005).Theodouroflive and response has been very useful in developing
PC males has been shown to be attractive to both monitoringand controltechniques for otherinsects
sexes of conspecifics and attributed to an aggrega- (Hardee 1982; Klassen et al. 1982; Jutsum and
tion pheromone (1R,2S)-1-methyl-2-(1-methylethe- Gordon1989;Burkholder1990;RidwayandInscoe
nyl)-cyclobutaneaceticacid,emittedbyvirginmales 1990; Silverstein 1990; Neilsen and Jensen 1993;
ofthespecies,alsoknownasgrandisoicacid(GA) Smart et al. 1994). Production of GA may be
(Eller and Bartelt 1996). The emission of a male- important throughout the lifetime of male PC; in
produced aggregation pheromone by PC has also laboratory studies they continue to mate until they
been described inotherweevilspecies, notably the die (Johnson and Hays 1969). Since many factors
boll weevil (Anthonomus grandis Boheman; regarding the aggregation pheromone produced by
Coleoptera:Curculionidae)(Tumlinsonetal.1969), PCremainunknown,itisimportanttocharacterise
©2014EntomologicalSocietyofCanada
Hocketal. 3
the behaviour of this insect regarding its naturally resulting in consistent GA production; EAGs
produced pheromone component(s) in order to were conducted in order to determine (6) if PCs
develop better biological pest management strate- respondedelectrophysiologicallytoGAaswellas
gies for this insect. Many things can affect toGrandLurecomponents.
the response to, and release of, pheromones, as is Plum curculios have been shown to prefer the
seeninmanyotherweevilspecies.Forexample,sex odour of five males over one male (Leskey and
has been shown to affect the banana weevil Prokopy 2001). We therefore expected female
(Cosmopolites sordidus (Germar)) (De Graaf et al. attractiontolivemalestobegreatestbetweentwo
2005), where more females were attracted to and eight mature males (Leskey and Prokopy
pheromones than males. Amount of pheromone 2001). We also predicted that virgin and mated
producedisafactorintheresponseofthecigarette females would exhibit a similar attraction to
beetle (Lasiderma serricorne (Fabricius); Coleop- males, based on what was found in other olfact-
tera:Anobiidae)(Kuwaharaetal.1975)andtheboll ometer trials with PC (Akotsen-Mensah 2010).
weevil(Hardeeetal.1974),withmalesresponding Sexuallymaturemalesarecapableofmatingwith
to specific quantities. Age also influences the females with most matings occuring between
response of the boll weevil (Spurgeon 2003) with 16–20 days (Johnson and Hays 1969); PC have
pheromones being produced in larger quantities in been observed to take an average of 13 days to
younger insects; and mated status affects the rice mature (Smith 1957; Smith and Salkeld 1964).
weevil (Sitophilus oryzae (Linnaeus); Coleoptera: Based on the reproductive behaviour of this
Curculionidae)(PhillipsandBurkholder1981),with insect, itislikely thatmature males willbemore
matedmalesbeinglessattractivethanvirginmales. attractive tofemalesthanimmatureoroldmales.
Advancements in pest management strategies High humidity and temperatures in the presence
throughtheanalysisofpheromonecomponentsand of suitable fruit should also elicit a good pher-
by measuring behaviour and attraction to aggrega- omoneproductionbasedonthenaturalbehaviour
tionpheromoneshavebeenusedagainstotherwee- of the insect, which exhibits a greater activity
vilswithsuccess,withthebollweevilbeingaprime undertheseconditions(GarmanandZappe1929;
example (Tumlinson et al. 1969; Dickerson et al. SmithandFlessel1968;ButkewichandProkopy
1987; Spurgeon 2003). If complete analysis of the 1993; Chouinard et al. 1993, 1994). It is likely
PCaggregationpheromonecanbeachieved,similar that PCs respond to low doses of GA (Leskey
advancements would be possible for the control of and Prokopy 2001), since high doses do not
thispestaswell. result in an increased attraction (Prokopy et al.
The goal of this study was to characterise 2004). Plum curculios should also respond more
theolfactoryresponseofPCtothenaturalodours stronglytomixturesthatcontaingreateramounts
produced by the males (Eller and Bartelt 1996). of the positive enantiomer, since this is what
In addition, we also determined the conditions is produced naturally (Eller and Bartelt 1996).
resulting in optimum pheromone production as Both strains were studied, since they both
well as evaluated electroantennogram (EAG) attack pome and stone fruits (Quaintance and
responses to pheromone stimuli and related Jenne 1912) and produce the aggregation pher-
compounds. To this end bioassays as well as omone (Eller and Bartelt 1996), with the main
head-space collections and EAGs were con- differences between them being their geo-
ducted. Bioassays consisted of a vertical dual- graphical distribution (Quaintance and Jenne
choice still-air olfactometer where responses 1912),theirdiapause(obligateversusfacultative)
to conspecific odours were tested using PC in (Bobb 1952), and their reproductive incompati-
different physiological states. Experiments were bility (Zhang and Pfeiffer 2008; Zhang et al.
designed to determine: (1) the quantity of males 2010). In order to avoid fruitless interstrain mat-
needed to elicit a maximum attractive response ing, the two strains may respond differently to
from females, (2) the age of males that was the odoursofconspecificsandpheromonesinregards
most attractive to females, (3) the effect of toquantityorratio,butweexpectthatbothstrains
responder mating status, and (4) the effect of willrespondtothesametypeofodours(i.e.,both
strain. Head-space collections were aimed at strains will be able to detect GA and respond to
identifying (5) abiotic and biotic conditions conspecifics).
©2014EntomologicalSocietyofCanada
4 Can.Entomol. Vol.00,2014
Materialsandmethods experiment 2. In experiment 2 immature males
(less than two weeks old), mature males (two to
Bioassays
three weeks old), and old males (three to four
Plum curculio. PC from both strains were used.
weeksold)weretestedasodoursources.
Univoltine PC were obtained and maintained as
All females tested were virgin except for
described by Hock et al. (2013). Insects were
experiment3(virginversusmatedfemales),where
obtained from infested apples (Malus Miller
females from the “mated” modality were held
species (Rosaceae)) collected in late June early
with males for two weeks before experiments to
July 2009 from unsprayed orchards and kept
allowformating(Leskeyetal.2010).Allfemales
in emergence cages, with emerging adults col-
from the “mated versus virgin” experiment were
lected daily and immediately separated by sex
dissectedattheendoftheexperimenttoverifytheir
(Thompson 1932), transferred to overwintering
physiologicalstatus.Inordertoverifymaturityand
cages (Le Blanc 1992), and then placed under
sexual status, half of the females tested were
natural conditions throughout winter. Over-
randomlyselectedandplacedin70%ethanoland
wintered adults were removed from the cages
heldat4°C(Hoffmannetal.2004)untildissected.
the following spring and placed in 2-L plastic
Dissections were done as per Hoffmann et al.
containers with small apples and water (wetted
(2004), which involved examining the state of
cotton dental wick). The containers were placed
oocytes and the spermatheca. Although there was
in environmental control chambers at 25±2°C,
very little chance of females from the virgin
70% relative humidity, and 16:8 hour light:dark
modality being mated, a sample of these females
photoperiod to mimic optimal summer condi-
wasdissectedtovalidateresults.
tions (Amis and Snow 1985). A laboratory
population using some of the wild individuals
Olfactometer. The olfactometer is described in
collected in 2009 was also established as descri-
Hock et al. (2013) and consisted of a large
bed in Hock et al. (2013) using the procedure of
(inner Ø = 105mm, 50mm in height) round
Hoffmannetal.(2007).
Pyrex® glass container (Corning Inc., Corning,
Trials with multivoltine curculios were taken New York, United States of America). Two
from a laboratory population established at the lateral openings on the apex (inner Ø = 24mm,
AppalachianFruitResearchStation(Kearneysville, 60mmheight)weretheconnectors(leftandright)
West Virginia, United States of America) in 2001 for the odour jars while a central apical opening
and augmented annually with wild individuals, as (inner Ø = 24mm, 60mm height) was the site
perLeskeyetal.(2010).Adultswererearedinthe where PCs were introduced into the arena. Each
laboratory at 25±2°C, 14:10 hour light:dark 500mL Mason™ “odour jar” (Bernardin Ltd.,
photoperiodonadietofgreenthinningapplesand Richmond Hill, Ontario, Canada) contained either
water based on the methods of Amis and Snow male PCs (test odour) or air only (control odour)
(1985).Newlyemergedadultswereheldinmixed- (Tinzaara et al. 2007). Standard nylon mosquito
sexgroupsof100individuals,andwereallowedto screening was used to cover the lateral two open-
mate and lay eggs in thinning apples. Larvae ings of the olfactometer as well as the Mason
emerging from apples were placed in 500mL jars jars in order to prevent females from escaping
containing soil and shipped to the Institut de and reaching the odour source. Parafilm®M
recherche et de développement en agroenvironne- (Sigma-Aldrich Canada Ltd., Oakville, Ontario,
ment (IRDA; St-Hyacinthe, Québec, Canada). Canada) was used to secure the inverted Mason
Jarscontainingthemultivoltinepupaewereplaced jarstotheolfactometer.
in environmental control chambers at 25±2°C,
70%relativehumidity(16:8hourlight:darkphoto- Experimental conditions. Tests were conducted
period). Emerging adults were collected daily, asdescribed inHocketal.(2013)inanobserva-
separated by sex and held under the same condi- tion room held at 25±2°C and 70% relative
tionsastheunivoltinestrain. humidity (Smith and Flessel 1968; Racette
All female PCs used as “test PC” in experi- et al. 1991; Chouinard et al. 1993). A red filter
mentsweresexuallymature,aswereallmalePCs (LEE Filter, red primary no. 106®, Son-Art
used as odour sources with the exception of Production, Saint-Hyacinthe, Québec, Canada)
©2014EntomologicalSocietyofCanada
Hocketal. 5
was used to cover a neon light (40W); the sole the time of arrival at the destination (personal
source of light during experiments since PC are observations).
not perturbed by red light (Prokopy et al. 1995).
At the beginning of each trial one female was Male PCs. For the constant versus random flow
introduced into the olfactometer and left for rate experiment, four-day-old virgin multivoltine
30 minutes, after which its position was noted males were used and collections were done
and the female removed; only insects found everyfourdaysforadurationof36days.Forall
within a radius of 10mm or within the tubes other experiments, male PCs were 15 days old
leading to the odour jars (e.g., test or control and collections were collected daily for five
odour zones) were used for statistical analysis; days. There were three replicate jars (with eight
other positions were considered as no choice maleseach)foreachmodality.
having been made and were disregarded (aver-
age of 25%) (Altuzar et al. 2007; Tinzaara et al. Head-space collection procedure. Head-space
2007; Akotsen-Mensah 2010). Each female was volatiles were collected from eight multivoltine
considered one replicate, and odour zones were virgin males introduced into six 1-L, four-necked
glass containers (Fig. 1A, B) and provided with
randomised after each replicate. The average
eight green thinning apples, except in the “Plum”
number of replicates was 17, however occasion-
experiment were either eight Stanley plums or
ally replicate number was higher. Also, because
nothing was given (control). Air was drawn into
there were occasional deaths of PCs during rear-
the container through 6–14 mesh activated char-
ing within the selected age groups, and because
not all PCs responded during trials, there were coal (Fisher Scientific, Pittsburgh, Pennsylvania,
times when replicate number was lower. Odours UnitedStatesofAmerica),andoutofthecontainer
tested were virgin males of different ages and of through a trap (outer Ø 150×15mm) containing
Super Q (0.200g each; Alltech Associates Inc.,
differentquantities.
Deerfield, Illinois, United States of America) by
vacuum (~1L/minute per collection tube×two
Statistical analysis. SPSS statistical software
tubes per container). Volatiles were aerated con-
(SPSS Inc., 2006, Chicago, Illinois, United States
tinuouslyfor24hoursat25±2°Cand14:10hour
ofAmerica)wasusedtoanalysealldata.Compar-
light:dark photoperiod. Volatile collections from
isons between choices (test versus control)
each of the two sample tubes per container were
of the number of PC were analysed using a two-
tailed χ2 test (P<0.05), as were the responses eluted daily with 2mL methylene chloride and
immediately stored in a freezer at −30°C. Each
between experimental modalities examined (e.g.,
sampletubewassubsequentlyrinsedwithanaddi-
female response to test odours of immature males
tional4mLofmethylenechloride.
versus mature males versus old males); a Yates
continuity correction was applied when necessary Sampleswereanalysedusinggaschromatography-
(SiegelandCastellan1988).Comparisonsbetween mass spectrometry (GC-MS). Electronic impact
experiments (e.g., responses of univoltine mated (EI) GC-MS was conducted on a HP 6890 GC
females versus multivoltine mated females to test coupled to a HP 5973 Mass Selective Detector
odours) or between strains were done using a using an DB-WAXETR capillary column
Mann–WhitneyUtest(P<0.05). (60000×0.25mm ID, 0.25mm film-thickness,
J&W Scientific Inc., Folsom, California, United
Head-spacecollections
States of America). Oven temperature was set at
Head-spacecollectionsweredoneusingmulti- 50°C for 2 minutes, then programmed to 230°C
voltine PC, since the tests were conducted at the at15°C/minutesandheldfor15minutes.A70eV
United States Department of Agriculture- electronbeamwasemployedforsampleionisation
Agricultural Research Service (Kearneysville)
andheliumwasusedascarriergas.
during the winter months of January–March,
when no wild univoltine PCs were available due Experimental parameters. Conditions associated
todiapause.Also,shippinglaboratory-rearedPCs with three of the six collection jars were mani-
overlongdistances,particularlyduringthewinter, pulated while the other three collection jars
usuallyresultsinlossoflargenumbersofPCsby were held under standard (control) conditions.
©2014EntomologicalSocietyofCanada
6 Can.Entomol. Vol.00,2014
Fig.1.Dualsource(laboratory)head-spacecollectionapparatus.(A)Schematic,(B)actualsetup.
Controls included eight virgin male PCs with humidity combination (20°C at 25% relative
eight apples and water (wetted cotton wicks) humidityversus30°Cat75%relativehumidity),
held at 25±2°C at 75% relative humidity, no and harbourage inclusion (folded paper versus
harbourage, standard position for collection no folded paper) (Table 1). All of the above
tubes, and continuous collections at the normal weretestedinordertodetermineifalteringthese
flow rate of 1L/minute (for each tube). Experi- factors improved pheromone production. For
mental conditions evaluated included: random example, it has been shown that PCs increase
versus constant airflow, flow rate (0.5L/minutes activity, movement, and oviposition at higher
versus 1L/minute), collection tube strata/posi- levels of relative humidity (Hoyt et al. 1983;
tion (low versus standard), humidity (15% ver- LeBlancetal.1984;Racetteetal.1991;Chouinard
sus 75%, and 25% versus 75%), temperature/ et al. 1993, 1994; Prokopy and Wright 1998;
©2014EntomologicalSocietyofCanada
Hocketal. 7
Table 1. Average GA peak in each modality from each collection, as well as the highest magnitude GA peak
detectedforeachmodality.
Head-spacecollections
Averagepeak Highest Numberofcontainerswhere
Modality Description magnitude* peak GAwasdetected
Extractions Random 8.48 90.00 3
Constant 3.21 30.00 3
Flowrate 0.5L/minute 0.14 0.24 2
1L/minute 1.83 8.88 2
Collectiontubestrata Lowerposition 3.16 13.94 3
Standardposition 3.68 21.57 2
HumidityI 15% 2.53 18.17 3
75% 10.73 41.97 3
HumidityII 25% 1.71 1.71 1
75% 8.00 22.46 3
Temperature 20°C(25%relativehumidity) 7.58 13.39 1
30°C(75%relativehumidity) 2.75 4.38 2
Foodsource 8plums 17.94 46.78 2
0plums 0.86 0.86 1
Harbourage Foldedpaper(refuge) 0.17 1.20 1
Control(norefuge) 0.30 0.42 2
Note:*TakenonlyfromthosecontainerswhereGAwasfoundtobeproduced(maximumofthreecontainerspermodality).
Dixon et al. 1999; Prokopy et al. 1999) and therefore only multivoltine PC were used for
warmer temperatures. Low temperatures (⩽6°C) the same reasons as previously listed for head-
arrest development (Sarai 1969; Piñero and Pro- spacetrials.
kopy 2004), and temperatures between 15°C and
19°C tend to immobilise or reduce PC move- Female PCs. Multivoltine PCs used in EAG
ment, while temperatures between 20°C and trialsweretreatedasdescribedabove(bioassays)
30°C result in increased PC activity and flight (Leskey et al. 2010). Test subjects were fed,
(Chouinard et al. 1993, 1994; Prokopy and sexually mature females aged 14–21 days, since
Wright 1998; Dixon et al. 1999; Prokopy et al. sexually mature females are considered to be the
1999; Leskey and Prokopy 2002). It is possible most damaging portion of the population and
that an increase in activity may be related to an give statistically higher EAG responses com-
increaseinpheromoneproductioninmales. paredtomales (Leskey etal.2009,2010). There
were four replicates per female, with seven
Statistical analysis. SPSS statistical software was females in the high dose modality, and six
used to analyse all data. Each trial/modality was females for the low dose modality and also for
analysed by performing a Wald’s statistic logistic thetrialscomparingGA.
regression (P<0.05) to determine if any of the
manipulationsinfluencedpheromoneproduction. Odour sources. Odour sources included the syn-
thetic volatile trans-2-hexenal (T2H) and volatiles
Electroantennogramtrials collected from “Stanley” plum (at 21mm fruit)
Electroantennograms trials were conducted found previouslyto be highly stimulating (Leskey
at the United States Department of Agriculture- et al. 2009, 2010), both of which were used as
Agricultural Research Service (Kearneysville, standards. The “Stanley” plum standard was pro-
West Virginia, United States of America) since duced according to the methods described in
the equipment for conducting EAG trials of PC Leskey et al. (2010). Other synthetic volatiles
was only available there. EAG studies werecon- used were the positive enantiomer of grandisoic
ducted at the same time as head-space trials, acid(+GA)andtheracemicmixtureofgrandisoic
©2014EntomologicalSocietyofCanada
8 Can.Entomol. Vol.00,2014
acid (RGA), all coupled with a dichloromethane each time. After four re-crystallisation steps, the
(DCM) solvent control. The RGA contains equal solid was dissolved in CH C and the negative
2 l2
amounts of the positive and negative enantiomers enantiomer of GA was extracted with NaHCO .
3
(50:50), while +GA contains 71% +GA. The The aqueous layer was washed with CH C and
2 l2
+GA was made from RGA, which was obtained neutralisedwithaq.HCl10%,whichprecipitates
fromgrandisolthroughoxidationperformedatUni- GA. The latter was extracted with CH C , dried
2 l2
versityofQuebecatChicoutimi,Québec,Canada. with MgSO4 and evaporated under reduced
pressure.Themixturewasacidifiedwithaq.HCl
Procedure for oxidation of racemic grandisol.
10% and GA was extracted with CH C . The
2 l2
Grandisoic acid was prepared by sequential
organic phase was washed with aq. HCl 10%,
oxidation of grandisol, the active ingredient of
driedwithMgSO andevaporatedunderreduced
4
commercial GrandLure (Bedoukian Research,
pressure.
Danbury, Connecticut, United States of America).
N-methylmorpholine (NMO, 3.40g, 29.0mmol) Three different lurescontaining grandisol (from
which GA is made) were also tested and obtained
was added to a dried solution of 2.24g of grand-
isol (14.5mmol) in methylene chloride (CH Cl , fromChemTica(SanJose,CostaRica).Theselures
2 2
25mL). The mixture was chilled with ice water, were:GrandLureI(cis)-1-methyl-2-(1-methylethe-
nyl)cyclobutaneethanol, GrandLure II (Z)-2-(3,3-
after which the catalytic amount of tetrapropyl-
dimethylcyclohexylidene) ethanol and GrandLure
ammonium perruthenate (TPAP, 0.255g, 0.726
III/IV (Z/E)-(3,3-dimethylcyclohexylidene)acetal-
mmol) was added to a dried solution of grandisol
dehyde. All synthetic volatiles were evaluated at
(CH C ). The mixture was stirred for 1 hour at
2 l2
two concentrations: 0.010 and 0.001g/mL diluted
room temperature or until the reaction was com-
inDCM.
plete, as shown by using thin-layer chromato-
graphy. The solution was evaporated under
reducedpressuretogiveablackoilyresidue. Electroantennogram setup. Electroantennogram
experiments were conducted as in Leskey et al.
The second oxidation step was conducted (2009, 2010). Subject females were immobilised
by dissolving crude grandisal in a mixture of
in a custom polycarbonate insect holder. The
t-butanol(30mL),water(5mL),and2-methyl-2- indifferent electrode was filled with a diluted
butene(31mL).Themixturewaschilledto0°C.
reference electrode solution (diluted from 4.0 M
An aqueous solution (20mL) containing sodium
KCl-saturated AgCl to 0.4 M). The electrode
chlorite (2.36g, 26.1mmol) and monosodium
wastheninsertedthroughaportinthetopofthe
phosphate (3.80g, 27.6mmol) was added drop-
insect holder through the exposed membrane
wiseoveraperiodof10minutes.Themixturewas attached to the ventral cervical sclerite between
vigorously agitated for a period of 30 minutes. the thorax and the head. The recording electrode
Work-up was performed by evaporating the was similarly filled with electrode solution and
solvent and adding 50mL of NaOH N. The
2 inserted through a port at the rear of the holder
mixture was washed with CH C (4×50mL).
2 l2 into the mid-point (between antennomeres two
Theaqueousphasewasacidifiedwithaq.HCl10%
and three) of the immobilised four-antennomere
to pH 4. The product was extracted with CH Cl
2 club. This region of the antenna has the highest
(5×50mL) and dried with MgSO . Evaporation
4 concentration of potential olfactory receptors
afforded1.90gofRGA(78.0%yield). (AlmandHall1986).Theinsectholderwasthen
nested into a secondary polycarbonate slide-
Enantiomeric purification. Enantiomeric purifi-
frame (10mm width×6.0mm height×110mm
cation was performed by co-crystallisation of
length) to permit insertion of polished tungsten
GA; 1g of GA was dissolved in hot ethanol-
electrodes (Ø = 0.2×140mm length) into the
water mixture (50:50) and 0.965g of quinine
filledglasselectrodes.
was added. The solution was allowed to chill in
a cold room (−18°C) for 24hours. The crystals BaselineoutputsignalfromantennaeofPCwas
obtained were filtered and re-crystallised three allowedtostabilisefor10minutes.PCexhibiting
other times in a hot ethanol-water mixture output baseline variation (noise) greater than
(50:50). The mother liquors were combined ±25mVwerenottested(10%ofthosemounted).
©2014EntomologicalSocietyofCanada
Hocketal. 9
Fig.2.FemaleresponsetotheodourofdifferentnumbersofmalesforbothunivoltineandmultivoltinePC.Error
bars=±SE, n = number or replicates, * =significant differences between test and control odours using χ2 test
(P<0.05).
Afterbaselinestabilisation, theinsectholderwas Statisticalanalysis.SPSSstatisticalsoftwarewas
insertedintoacylindricalport(Ø = 16mm)atthe used to analyse all data. The EAG response data
terminusofamovingairstream,andcleanairwas from each individual were analysed using the
passedacrosstherecordedantennaat1L/minute. GLM procedure for mixed models to construct
Astimuluscartridgewaspreparedforeachodour analysisofvariancetablesformeanamplitudeof
stimulus.Inbrief,0.05mLsolutionfromthe8mL response (mV) among all individuals evaluated.
parent extract was dispensed onto a filter paper The EAG sensitivity model evaluated the effect
strip (Whatman Grade three filter paper, 75×6 ofodour stimulus with replicate used as a block-
mm, Whatman Inc., Piscataway, New Jersey, ing factor. When the GLM indicated significant
UnitedStatesofAmerica).Afterevaporation,the differences between multiple odours, multiple
strip was loaded into a glass Pasteur pipette and comparisons were calculated using Tukey’s
mountedona10mLsyringe.Arotationof2mL honestlysignificantdifference(P<0.05).
puffsofeachtestedstimuluswasinjectedbyhand
into the clean air stream through an orifice 150 Results
mm upwind from the antenna at a 30 second
interval. Four replicates were performed per Bioassays
insect, yielding a 12 minute total trial time for Response of virgin females to different numbers
each responder. A total of seven females were of males. For (virgin) univoltine PC and multi-
evaluatedforresponsestoeachofthehighdoses voltine PC, significantly more females responded
of stimuli while six females were evaluated for to males compared with the control only for the
responses to the low dose and GA trials, twomalemodality(univoltine:χ2 = 9.941,df = 1,
respectively. P = 0.002; multivoltine: χ2 = 4.172, df = 1,
Inputsignalswereamplifiedandreceivedbya P = 0.041) (Fig. 2). Results for the response
USB-1608FSdataacquisitionunit(Measurement of (virgin) females to one male (univoltine:
Computing Corporation, Norton, Massachusetts, χ2 = 0.692, df = 1, P = 0.405; multivoltine:
UnitedStatesofAmerica).Signalswerepassedto χ2 = 0.043, df = 1, P = 0.835), and five males
a computer-based analytical program (DasyLab (univoltine:χ2 = 1.333,df = 1,P = 0.248)versus
9.0; Dasytec USA, Amherst, New Hampshire, the control revealed no significant attractive
United States of America) for interpretation and response. There was however a significant repul-
recording of output. Output samples were taken sionformultivoltinefemalestowardsthefivemale
andrecordedatarateof31samplespersecond. modality(χ2 = 5.400,df = 1,P = 0.020).
©2014EntomologicalSocietyofCanada
10 Can.Entomol. Vol.00,2014
Fig. 3. Female response to the odour of different ages of males for both univoltine and multivoltine. Error
bars=±SE, n = number or replicates, * =significant differences between test and control odours using χ2 test
(P<0.05),x =significantdifferencesbetweenstrainsusingMann–WhitneyUtest(P<0.05).
Fig. 4. Virgin and mated female response to the odour of two males for both univoltine and multivoltine PC.
Error bars =±SE, n = number or replicates, * = significant differences between test and control odours using
χ2test(P<0.05).
Response of females to different ages of males. males: χ2 = 1.316, df = 1, P = 0.251, old males:
Since two males produced the most attractive χ2 = 1.143, df = 1, P = 0.285) (Fig. 3). The
odour for both strains of PC, this was the quan- response of females to mature males differed sig-
tity used as an odour source for the remaining nificantlybetweenstrains(Z =−2.151,P = 0.031)
experiments. Experiments with male emitters withunivoltinefemalesbeingmoreresponsivethan
of different ages revealed that for univoltine multivoltinefemales.
PC, only mature males were significantly attrac-
tive (χ2 = 14.727, df = 1, P<0.001) versus the Mated versus virgin female response to two
control (Fig. 3). For multivoltine PC, there was males. Only virgin females of both strains were
no significant difference for any of the modalities significantly attracted to two males (univoltine:
(Fig. 2) when compared with the controls (imma- χ2 = 4.000 df = 1, P = 0.045; multivoltine:
ture males:χ2 = 1.800, df = 1, P = 0.180;mature χ2 = 4.455,df = 1,P = 0.035)(Fig.4).
©2014EntomologicalSocietyofCanada