Sex Pheromone of HorseChestnut Leafminer Cameraria ohridella and Its Use in a Pheromone-Based...

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Journal of Chemical Ecology [joec] pp751-joec-459300 February 18, 2003 19:41 Style file version June 28th, 2002

Journal of Chemical Ecology, Vol. 29, No. 2, February 2003 (©C 2003)

SEX PHEROMONE OF HORSE-CHESTNUT LEAFMINERCameraria ohridellaAND ITS USE IN A PHEROMONE-BASED

MONITORING SYSTEM

B. KALINOV A,1,∗ A. SVATOS,1,2 J. KINDL,1 O. HOVORKA,1 I. HRDY,1

J. KULDOVA,1 and M. HOSKOVEC1

1Department of Natural Products – Insect Chemistry GroupInstitute of Organic Chemistry and Biochemistry

Academy of Sciences of the Czech RepublicFlemingovo nam. 2, 166 10 Prague 6, Czech Republic

2Max Planck Institute of Chemical EcologyWinzerlaer Str. 10, 07745 Jena, Germany

(Received February 12, 2002; accepted October 5, 2002)

Abstract—Gas chromatography combined with electroantennographic detec-tion (GC-EAD), electroantennography (EAG), and wind-tunnel and field ex-periments were used to reinvestigate the composition ofCameraria ohridella(Lepidoptera, Gracillariidae, Lithocolletinae) sex pheromone. The GC-EAD ex-periments showed one EAD-active area corresponding to the major pheromonecomponent, (8E,10Z)-tetradeca-8,10-dienal. The EAG experiments proved that(9E)-tetracedecenal and stereoisomers of (8E,10Z)-tetradeca-8,10-dienal ex-hibited significant electrophysiological activity and could, therefore, be con-sidered as possible minor pheromone components. However, wind-tunnel andfield experiments demonstrated that none of these compounds affect the efficacyof the main pheromone component. A monitoring system based on (8E,10Z)-tetradeca-8,10-dienal was developed and used to study the flight activity ofC. ohridella.

Key Words—Pheromone, (8E,10Z)-tetradeca-8,10-dienal, Camerariaohridella, Lepidoptera, horse-chestnut leafminer, antennal sensitivity, fieldexperiments, monitoring.

∗ To whom correspondence should be addressed. E-mail: [email protected]

387

0098-0331/03/0200-0387/0C© 2003 Plenum Publishing Corporation

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388 KALINOV A ET AL.

INTRODUCTION

Cameraria ohridellaDeschka et Dimić (Lepidoptera, Gracillariidae, Lithocol-letinae), a species recently found and described in Macedonia (Simova-Toˇsicand Filov, 1985; Deschka and Dimić, 1986), has spread almost everywhere incentral Europe and is moving towards the west (Puchberger, 1990; Holzschuhand Krehan, 1992; Butin and Führer, 1994; Szabóky, 1997; Tomiczek, 1997;Krehan, 1997; Tomiczek and Krehan, 1998;Sefrova and Lastuvka, 2001). Thistiny leafminer selectively infests horse-chestnut trees (Aesculus hippocastanum,Hippocastanaceae).

Females ofC. ohridellaproduce a sex pheromone to attract males for mating(Svatos et al., 1999a). The major sex pheromone component was recently identifiedas (8E,10Z)-tetradeca-8,10-dienal [8E10Z-14:Al] (Svatos et al., 1999b). In thisstudy, we reinvestigated the pheromone with particular attention to EAG activecompounds that could be considered as minor pheromone components. Using anovel pheromone-based monitoring system, we describe the pattern ofC. ohridellaflight activity.

METHODS AND MATERIALS

Insects.Horse-chestnut leaves infested with overwinteringC. ohridellapu-pae were field-collected in autumn. Collected leaves were kept outdoors. After thefirst freezing period, leaves were placed in nylon cages in the laboratory, and adultswere allowed to emerge (temperature 20–23◦C, 50% relative humidity, 16L:8Dphotoperiod regime). Emerged adults were kept individually in 5-ml glass vials toprevent mating. Pheromone glands of 1- to 2-day-old virgin calling females wereexcised and extracted for 1 hr with hexane (5µl/gland). The EAGs were recordedfrom antennae of 1- to 2-day-old males kept at 5◦C. In behavioral experiments,1- to 2-day-old males kept at 20–23◦C, 16L:8D photoperiod and 50% relative hu-midity were flown in a wind tunnel. Field experiments were performed during the2000–2001 seasons.

Scanning Electron Microscopy.For scanning electron microscopy (SEM),male antennae and female abdominal tips were dehydrated for 15 min each inan ascending ethanol series starting at 70% and in acetone, mounted on stubs,sputter-coated with gold (300A), and viewed in a scanning electron microscope(Jeol 6300, Jeol Ltd., Akishima, Japan).

EAG Recordings.The whole animal preparation (Svatoˇs et al., 1999a,b),Ag–AgCl electrodes, CyberAmp 320 amplifier (Axon Instruments) equipped withAI402 probe (Axon Instruments), PC with A/D conventer board (Metrabyte DAS-16), and Stand-Alone Enhanced Data Acquisition System (Version 3.0, RUN Tech-nologies) were used for EAG recordings.

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HORSE-CHESTNUT LEAFMINER SEX PHEROMONE 389

EAG PROFILES

The saturated and monounsaturated C12 and C14 alcohols, acetates, and alde-hydes (12:Ac, 12:OH, 12:Al, 14:Ac, 14:OH, 14:Al; purity 99%) were dissolved inhexane. One microgram of the respective compound was loaded on a filter paperdisk (1 cm diam.), hexane was evaporated, and the loaded disk was placed into aPasteur pipet, sealed with Parafilm, and stored in a freezer until use. To achievestimulation, a solenoid-activated odor delivery system was used to divert a continu-ous clean airstream (charcoal filtered and humidified, velocity 1 liter/min) throughthe loaded Pasteur pipet (stimulus duration: 0.5 sec) and then over the antenna ofthe fixed moth. Maximum negative deflections of EAG potentials were recorded.Successive stimuli were separated by 5-min intervals to allow the antennae torecover from the previous stimulation. The antennal sensitivity and its possiblechange during the experiment were checked by standard stimulation [1µg of(10Z)-tetradec-10-en-1-yl acetate]. Stimuli were applied to the antenna randomlyto prevent systematic errors caused by previous stimulations. Data from six prepa-rations with approximately constant responses to standard stimulation within theexperimental session were evaluated: EAG mean values were calculated for eachcompound. Means were plotted against tested chemical identities to create EAGprofiles.

EAG Dose–Response Experiments.The test compounds were diluted indecadic steps in hexane. Pasteur pipets were loaded with aliquots of 10µl (theamount of substance in the pipets ranged between 0.1 and 1µg). The test sub-stances were presented to the antenna at all dose levels, starting with the lowestones. The stimuli were presented with an interval of 2 min. Where reasonable, theEAG data were analyzed using one-way ANOVA followed by Dunnett’s multiplecomparison test (α = 0.05).

GC-EAD Experiments.In GC-EAD experiments, intact antennal prepara-tions served as biological detectors for the HP 5890A gas chromatograph. Threecolumns with different polarities: DB-Wax, DB-5, and DB-1 (30 m length, 0.25 ID,film thickness 0.25µm; J & W Scientific) were used. The inlet and FID detectortemperatures were set at 200◦C and 260◦C, respectively. Samples were injectedsplitless. Helium was used as a carrier gas. The temperature program used forthe analyses: 70◦C for 2 min, 4◦C/min to 140◦C, 5◦C/min to 220◦C, 20◦C/min to280◦C, and then 5 min at this temperature. The column was split at the end by a four-arm splitter (Graphpack 3D/2). The first arm of the splitter was linked to the originalcolumn, the second arm was connected to the FID detector by means of 0.5 m ofa deactivated silica column (0.33 mm id), and the third arm (0.5 m of deactivatedsilica column) led through a transfer line (heated up to 180◦C) to the outside of theGC (Effluent Conditioning Assembly, Syntech, Hilversum, The Netherlands). Thethird arm ended in a mixing chamber, from which a continual airstream (1 liter/min)was blowing onto the antennal preparation via a PTFE-coated stainless steel flow

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tube. Makeup gas (20 ml/min, N2) was introduced through the fourth arm of thesplitter to prevent condensation.

Chemicals.The pheromone, 8E10Z-14:Al, and its geometric isomers weresynthesized in our laboratory (Hoskovec et al., 2000). Synthetic pheromone stan-dards were purchased from Pherobank, IPO-DLO, Wageningen, The Netherlands.

Wind-Tunnel Experiments.The flight tunnel consisted of a rectangular tube(clear Plexiglas) 186 cm long. Its upwind and downwind ends were closed witha mesh screen (2-mm openings). Charcoal filtered and humidified air was pushedthrough the tunnel by means of four ventilators creating an airspeed of 0.25 m/sec.The flight tunnel was diffusely illuminated from above with a light intensity ofabout 1000 lux. The stimuli were applied on a filter paper disk (1 cm diam.)positioned in a holder fixed in the center of the upwind mesh. The plume parametersand orientation were checked periodically using TiCl4 smoke. The experimentswere performed during the first 2 hr of the photophase. Prior to an experiment,males were placed individually into clean glass tubes (10 cm long, 4 cm diam.),closed by nylon cloth. These release cages were kept in an illuminated quiet placeuntil use (at least 15 min). The stimulus source was set in place, and the release cagewas then opened and placed into a holder positioned in the central part of the flightcompartment near the bottom. In this position, the male inside the cage was stilloutside the odor plume and usually did not respond. Then, the holder was movedup into the odor plume, and the male’s behavior was observed during the following5 min. Males that did not respond (no antennal movement, walking, wing fluttering,or taking off) were considered nonresponding. The behavior of responding maleswas classified into four categories: (1) activation (walking, antennal movements,and wing fanning), (2) take off, (3) oriented flight, and (4) source localizationand landing. Each male was tested only once. Twenty-five males were flown infive replicates to each treatment. An extract of three pheromone glands servedas a standard stimulus and was presented at the beginning of every experiment.When 4 of 5 males responded to the standard stimulus, other treatments weretested. After each experimental session, the glass tubes and holders were washedwith light petroleum and oven-dried at 200◦C. The data were analyzed byχ2 test(α = 0.05).

Field Experiments.All field experiments were performed in Prague, CzechRepublic. In the majority, delta traps (Biolatrap Delta 155; ZD Chelˇcice, CzechRepublic) containing sticky paper inserts (200× 80 mm), covered with LonameltPS 3199/05 glue (Vetox, Prague, Czech Republic) were used. Traps were placedwithin the canopy of horse-chestnut trees at 1–2 m above the ground. Filter paper,PE foil, and rubber septa (Thomas Scientific, Swedesboro, New Jersey, USA;Cat. No.1780-J07) were used as pheromone dispensers. Filter paper and PE foildispensers were replaced daily, and rubber dispensers were replaced every 14 days.Traps were rotated daily to eliminate any position effect (Latin-square design)(Perry et al., 1980). Sticky inserts were replaced when necessary. The data were

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analyzed by two-way (position and treatment) ANOVA (α = 0.05) in all cases,except for monitoring experiments.

RESULTS

Scanning Electron Microscopy.Scanning electron microscopy (SEM) of fe-male abdominal tips exposed during calling behavior revealed that the sexpheromone gland is associated with the intersegmental membrane between theeighth and ninth segments and the papillae anales. The gland is located dorsallyin the intersegmental membrane (Figure 1A).

Olfactory sensilla were found over the whole annular surface of long flagellateantennae interspaced between regularly arranged scale rows (Figures 1B,C). SEMof male and female antennae showed that male antennae possess male-specificolfactory sensilla trichodea, presumably specialized for pheromone perception.

GC-EAD and EAG Experiments.The GC-EAD analyses of pheromone glandextracts consistently showed only one EAD response (Figure 2A). The respective

FIG. 1. (A) Scanning electron micrographs (SEM) of exposed pheromone gland and papillaannales; (B) scanning electron micrograph (SEM) of two annuli of flagellate male antennacovered by scales and olfactory sensillae; (C) SEM of male-specific sensilla trichodea.

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FIG. 1. Continued.

compound eliciting the EAG response was not FID- or MS-detectable, even thoughhighly concentrated extracts were analyzed (>100 FE). The Kovats’ indices ofthe EAD active peak obtained on three different GC columns matched with theretention times of 8E,10Z-14:Al (Table 1). No GC-EAD evidence about the even-tual minor pheromone component(s) was obtained. The GC-EAD recordings with

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FIG. 2. Sections of GC-EAD/FID traces: (A) pheromone gland extract (ca. 10 FE); (B) 1µgof a solution of the four synthetic stereoisomers of 8, 10–14:Al; (C) 1 ng of 8E10Z-14:Al;(D) 1 µg of 9E-14:Ald. A mixture of C9–C22 hydrocarbons was coinjected with testedsamples to help peak identification.

synthetic standards revealed also the EAD activity for other stereoisomers of 8,10–14:Al (Figure 2B). The EAG activity of theEZ stereoisomer (Figure 2C) wasfollowed by ZZ, EE, andZE, respectively. As can be seen in Figure 3, theEZdose–response curve exhibited a typical sigmoidal shape with a threshold dose of0.1 ng/pipet (one-way ANOVA, Dunett’s multiple comparison test,α = 0.05) and

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TABLE 1. KOVATS’ I NDICESa OF EAD PEAK IN C. ohridellaFEMALE EXTRACTS

AND SOME OTHER SYNTHETIC COMPOUNDS

CompoundsFemale extractc

GC phaseb (ca. 3 FE) 12:Ac 14:Ald 14:OH 8E10Z-14:Alc

DB-1d 1623.3 1623.9DB-5d 1674.4 1605.9 1610.8 1675.4 1674.3DB-WAXe 2031.2 2031.8

aBased on saturated hydrocarbons.bJ & W Scientific, 30 m× 0.25 mm, film thickness 0.25µm.cFor EAD trace.d170◦C.e140–240◦C at 5◦C/min.

saturation above 100 ng/pipet. TheZZ curve was less sigmoidal, reached lowermaximum EAG values, and was shifted by one order of magnitude to higher dosesthan theEZ isomer (threshold dose 1 ng). TheEEandZEcurves were much flatter.TheEE isomer exhibited the highest threshold dose (10 ng) and lowest saturationamplitude.

EAG Profile Spectra.The EAG profile spectra showed a pattern compatiblewith 8E,10Z-14:Al as the main pheromone component and provided no cluesabout the prospective minor ones. Among linear saturated C12 and C14 standards,

FIG. 3. The dose-response curves of the four stereoisomers of 8, 10–14:Al. Each valuerepresents mean from six antennal preparations and standard error of mean.

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FIG. 4. Male EAG responses to 1µg of saturated C12 and C14 alcohols, acetates, andaldehydes (mean+ standard error of mean,N = 6). Asterisks represent significantly higherEAG response than to hexane (one-way ANOVA,α = 0.05; hex= hexane).

aldehydes elicited higher EAG responses than alcohols or the corresponding ac-etates. Tetradecanal (14:Al) elicited higher EAG responses than dodecanal (12:Al)and dodecyl and tetradecyl acetates (12:Ac, 14:Ac) and alcohols (12:OH, 14:OH).12:Ac gave higher responses than 14:Ac (Figure 4). In the monounsaturated C14 se-ries, EAG activity increased gradually when the double bond was moved from posi-tion 2 to position 7, remained high between positions 8 and 10, and decreased againwhen the double bond was moved to the penultimate position (Figure 5). Maxi-mum EAG activity was observed with the double bond in position 9. TheE-isomer,9–14:Al (9E-14:Al) was more active than the correspondingZ isomer (9Z-14:Al)

FIG. 5. Male EAG responses to tetradecenals with different double bond positions(mean+ standard error of mean,N = 6; hex= hexane).

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FIG. 6. Male EAG responses to three thermodynamic mixtures of tetradecadienals withdienic system in positions 9,11; 7,9, and 8,10 (mean+ standard error of mean,N = 6;hex= hexane).

and in GC-EAD experiments elicited significant EAD activity (Figure 2D). Fromconjugated tetradecadienals with carbon–carbon double bonds in positions 7,9,8,10, and 9,11, male antennae were the most sensitive to 8,10-14:Al (Figure 6).

Wind Tunnel Experiments.In wind-tunnel experiments, 8E10Z-14:Al alonewas attractive toC. ohridella males in extremely low doses. Males respondedreadily to 0.1 pg loaded onto a filter paper disk. 8E10Z-14:Al (1 pg) elicited amale behavioral response comparable to that evoked by an extract of 3 femaleequivalents (FE) (Figure 7).

FIG. 7. Male behavioral responses to pheromone gland extract (3 FE; black bar) and to0.1, 1 and 10 pg of synthetic 8E10Z-14:Al (see legend). X-axis: the respective behavioralcategory. Y-axis: percentage of males displaying the behavior.

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TABLE 2. ATRACTIVENESS OFMIXTURES OF8E10Z-14:Al AND 9E-14:Al TO

Cameraria ohridellaMALES IN FIELD EXPERIMENTSa

Pheromone–9E-14:Al ratio

1:0 1:1 5:1

Catches/trap 386.6± 58.8 371.7± 29.2 389.6± 30.5(mean± SEM)

a Delta traps, dispenser= 1 cm2 of PE–Aluminum foil, sticky inserts replaced daily, Latin-square design, 2 replicates. Loadings: 1:0 ratio= 5 ng of 8E10Z-14:Al; 1:1 ratio= 5 ngof 8E10Z-14:Al and 5 ng of 9E-14:Al; 5:1 ratio= 5 ng of 8E10Z-14:Al and 1 ng of 9E-14:Al (mean catches per trap and day± standard error of mean, 2 replicates). Differenttreatments were not statistically different (two-way ANOVA,α = 0.05).

Neither 9E-14:Al nor stereoisomers of 8E10Z-14:Al attracted males whenpresented alone in wind-tunnel or field experiments (data not shown). When theywere presented in 1:1 or 10:1 mixtures with the pheromone (10 pgEZ), they didnot significantly affect the effectiveness of the main pheromone (Tables 2 and3), suggesting that they do not take part in the sexual communication system ofC. ohridella.

TABLE 3. BEHAVIORAL RESPONSES OFC. ohridellaMALES TO 1:1AND 10:1 BLENDS OF10 pgOF 8E10Z-14:Al AND 10 OR 1 pgOF

RESPECTIVEISOMERS INWIND-TUNNEL EXPERIMENTS

Responding males (%, mean± SEM)

EE ZE ZZ Standard stimulus

E Z–isomer= 1:1No reaction 0± 0 0± 0 0± 0 0± 0Activation 100± 0 100± 0 100± 0 100± 0Take off 88± 4.89 96± 4.00 88± 4.89 96± 4.00Oriented flight 84± 4.00 88± 4.89 72± 4.89 72± 4.89Localization 80± 0 80± 6.33 68± 4.86 72± 4.89

EZ–isomer= 10:1No reaction 0± 0 0± 0 0± 0 0± 0Activation 100± 0 100± 0 100± 0 100± 0Take off 92± 4.89 96± 4.00 96± 4.00 100± 0Oriented flight 84± 4.00 80± 6.32 92± 8.00 92± 4.89Localization 80± 0 76± 4.0 76± 7.48 80± 6.32

a One or 10 pg of the respective isomer was added to 10 pg of 8E10Z-14:Al andcompared with a standard stimulus (10 pg of 8E10Z-14:Al). Mean percentages ofresponding males by the respective behavior+ standard error of mean (N = 25).No significant differences between various treatments and the standard stimuluswas observed (χ2 test;α = 0.05).

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FIG. 8. The effect of pheromone dose on trap effectiveness (delta trap, pheromone wasloaded on red rubber septa). The number of caught males was counted after 3 hr of trapexposure (10:00–13:00 hr; mean+ standard error of mean,N = 12).

Monitoring System.Traps baited with 8E10Z-14:Al, were remarkably at-tractive. Their efficacy depended on the pheromone dose loaded onto the dis-penser. Figure 8 shows the effect of pheromone doses ranging from 50 ng to 10µgof 8E10Z-14:Al per dispenser on trap catches (tested in August 2001 in Matˇej,Prague 6). In some traps, moths were attracted by a dose as small as 10 ng. Malesof C. ohridellawere active mainly during the period from 06:00 to 18:00 hr with apeak between 10:00 and 12:00 hr (tested on August 8, 2001, inCimice; Figure 9).

FIG. 9. The circadian activity ofC. ohridellamonitored by delta traps baited with 100 ngof 8E10Z-14:Al.

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FIG. 10. Flight activity ofC. ohridella monitored by delta traps baited with 100 ng of8E10Z-14:Al loaded on red rubber septa. Sticky inserts were exposed two times per weekfor 24 hr, pheromone dispensers were replaced every 14 days. (A) year 2000 flight season;(B) year 2001 flight season.

The pattern of flight activity ofC. ohridella estimated by pheromone trapcatches was influenced by climatic conditions during the respective season.Figure 10 shows the flight activity ofC. ohridella and temperature recorded attwo localities (Cimice I and II; Prague 8) during the 2000 and 2001 seasons. Dur-ing the 2000 season (Figure 10A), the flight activity exhibited two distinct peaks(May 5 and June 24) separated by a period without any catches (2–3 weeks). Thesecond decline exhibited a smaller secondary peak on July 4. Considering the timeof egg, larval, and pupal development, the two main peaks likely correspond totwo generations. The whole season was relatively warm. The situation was slightlydifferent the following year (Figure 10B). In 2000, the first moths appeared at theend of April, and trap catches of the first generation reached two maxima on May14 and 25. Individuals of the second generation appeared at the beginning of July,created two peaks on July 13 and August 3 and reached almost zero around August15. At the end of August, and in September and October, several smaller activitypeaks occurred. The relative increase of the second generation over the first wasmore pronounced than in 2000. The third generation was detected during the periodof late August and September. The 2001 season was cooler than that of 2000.

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DISCUSSION

SEM. The compact gland structure found inC. ohridellalocated dorsally inthe intersegmental membrane between the 8th and 9th abdominal segments is quitecommon among Lepidoptera. Families where it is found include the Ageriidae,Olethreutidae, Tortricidae, Noctuidae, Pyralidae, Gelechiidae, and others (Percy-Cunningham and MacDonald, 1987). The observed antennal dimorphism betweensexes also is widespread among Lepidoptera. The male-specific sensilla trichodeaof C. ohridellapresumably contain pheromone receptor cells, since male but notfemale antennae respond to the pheromone (data not shown).

Pheromone.Our data confirmed that 8E10Z-14:Al is the main pheromonecomponent ofC. ohridella.(Svatos, 1999b; Francke et al., 2003). None of the testedcompounds synergized or inhibited the attractiveness of 8E10Z-14:Al, althoughthey were EAG active. However, the EAG itself does not necessarily indicate that acompound is part of a pheromonal blend, since compounds chemically similar to apheromone could elicit significant EAG responses (Roelofs, 1977, 1984; Löfstedtand Lofquist, 1983). The prospect thatC. ohridella employs a one-componentpheromone system fits with the pattern of EAG response profiles reflecting maleantennal specificity. On the other hand, minor pheromone components are usuallypresent in considerably lower amounts than the major one(s), and male antennaeare less sensitive to them due to fewer olfactory receptor neurons devoted to theirdetection (Roelofs, 1977, 1984; Löfstedt and Löfquist, 1983). Therefore, there stillis a theoretical possibility thatC. ohridellaminor pheromonal component(s) existbut are present in amounts not detectable by available analytical instrumentation.Many pheromones originally identified as a single component proved to be mul-ticomponent blends when reinvestigated with better analytical equipment.

The great majority of lepidopteran sex pheromones are multi-, rather thansingle-component blends (Arn,), although one-component pheromones also exist(i.e., inLymantria dispar) (Bierl et al., 1970; Cardé et al., 1977). Among the eightpheromones so far investigated in the family Gracillariidae (subfamily Gracillari-inae), two are based on a single component. InAcrocercopssp. and inGracillariaelongella, 8E10E-14:Al and 10Z12E-16:Al, respectively, were reported as singleattractants for conspecific females (Ando et al., 1987; Priesner, 1985).

Pheromone-Based Monitoring System.The high efficacy of the main com-pound allowed the successful employment in pheromone-based monitoring sys-tems. From available trap systems (Cardé and Elkinton, 1984), the delta stickytraps were selected because of low cost and operational simplicity. However, theirsuitability is limited, since the sticky surface might become saturated or loose itsstickiness upon aging. Delta traps used in our experiments retain a maximum of ap-proximately 600–700 moths per sticky insert. Thus, in a monitoring system, whenthe population density is high, saturation could disorder the relationships betweentrap catch and population density unless sticky inserts are changed frequently or

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traps are baited with very low dosages of the attractant. The pheromone doseaffects the trap efficiency considerably. Generally, a high rate of pheromone emis-sion produces an increase in trap catches (Cardé and Elkinton, 1984), although trapattractiveness may be weak at high release rates. The effect of reduced responsive-ness is especially pronounced inG. molesta, a species that exhibits attraction andmating responses to a comparatively narrow range of pheromone concentrations(Carde et al., 1975; Baker and Cardé, 1979; Baker and Roelofs, 1981). Similartrends presumably exist in other species, but are not pronounced inC. ohridella,where trap catches increased over 4 orders of magnitude and inhibitory effectswere only observed at high doses (over 10µg/dispenser). The increase of trapcatches with high pheromone loadings might be attributed to increased activespace of the trap. Higher doses likely attract males from longer distances. Dataon the estimation of trap active space forC. ohridellaare not available.Sefrovaand Lastuvka (2001), who studiedC. ohridelladispersal in Europe for 10 yearsand estimated the invasion rate to be 60–90 km annually, considered air currenttransport as the most important factor for the dispersal of this species (Sefrova andLastuvka, 2001). The invasion rate, however, tells nothing about how far a male canactively fly while searching for a mate, since the dispersal behavior of pheromone-responsive and nonresponsive insects might differ and males can disperse muchfarther than females (Cardé, 1979). High release rates, thus, may lure males froma considerable distance, thereby causing migration of populations from outsidethe investigated area. Therefore, such trap catches may not accurately reflect therespective population density and infestation level.

Rubber septa are the most common dispensers for controlled pheromonerelease currently used in monitoring traps (Daterman, 1982; Vrkoˇc et al., 1988).However, they do not release pheromones at a constant rate over time or at differenttemperatures or wind speeds (Zeoli et al., 1982; Cardé and Elkinton, 1984; Vrkoˇcet al., 1988; Horák et al., 1989; Streinz et al., 1993; Svatoˇs, unpublished data).The evaporation rate of pheromones from the red rubber septa used in our studydecreased over 50% within 14 days of exposure (data not shown). Therefore,periodic replacement was necessary to ensure trap efficacy. Replacement was alsorequired because pheromones with conjugated double bonds are generally lessstable and undergo photooxidation and isomerization (Ideses et al., 1982; Brownand McDonough, 1986). Isomeric purity is critical for attraction in some species(Vrkoc et al., 1988; Horák et al., 1989). Apparently this is not the case withC. ohridella, where addition of theZE, ZZandEE isomers of 8E10Z-14:Al doesnot significantly affect the attractiveness for males of theEZ isomer.

The circadian rhythm observed in male flight activity corresponds closelyto the circadian rhythm of calling behavior in females (Svatoˇs et al. 1999a).The dynamic of pheromone trap catches confirmed two to three generations ofC. ohridella in Prague. The data show that the abundance ofC. ohridella doesnot increase dramatically during the season as was originally expected (Deschka,

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1993, 1995). The second generation was the most abundant, while the third gener-ation, when present, was much smaller and did not reach the level of the first one.Paradoxically, the number of generations was lower in the warmer year, 2000, thanin the following, colder 2001. One factor that could be responsible for this effectmight be food availability. A significant increase in larval mortality was reportedwhen mines on a leaf started to coalesce (Heitland and Freise, 2001). In 2000, theinfestation level was much higher, and the second generation might have experi-enced considerable food shortage during development (due to mine coalescence),resulting in a higher ratio of pupae entering diapause and, thus, elimination of asignificant third generation.

The number ofC. ohridellagenerations found by pheromone trapping differsfrom the data of Skuhravý (1998), who reported four to five overlapping genera-tions from the Czech Republic. However, the existence of so many generations peryear is unlikely considering the time required for egg, larval, and pupal develop-ment (Heitland et al., 2000; Grabenweger and Grill, 2000; Laˇstuvka andSefrova,unpublished observations). On the other hand, our phenological data agree withthose reported from other areas in Europe (Deschka and Dimić, 1986; Pschorn-Walcher, 1994; Hellrigl, 1999; De Prins and Puplesiene, 2000).

In summary 8E10Z-14:Al was confirmed as the main pheromone componentin Cameraria ohridella. No minor components were found. The scope and lim-itations of pheromone sticky traps were discussed. Up to three generations weredetected in the Czech Republic by means of trapping of males.

Acknowledgments—We thank Jarmila Titzenthalerová and Ondˇrej Blazek for skillful assistance.We also thank Dr. Frantiˇsek Weyda and Marie Nebesárova from the Entomological Institute of Academyof Sciences of the Czech Republic for their kind assistance with SEM. Special thanks are given toDr. Robert E. Doolittle (Gainesville, Florida) for proofreading of the manuscript.

The study was supported by European Union in the 5th Framework program QLK5-CT-2000-01684 (CONTROCAM); however, its support does not necessarily reflect the commission’s view andin no way anticipates its future policy in this area. The study was partially supported by research projectZ4-055-905 from Academy of Sciences of the Czech Republic.

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