J. Appl. Entomol.

ORIGINAL CONTRIBUTION Sex pheromone of the , abietaria (: Geometridae), a pest of cones H. L. Wang1, G. P. Svensson1, J. Jakobsson1, E. V. Jirle1, O. Rosenberg2, W. Francke3, O. Anderbrant1, J. G. Millar4 &C.Lofstedt€ 1

1 Department of Biology, Lund University Lund, Sweden 2 The Forestry Research Institute of Sweden, Skogforsk Uppsala, Sweden 3 Institute of Organic Chemistry, University of Hamburg Hamburg, Germany 4 Department of Entomology, University of California Riverside, CA, USA

Keywords Abstract electrophysiology, field tests, gland extract € analysis, pest management, spruce seed The sex pheromone of the cloaked pug moth, Eupithecia abietaria Gotze, orchard an important cone-feeding pest in spruce seed orchards in Europe, was investigated. Chemical and electrophysiological analyses of pheromone Correspondence gland extracts of female and analogous analyses of synthetic Christer Lofstedt€ (corresponding author), hydrocarbons and epoxides of chain length C19 and C21 revealed Department of Biology, Lund University, SE- (3Z,6Z,9Z)-3,6,9-nonadecatriene (3Z,6Z,9Z-19:H) and 3Z,6Z-cis-9,10- 223 62 Lund, Sweden. E-mail: [email protected] epoxynonadecadiene (3Z,6Z-cis-9,10-epoxy-19:H) as candidate phero- mone components, which were found in a gland extract in a ratio of Received: May 9, 2014; accepted: August 20, 95 : 5. In field trapping experiments, conspecific males were only 2014 attracted to a combination of 3Z,6Z,9Z-19:H and the (9S,10R)-enantiomer of 3Z,6Z-cis-9,10-epoxy-19:H. The (9R,10S)-enantiomer was not attrac- doi: 10.1111/jen.12167 tive, which is in agreement with studies on other Eupithecia , for which males have only been attracted by the (9S,10R)-enantiomer of ep- oxides. Subsequent experiments showed that E. abietaria males were attracted to a wide range of ratios of the two active compounds and that trap catches increased with increasing dose of the binary blend. A two- component bait containing 300 lg3Z,6Z,9Z-19:H and 33 lg of the (9S,10R)-enantiomer of 3Z,6Z-cis-9,10-epoxy-19:H was efficient for moni- toring E. abietaria in spruce seed orchards in southern Sweden, where this species has probably been overlooked as an important pest in the past. With sex pheromones recently identified for two other moths that are major pests on spruce cones, the spruce seed moth, strobilella L., and the spruce coneworm, Dioryctria abietella Denis & Schiffermuller,€ phero- mone-based monitoring can now be achieved for the whole guild of cone- feeding moths in European spruce seed orchards.

uted throughout Eurasia and has been observed on Introduction several coniferous species, such as , Picea The moth Eupithecia in the family Geometridae abies, Pinus cembra and (Mironov 2003). is one of the largest genera in the Lepidoptera, with Larvae feed on the seeds within ripening cones or on approximately 1500 described species (Mironov and soft cone tissue (Spessivtseff 1924; Mironov 2003), Galsworthy 2012). Larvae of many Eupithecia moths which results in accumulation of excrement in feed on reproductive parts of their host , and characteristic frass webs on the exterior of infested several species are considered destructive pests. The cones. Such webs are also produced by larvae of cloaked pug moth, Eupithecia abietaria Gotze,€ is distrib- the spruce coneworm, Dioryctria abietella Denis &

352 J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. H. L. Wang et al. Sex pheromone of the cloaked pug moth

Schiffermuller,€ which is considered a major pest in Materials and Methods spruce seed orchards in northern Europe (Rosenberg and Weslien 2005). Due to the difficulties in distin- guishing frass webs produced by larvae of D. abietella and E. abietaria, the latter species has probably been In July–August 2010, cones of Norway spruce Picea overlooked in the past as a potentially important pest abies (L.) infested by E. abietaria were collected in a of spruce cones, as noted by Spessivtseff almost a cen- spruce seed orchard near Gringelstad (55°560N, tury ago (1924). Indeed, a more recent assessment of 14°060E), Skane Province, Sweden, and placed in an damage caused by cone-feeding moths in a spruce environmentally controlled chamber. Initially, these seed orchard in Sweden showed that a large propor- cones were stored at 18°C under a 12:12 L:D cycle. To tion (70–80%) of the damaged cones was infested by induce diapause of moth larvae, the temperature was E. abietaria (Rosenberg and Weslien 2005). slowly decreased during the following 3 months to Like most moths, Eupithecia species rely on female- reach a minimum of 5°C, and the light regime was produced sex pheromones for mate finding, but in simultaneously changed to reach a final cycle of spite of the species richness of the genus, detailed sex 8:16 L:D. During the following spring, cones were pheromone analyses have so far been restricted to moved to another chamber with a 12°C and two species: E. assimilata Doubleday in Europe and 12:12 L:D regime and larvae exited the cones, E. annulata Hulst in North America (Wong et al. pupated and emerged as adults under these condi- 1985; Millar et al. 1991; Campbell et al. 2007). tions. Adult moths were collected as soon as they Females of both species produce (3Z,6Z)-cis-9,10- appeared, separated by sex and kept individually in epoxyheneicosadiene (3Z,6Z-cis-9,10-epoxy-21:H), small plastic tubes until used in electrophysiological and males are attracted only by the (9S,10R)-enantio- and chemical analyses. mer of this epoxide, whereas the (9R,10S)-enantio- mer antagonizes attraction of E. assimilata but not Chemicals E. annulata males (Millar et al. 1991; Campbell et al. 2007). Male E. annulata are also attracted by the The polyenic hydrocarbons (3Z,6Z,9Z)-3,6,9-nona- (9S,10R)-enantiomer of 3Z,6Z-cis-9,10-epoxy- decatriene (3Z,6Z,9Z-19:H) and (3Z,6Z,9Z)-3,6,9- eicosadiene (3Z,6Z-cis-9,10-epoxy-20:H), but this heneicosatriene (3Z,6Z,9Z-21:H) were prepared from compound was not found in gland extracts of conspe- linolenic acid according to Underhill et al. (1983). A cific females (Millar et al. 1991). In addition, second batch of 3Z,6Z,9Z-19:H used in field trials was (3Z,6Z,9Z)-3,6,9-heneicosatriene (3Z,6Z,9Z-21:H) synthesized as described by Wang and Zhang (2007). was detected in pheromone gland extracts of E. annu- Enantiomerically enriched samples of (6Z,9Z)-cis-3,4- lata females, but was not attractive to males when epoxy-6,9-nonadecadiene (6Z,9Z-cis-3,4-epoxy-19:H) tested in the field (Millar et al. 1991). Moreover, in were prepared according to Szocs} et al. (1993). The screening studies in Europe and North America, syntheses of (3Z,9Z)-cis-6,7-epoxy-3,9-nonadecadiene males of several other Eupithecia species have been (3Z,9Z-cis-6,7-epoxy-19:H) and its bishomologue captured in traps baited with C19 and C21 hydrocar- (3Z,9Z)-cis-6,7-epoxy-3,9-heneicosadiene (3Z,9Z-cis- bons and epoxides, but generally in low numbers, 6,7-epoxy-21:H) followed the procedure described by and no chemical analyses of gland extracts from Mori and Brevet (1991). Finally, optically active sam- females or electrophysiological studies have been per- ples of (3Z,6Z)-cis-9,10-epoxy-3,6-nonadecadiene formed on these species (Wong et al. 1985; Szocs} (3Z,6Z-cis-9,10-epoxy-19:H) and its bishomologue et al. 1987, 2002; Ostrauskas 2004). (3Z,6Z)-cis-9,10-epoxy-3,6-heneicosadiene (3Z,6Z-cis- An efficient sex pheromone lure could provide a 9,10-epoxy-21:H) were prepared according to Mori tool for monitoring the abundance of E. abietaria in and Ebata (1986). European spruce seed orchards, where this species To provide sufficient amounts of stereochemically has been neglected as a potentially important pest in pure material for field trials, (3Z,6Z,9S,10R)-9,10- the past. As part of a research programme to elucidate epoxy-3,6-nonadecadiene (3Z,6Z-9S,10R-epoxy-19:H) sex pheromone communication in the guild of seed- and its enantiomer 3Z,6Z-9R,10S-epoxy-19:H were syn- feeding moths associated with Swedish spruce seed thesized from (2Z,5Z,8Z)-1-tert-butyldimethylsilyloxy- orchards, we report here the identification of a two- 2,5,8-undecatriene 1 (Raylo Chemicals, Edmonton, component sex pheromone of E. abietaria, using data Alberta, Canada) as previously described (Wong et al. from chemical, electrophysiological and behavioural 1985); however, the enantiomeric purities of the analyses. epoxyalcohol intermediates 3 were increased by

J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. 353 Sex pheromone of the cloaked pug moth H. L. Wang et al. formation and recrystallization of their dinitrobenzo- physiological recordings and coupled GC-MS ate derivatives, as described by Mori and Ebata (1986) analyses. (fig. 1). After hydrolysis, (2S,3R)-2-hydroxymethyl- 3-[(2Z,5Z)-2,5-octadien-1-yl]oxirane [(2S,3R)-5] was Electrophysiological analyses obtained in 98.1% enantiomeric excess (ee), and (2R,3S)-5 was obtained in 97.1% ee, as determined by Gas chromatography with simultaneous flame ioniza- analysis on a b-Dex 225 chiral stationary phase col- tion detection (FID) and electroantennographic detec- umn (30 m 9 0.25 mm i.d., 0.25 l film thickness; tion (EAD) was used to identify compounds eliciting Supelco Inc., Bellefonte, PA, USA) with a temperature responses from antennae of male E. abietaria. Phero- programme of 50°C/1 min, 2°C/min to 220°C. The mone gland extracts of females or mixtures of syn- enantiomers of 5 were separated to baseline on this thetic C19 and C21 hydrocarbons and epoxides were column ((2R,3S)-5: 55.32 min; (2S,3R)-5: used as test stimuli. The head of a male moth, includ- 56.10 min), whereas they did not resolve on a Cyclo- ing both antennae with their tips cut off, was dex B column (J&W Scientific, Folsom, CA, USA). mounted to a PRG-2 EAG probe (109 gain) (Syntech, These intermediates were then transformed (as out- Kirchzarten, Germany) using Blagel conductive gel lined in fig. 1) to 3Z,6Z-cis-9S,10R-epoxy-19:H and (Cefar, Malmo,€ Sweden). The GC effluent passed 3Z,6Z-cis-9R,10S-epoxy-19:H with 98.1% and 97.3% through a heated transfer line set at 255°C and was ee, respectively. mixed with charcoal-filtered and humidified air (9Z,11E)-tetradecadienyl acetate (overall purity before being passed over the antennal preparation, >97% and >98% isomeric purity) was purchased which was placed 5 mm from the glass tube outlet. from Bedoukian Research Inc., Danbury, CT, USA. Samples were injected into an Agilent 7890A gas (3Z,6Z,9Z,12Z,15Z)-pentacosapentaene [overall pur- chromatograph (Agilent Technologies, Palo Alto, CA, ity >96% by gas chromatography–mass spectrometry USA), equipped with a polar HP-INNOWax column (GC-MS)] was a gift from Dr. J.M. Chong, Depart- (30 m 9 0.25 mm i.d., 0.25 lm film thickness; J&W ment of Chemistry, University of Waterloo, ON, Scientific, Agilent Technologies, Santa Clara, CA, Canada. USA). Hydrogen was used as carrier gas at a flow rate of 1 ml/min. The injector temperature was 225°C, and samples were injected in splitless mode. A split at Extraction of pheromone glands of female moths the end of the column allowed division of the GC The pheromone glands of 2- to 3-d-old female E. abie- effluent to the FID and the EAD. The column taria were dissected 2–4 h into the scotophase and temperature was maintained at 80°C for 1 min extracted in hexane (10 ll/gland) at room tempera- after injection, then increased by 5°C/min or by 10°C/ ture for 30 min. The extracts were used for electro- min to 220°C, with a final hold of 10 min at this temperature. The GC-EAD Pro version 4.1 software (Syntech, Kirchzarten, Germany) was used for data analysis.

Chemical analyses The pheromone gland extracts were analysed using a Hewlett-Packard 5972 mass-selective detector, cou- pled to a Hewlett-Packard 5890II gas chromatograph equipped with an HP-INNOWax column (dimensions as above). The temperature was maintained at 80°C for 1 min after injection, then increased by 5°C/min to 220°C, with a final hold of 10 min at this tempera- ture. Injector and transfer line temperatures were 230 and 280°C, respectively, and helium was used as the carrier gas. Samples were injected in splitless mode Fig. 1 Synthesis of enantiomers of 3Z,6Z-cis-9,10-epoxynonadecadiene. (a) p-toluenesulphonic acid, MeOH; (b) Sharpless asymmetric epoxida- and eluted at a constant flow rate of 0.8 ml/min. The tion; (c) 3,5-dinitrobenzoyl chloride, pyridine; recrystallize; (d) Na2CO3, components eliciting antennal responses were identi- MeOH; (e) p-toluenesulphonyl chloride, pyridine; (f) Dioctyllithium cup- fied through comparison of retention times and mass rate, ether. spectra with those of synthetic standards. Selected ion

354 J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. H. L. Wang et al. Sex pheromone of the cloaked pug moth monitoring (SIM) was used to monitor the ratio of the orchards was investigated. Traps baited with 300 lg characteristic ions from the trace epoxynonadeca- 3Z,6Z,9Z-19:H and 33 lg3Z,6Z-9S,10R-epoxy-19:H diene according to Millar (2000). were placed in three orchards in Skane province. The orchards were located in Maglehem (55°470N, 14°100E), Gringelstad (55°550N, 14°60E, same as Field experiments above) and Maltesholm (55°540N, 13°590E, same as To check the attractiveness of candidate compounds, above). In each of the orchards, three traps were hung field bioassays were performed in 2012 and 2013 in on spruce branches approximately 5 m above ground. two Swedish spruce seed orchards: Albrunna Traps were deployed on May 21st and checked weekly. (59°300N, 17°320E), Uppland Province, and Maltes- Baits were replaced on the 25th of June, and the holm (55°540N, 13°590E), Skane Province. Transpar- experiment was terminated on July 29th. For compari- ent plastic delta traps with sticky inserts (Csalomon, son, the flight of D. abietella was monitored in the same Budapest, Hungary) were used for capturing E. abie- orchards, using the same protocol with the exception taria males. Test solutions were prepared in hexane, that the D. abietella baits were not replaced during the and dispensers were produced by loading 100 llofa trapping period. The traps for D. abietella were baited solution on red rubber septa (11 9 5 mm, #224100- with a mixture of 100 lg(9Z,11E)-tetradecadienyl 020; Wheaton Science Products, Millville, NJ, USA). acetate and 1000 lg(3Z,6Z,9Z,12Z,15Z)-pentacosa- Traps were hung on spruce branches 1.5–4 m above pentaene on rubber septa (Lofstedt€ et al. 2012). ground and at least 10 m apart. Five replicates per Trapped Eupithecia moths were identified based on treatment were used in all tests, and a randomized size and wing colouration according to Spessivtseff block design was applied, with each row of trees being (1924) and Skou (1986a). Another geometrid, Epirr- a block and with the different treatments randomized hoe tristata L., was also attracted to traps. This species within each row. Each experiment was conducted for is typically darker than E. abietaria, but some trapped about a month, and traps were checked once per individuals appeared pale, especially at the end of the week. field season, and to confirm species identification of In the first experiment, conducted in both orchards trapped moths, genital preparations were examined in 2012, 3Z,6Z,9Z-19:H, 3Z,6Z-9S,10R-epoxy-19:H, for a subset of captured specimens for different treat- and 3Z,6Z-9R,10S-epoxy-19:H were tested. Com- ments. The abdomen was boiled in 8% aqueous KOH pounds were applied individually, as binary blends in for 1.5 h, or placed in 70% ethanol for at least a 95 : 5 ratio of hydrocarbon and epoxide enantio- 10 min, to dissolve adipose tissue. Genital characters mer, or a 1 : 1 ratio of the epoxide enantiomers, or as were identified according to Skou (1986b) as well as a ternary blend in a 95 : 2.5 : 2.5 ratio of hydrocar- the identification keys for E. abietaria, the similar- bon, (9S,10R)-enantiomer and (9R,10S)-enantiomer. looking Diakonoff, and tris- For all treatments, the total dosage was 100 lg per tata (available at www.lepiforum.de/lepiwiki.pl.). septum. This experiment was repeated at both sites in 2013. The second experiment, conducted in Albrunna Results in 2012, included eight different ratios of 3Z,6Z,9Z-19: H and 3Z,6Z-9S,10R-epoxy-19:H. For all treatments, Electrophysiological and chemical analyses the total dosage was 100 lg per septum. This experi- ment was repeated at both sites in 2013. Finally, a The initial GC-EAD screening using various combina- third experiment, conducted in Albrunna in 2013, tions of synthetic hydrocarbons and epoxides showed tested five different doses of a 100 : 11 blend of the that only two of our synthetic standards, 3Z,6Z,9Z-19: hydrocarbon and the (9S,10R)-enantiomer of the H and 3Z,6Z-cis-9,10-epoxy-19:H, elicited consistent epoxide, at a total dosage of 11.1, 33.3, 111, 333 and responses from antennae of male E. abietaria (fig. 2a). 1111 lg per septum. For each experiment, trap catch The 6Z,9Z-cis-3,4-epoxy-19:H, eluting between 3Z,9Z- data were pooled for each trap and log (x + 1)-trans- cis-6,7-epoxy-19:H and 3Z,6Z-cis-9,10-epoxy-19:H, formed before applying one-way ANOVA, followed by was tested separately but did not elicit an EAD multiple comparisons adjusted according to the Bon- response. When pheromone gland extracts from ferroni post hoc test, to compare catches among treat- female moths were used as stimuli for GC-EAD analy- ments. All statistical tests were performed using SPSS ses, an extract constituent with the same retention version 19 (SPSS Inc. Chicago, IL, USA). time and mass spectrum as 3Z,6Z,9Z-19:H consistently In 2014, the efficiency of the two-component pher- elicited responses from antennae, whereas anten- omone for monitoring E. abietaria in spruce seed nal responses at the expected retention time of the

J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. 355 Sex pheromone of the cloaked pug moth H. L. Wang et al.

(a)

Fig. 2 Coupled gas chromatographic-electro- (b) antennographic (GC-EAD) analyses of (a) mix- tures of synthetic C19 and C21 hydrocarbons and epoxides, and (b) a female Eupithecia abietaria pheromone gland extract. For each subfigure, the upper trace is the GC trace and the lower trace is the antennal response of a male E. abietaria. Note that the column tem- perature was increased by 10°C/min for the analysis of synthetic compounds, but at 5°C/ min for the analysis of the gland extract, resulting in different retention times for the same compounds in the two subfigures. epoxide were small or absent. No other antennal Field experiments responses were elicited by any other components of gland extracts (fig. 2b). Gas chromatography–mass In the first field bioassay, E. abietaria males were only spectrometry analysis of the gland extracts confirmed significantly attracted to the combination of 3Z,6Z,9Z- that females produce 3Z,6Z,9Z-19:H and a trace of an 19:H and 3Z,6Z-9S,10R-epoxy-19:H (fig. 3). Traps bai- epoxynonadecadiene. The primary structure of this ted with the hydrocarbon and the (9R,10S)-enantio- compound was identified as 3Z,6Z-cis-9,10-epoxy-19: mer as individual components, or the hydrocarbon H by comparing: (i) its retention time with those of and a racemic blend of the epoxide enantiomers, were three reference epoxynonadecadienes with the epoxide in different positions, that is 3Z,9Z-cis-6, 7-epoxy-19:H, 6Z,9Z-cis-3,4-epoxy-19:H and 3Z,6Z- cis-9,10-epoxy-19:H and (ii) the ratio of the character- istic ions at m/z 79, 93 and 108, which differs among the three epoxynonadecadienes (Millar 2000). Gland extracts also contained 3Z,6Z,9Z-21:H based on GC retention time and mass spectrum matches with those of a synthetic reference. Very few females emerged from the cones, and analyses of variation in the ratio of 3Z,6Z,9Z-19:H and 3Z,6Z-cis-9,10-epoxy-19:H between individuals could not be performed. It was also not possible to determine which enantiomer of the latter compound was produced by females because it has not been possible to resolve the enanti- omers on any commercial or custom-made chiral sta- tionary phase GC columns (Millar 2000). The ratio in + Eupithecia abietaria a composite sample from seven female glands was Fig. 3 Mean catches ( SEM) of male in traps bai- ted with different combinations of 3Z,6Z,9Z-nonadecatriene and enanti- 95 : 5, and this blend ratio was selected as a treat- omers of 3Z,6Z-cis-9,10-epoxynonadecadiene. Maltesholm, Sweden, 30 ment for the first field trapping experiment. The May–20 June 2012 (n = 5). Bars with different letters indicate signifi- amount of the triene 3Z,6Z,9Z-19:H was estimated to cantly different catches [ANOVA on log(x + 1)-transformed data, followed be approximately 1 ng/female equivalent. by multiple comparisons according to the Bonferroni post hoc test].

356 J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. H. L. Wang et al. Sex pheromone of the cloaked pug moth not attractive. The same pattern of catches was observed at both sites in both years, but in the Albrunna experiment in 2013, only two males were trapped (both with the combination of hydrocarbon and (9S,10R)-enantiomer). In the second experiment at Albrunna in 2012, males were attracted to a wide range of ratios of 3Z,6Z,9Z-19:H and the (9S,10R)- enantiomer of the epoxydiene, with highest catches in traps baited with 75–95% of the hydrocarbon, but no catches when either compound was tested on its own (fig. 4). This experiment was repeated at both sites in 2013, but too few males were trapped for any meaningful statistical analyses, although catch pro- files were similar to those obtained in 2012, that is, Fig. 5 Mean catches (+S.E.M.) of male Eupithecia abietaria in traps bai- attraction to a wide range of ratios with highest num- ted with different doses of a 100 : 11 ratio of 3Z,6Z,9Z-non-adecatriene Z Z S R – bers caught using a 75 : 25 ratio of hydrocarbon to and 3 ,6 -9 ,10 -epoxynonadecadiene. Albrunna, Sweden, 18 June 5 August 2013 (n = 5). Bars with different letters indicate significantly dif- epoxide (data not shown). In the third experiment, ferent catches [ANOVA on log(x + 1)-transformed data, followed by multi- using different doses of a 100 : 11 blend of hydrocar- ple comparisons according to the Bonferroni post hoc test]. bon and the (9S,10R)-enantiomer, the 111 lg dose, which was similar to the 100 lg dose used in the first two experiments, attracted significantly fewer males of July. In two of the orchards, catches never than the 1111 lg dose, whereas the 333 lg dose was exceeded six moths per trap per week, whereas in not significantly different from either the 111 or the Maglehem, there was a pronounced increase in the 1111 lg dose (fig. 5). No males were caught in traps first half of July, and numbers peaked at more than baited with doses of 11 lgor33lg in this experi- 25 male moths per trap on the 9th of July. Catches of ment. D. abietella were consistently below four moths per Monitoring the flight of E. abietaria in 2014 using trap per week in all three orchards, with the exception the 333 lg dose of the two-component pheromone of one single occasion in Gringelstad in mid July revealed clear differences in abundance between (fig. 6b). three orchards (fig. 6a). Starting with very low trap After an extended period in the sticky traps, colours catches in late May/early June, abundance increased and wing patterns of trapped specimens may be through June and then decreased in the second half obscured, making species identification difficult. To corroborate the results, analyses of genitalia were performed on 38 males that were caught in traps bai- ted with mixtures of 3Z,6Z,9Z-19:H and 3Z,6Z- 9S,10R-epoxy-19:H and had been identified as E. abie- taria based on gross morphology. Of these 38 males, 35 individuals (92%) were positively assigned to E. a- bietaria based on examination of the genitalia, one was assigned to Epirrhoe tristata (3%), and the remain- ing two individuals (5%) could not be conclusively identified. Specimens that were not positively identi- fied as E. abietaria are not included in the trap catches presented above.

Discussion The results from GC-MS, GC-EAD and field trapping + Eupithecia abietaria Fig. 4 Mean catches ( SEM) of male in traps bai- experiments suggest that E. abietaria uses a two- ted with different ratios of 3Z,6Z,9Z-nonadecatriene and 3Z,6Z-9S,10R- epoxynonadecadiene. Albrunna, Sweden, 5 July–6 August 2012 (n = 5). component sex pheromone consisting of 3Z,6Z,9Z-19: Bars with different letters indicate significantly different catches [ANOVA H and 3Z,6Z-cis-9,10-epoxy-19:H. Our GC-EAD on log(x + 1)-transformed data, followed by multiple comparisons screening studies using synthetic hydrocarbons and post hoc according to the Bonferroni test]. epoxides of chain length C19 or C21 revealed consis-

J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. 357 Sex pheromone of the cloaked pug moth H. L. Wang et al.

(a) which attracted significant numbers of males at both study sites (fig. 3), and this pattern also was observed when the experiment was repeated in 2013 at the southern site. With combinations of the (9R,10S)- enantiomer or both enantiomers of the epoxide with 3Z,6Z,9Z-19:H, catches were significantly reduced. It is not known whether males can detect the (9R,10S)- enantiomer, and our experimental design could not distinguish between alternative explanations for the lower catches when using the racemic mixture vs. the (9S,10R)-enantiomer. The (9R,10S)-enantiomer may antagonize responses of males, thus decreasing their attraction to traps, which has been observed for the congener E. assimilata (Campbell et al. 2007). Alter- natively, the compound may not be detected by males, and in that case, the resulting perceived 95 : 2.5 ratio of 3Z,6Z,9Z-19:H and 3Z,6Z-9S,10R- epoxy-19:H may be less attractive than the 95 : 5 (b) ratio of these compounds, as suggested by trapping data from the second field experiment in which fewer males were attracted to a 98 : 2 ratio compared to a 95 : 5 ratio (fig. 4). The ratio experiment in Albrunna in 2012 showed that E. abietaria males are attracted to a range of mix- tures of the two active compounds (fig. 4). In the third field experiment, a 10-fold higher dose of the 90 : 10 blend compared to what had been used previ- ously, i.e. the 1111 lg compared to the 111 lg dose, resulted in a significant increase in trap catches (fig. 5), suggesting that the low catches in the early tests may have been due in part to using suboptimal doses. Numerically, the 1111 lg dose was more than three times as attractive as the 111 lg dose, whereas the 333 lg dose was in between. Monitoring of E. abietaria in 2014 using the 333 lg dose confirmed that this bait is indeed efficient for trapping of E. abie- Fig. 6 Monitoring of Eupithecia abietaria (a) and Dioryctria abietella (b) taria. The monitoring experiment revealed clear dif- in three spruce orchards in southern Sweden using pheromone-baited traps. Trap catches are means of weekly catches in three traps per orch- ferences in male moth abundance over the season ard. E. abietaria traps were baited with 300 lg3Z,6Z,9Z-nonadecatri- and between catches in the different orchards. Thus, ene and 33 lg3Z,6Z-9S,10R-epoxynonadecadiene. Dioryctria abietella low catches should correctly reflect a low number of traps were baited with a mixture of 100 lg(9Z,11E)-tetradecadienyl ace- sexually mature male moths, whereas in an area with tate and 1000 lg(3Z,6Z,9Z,12Z,15Z)-pentacosapentaene. a high abundance, like in the Maglehem orchard, trap catches are high. Interestingly, we trapped many more E. abietaria than D. abietella. The two-compo- tent male antennal responses to 3Z,6Z,9Z-19:H and nent pheromone of D. abietella was identified in a € 3Z,6Z-cis-9,10-epoxy-19:H, but not to any C21 com- previous study (Lofstedt et al. 2012), it is similar to pound (fig. 2a). Subsequent GC-MS analysis showed the pheromone of the congeneric American species that extracts of pheromone glands of females con- D. abietivorella (Millar et al. 2005), and is considered tained 3Z,6Z,9Z-19:H and small amounts of 3Z,6Z-cis- an efficient pheromone for monitoring. 9,10-epoxy-19:H, although the antennal response to Limited data are available regarding pheromone this compound was weak or absent (fig. 2b). In the communication in Eupithecia moths. The two congen- first field trial in 2012, a combination of 3Z,6Z,9Z-19: ers previously studied in detail (E. annulata and

H and 3Z,6Z-9S,10R-epoxy-19:H was the only lure E. assimilata) use C20 or C21 hydrocarbons and epox-

358 J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. H. L. Wang et al. Sex pheromone of the cloaked pug moth ides as sex attractants (Millar et al. 1991; Campbell ther pheromone studies as well as control measures to et al. 2007), and E. abietaria is the first species in the reduce seed losses in orchards. genus which has been shown to use C19 compounds. In addition, attraction of males to C and C hydro- 19 21 Acknowledgements carbons or epoxides has been observed in screening studies, including E. ravocostaliata Packard (Wong We thank the owners and managers of the seed orch- et al. 1985), E. satyrata dodata Taylor (Wong et al. ards for collaboration during the field experiments. 1985), E. subnotata Hubner€ (Szocs} et al. 1987) and Funding for this study was provided by the Swedish E. vulgata Haworth (Szocs} et al. 2002; Ostrauskas Research Council Formas as part of the project Phero- 2004). These species probably use additional com- mones for monitoring and control of insects in spruce seed pounds in their sexual communication systems orchards (229-2009-1359). because studies on other geometrids have revealed that long-chain hydrocarbons and epoxides (known References as type II pheromone compounds) are commonly used in blends as sex pheromones (Ando et al. 2004). Ando T, Inomata SI, Yamamoto M, 2004. Lepidopteran sex 3Z,6Z,9Z-19:H has been reported as a sex pheromone pheromones. In: Topics in Current Chemistry, Vol. 239. component or attractant for more than 40 species, Ed. by Schulz S, Springer, Berlin, Germany, 51–96. including the mottled umber, Erannis defoliaria Campbell CAM, Tregidga EL, Hall DR, Ando T, Yamamoto (Clerck) (Szocs} et al. 1993), the autumn gum moth, M, 2007. Components of the sex pheromone of the cur- Mnesampela privata (Guenee) (Steinbauer et al. 2004), rant pug moth, Eupithecia assimilata, a re-emergent hop – the fall cankerworm moth, Alsophila pometaria (Harris) pest in UK. Entomol. Exp. Appl. 122, 265 269. (Wong et al. 1984) and the Japanese giant looper, Asc- El-Sayed AM, 2014. The pherobase: database of otis selenaria cretacea Butler (Witjaksono Ohtani et al. pheromones and semiochemicals. URL http://www. 1999), for which the pheromone identifications were pherobase.com. Lofstedt€ C, Svensson GP, Jirle EV, Rosenberg O, Roques A, rigorously supported by electrophysiological and Millar JG, 2012. (3Z,6Z,9Z,12Z,15Z)-pentacosapentaene chemical analyses of the female-produced com- and (9Z,11E)-tetradecadienyl acetate: sex pheromone of pounds. Racemic 3Z,6Z-cis-9,10-epoxy-19:H or the the spruce coneworm Dioryctria abietella (Lepidoptera: corresponding enantiomers have been reported as a Pyralidae. J. Appl. Entomol. 136, 70–78. sex attractant or sex pheromone component for nine Millar JG, 2000. Polyene hydrocarbons and epoxides: a species (summarized in El-Sayed 2014). second major class of lepidopteran sex attractant phero- Seed orchards have been established in many Euro- mones. Annu. Rev. Entomol. 45, 575–604. pean countries for production of genetically improved Millar JG, Giblin M, Barton D, Wong JW, Underhill EW, seeds for reforestation, but the seed harvest in such 1991. Sex attractants and sex pheromone components of orchards can be greatly reduced by cone-infesting noctuid moths Euclidea cuspidea, Caenurgina distincta, and insects (Turgeon et al. 1994). Identification of sex geometrid moth . J. Chem. Ecol. 17, pheromones of these cone pests, and subsequent 2095–2111. development of pheromone-based methods for popu- Millar JG, Grant GG, McElfresh JS, Strong W, Rudolph C, lation monitoring and control, could help to predict Stein JD, Moreira JA, 2005. (3Z,6Z,9Z,12Z,15Z)-penta- and limit their damage to cones in orchards. In Eur- cosapentaene, a key pheromone component of the fir ope, two major pests of spruce cones are the spruce coneworm moth Dioryctria abietivorella. J. Chem. Ecol. seed moths, Cydia strobilella L., and D. abietella (Seifert 31, 1229–1234. et al. 2000; Rosenberg and Weslien 2005), and we Mironov V, 2003. The geometrid moths of Europe. Vol. 4: have recently characterized the two-component sex II (Perizomini and ). Apollo – pheromones of those species (Wang et al. 2010; Books, Stenstrup, Denmark, 1 463. Lofstedt€ et al. 2012; Svensson et al. 2013). With the Mironov VG, Galsworthy AC, 2012. A generic level review identification of the sex pheromone of E. abietaria,we of Eupithecia Curtis and some closely related genera now have a basis for implementing pheromone-based based on the Palaearctic, Nearctic and Oriental fauna (Lepidoptera, Geometridae, Larentiinae). 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360 J. Appl. Entomol. 139 (2015) 352–360 © 2014 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH.