Agricultural and Forest Entomology (2002) 4, 1±4
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Sex pheromone of apple fruit moth Argyresthia conjugella (Lepidoptera, Argyresthiidae)
Gunnhild Jaastad*, Marie Bengtsson², Peter Anderson², Sverre Kobro³, Geir Knudsen*, and Peter Witzgall²
1 The apple fruit moth Argyresthia conjugella Zell. (Lepidoptera: Argyresthiidae) is the most important pest of apple in Scandinavia. It invades apple orchards and can destroy an entire crop during years of poor flowering and fruitsetting of its principal host, mountain ash Sorbus aucuparia. We investigated the female sex pheromone of apple fruit moth in order to develop a reliable lure, which can be used to detect migration of apple fruit moth into orchards and thus to avoid preventive insecticide sprays. 2 Pheromonal compounds obtained by solvent extraction of excised A. conjugella female pheromone glands were identified by coupled gas chromatography/ electroantennography and gas chromatography/mass spectrometry. Two compounds (Z)-11-hexadecenyl acetate, and the analogous alcohol (Z)-11-hexadecen-1-ol, elicited a strong response from male antennae. (Z)-11-hexadecenyl acetate was highly attractive in field trapping tests, whereas as little as a 1%-addition of (Z)-11-hexadecen-1-ol strongly reduced male attraction. 3 (Z)-13-octadecenyl acetate, a previously reported sex attractant, had no effect on A. conjugella male attraction.
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*The Norwegian Crop Research Institute, Ullensvang Research Station, 5781 Lofthus, Norway, yDepartment of Crop Science, Swedish University of Agricultural Sciences, 230 53 Alnarp, Sweden and zPlant Protection Centre, Department of Entomology and Nematology, 1432 AÊs, Norway
Introduction
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Keywords Apple orchard, Argyresthia conjugella, chemical identification, field trapping, sex pheromone, Sorbus aucuparia.
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Apple fruit moth, Argyresthia conjugella Zell. (Lepidoptera: Argyresthiidae), is the most important pest of apple in Scandinavia (Ahlberg, 1927). The primary host of A. conjugella is rowan (mountain ash) Sorbus aucuparia. However, flowering and fruitsetting of rowan is strongly cyclic (Sperens, 1997a,b). Populations of A. conjugella build up in forests during good fruiting years, until every second to fourth year, when too few rowan berries are available for egg-laying. Females of A. conjugella then invade apple orchards, where the entire crop can be destroyed (Ahlberg, 1927; Kobro, 1995). In Scandinavian orchards, sprays with organophosphorus compounds are used to control A. conjugella. The
Correspondence: Dr Gunnhild Jaastad, Planteforsk Ullensvang, 5774 Lofthus, Norway. Tel. 47 53 67 12 22; e-mail: gunnhild.
[email protected]
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registration of these neurotoxic insecticides is currently under discussion. Mating disruption by pheromone, by contrast, allows environmentally safe control of another most important pest of apple, codling moth Cydia pomonella L. The latter method is used in area-wide programmes in Australia, Argentina, Italy and the USA (Waldner, 1997; Gut & Brunner, 1998; Thomson et al., 2001). The main argument against the use of mating disruption against codling moth in Scandinavia is that insecticide sprays are already required for control of A. conjugella, as biological methods are not available. There is, therefore, a need to develop pheromone-based monitoring and control techniques for this species. A forecasting method has been established in Norway, which is based on estimates of population densities in relation to the abundance of rowan berries (Edland, 1974). However, the excessive damage done by apple fruit moth leads most farmers to apply routine sprays even during years when immigration pressure is presumably low.
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Methods Last-instar larvae of A. conjugella were collected from rowan berries in Southern Norway, and were allowed to overwinter outside. Adults emerged from mid-June to midJuly, were sexed daily and kept in Plexiglass cages (33 33 33 cm), under the natural photoperiod and temperature regime (Scania, Sweden). Pheromone glands of 2- to 5-day-old females were extracted in batches of 25±81 (n 14). Forcefully protruded glands were dissected with forceps, collected in a reactive vial held in liquid air ( 185 C), and then extracted with 7 mL redistilled heptane at c. 20 C for 1 min. The solvent was reduced to c. 3 mL under a stream of nitrogen, and the extracts were either analysed immediately or stored in sealed glass capillaries at 19 C. Compounds in gland extracts were identified using a Hewlett Packard 5970B (Hewlett-Packard, Palo Alto, CA, U.S.A.) mass spectrometer (MS) with electron impact ionization interfaced with a Hewlett Packard 5890 gas chromatograph (GC), equipped with a polar DB-Wax column (30 m 0.25 mm; J & W Scientific, Folsom, CA, U.S.A.). In addition, retention times of the identified gland compounds were compared with synthetic compounds on a Hewlett Packard 5890 GC using flame ionization detection and DB-Wax and non-polar SE-54 columns (25 m 0.32 mm; Kupper & Co., Bonaduz, Switzerland). Oven temperature was programmed from 80 C (for 2 min) at 10 C/min to 220 C (for 10 min). A Hewlett-Packard 6890 GC with an HP-Innowax column (30 m 0.25 mm) was interfaced with an apparatus for electroantennographic detection (GC-EAD). The column was split, allowing simultaneous recordings from the flame
ionization (FID) and the electroantennographic detector (EAD) (Arn et al., 1975), using GC-EAD software (Syntech, Hilversum, the Netherlands). GC-EAD recordings were done with synthetic compounds, and seven female extracts of 25±54 glands. One arm of the split column led into a glass tube (i.d. 8 mm), which was continuously flushed with a charcoalfiltered and humidified air stream (0.5 L/min). Apple fruit moth male antennae were mounted between two glass electrodes (containing Beadle-Ephrussi Ringer solution) 0.5 cm from the end of this glass tube and 30 cm from the EAD-outlet of the GC. One electrode was connected to the ground and the other to an amplifier (Syntech, ). The GC was operated in splitless injection mode and the oven was programmed from 50 C (for 2 min), at 10 C/min, to 220 C (for 10 min). The EAD-outlet temperature was 220 C and the split ratio between FID and EAD was approximately 1 : 1. For field trapping tests, synthetic compounds disolved in hexane were formulated on grey rubber septa (ABS, Dietikon, Switzerland). Tetra traps (Arn et al., 1979) baited with (Z)-11-hexadecenyl acetate (Z11±16Ac), and with blends of Z11±16Ac and (Z)-11-hexadecen-1-ol (Z11±16OH) or (Z)-13-octadecenyl acetate (Z13±18Ac), were hung at c. 150 cm on green rowan branches at Ullensvang and AÊs, Norway. Traps within one replicate were 5 m apart, and were inspected every 2±5 days. Time traps, baited with 10 mg Z11±16Ac on rubber septa, consisted of 12 cylindrical chambers covered with sticky paper. Each chamber was exposed during 1 h (Sñtre, unpublished data). The chemical and isomeric purity of synthetic compounds (Pherobank, Research Institute for Plant Protection, Wageningen, the Netherlands) was > 99.6%. The number of males trapped was transformed to log(x 1) and tested statistically using an analysis of variance (ANOVA) and a Tukey test.
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Availability of a sensitive and reliable monitoring lure will allow accurately monitoring of Argyresthia conjugella dispersal and thus avoid preventative sprays during years when migration into apple orchards is negligible. In order to develop a reliable monitoring lure, we chemically identified compounds produced in sex glands of A. conjugella females and determined their effect on male attraction in field trapping tests.
Results Compounds identified from A. conjugella female pheromone gland extracts are shown in Table 1. Female glands contained on average 1.1 0.4 ng Z11±16Ac (n 14).
Table 1 Compounds identified from pheromone gland extracts of apple fruit moth females A. conjugella, by GC, GC-MS and GC-EAD
Short form
Relative amounts
Male antennal response (GC-EAD)3
Dodecyl acetate Tetradecyl acetate Hexadecyl acetate (Z)-11-Hexadecenyl acetate (Z)-11-Hexadecen-1-ol Octadecyl acetate (Z)-13-Octadecenyl acetate Eicosyl acetate
12Ac 14Ac 16Ac Z11±16Ac Z11±16OH 18Ac Z13±18Ac 20Ac
1 16 100 271 3 50 ND2 56
± 0.29 0.23 1 0.42 ± ± ±
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Compound
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Average gland content 1.1 0.4 ng Z11±16Ac/female (n 14). Not detected (< 0.1%). 3 Relative to the response to Z11 16Ac (n 7). 2
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Sex pheromone of apple fruit moth
Z11±16Ac Z11±16OH
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Compound
1001
Number of males trapped 283 a 1
Isomeric purity 99.8% Isomeric purity 99.1%
1002
251 ab
1001 5
99 b
1001 20 39 c
1001 100 2d
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Table 3 Field attraction of A. conjugella males to the main pheromone compound Z11±16Ac or blends of Z11±16Ac with the minor gland component Z11±16OH (N 20), Ullensvang and AÊs (Norway), 30 June to 7 July 2000. Numbers followed by different letters are statistically different (Tukey test, P 0.05)
Z11±16Ac alone. The addition of 12Ac, 14Ac, 16Ac and 18Ac, at the proportions present in gland extracts, to the 100 : 1 blend of Z11±16Ac and Z11±16OH did not increase attractivity (d.f. 49, P < 0.001, F 8.75).
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Discussion
Chemical analysis and field trapping showed that the main pheromone component of apple fruit moth A. conjugella is Z11±16Ac. This is the first identification of a sex pheromone in an argyresthiid moth (Arn et al., 1992, 2000). A blend of Z11±16Ac and Z13±18Ac has been reported as a sex attractant for A. conjugella males (Booij & Voerman, 1984). This study shows, however, that Z13±18Ac, which we did not detect in female gland extracts, does not augment the attraction of males (Tables 1 and 2). Lack of species-specificity is an obstacle for the routine use of Z11±16Ac as a trap lure for detection and monitoring. This compound also attracts A. sorbiella, which feeds on mountain ash leaves, and the apple blossom moth A. cornella (Arn et al., 1992, 2000). Determination of Argyresthia species requires thorough taxonomic knowledge. However, A. cornella is rarely found in Scandinavian orchards and the occurrence of A. sorbiella is restricted to forest habitats. Traps baited with Z11±16Ac are highly attractive to A. conjugella males and infestation of apple can be excluded in the absence of trap captures in orchards. It is, however, essential to avoid contamination of Z11±16Ac with its analogous alcohol Z11±16OH, which is a potent attraction antagonist (Table 3). A more specific trap for A. conjugella may be obtained by blending Z11±16Ac with additional, minor gland compounds, which may have a synergistic effect on A. conjugella, and an antagonistic effect on other species. Some compounds may have been overlooked in female A. conjugella glands, because of the overall small amounts of pheromone produced. Gland extracts of a larger number of females, or field-screening tests with various compound blends are needed for the development of a more specific attractant blend.
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The presence of the minor gland compounds 12Ac and Z11±16OH was verified by comparison of retention times on two GC columns and the male antennal response to synthetic and authentic compounds. The main compound Z11±16Ac consistently elicited the strongest male antennal response in GC-EAD recordings (n 7). The diel periodicity of A. conjugella male attraction to synthetic pheromone, Z11±16Ac, was investigated with time traps (n 4) over 28 days. Captures in these traps showed that the males were on their wings at dawn, from 03.00 to 08.00 hours. During this period, light intensity close to the time traps ranged from 20 to 1800 lux, and temperatures from 5 to 16 C. Peak attraction was recorded between 05.00 and 06.00 hours. Visual observations showed that most females were calling between 05.00 and 07.00 hours. Calling females raised their wings slightly, and extruded the ovipositor. Calling females, however, can be mistaken for resting females, which assume the `head-standing' position, typical for Argyresthiidae. In this position, the head points downward and the hind legs are kept close to the abdomen. Contrary to calling females, the ovipositor is not visible in resting females. During courtship, a male landed close to a calling female and approached her from behind. In a side-by-side position, the male bent his abdomen with extruded claspers and hairpencils and touched the female's abdomen. Successful courtship was followed by copulation, which lasted for 50.4 2.6 min (n 5) in the field. Extracts from up to 50 male hairpencils were analysed using GC-EAD. These extracts were delivered to female antennae. They did not show the presence of any active compounds. Field trapping tests showed that the main pheromone compound Z11±16Ac is attractive on its alone. Z13±18Ac, which was not detected in gland extracts, was not attractive on its alone and did not have an effect on male attraction to Z11±16Ac (Table 2). Isomeric purity of Z11±16Ac was not critical for male attraction, a batch containing almost 1% of the E-isomer was suitable for male attraction (Table 3). In contrast, male attraction was reduced by a 5% addition of Z11±16OH, which was present in all gland extracts (Tables 1 and 3). Another trap test (Ullensvang, n 10) was done to determine the effect of small amounts of Z11±16OH, and the attractivity of the full blend of compounds identified from female glands (see Table 1). A blend of 100 mg Z11±16Ac and 1 mg Z11±16OH captured 384 males, a 100 : 0.1 blend captured 508 males, and 754 males were captured with
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Table 2 Field attraction of A. conjugella males to the main pheromone compound Z11±16Ac and/or Z13±18Ac (n 10), Ullensvang (Norway), 9±30 June 2000. Numbers followed by different letters are statistically different (Tukey test, P 0.05)
Compound Z11±16Ac Z13±18Ac
Number of males trapped
mg/trap 10
10 2
133 a
126 a
10 10 95 a
2 10 1b
10 2b
Acknowledgements This study was supported by the Norwegian Department of Agriculture, the Foundation for Strategic Environmental
# 2002 The Royal Entomological Society, Agricultural and Forest Entomology, 4, 1±4
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References Ahlberg, O. (1927) RoÈnnbaÈrsmalen, Argyresthia conjugella Zell. En redogoÈrelse foÈr undersoÈkningar aÊren 1921±26. Meddel, no. 324. Centralanstalten foÈr foÈrsoÈksvaÈsendet paÊ jordbruksomraÊdet, Lantbruksentomologiska avdelningen, Stockholm. Arn, H., Rauscher, S. & Schmid, A. (1979) Sex attractant formulations and traps for the grape moth Eupoecilia ambiguella Hb. Mitteilungen der Schweizer Entomologischen Gesellschaft, 52, 49±55. Arn, H., StaÈdler, E. & Rauscher, S. (1975) The electroantennographic detector ± a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Zeitschrift fuÈr Naturforschung, 30c, 722±725. Arn, H., ToÂth, M. & Priesner, E. (1992) List of Sex Pheromones of Lepidoptera and Related Attractants, 2nd edn. International Organization for Biological Control, Montfavet. Arn, H., ToÂth, M. & Priesner, E. (2000) The Pherolist. http:// www.pherolist.slu.se. Booij, C.J.H. & Voerman, S. (1984) (Z)-11-Hexadecenyl compounds as attractants for male Microlepidoptera of the subfamilies Argyresthiinae, Glyphipteryginae, and Crambinae. Entomologia Experimentalis et Applicata, 36, 47±53.
Edland, T. (1974) The rowan-berry moth (Argyresthia conjugella Zell.). Prognosis of attack on apple. Methods and results. A preliminary report. Gartneryrket, 64, 524±532. Gut, L.J. & Brunner, J.F. (1998) Pheromone-based management of the codling moth (Lepidoptera: Tortricidae) in Washington apple orchards. Journal of Applied Entomology, 15, 387±405. Kobro, S. (1995) Attack by the apple fruit moth in 1994. Gartneryrket, 11, 19±21. Sperens, U. (1997a) Long-term variation in, and effects of fertiliser addition on, flower, fruit and seed production in the tree Sorbus aucuparia (Rosaceae). Ecography, 20, 521±534. Sperens, U. (1997b) Fruit production in Sorbus aucuparia L. (Rosaceae) and pre-dispersal seed predation by the apple fruit moth (Argyresthia conjugella Zell.). Oecologia, 110, 368±373. Thomson, D., Brunner, J., Gut, L., Judd, G. & Knight, A. (2001) Ten years implementing codling moth mating disruption in the orchards of Washington and British Columbia: starting right and managing for success! IOBC WPRS Bulletin, in press. http://phero.net/iobc/hohenheim/bulletin/thomson.html. Waldner, W. (1997) Three years of large-scale control of codling moth by mating disruption in South Tyrol, Italy. IOBC WPRS Bulletin, 20, 35±44.
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Research (MISTRA), and the Swedish Council for Forestry and Agricultural Research (SJFR).
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Accepted 17 March 2002
# 2002 The Royal Entomological Society, Agricultural and Forest Entomology, 4, 1±4
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