138 Naturwissenschaften 86 (1999) Q Springer-Verlag 1999

Naturwissenschaften 86, 138–140 (1999) Springer-Verlag 1999

Inhibition of Sexual Attraction and Mating byPheromone Enantiomers in Male LasiodermaserricorneAnna Levinson, Hermann LevinsonMax-Planck-Institut für Verhaltensphysiologie, D-82319 Seewiesen, Germany

Received: 15 July 1998 / Accepted in revised form: 2 November 1998

Correspondence to: A. Levinson

Introduction

Female tobacco beetles (Lasiodermaserricorne F., Anobiidae) are knownto emit a sex pheromone whose maincomponent is 4,6-dimethyl-7-hydrox-ynonan-3-one (named serricornin)which attracts males of the same spe-cies and stimulates them to mate andreproduce [1–3]. Its predominant ac-tive constituent has been found to bethe 4S,6S,7S (threo)-enantiomer ofserricornin [4] whose stereochemicalstructure is shown in Fig. 1.Since it was intriguing that male L.serricorne are not attracted to a ra-cemic mixture of all (threo)-and (ery-thro)-enantiomers of serricornin ap-plied at doses ranging from 10–5 to10–1 mg [5–7], we investigated the be-havior and olfactory responses ofmale tobacco beetles to single enan-tiomers of serricornin and to4S,6S,7S–serricornin in combinationwith certain enantiomers of the lat-ter.

Behavior Tests

The olfactory responses of 80 un-mated male L. serricorne (22–25 daysafter pupal-adult ecdysis) were testedin eight repetitions per dose, enan-tiomer and enantiomeric combina-tion, for a period of 15 min. A givenenantiomer or enantiomeric combina-tion was volatilized from the centralregion (diameter approx. 1 mm) of ahorizontal filter paper arena (diam-eter approx. 75 mm) conditioned at30B0.17 C and approx. 800 lux, while

Table 1. Degree of inhibition of pheromone activity exerted by some enantiomers of serricor-nin in presence of 4S,6S,7S-serricornin. Inhibition due to a given enantiomer is expressed aspercentage of suppressing pheromone activity (%) caused by 10P3 mg of 4S,6S,7S-serricornin.The latter was found to induce an average of 98 (B2)% attraction to the pheromone source,310 (B6) sec aggregation time and 34 (B4) mating attempts of unmated male tobacco beetles.The pheromone and a proportional amount of an enantiomer were simultaneously volatilizedfrom the centre of an olfactometer arena

Ratio of Simultaneous release ofSSS1 :enantiomer SSS1 :SRR2 SSS1 :SRS3 SSS1 :SSR4

% %1:0 P P P1 :0.01 no inhibition no inhibition 71 :0.1 no inhibition no inhibition 641 :1 no inhibition 42 981 :10 no inhibition 55 100

1 4S,6S,7S – serricornin, 10P3 mg2 4S,6R,7R – serricornin3 4S,6R,7S – serricornin4 4S,6S,7R – serricornin

Fig. 1. Relationship between stereochemicalstructure and inhibition of pheromone activi-ty by (threo)-serricornins (a,b) and (erythro)-serricornins (c,d) in male tobacco beetles, asmeasured by behavioral tests and electrophy-siological recordings. a) Main sex pheromonecomponent. b) Inactive as pheromone antag-onist. c) Strong pheromone antagonist. d)Moderate pheromone antagonist

ten male tobacco beetles were re-leased at the periphery of the arena.The behavior tests, which were partof a more comprehensive study [8],yielded the following results (Ta-

ble 1). The marked sex pheromoneactivity – namely maximal attraction,aggregation, and mating attempts in-duced in unmated male tobacco bee-tles by 4S,6S,7S–serricornin – wasstrongly inhibited by 4S,6S,7R–serri-cornin, moderately restrained by4S,6R,7S–serricornin, and not sup-pressed by 4S,6R,7R–serricorninwhen exposed to ratios of 1 :10, 1 :1,and 1 :0.1 of 4S,6S,7S–serricornin tothe above enantiomers (Table 1,Fig. 1). Surprisingly, male L. serri-corne also displayed strong avoidancereactions upon approaching thesource from which the blends of4S,6S,7S–serricornin and 4S,6S,7R–serricornin (1 :10, 1 : 1 or 1 :0.1) werereleased to the ambient atmosphere.The subdued responsiveness of maletobacco beetles to racemic serricorninand to 1 :1 or 1 :10 blends of4S,6S,7S–serricornin and the above(erythro)-enantiomers can thus be ex-plained by the occurrence of4S,6S,7R– and 4S,6R,7S–serricorninin the racemate.

Electrophysiological Recordings

Olfactory stimuli were produced bypassing two successive air puffs of20 ml/0.5 s (interval: 0.1 s) through 10mg-plugs of purified cotton wool im-pregnated by the enantiomers anddoses as presented in Table 2. Recep-tor potentials (extracellular DC) andnerve impulse activity (extracellularAC) were recorded by glass electro-

Naturwissenschaften 86 (1999) Q Springer-Verlag 1999 139

Table 2. Influence of reciprocal stimulation by 4S,6S,7S-serricornin and 4S,6S,7R-serricorninon the antennal pheromone receptors of male Lasioderma serricorne. Receptor potentials (ex-tracellular DC) and nerve impulse activity (extracellular AC) were recorded by thin glass elec-trodes (tip diam. F2.6 mm) following exposure of the long sensilla basiconica on the antennalapex of male L. serricorne to two successive air-borne stimuli providing first 4S,6S,7S-serricor-nin and then 4S,6S,7R-serricornin or vice versa. The tabulated data represent average valuesand relevant standard deviations (B) recorded from the antennal tip of 10 male tobacco bee-tles

Stimulus 1 2 1 2 1 2

SSS, mg SSR, mg mVa mVa nb nb

10P1 0 3.2B0.4 P 15.8B4.6 P10P1 10P1 3.1B0.4 0.5B0.1 14.6B3.8 2.5B0.410P1 10P2 3.3B0.6 0.0 14.8B4.2 0.0SSR, mg SSS, mg

10P1 0 2.9B0.2 P 12.5B3.0 P10P1 10P1 2.8B0.3 0.6B0.1 13.8B3.4 3.2B0.210P1 10P2 3.0B0.4 0.0 12.8B2.1 0.0

a average receptor potentialb average number of nerve impulses

des (tip diameter approx. 2.6 mm)containing Beadle-Ephrussi’s Ringersolution which were inserted near thebase of the long sensilla basiconicaoccurring on the apex of the antennaeof male L. serricorne. The indifferentelectrode was maintained in the he-molymph, inserted to the thin cuticlebetween head and thorax.A dose-dependent inhibition of phe-romone perception by the long olfac-tory pegs on the antennal apex ofmale L. serricorne due to stimulationby either 4S,6S,7S–serricornin (sexpheromone) and subsequently4S,6S,7R–serricornin (pheromone an-tagonist) or vice versa is clearly evi-dent (Table 2). Stimulation of singlecells of the antennal sensilla basiconi-ca by l0–1 mg 4S,6S,7S–serricornin re-sulted in receptor potentials of3.1–3.3 mV and 14.6–15.8 nerve im-pulses, while successive stimulation ofthe above olfactory cells by 10–1 mg4S,6S,7R–serricornin caused approx.84% lower receptor potentials andabout 71% less nerve impulses thaninitial stimulation by the same dose of4S,6S,7S–serricornin. Generation ofreceptor potentials and nerve im-pulses was completely interrupted bystimulation of the olfactory cells by10–1 mg 4S,6S,7S–serricornin followedby 10–2 mg 4S,6S,7R–serricornin.Interestingly, reversal of the abovesequence of stimuli produced similar

results. Stimulation of the olfactorycells by 10–1 mg 4S,6S,7R–serricornininduced receptor potentials of2.8–3.0 mV and 12.5–13.8 nerve im-pulses, while successive stimulationby 10–1 mg 4S,6S,7S–serricornin re-sulted in about 79% lower receptorpotentials and about 75% less nerveimpulses than the initial stimulus of10–1 mg 4S,6S,7R–serricornin. More-over, stimulation of the olfactory cellsof the long sensilla basiconica of maletobacco beetles by 10–1 mg4S,6S,7R–serricornin and subsequent-ly by 10–2 mg 4S,6S,7S–serricornin re-sulted in total suppression of both re-ceptor potentials and nerve impulseactivity (Table 2). These results indi-cate that the main pheromone com-ponent of L. serricorne, namely4S,6S,7S–serricornin and the inhibito-ry 4S,6S,7R–serricornin are perceivedat the same site and by the same typeof receptor cell of the long olfactorypegs of male tobacco beetles. Bothexcitatory stimuli cause mutual adap-tation disrupting the generation of re-ceptor potentials and nerve impulses.This is in accordance with findings ofKaissling [9] suggesting that special-ized pheromone cells have only a sin-gle type of receptor molecule.Figure 1 summarizes the relationshipbetween stereochemical structure andolfactory responsiveness of male L.serricorne. Behavioral and electro-

physiological studies have shown thatthe sex pheromone activity of4S,6S,7S–serricornin for male L. serri-corne is strongly antagonized by4S,6S,7R–serricornin, moderatelysuppressed by 4S,6R,7S–serricornin,and not affected by 4S,6R,7R–serri-cornin. The olfactory interference be-tween 4S,6S,7R–serricornin and4S,6S,7S–serricornin is certainly dueto the effect of the steric change atthe asymmetric center at C-7, i.e. themirror image position of hydrogenand hydroxyl around C-7 occurring inthe inhibitory enantiomer. The sub-dued antagonistic interaction be-tween 4S,6R,7S–serricornin and4S,6S,7S–serricornin probably resultsfrom the olfactory effect of the re-versed position of hydrogen andmethyl at the asymmetric C-6. In viewof the close structural similarity be-tween 4S,6S,7R–serricornin and4S,6S,7S–serricornin as well as the de-pendence on a certain ratio betweenthe two enantiomers, in order tocause interruption of pheromone per-ception and responsiveness, one istempted to explain these effects bycompetitive inhibition. Chiral inhibi-tion of pheromone perception doesnot seem to be frequent among in-sects; most of the investigated speciesare known to utilize one or more phe-romone enantiomers, while their anti-podes are either favorable or not ob-structive to pheromone perception[10].

Acknowledgements The authors aremost grateful to Dr. Zili Ren (NankaiUniversity, Tientsin, People’s Repub-lic of China) for performing some ofthe electrophysiological recordingsand to Prof. Dr. K. Mori (ScienceUniversity of Tokyo, Japan) and toDr. T. Chuman (Japan Tobacco andSalt Public Corporation, Yokohama)for kindly providing the enantiomersused in this study.

1. Chuman T, Kohno M, Kato K, NoguchiM (1979) 4,6-Dimethyl-7-hydroxynonan-3-one, a sex pheromone of the cigarettebeetle (Lasioderma serricorne F.). Tetra-hedron Lett 25 :2361–2364

2. Chuman T, Kato K, Noguchi M (1979)Synthesis of (B)-serricornin, 4,6-dime-thyl-7-hydroxynonan-3-one, a sex phero-

140 Naturwissenschaften 86 (1999) Q Springer-Verlag 1999

mone of the cigarette beetle (Lasiodermaserricorne F.). Agric Biol Chem43 :2005–2006

3. Coffelt JA, Burkholder WE (1972) Re-productive biology of the cigarette bee-tle, Lasioderma serricorne. 1. Quantita-tive laboratory bioassay of the female sexpheromone from females of differentages. Ann Entomol Soc Am 65 :447–450

4. Mori M, Chuman T, Kohno M, Kato N,Noguchi M, Nomi H, Mori K (1982) Ab-solute stereochemistry of serricornin, thesex pheromone of the cigarette beetle, asdetermined by the synthesis of its(4S,6R,7R)-isomer. Tetrahedron Lett23 :667–670

5. Levinson HZ, Levinson AR, Francke W,Mackenroth W, Heemann V (1981) Thepheromone activity of anhydroserricor-nin and serricornin for male cigarettebeetles (Lasioderma serricorne F.). Na-turwissenschaften 68 :148–149

6. Levinson AR, Levinson HZ (1986) An-tagonized pheromone responses of maletobacco beetles (Lasioderma serricorneF.) due to erythro-diastereoisomers of4S,6S,7S-serricornin. Naturwissenschaf-ten 73 :36–37

7. Levinson AR, Levinson HZ (1986) Ste-reomeric inhibition of pheromone re-sponses in male tobacco beetles. In: Useof pheromones and other semiochemicals

in integrated control. OILB-SROP Ab-stracts 40–41

8. Levinson HZ, Levinson AR (1987) Phe-romone biology of the tobacco beetle(Lasioderma serricorne F., Anobiidae)with notes on the pheromone antagonismbetween 4S,6S,7S- and 4S,6S,7R-serricor-nin. J Appl Entomol 103 :217–240

9. Kaissling K-E (1987) R.H. Wright Lec-tures on Insect Olfaction (ed. K. Col-bow), 75 pgs. Simon Frazer University,Burnaby, BC, Canada

10. Mori K (1989) Synthesis of optically ac-tive Pheromones. Tetrahedron 45 :3233–3298

Naturwissenschaften 86, 140–143 (1999) Springer-Verlag 1999

The Effect of a Strong Magnetic Field onMonarch Butterfly (Danaus plexippus)Migratory BehaviorSandra M. Perez1, Orley R. Taylor, Rudolf JanderDepartment of Entomology, University of Kansas, Lawrence, KS 66045,USA

Received: 15 June 1998 / Accepted in revised form: 6 November 1998

Electronic Supplementary MaterialMonarchs roosting in the mountains ofMexico (see our homepage at:http://link.springer.de/journals/nawi)

Correspondence to: S.M. Perez

1 Department of Ecology and EvolutionaryBiology, University of Arizona, Tucson, AZ85721, USA

Monarch butterflies from across thecentral and eastern United States andCanada converge yearly on overwin-tering sites in central Mexico [1–3].On clear days the directional orienta-tion of migrating monarchs is guidedby the use of a sun compass [4]. Po-larized light perception and orienta-tion may also be incorporated intothis compass [5]. However, even oncompletely overcast days, when thesun’s position and related celestialcues are unavailable, monarchs main-

tain a normal southwesterly orienta-tion [6, 7]. Therefore, in addition to asun compass [4, 5, 8], monarchs musthave a backup mechanism of orienta-tion such as a magnetic compass thatcan be used when the sun is hidden.We tested the butterflies’ sensitivityto magnetic fields, a requirement ofany magnetic compass orientation, byexposing subjects to a strong magnet-ic field [9–13] and measuring theirsubsequent flight direction. Experi-mental butterflies were completelydisoriented by the magnetic treat-ment. In contrast, sham control but-terflies continued to direct their bod-ies toward the southwest, as did natu-ral control butterflies. This study de-monstrates for the first time thatmonarch butterflies are sensitive tomagnetic fields and suggests thatmagnetic field perception is somehowincorporated into their transcontinen-tal migratory navigation system.

Magnetic compass orientation isknown in species of migratory andhoming animals from various taxa in-cluding the order Lepidoptera[14–19]. Use of magnetic compassorientation necessarily calls for a sen-sory mechanism receptive to magnet-ic fields. One of the most commonlyproposed mechanisms is a magnetite-based sensitivity to the earth’s weakmagnetic field [14, 15, 17, 20]. The mi-neral magnetite is present in manyspecies known to use magnetic orien-tation [21]. Although details of thesensory mechanism involving this mi-neral are only now beginning to cometo light [20], it has been suggestedthat the magnetite particles act as bio-synthesized compass needles. In thisproposed mechanism, the internalcompass needles are deflected by theearth’s magnetic field, and the ani-mals are sensitive to these minute de-flections. Monarchs have previouslybeen shown to contain magnetite[22–24]. Although suggestive, thepresence of magnetite alone does notconstitute evidence of magnetic per-ception. We tested experimentally fora sensitivity to magnetic fields by de-termining whether exposure to astrong magnetic field [9, 10, 12, 13] af-fects orientation in migratory mon-archs.We captured approximately 300 mi-grating monarchs from middle to lateSeptember 1997 at known migratoryroosting sites along the Kansas Riverand wetlands in northeastern Kansas,USA (39.967N, 95.237W). We held the