Journal of Chemical Ecology, Vol. 16, No. 3, 1990

SUNFLOWER AROMA DETECTION BY THE HONEYBEE

Study by Coupling Gas Chromatography and Electroantennography

D E N I S T H I E R Y , I J E A N M A N U E L B L U E T , 1

M I N H - H A P H A M - D E L I ~ G U E , 1 P A T R I C K ETI t~VANT, 2 and

C L A U D I N E M A S S O N l

ILaboratoire de Neurobiologie Comparde des lnvert~brds, INRA-CNRS UA 1190, 91440 Bures sur Yvette, France

2Laboratoire de Recherches sur les Ardmes, INRA Dijon Cedex 21034, France

(Received August 15, 1988; accepted April 14, 1989)

Abstract--Combined electrophysiological recordings (EAG) and gas chro- matographic separation were performed in order to investigate which volatile chemical components of a sunflower extract could be detected by honeybee workers and thus are likely to trigger the foraging behavior. A direct coupling device allowed for the stimulation of the antennal receptors with individual constituents of a polar fraction of the flower aroma shown to be attractive to bees. More than 100 compounds were separated from the extract. Twenty- four compounds elicited clear EAG responses. These compounds were iden- tified by mass spectrometry (electronic impact and chemical ionisation). Both short- and long-chain aliphatic alcohols, one short-chain aliphatic aldehyde, one acid, two esters, and terpenic compounds were found to stimulate the antennal receptors. Six compounds identified in previous behavioral experi- ments were found to exhibit EAG activity. The chemicals screened by this method may be used for recognition of the plant odor and the selective behav- ior of honeybees.

Key Words--Insect-plant relationships, olfaction, honeybee, Hymenoptera, Apidae, sunflower, allelochemicals, coupling GC-EAG.

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0098-0331/90/03000701 $06.00/0 �9 1990 Plenum Publishing Corporation

702 TmZRY ET AI~.

INTRODUCTION

Chemical analyses of plant volatile extracts of various species showed a large diversity among different chemical classes. More than 50 compounds were found in artichokes (MacLeod et al., 1982), cotton (Hedin, 1976), Ranunculaceae (Pellmyr et al., 1984), maize (Buttery et al., 1978), about 150 in orchid flowers (Borg-Karlson and Tengo, 1986), and between 80 and 250 in different geno- types of sunflower (Etirvant et al., 1984; Pham Del~gue et al., 1988). More- over, these complex blends can fluctuate in quality and quantity according to plant phenology (Maarse and Kepner, 1970; Thompson et al., 1971; Hedin, 1976; Pham Del~gue et al., 1988, Hamilton-Kemp et al., 1988), and also in response to climatic factors and soil characteristics (Robacker et al., 1982). One question arising is how phytophagous insects process the chemical information available and adapt their behavior to such complexity and variation of plant aromas (Masson, 1983).

In natural conditions, honeybee foragers are attracted to sunflower crops and are able to distinguish between different genotypes (Parker, 1981; Pham- Del~gue et al., 1985). In a previous work, Pham-Delbgue et al. (1986) condi- tioned bees to a sunflower extract and offered them a choice between the con- ditioning scent and different fractions of this extract. This work established the attractiveness of a fraction of the sunflower extract containing mainly the polar constituents and led to the identification of a limited number of molecules pos- sibly involved in the recognition of the total aroma by bees. Electroantennogram responses (EAG) at the outlet of a gas chromatograph (GC) were recorded in order to determine which compounds of the polar fraction are detected by the olfactory receptor cells and hence are likely to be used as chemical cues to elicit the orientation of bees.

Most of the GC-EAG methods described in the literature for screening the olfactory activity of chemical substances are based on a trapping of these prod- ucts at the outlet of the GC and a subsequent flushing of the trapped material onto the biological preparation (Moorhouse, 1969; Wallbank and Wheatley, 1979; Lrfstedt et al., 1983). Such a technique is not practical when large num- bers of constituents from plant extracts have to be tested for EAG activity. We therefore used a direct coupling of EAG and GC based on the same principles as those developed by Am et al. (1975) and Wadhams (1982) for pheromones or by Guerin and Stadler (1984) for plant odors.

METHODS AND MATERIALS

Plant Extract. Ten sunflower heads were extracted in a Soxhlet apparatus with dichloromethane. Volatile constituents were removed from the resultant concentration by a high-vacuum cold-finger distillation. Volatile materials con-

SUNFLOWER AROMA 703

tained in the cold traps and condensed on the cold finger were taken up in dichloromethane and concentrated. The extract was then separated into polar and apolar fractions by column chromatography on silica gel. The column was eluted with purified pentane (apolar fraction) and purified ether (polar fraction). The polar fraction was stored under nitrogen in sealed glass tubes at - 2 0 ~ The entire procedure was described elsewhere (Eti6vant et al., 1984).

Insect Material. Worker honeybees (Apis mellifera ligustica, L.) emerging in June were kept in an incubator at 33~ and were fed with a piece of candy sugar and water. Electrophysiological recordings were performed when the bees were 8 days old (number of bees: 10).

Direct Coupling between Gas Chromatography and Electroantenno- graphic Apparatus. Gas chromatography (GC) was performed on a Girdel 3000 gas chromatograph equipped with a J&W retrofit on-column injector (Jennings and Takeoka, 1984). A 30-m long fused silica megabore column (0.53 mm ID, J&W DB5, thickness 1.5 #m) was used for the separations. A deactivated fused silica column (5m) was connected to the megabore column as a retention gap (Grob et al., 1985). Helium was used as carrier gas with a velocity of 28 cm/sec. Samples of 0.4 #1 were injected at room temperature into the precol- umn, which was then pushed inside the oven. After 3 rain at 40~ the tem- perature was linearly raised at 3 ~ up to 250~ At the exit of the column, the effluent was split into two parts: one directed onto the insect antenna (2/3), the other sent to the flame ionization detector (1/3). The splitter was a Y-shaped vitreous silica outlet splitter (SGE Ltd.) without gas makeup hdded. To avoid variations of the split ratio with temperature, the splitter and the two silica lines were maintained at a constant temperature of 250~

The molecules eluted from the column were blown onto the antenna in a humidified stream of purified air (speed 20 cm/sec, internal diameter of the glass tubing -=- 0.8 cm). The distance between the outlet of the silica line and the outlet of the tubing was set at 3 cm (ca. 4 cm from the antenna).

The relative concentration of each stimulating compound was expressed as a percentage of the sum of all the peaks after subtraction of the solvent peak area.

Electroantennograms (EAGs) were recorded by an electrode covering the cut tip of the left antenna of each bee. The indifferent electrode was gently inserted at the base of the same antenna. The bees were maintained inside a Perspex block adjusted to the bee. Glass electrodes were filled with a physio- logical saline solution (NaC1, 6.5 g/liter; KC1, 0.25 g/liter; CaC12, 0.3 g/liter; Na2CO3, 0.2 g/liter). An Ag-AgC1 wire inserted into each electrode connected the antenna with the recording device: input probe, preamplifier (Carrack PB 181-t 1), and differential oscilloscope (Tektronix 5A22N). EAG amplitudes were measured on the oscilloscope screen and expressed in milli- volts. The impedance of the preparations used was lower than 2 • 106 ~. Changes in the sensitivity of the preparation were checked regularly with a

704 TmERV ET AL.

standard stimulus (1 ml of air flushed into the air stream through a pipet con- taining a filter paper soaked with 20/xl of hexanol in paraffin oil, dilution 1% v/v). Olfactory stimulations by the standard were delivered at least four times during each GC ran. Compounds eliciting responses in more than eight of 10 preparations were considered as effective stimuli, others were ignored. Since the physiological significance of hyperpolarizing EAGs is questionable, the mean value of EAGs was calculated on the basis of depolarizations exclusively; confidence intervals (CI) are calculated as follows: t x (SD)/~n where SD is the standard deviation and t is the value of the t test at P < 0.05.

Chemical identifications of peaks eliciting significant EAG responses were performed using a coupled GC-MS device (GC Girdel 300, R 10/10 Nermag). Identifications were based on electron-impact ionization (EI) (70 eV). Chemical ionization (CI-NH;-) was used to confirm the molecular weights. The mass spectra were confirmed by injection of standard compounds when available. Confidence in identifications is given in Table 1.

RESULTS

More than 100 compounds were detected in the polar fraction of the sun- flower extract. The separation of all the compounds was obtained within 90 rain. As an example, in Figure 1 are shown EAG recordings superimposed on the FID signal during a 13-rain period of chromatography. The response to the standard stimuli was rather constant (mean = - 1.7 mV, CI = -0 .15 mV). The decrease of the EAG amplitude to the standard stimuli during the period of chromatography was small (less than 20%). Therefore, no correction was applied to the observed values of the EAGs.

From the 10 individuals for which complete recordings were obtained, a total number of 529 responses were observed during the chromatography: 95 % of them were depolarizations (varying from - 0 . 2 mV to - 1 . 9 mV), and 5% were hyperpolarizations (varying from +0.2 mV to +2 mV). The responses to the different constituents were found throughout the chromatogram (Figure 2), except at the end when nonvolatile products were eluted (Figure 3). A larger number of responses occurred in the middle part of the chromatogram (retention times between 30 and 70 min) when compared to the remaining periods (Figure 2). Hyperpolarizations occurred throughout the period of analysis and were not related to particular peaks. The hyperpolarizations are evenly distributed along the chromatogram (X 2, p < 0.02), and 85 % of these hyperpolarizations occurred for only four individuals. The number of hyperpolarizations recorded from each bee could not be correlated with the sensitivity of each bee (mean value of the depolarizations recorded in each antenna) (r 2 = 0.20).

SUNFLOWER AROMA 705

TABLE 1. IDENTIFICATION OF CONSTITUENTS OF SUNFLOWER POLAR FRACTION RELEASING E A G

RESPONSES IN THE HONEY B E E f

Amount Peak MW (% of Frequency Mean LAG

number Molecule CI--NH3 ~ extract) of responses ~ in mV + CI J

1 1-Pentene-3-ol (a) 0.06 * - 0 . 5 + 0.4 2 3-Methyl-l-butanol (a) 88 0.06 * - 0 . 9 + 0.4 3 trans-2-Hexenal (a) 98 0.2 *** - 0 . 6 + 0.2 4 1-Hexanol (a) 102 0.07 ** - 0 . 6 + 0.3 5 cis-Thujenol (c) 152 0.01 * - 0 . 5 + 0.3 6 l~8-Cineole (eucalyptol) (a) 154 0.5 * - 0 . 9 + 0.5 7 4-Thujanol (a) 154 1.2 * - 0 . 5 + 0.3

(trans-sabinene hydrate) 8 Myrtenal and myrtenol (a) 150 0.8 ** - 0 . 5 + 0.3

152 9 Unknown 166 0.08 ** - 0 . 3 + 0.1

10 2,3,3-Trimethyl-epoxy (c) 168 0.07 ** - 0 . 4 + 0.2 cyclopentyl acetaldehyde

11 Bomyl acetate (a) 196 1.4 ** - 0 . 5 + 0.3 12 Unknown 180 0.7 *** - 0 . 5 + 0.3 13 Unknown 188 0.06 * - 0 . 4 + 0.3 14 /3-Elemene (a) 204 0.3 ** - 0 . 6 + 0.6 15 Vanillin (a) 152 0.6 ** - 0 . 4 + 0.3 16 Branched CE0 methyl ester 186 1.2 ** - 0 . 5 + 0.3 17 Germacrene D (a) 204 0.06 * - 0 . 4 + 0.2 18 Propiovanillone (a) 186 1.4 ** - 0 . 5 + 0.3 19 Caryophylene oxide (a) 220 0.3 ** - 0 . 8 + 0.2 20 Sesquiterpenic alcohol 220 3.9 ** - 0 . 3 + 0.3 21 4-Hydroxy-2-methoxy (b) 178 1.2 *** - 0 . 4 + 0.2

cinnarnaldehyde 22 Unknown 266 0.07 ** - 0 . 5 + 0.2 23 Tetradecanol (d) 0.3 *** - 0 . 5 + 0.2 24 Hexadecanoic acid (a) 256 2.9 * - 0 . 3 + 0.3

Relative intensities of the eight major ions of unidentified peaks: 9: 43(100), 41 (72), 55(65), 83(44), 39(38), 67(36), 95(35), 99(34); 12: 41(100), 93(85), 39(84), 53(58), 105(53), 121(50), 91(46), 120(38); 13: 108(100), 41(54), 93(47), 95(37), 43(29), 39(27), 109(26), 55(23); 22: 123(100), 43(90), 41(12), 124(11), 55(6), 39(4), 77(4), 79(3).

bLetters in parenthes~s indicate the reliability of identification: (a) EI mass spectrum identical with literature spectra, MW confirmed by CI--NH3 + and identity confirmed by injection of standard compounds; (b) EI mass spectrum identical with literature spectra, MW confirmed by CI--NH3; (c) EI mass spectrum in agree- ment with literature spectra based on eight major peaks, MW confirmed by CI--NH3; (d) El mass spectrum in agreement with literature spectra based on eight major peaks. The molecular weight is the result of the chemical ionization (reported when available).

CThe frequency of responses is calculated from the number of antenna responding to the same stimulation: * = 80%, ** > 80%, *** = 100%.

dMean calculated from depolarizations, CI is the confidence intervat = standard error, t = 0.05).

7 0 6 THIERY ET AL.

1

2

EAG

( in mY)

I I

I L ~ ~ - EAG

~ h ~ ~ F I D

1 rain.

Fm 1. Thirteen-minute sequence of the coupled signal synchronously recorded from the flame ionization detector (FID) and from a honeybee antenna (EAG). Standard is 1 ml of air odofized with hexanol 10 : 2 (v/v) in paraffin oil.

Twenty-four chemicals were found to stimulate the antennae for more than eight individuals. Amplitudes of the depolarizations recorded with these 24 compounds are plotted Figure 3. The total amount of these products account for 17% of the volatile constituents of the extract. The relative concentrations of these products range from 0.01% (thujenol cis) to 3.9 % (sesquiterpenic alco- hol, peak 20). Four compounds were commonly detected by all bees [trans-2- hexanal, peak 12 (unknown), peak 20 (sesquiterpenic alcohol), peak 22 (unknown); Figure 3, Table 1]. The variability of the amplitude of the recorded responses did not seem to be related to the retention time of the products: the higher variability observed (CI higher than 0.4 mV) occurred both at the begin- ning and the end of the experiment with 1-pentene-3-ol, 3-methyl-l-butanol, 1,8-cineole, and t3-elemene.

The chemical identification of the 24 compounds found to be active in this experiment is given in Table 1. As expected, most of the stimulating com- pounds are oxygenated compounds belonging to different chemical classes. Fourteen of these identifications were confirmed by comparison with standard

NB OF EAG

RESPONSES

70-

(30-

,50-

40-

30.

20

10

0 m

m , . . m l m - - , , -

i0 20 30 40 50 60

I

L 70

l i

80 90

DURATION OF THE GC SEPARATION (in rain.)

F[o. 2. Distribution of the EAO(s) recorded during the gas chromatographic separation

of the polar fraction from a sunflower extract. White bars = depolarizations; black bars = hyperpolafizations.

t 20

1 ~ O8

MEAN EAG

(my)

11 tl I1 FIG. 3. Mean EAG responses of the honeybee antennae to compounds constitutive of a sunflower polar fraction. Numbers correspond to peak releasing responses in more than 80 % of the recorded antennae. Thick bars represent the mean EAG, thin bars represent confidence intervals. Identification of chemicals is given in Table 1.

708 THIERY ET AL.

compounds; four of them were identified by comparison to published mass spectra; six compounds could not be identified precisely, although there is evi- dence that peaks 16 and 20 are a branched Cl0 methyl ester and a sesquiterpenic alcohol, respectively. The mass spectra of the four remaining products are listed in Table 1.

DISCUSSION

In a previous comparative study between the Soxhlet extraction technique and headspace sampling methods, Eti6vant et al. (1984) stressed the validity of both methods for sunflower odor analysis. The Soxhlet extraction procedure has been preferred according to the higher concentration of chemicals collected as compared to air sampling. In the present work, more than 100 compounds have been separated from a polar fraction of sunflower solvent extract. From the same extract, Eti6vant et al. (1984) separated a polar fraction of 47 compounds from a total of 84 products occurring; 31 polar compounds were identified. These differences may result from several reasons: our polar-apolar fractioning procedure could have been performed in slightly different conditions, degra- dation compounds may have appeared even if the initial extract was sealed in glass tube under nitrogen. The performance of our GC separation was higher, since we used a different bonded phase and an "on-column injector" that avoid thermal degradations and favors high-boiling compounds when compared to split-splitless injectors (Jennings, 1984; Jennings and Mehran, 1986).

In honeybees, detection of odors is thought to be achieved by olfactory receptor cells with broad overlapping quality spectra (Lacher, 1964; Vareschi, 1971). Since the EAG is believed to represent a summation of the receptor potentials of the responding cells (Kaissling, 1971), the exposure of antennal receptor cells to repeated stimuli may provoke an adaptation and a subsequent decrease of the EAG amplitude. Therefore the EAG activity needs to be inter- preted with care. However, the slight decrease in response observed to the stan- dard during the experiments suggests that the adaptation of receptors was limited, which may ensure the validity of such a screening.

In the present work, we used 8-days-old bees, which were grown in similar olfactory environment. Foraging bees (usually older than 8 days) may present differences in olfactory sensitivity because of age and also previous experience (Arnold and Masson, 1980; Masson and Arnold, 1984). Thus the spectrum of responses to the constituents of the sunflower odor might be different according to the age.

The EAGs recorded from the components of the polar fraction of the sun- flower extract presented at a single concentration demonstrate that as many as 24 products were detected by the antennal receptor ceils. The first four corn-

SUNFLOWER AROMA 709

pounds that release significant responses are only minor constituents of the extract (ranging from 0.06% to 0.2% of the extract). These are a short-chain aldehydes and alcohols (1-penten-3-ol, 3-methyl-l-butanol, 2-hexenal (trans), and 1-hexanol), which are common plant compounds also found in alfalfa flow- ers (Buttery et al., 1982), red clover flowers (Buttery et al., 1984), and ophrys flowers (Borg-Kaflason and Tengo, 1986). Two of these compounds (3-methyl- 1-butanol and 1-hexanol) are present in the alarm pheromone of honeybees and are highly effective in releasing alarm behavior (Collins and Blum, 1983). Tetradecanol is also mentioned as a volatile component of orchids flowers (Bergstr6m, 1978). Ten of the 24 compounds detected by bees are terpenes and sesquiterpenes. Volatile terpenic compounds seem to occur frequently in flower aromas of the compositae (Etidvant et al., 1984; Flath et al., 1985; Buttery et al., 1986). These chemicals may be partly involved in flower recognition by bees (Waller et al., 1974; Pham Del~gue et al., 1986). Caryophyllene oxide is probably a degradation product that may have appeared in the stored extract. It is nevertheless striking that bees are particularly sensitive to this product. This compound, identified in cotton plant extracts (Hedin, 1976), was also reported as releasing weak EAG responses in the boll weevil (Dickens, 1984). Some high-boiling-point compounds (e.g., linoleic acid) also elicited EAG responses in the bees. Their occurrence as olfactory stimulants may be due to the heating of these compounds in the gas chromatograph. Since they have very low vola- tility in natural conditions, it is doubtful whether they are important in olfactory communication and plant odor recognition.

The results presented here show a pattern of compound detection different from that found using foraging behavior experiments (Pham-Del~gue et al., 1986). The 24 chemicals that elicited reproducible responses in our experiment are probably good candidates to be involved in the recognition of sunflower aroma by bees. Of these 24 compounds, more attention should be paid to the six compounds identified in the behavioral experiments mentioned above; namely, bornyl acetate, vanillin, propiovanillone, the products corresponding to peaks 9 and 10, and the branched methyl ester (peak 16), previously misi- dentified as methyl caprate.

In conclusion, coupling GC and EAG recordings allowed for rapid screen- ing for components of the complex volatile mixtures in order to determine which of them might be used in olfactory recognition. However, the present results should be followed by behavioral tests to stress the role of the active compo- nents in blends. This approach can be useful for the tentative identification of a reduced chemical blend used by the bees for complex odor recognition. A better understanding of the role played by some component chemicals of the sunflower odor in the olfactory process could lead to their use in plant breeding programs in order to enhance the attractiveness of the plant and thus ensure pollination.

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Acknowledgments--The authors are indebted to Jean Luc Le Quer6, INRA Dijon, France, for the identification of chemicals.

REFERENCES

ARN, H., ST,~DLER, E., and RAUSHER, S. 1975. The electroantennographic detector--a selective and sensitive tool in the gas chromatographic analysis of insect pheromones. Z. Naturforsch. 30c:722-725.

ARNOLD, G., and MASSON, C. 1980. Postnatal maturation of the olfactory system in the workerbee. Effect of early social deprivation. Olfaction Taste 7:279.

BERGSTROM, G. 1978. Role of volatile chemicals in ophrys-pollinator interactions, pp. 207-230, in J. B. Harborne (ed). Biochemical Aspects of Plant and Animal Coevolution. Academic Press, New York.

BORG-KARLSON, A.K., and TENGO, J. 1986. Odor mimetism? Key substances in Ophrys lutea pollination relationships (Orchidacea: Andrenidae). J. Chem Ecol. 12:1927-1941.

BUTTERY, R.G., LING, L.C., and CHAN, B.G. 1978. Volatiles of corn kernels and husks: Possible corn ear worm attractants. J. Agric. Food Chem. 26:865-869.

BUTTERY, R.G., KAMN, J. A., and LING, L.C. 1982. Volatile components of alfalfa flowers and pods. J. Agric. Food Chem. 30:739-742.

BUTTERY, R.G., KAMM, J.A., and LING, L. C. 1984. Volatile components of red clover leaves, flowers and seed pods: Possible insect attractants. J. Agric. Food Chem. 32:254-256.

BUTTERY, R.G., MADDOX, D.M., LIGHT, D.M., and LING, L.C. 1986. Volatile components of yellow starthistle. J. Agric. Food Chem. 34:786-788.

COLLINS, A.M., and BLUM, M.S. 1983. Alarm responses caused by newly identified compounds derived from the honey bee sting. J. Chem. Ecol. 9:57-65.

DICKENS, J.C. 1984. Olfaction in the boll weevil, Anthonomus grandis BOH. (Coleoptera: Cur- culionnidae): Electroantennogram studies. J. Chem. Ecol. 10: 1759-1785.

ETII~VANT, P.X., AZAR, M., PHAM-DELI~GUE, M. H., and MASSON, C. 1984. Isolation and iden- tification of volatile constituents of sunflowers (Helianthus annuus L.) J. Agric. Food Chem. 32:503-509.

FLATH, R.A., MON, R., and ROGERS, C.E. 1985. Volatile constituents of oilseed sunflower heads (Helianthus annuus L.), pp. 265-273, in T.E. Acree and D.M. Soderlund (eds.). Semiochem- istry: Flavor and Pheromones. W. De Gmyter, Berlin.

GROB, K., JR, KARRER, G., and RIEKKOLA, M.L. 1985. On-column injection of large sample vol- umes using the retention gap technique in capillary gas chromatography. J. Chromatogr. 334:129-155.

GUERIN, P.M., and STADLER, E. 1984. Carrot fly cultivar preferences: Some influencing factors. Ecol. Entomol. 9:413-420.

HAMILTON-KEMP, T.R., ANDERSEN, R.A. , RODRIGUEZ, J.G., LOUGHRIN, J.H., and PATTERSON, C.G. 1988. Strawberry foliage headspace vapor components at periods of susceptibility and resistance to Tetranichus urticae Koch. J. Chem. Ecol. 14:789-796.

HEDIN, P.A. 1976. Seasonal variations in the emission of volatiles by cotton plants growing in the field. Environ. Entomol. 5:1234-1238.

JENNINGS, W. 1984. State-of-the-art gas chromatography: Instrumental design features. J. Chro- matogr. Sci. 22:129-135.

JENNINGS, W., and MEHRAN, M.F. 1986. Sample injection in gas chromatography. J. Chromatogr. Sci. 24:34-40.

JENNINGS, W., and TAKEOKA, G. 1984. State-of-the-art fused silica capillary gas chromatography:

SUNFLOWER AROMA 711

Flavor problem application, pp. 63-75, in P. Schreier (ed.). Analysis of Volatiles. Methods and Applications. W. de Gmyter.

KAISSLING, K.E. 1971. Insect olfaction, pp. 351-431, in L.M. Beidler (ed.). Handbook of Sensory Physiology, Vol. 4. Springer-Verlag, Berlin,

LACHER, V. 1964. ElektrophysioIogische Untersuchungen an einzelnen Rezeptoren ffir Gemch, Kohlendioxyd, Luftfeuchtigkeit und Temperatur auf den Antennen der Arbeitsbiene und der Drohne. Z. Vergl. Physiol. 48:587-623.

LOFSTEDT, C., VAN DEN PENS, J.N.C., LOFQVIST, J., and LANNE, B.S. 1983. Sex pheromene of the cone pyralid Dioryctria abietella. Entomol. Exp. Appl. 34:20-26.

MACLEOD, A.J., PIERIS, N.M., and GONZALES DE TROCONIS, N. 1982. Aroma volatiles of Cynara scolymus and Helianthus tuberosus. Phytochemistry 21:1647-1651.

MAARSE, H., and KEPNER, R.E. 1970. Changes in composition of volatile terpenes in Douglas fir needles during maturation. J. Agric. Food Chem. 18:1095-1099.

MASSON, C. t983. Rrle des mddiateurs chimiques d'origine animale et v~g6tale dans la pollini- sation, pp. 25-37, in C.R. 5th Symposium int. pollinisation, INRA PuN.

MASSON, C., and ARNOLD, G. 1984. Ontogeny, maturation and plasticity of the olfactory system in the workerbee. J. Insect Physiol. 30:7-14.

MOORHOUSE, J.E., YEADON, R., BEEVOR, P.S., and NESBITT, B.F. 1969. Method for use in studies of insect chemical communication. Nature 223:1174-117.

PARKER, F.D. 1981. How efficient are bees in pollinating sunflowers? J. Kans. Entomol. Soc. 54:61-67.

PELLMYR, O., GROTH, I., and BERGSTROM, G. 1984. Comparative analysis of the floral odors of Actaea spicata and A. erythrocarpa (Ranunculaceae). Nov. Acta Reg. Soc. Sci. Upsal. V.C 3:t57-160.

PHAM-DELt'GUE, M. H., FONTA, C., MASSON, C., and DOUAULT, P. 1985. l~tude comparre du comportement de butinage d'insectes pollinisateurs (abeilles domestiques Apis mellifica L. et bourdons Bombus terrestris L.) sur les ligndes parentales d'hybrides de toumesol Helianthus annuus L. Acta Oecol. Oecol. Applic. 6:47-67.

PHAM-DEL~GUE, M. H., MASSON, C., ETIt~VANT, P., and AZAR, M. 1986. Selective olfactory choices among food scents by honeybees, a study by coupled associative conditioning and chemical analysis. J. Chem. Ecol., 12:781-793.

PHAM-DELI'GUE, M. H., E'rII~VANT, P., GU1CHARD, E., and MASSON, C. 1988. A study of sunflower volatiles invoved in honeybee discrimination among genotypes and flowering stages. J. Chem. Ecol. 15:329-343.

ROBACKER, D.C., FLOTTUM, P.K., SAMMATARO, D., and ERXCKSON, F.H., 1982. Effects of cli- matic and edaphic factors on soybean flowers and on the subsequent attractiveness of the plant to honey bees. Field Crop Res. 122:267-278.

THOMPSON, A.C., BAKER, D.A., GUELDNER, R.C., and HEDIN, P.A. 1971. Identification and quan- titative analysis of the volatile substances emitted by maturing cotton in the field. Plant Phys- iol. 48:50-52.

VARESCHI, E. 1971. Duftunterscheidung bei der Honigbiene-Einzelzell-Abteitungen und Verhal- tensreaktionen. Z. Vergl. Physiol. 75:143-173.

WALLBANK, B.E., and WHEATLEY, G.A. 1979. Some responses of cabbage root fly (Delia bras- sicae) to allyl isothhiocyanate and other volatile constituents of cmcifers. Ann. Appl. Biol. 91:1-12.

WALLER, G.D., LOPER, G.M., and BERDEL, R.L. 1974. Olfactory discrimination by honeybees of terpenes identified from volatiles of alfalfa flowers. J. Apic. Res. 13:191-197.

WADAMS, L.J. 1982. Coupled gas chromatography single cell recording: A new technique for use in the analysis of insect pheromones. Z. Naturforsch. 37c:947-952.