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The electroantennogram (EAG) as an assay for the reception of odours by the gypsy moth
J. Insect Physial., 1965,Vol. 11, pp. 1573zo 1584. Pergmwn Press Ltd.
Printed in Great Britain
THE ELECTROANTENNOGRAM (EAG) AS AN ASSAY FOR THE RECEPTION OF ODOURS BY THE GYPSY MOTH BRUNHILD
STURCKOW
Entomology Research Division, Agricultural Research Service, USDA, Beltsville, Maryland (Received 14 June 1965) Abstract-The electroantennogram (EAG), an overall electrical response of the insect antenna to an adequate stimulus, has been used as a testing method for the reception of odours. Synthetic sex lures for the male gypsy moth Porthetriu dispar (IO-acetoxycis-7-hexadecen-l-01 and 12-acetoxy-cis-9-octadecen-l-01) were assayed electrophysiologically and behaviourally, and their effectiveness was compared with that of a natural sex pheromone extract derived from female gypsy moths, and with sexually neutral compounds. INTRODUCTION
THE RESPONSE of the male gypsy female was studied behaviourally by physiological (acetoxy-cis-hexadecenol) cannot be compared directly with obtained method. As
moth to the natural sex pheromone of the SCHNEIDER (1963). He also included electroisomers of a synthetic sex attractant JACOBSON However, results those from the lure since they were long as the knowledge of the relation between limited, stimulation-response compared directly obtained by the same the behavioural criteria used within each method should be comparable.
extended to assay both the of acetoxy-cis-hexadecenol gyplure (acetoxy-cis-octadecenol), another synthetic JACOBSONand JONES A few behavioural suitable technique could not be developed with the relatively
odours. antennae of are lacking, receptor potentials excited sense cells. nervous
of the receptor
Considered
activity exists, 1573
Since interneurons detail, it also contains contain the
1574
BIWNHILD STURCKOW
sum phenomenon of each type of potential derived from the central cells. However, the influence of the last two factors on the shape and kinetics of the EAG in general is negligible. MATERIALS
AND METHODS
From a total of 50 male pupae * of Porthetria dispar L. 16 moths, 3-4 days old, tests 31 moths were used for the electrophysiological tests. For the behavioural were used 2-5 days after emergence. All tests were made during the month of December. The air in the testing room was kept as clean as possible by ventilation. The temperature ranged from 22” to 25°C. The following materials were provided by Mr. Martin Jacobson (of this Division) as stock solution in hexane: 1. Crude natural pheromone (20 mg extract of 500,000 female abdominal tips, JACOBSONet al., 1961) 1 female unit = approximately 4 x 1O-2 pg. The stock solution contained 100 female units/O*5 ml hexane. 2. A synthetic sex lure (lo-acetoxy-cis-7-hexadecen-I-ol), D- and L-isomers and the racemic mixture (JACOBSON, 1962). Each stock solution contained 100 pg/O.5 ml hexane. The mol. wt. is 298. 3. A synthetic sex lure, gyplure (12-acetoxy-cis-9-octadecen-I-ol), n-isomer (JACOBSON and JONES, 1962). The stock solution contained 100 pgjO.5 ml hexane. The mol. wt. is 314. The stock solutions were diluted so that the same volume (5 ~1) could be used Cartridges loaded with hexane, for each test, except for each highest concentration. ammonia, and amyl acetate and an empty cartridge were used for comparison or as a control. In each test series, two compounds were tested alternately in 4 or 5 steps, each 10 times higher in concentration. In order to reduce the error by coupling effects, the sequence of the compounds in each concentration step was changed at random, for example: a b, b’ a’, b” a”, a”’ b”’ etc. Each substance was tested electrophysiologically in three or more series and behaviourally in one series as shown in Table 1. Electrophysiological
tests
Each moth was mounted on a piece of cardboard so that wing, head, and leg movements were prevented without inhibition of breathing. The antenna not used in the experiment was glued backward, the other was fixed forward between 2 electrodes. One of the electrodes (a capillary) was slipped over the tip of the antenna, the other (a needle) was inserted into its base. Experiments of l-2 hr duration were possible under these conditions. The stimulus, a sudden odorous puff, was directed into the basket-shaped antenna behind which an exhaust duct carried off the contaminated air. The movement of the plunger of a 50 ml syringe provided a measured volume of air which * The pupae were supplied by Mr. Thomas McIntyre, Methods Improvement Laboratory, Plant Pest Control Division, Agricultural Research Service, USDA, Falmouth, Mass.
THE EAG AS AN ASSAY FOR THE RECEPTION
OF ODOURS BY THE GYPSY MOTH
1575
was blown through a glass cartridge containing the test material on a piece of filter paper (Whatman No. 2). Since the same volume of solution was used for each test, the evaporating area on each filter paper was the same. The syringe was filled with air from outside of the stimulation system by means of a two-way cock. Three to seven EAG’s were made with each cartridge at intervals of 5.8 set _+0.3 standard error based on the measurement of 100 consecutive tests. Within this time the EAG potential had always returned to the base line. The cartridge was loaded outside of the room just before the test; loading and corking took 3 min. The use of ground-glass joints permitted rapid interchange of the cartridges. The platinum electrodes were connected to a DC amplifier (Grass, Model P67*. 4 junction potential, partly due to different electrodes (needle and capillary) and partly due to the structure of the antenna (as seen from measurements without antenna), was balanced out by the amplifier control. A light signal, indicating the release but not the duration of the stimulus, was activated by a foot control. The length of the puff from the hand-driven syringe was approximately 500 milliseconds. A Grass”* camera registered the results on paper film. The first of several EAG’s at each concentration were rephotographed and superimposed in printing with the aid of orientation marks. The number assigned to each antenna was carried over to the legend of the figures to make a comparison between EAG’s of the same antenna possible. The height of the on-effect (SCHNEIDER, 1962) of the first and second EAG in a series of tests with the same cartridge was used for the preparation of the stimulation-response curve expressed in arbitrary units.
Behavioural tests of at least 30 set Each moth was kept in an individual cage. Wing vibration was chosen as a positive criterium of sexual excitement. At about sunrise males, 2-5 days old, began to vibrate their wings, move about, and fly even without an odorous stimulus. They continued this activity interrupted by longer or shorter intervals throughout the day. Therefore, the tests were made from 4 to 8 a.m. in diffuse light when the moths were relatively quiet. The stimulus was applied to both antennae of an unrestrained moth from the same distance, with the same applicator, and in the same sequence as in the electrophysiological tests. The interval between tests was 20 min. Each filter paper was utilized for six puffs, the sequence of which was noted and altered in the next round. All moths were tested against all concentrations of all compounds (see Table 1). Sometimes the moths reacted to the mechanical component of the stimulating puff with tonic immobility which disappeared after l-5 min. The response to the * Mention of this proprietary product does not necessarily imply its endorsement by the U.S. Department of Agriculture.
BRUNHILDST~&CKOW
1.576
TABLE la-ELECTROPHY~IOLOGICALTESTS
Substance Natural lure extract
DL-Acetoxy-cis-hexadecenol
Antenna record number
Coupled with (all isomers refer to acetoxyhexadecenol) L-isomer L-isomer D-isomer D-isomer gyplure
2 4 3 4 5
L-isomer L-isomer gyplure amyl acetate iso-amyl acetate
8 9 9 11 11
D-isomer D-isomer gyplure and DL-aCetOXy-
L-Acetoxy-cis-hexadecenol
3 6 6 And see under natural lure extract hexadecenol
D-Acetoxy-cis-hexadecenol
See under natural lure extract and L-acetoxyhexadecenol
Gyplure
See under natural lure extract, DL-acetoxyhexadecenol, and L-acetoxyhexadecenol
iso-Amy1 acetate
6 amyl amyl 8 9 amyl 10 amyl And see under DL-acetoxyhexadecenol
Amy1 acetate
See under DL-acetoxyhexadecenol
acetate acetate acetate acetate
and iso-amyl acetate
TABLE 1b-BEH.4vIouR.4L TESTS L-Acetoxyhexadecenol coupled with D-acetoxyhexadecenol DL-Acetoxyhexadecenol coupled with gyplure Natural lure extract coupled with D-acetoxyhexadecenol iso-Amy1 acetate coupled with amyl acetate
odorous component of the puff was stored over this period as evidenced by differences between tests with air and with lures. For this reason the moths were observed up to 7 min after the application of the stimulus. RESULTS
Electrophysiological The first response in a series of tests with the same stimulus was usually stronger than the following ones, which remained equal for up to ten responses
FIG.
1.
Superimposition
of the first 4 EAG’s in a series of tests. (a) amyl acetate (11) 4 x 103. (b) gyplure (5) lo.* * NOTE FOR ALL FIGURES. The concentration of the stimulus compound is given in pg. The record number of the antenna is enclosed in parenthesis. Horizontal mark: 1 set; vertical mark: 2 mv (no dots), 4 mv (2 dots). The circle is a register The horizontal line above the EAG’s indicates mark used for the superimposition. the release of the stimulus.
FIG. 2. Different antennae excited by the same stimulus. (a) natural lure extract 5 x 10-2; I (S), 2 (2). (6) L-acetoxy-cis-hexadecenol 1; I (6), 2 (2), 3 (8). (c) iso-amyl acetate 4 x 102; I (6), 2 (ll), 3 (8). (d) amyl acetate 4 x 102; 1 (6), 2 (ll), 3 (8).
FIG. 3. Antenna hexadecenol; I to 4 (c) control with air. as in d. The voltage
8 stimulated in two series: a, b and d, e. (a) L-acetoxy-cis10d2 to 1O1. (b) nbacetoxy-cis-hexadecenol; I to 4 as in a. (d) iso-amyl acetate; I to 3 4 to 4 x 102. (e) amyl acetate; I to 3 mark equals in this case 4 mV.
.. -.
*“_I-
-_,_-_,_
.
-.L”
I ,-;:
*,.*
_/
.~l_“_x-__._
FIG. 4. Antenna 5 stimulated by the natural lure extract in comparison with a synthetic lure. (a) control with air. (b) natural lure extract; I to 4 5 x 10m4to 5 x 10-l. (c) gyplure; I to 4 lo-’ to 1O1. The voltage mark equals in this case 4 mV.
FIG. 5. Antenna 6 stimulated by the two optically active isomers of acetoxy-cishexadecenol. (a) control with air. (b) L-isomer; I to 5 10e3 to 10’. (c) D-isomer; I to 5 IO-3 to 101.
THE
1577
EAGAS AN ASSAYFORTHE RECEPTIONOF ODOURSBY THE GYPSYMOTH
depending on the intensity and quality of the stimulus (Fig. 1). After the group of the EAG’s dropped slowly. Sometimes the first relatively equal responses, response was not significantly higher than those following. In order to establish a reliable base for the evaluation of the tests, the first and second EAG of each series of tests were superimposed. Essential characteristics of the EAG were determined by the quality and intensity of the stimulus, the site of the electrodes, and the number and types of sensilla encountered by the stimulus (Figs 2 and 3). Therefore, a direct comparison between EAG’s from different antennae was approached with caution (see also SCHNEIDER, 1962). Different EAG’s were directly comparable if they were obtained from one antenna with the same placement of the electrodes, and with the same rise time, duration, and direction of the stimulating puff throughout the experiment, as in Figs 3 to 5. In order to compare the increase in the EAG with increasing concentrations between different antennae and different compounds, it was necessary to equate the height of the EAG’s derived from each antenna and each compound. This was done by adjusting the EAG’s derived from each highest concentration tested to 100 arbitrary units, and expressing their lower values in per cent. Two antennae were not stimulated by the highest concentration of amyl acetate (Table 2). In this case an adjustment to seventy-five arbitrary units was made. Fig. 6 and Table 2 show the stimulation-response curves obtained in this way. The tests with 2 rlO0
.
.
0
Y
CONTROL 10-S
-4
-3
-2
-1
1
lo+’
+!2
+a
MICRO(IRAM
FIG. 6. Means of the stimulation-response curves (electrophysiological tests). The response to air is taken as the control level. For the standard error and number in agreement. of replicates of all values see Table 2. 0 Means of the compounds Deviations from the means in agreement: 0 gyplure, o L-acetoxy-cis-hexadecenol, 0 iso-amyl acetate.
+4
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BRUNHILDST~~RCKOW
ammonia and hexane are not included because of their failure to produce an EAG, even at highest concentrations. The results were evaluated by the conventional analysis of variance with regard to the effectiveness of the synthetic lures and the natural lure extract. Since there were only three trials with gyplure on different antennae the samples used for each of the other compounds were reduced to three by the use of tables of random numbers. A correction and transformation of the values were indicated by the following characteristics : 1. The upper part of the S-shaped curves appeared to be slightly distorted by missing assays l-3 log units higher in concentration than those used. 2. The variance of the EAG showed a dependence on the mean (Fig. 6 and Table 2). TABLE ~-STANDARD ERRORANDNUMBEROF REPLICATES OF THE CURVESIN FIG. 6
Compound 1 Crude natural lure extract m-Acetoxy-cis-hexadecenol
+ 2.24 (5) * 1.59
f 5.48 (5) + 2.24
+ 2.74 (5) f 2.74
(‘5;
(is;
(5)
(5)
(5)
(5)
t 2.74 ;;
n-Acetoxy-cis-hexadecenol
&o-Amy1 acetate Amy1 acetate
5
*0 (5Y
L-Acetoxy-cis-hexadeceno1
n-Acetoxy-cis-octadecenol
Concentrations tested in increasing order (for actual amounts see Fig. 6) 2 3 4
F[ (3) *0 (5) ;p
(7) + 2.04 (4) * 3.54 (3) * 1.59 (5) f 1.94 (5)
f 2.99 (7) * 2.50 (4) f 7.64 (3) rf:5.00 (5) f 4.33 (5)
+ 3.45 (7) + 2.04 (4) _+2.89 (3) +_1.23 (5) + 6.02 (5)
f0 6; (‘4: ;; (13;
* Number of tests at each concentration in parenthesis.
3. Responses of a single antenna to different concentrations and compounds were treated in the analysis as responses of different antennae. However, these corrections were considered to be of minor importance and therefore were not applied. The comparison between the effectiveness of the 3 isomers of acetoxy-cishexadecenol and gyplure was of primary interest, see Table 3: 1. The analysis of variance indicates no difference between the means of the four synthetic lures -disregarding the differences in concentration, a significant difference between the effect of the four synthetic lures on the EAG is not demonstrable.
THE EAG AS AN ASSAY FOR THE RECEPTION OF ODOURS BY THE GYPSY MOTH TABLE ~-ANALYSIS Due to
1579
OF VARIANCE: EFFECTIVENESS OF THE FOUR SYNTHETIC LURES ss
DF
Compounds 38.33 Concentrations 74,227.09 Interaction (compound x concentration) 109.58 Sampling 896.67
3 4
12.77 18,556.77
12 40
9.15 22.41
Total
59
75,241.67
F
MS
1.39 2,028.06 -
P
in y. > 5 & 1 -
2. The differences between the means of the concentrations are significant-all four compounds show an influence by the concentration with respect to their effect on the EAG. Since there was no significant difference in the effectiveness of the synthetic lures, any of them could be compared with the natural lure extract; L-acetoxy-cishexadecenol was chosen for this comparison. The concentrations used in the calculation ranged from 10-4 to 1 pg. The corresponding values for the natural lure extract were taken from the stimulation-response curve of each antenna. The results are shown in Table 4:
TABLE 4-ANALYSIS
Due to
OF VARIANCE: EFFECTIVENESSOF L-ACETOXY-CZS-HEXADECENOLAND THE NATURAL LURE EXTRACT ss
DF
MS
F
P in y0 41
Compounds 7,680.OO Concentrations 28,628.33 Interaction (compound x concentration) 788.34 Sampling 650.00
1 4
7,680.OO 7,157.08
236.30 220.21
4 20
197.08 32.50
6.06 -
37,746.67
29
Total
1. The analysis of variance shows a significant difference between the means of the synthetic lure and natural lure extract-disregarding the differences in concentration, L-acetoxy-cis-hexadecenol and the natural lure extract influence the EAG differently. 2. The differences between the means of the concentrations are significantthe synthetic lure and natural lure extract show an influence by the concentration with respect to their effect on the EAG.
1.580
BRUNHILDST~RCKOW
3. A significant interaction exists between the means of the compounds and concentrations-the effective differences occurring with changes in the concentration depend on the compounds. n-Acetoxy-cis-hexadecenol had been determined to be the major attractive compound of the crude natural lure extract (JACOBSON et al., 1960, 1961). Therefore, a comparison between its effectiveness and that of the other lures on the basis of weight was justified. (The vapour pressure of all compounds could be expected to be approximately the same.) A comparison between the effectiveness of the lures and the amyl acetates was not feasible, since the vapour pressure of the amyl acetates appeared to be higher than that of the lures, as evidenced by cartridges which were used until exhausted. A legitimate comparison between the effectiveness of the different compounds may be based on the similar shape of the curves. The EAG’s incresased considerably for each compound within a relatively narrow range of increase in the stimulus, that is within 34 log units. Although the vapour pressure of both isomers of amyl acetate could not be ascertained, an approximate comparison between their effectiveness is possible based on their closely related boiling points (149°C for amyl acetate, 142°C for iso-amyl acetate). The stimulation-response curves of the two isomers were not significantly different. However, the shape and kinetics of the EAG’s differed distinctly (Fig. 2). Amy1 acetate evoked a characteristic on-effect and a regularly declining EAG, while the &o-isomer showed a slightly depressed and retarded on-effect, followed by a more or less steady level and a weak off-effect. Behavioural Males of the gypsy moth sexually excited by scent raise their antennae, Finally, they fly in occasionally clean them, vibrate their wings, and move about. the direction of the odorous source, which is in nature the fragrant female (SCHWINCK, 1955). When the male has found the female, he performs his display and copulation follows. BLOCK (1960) found that the male would begin his display from an artificially fixed position without the presence of a female and used this as a criterion for behavioural tests with sex lures in the laboratory. This display is a relatively high reaction level of copulatory behaviour which, however, could also be interpreted as an expedient behaviour because of the fixed position. In order to determine approximately the threshold at which flight and search begins, wing vibration of at least 30-set duration as a response to an odorous stimulus was chosen as the positive criterion for sexual excitement. Wing vibration of 30 set or longer was always connected with moving about. It generally lasted 1-5 min and would correspond to the fifth stage of sexual excitement noted by SCHWINCK (1955). Though the behavioural tests were of a preliminary nature, their results (Fig. 7) are about the same as those obtained electrophysiologically. The effect of the crude natural lure was stronger than the effect of the synthetic lures. Both forms of amyl acetate produced a few short wing flashes at their effective concentrations
THE
EAG
AS AN
ASSAY
FOR
THE
RECEPTION
OF
ODOURS
1581
BY THE GYPSY MOTH
(compare EAG’s); however, they failed to produce the typical wing fluttering of the sex lures. Ammonia and hexane caused no noticeable response even at much higher concentrations, as shown in Fig. 7.
dI
-4
-3
-2
-1
1
10+1
.2
+3
MICROGRAM
FIG. 7. Means of the stimulation-response curves (behavioural tests). Wing fluttering of 30 set or longer was considered as the positive criterion. The response to an air puff (9 trials with 31 moths, n = 279) is shown as control + standard error. The curves for the amyl acetates do not contain the short flashes of the wings mentioned in the text. DISCUSSION
Review of the method Excitement released by ascending and descending intensities of a stimulus results in the phenomenon of hysteresis. This is true for the activity of a single receptor cell (BURKHARDT, 1959) as well as for the activity of many receptor cells excited simultaneously (SCHNEIDER, 1963). Therefore, these tests were performed in ascending concentrations which show the least adaptation effect. The thresholds of the compounds had previously been determined with antennae which were not used for preparing the stimulation-response curves. A masking effect originating from the preceding test was excluded as far as possible by coupling one compound with different compounds in different test series and comparing their results (Table 1, and Figs 3-5), and by changing at random the succession of the compounds in each step. Although an adapting or masking effect could not be excluded completely in this type of assay, its influence was not pronounced as may best be seen by a comparison of tests with both amyl acetates (Fig. 2). These were made with the same concentration from 3 antennae: antenna No. 6 was stimulated first with normal- and then with iso-amyl acetate, while antennae Nos. 11 and 8 were stimulated inversely. There appeared to be no evidence that one compound significantly effected the other. The principal interest of the experiments was a comparison between the effectiveness of the natural lure extract and the synthetic lures. Studies with respect to the detailed relationship between stimulus and response were not possible
1582
BRUNHILDST~~RCKOW
Rise time, duration, and interval of the with the small number of moths available. stimulus were kept as uniform as possible because of their influence on the sensory response. The stimulation by an air puff from the hand-driven 50 ml syringe proved to be superior to the stimulation by an air flow from a pressure system. This superiority could have been due to the flow rate, which was about 6 I/min for the syringe and 0.36 to 3*6 l/min for the pressure system as measured with a flowmeter. The reliability of the stimulation method with the hand-driven syringe is evident from the uniform response to repeated puffs (Fig. 1). The electrophysiological tests with acetoxy-cis-hexadecenol were a repetition of tests made by SCHNEIDER (1963). The results of his tests and ours are in full agreement. Efiectiveness of the compounds The comparison between the effectiveness of the crude natural extract and the synthetic lures was based on the calculation that 1 female unit of the extract would have a weight of 4 x 10 -!z pg, since JACOBSONet al. (1961) obtained 20 mg of the crude attractive material from 500,000 females. The abdomens of 24-48 hr-old virgin females had been clipped and immediately dropped into the organic solvent (JACOBSON, personal communication). Although a loss of the active compound by enzymatic activity may be disregarded by this procedure, the accuracy of the calculation is questionable because of possible losses occurring in the separation and purification processes. In the female of BOW+ mori the amount of lure emitted equals approximately that of the stored lure (STEINBRECHT, 1964a, b). This result suggests that no storage of the active form of the lure takes place. Since STEINBRECHT does not mention a diurnal rhythm or other irregularities in the emission of the lure, an approximately equal content of lure for each clipped abdomen could also be expected in the case of the gypsy moth. If one assumes that the amount of lure per gypsy moth abdominal tip is 1 pg instead of the calculated 4 x 1O-2 pg, the significant difference between the effectiveness of the natural lure extract and the synthetic lures would not exist. This assumption would be a reasonable one in comparison with the amount of lure found in 24-48 hr-old virgin females of Bombyx mori (STEINBRECHT, 1964b). A biological assay to determine the effect of a single live female in comparison with that of a single female unit from an extract of insects proved to be active, appears to be the most practicable way to resolve the inconsistency between the effectiveness of the natural lure extract and the synthetic lures. Orientation by chemoreception It is generally accepted that a sensory the intensities of a stimulus in that range is found. In olfactory chemoreception
receptor is able to differentiate between in which its stimulation-response curve the differentiation between molecular
1583
densities is mainly used for orientation in the proximity of an odorous source (klinotaxis, tropotaxis, and finally osmotropotaxis, LINDAUERand MARTIN, 1963). Although it has been assumed that a male moth will be attracted by a female over a relatively great distance (6-12 miles), the principle of this attraction is not well understood. The extremely low thresholds of chemosensory reception in assays with the male of Bombyx mori (summarized by SCHNEIDER,1963) apparently explained this phenomenon by chemoreception. However, low thresholds could not be confirmed for either the synthetic lures or the natural lure extract of the gypsy moth. It is possible that orientation from afar requires only a stimulus of very low intensity which, when correlated by the insect with the direction of the wind, orients the insect without reference to the distance from the odorous source (anemotuxis, rhotaxis). This simultaneous evaluation of two different stimuli may explain attraction from a few hundred metres or even l-3 miles. The frequency of repeated stimuli may be more important in this case than the intensity. The continued production of the stimulus would be assured by the fragrant female. MINNICH (1926) was the first who stimulated a single labellar taste hair in the blowfly with a sugar solution and obtained a behavioural response. HODGSONet al. (1955) and EVANSand MELLON (1962) proved that in this case only two sense cells are excited, the sugar receptor and the water receptor. Although therefore, a sudden excitement of one or two single sense cells is capable of evoking a behavioural response, there must be, nevertheless, a limitation on the orientation curves the of insects by odour. At the threshold of the stimulation-response amount of material on the filter paper used in the experiments contained 1013 molecules for the synthetic lures and 5 x 1015 molecules for the amyl acetates. Only a part of these molecules were carried off with each puff, and only a part of those which were carried off impinged upon the adequate receptor sites on the antenna. Impingement on the receptor site is necessary for the excitation of chemoreceptive sense cells. OTTOSON(1956) obtained no response from the nasal mucosa of the frog after covering it with a plastic membrane, which impeded contact between the receptor sites and the stimulating molecules, but which transmitted infra-red radiation. studies of olfactory RICHTER (1962) calculated from electronmicroscopical organs of a fly larva that the total area of the olfactory pores in the cuticle measured only 5 per cent of the entire surface of the antenna1 tip, although this tip appeared to be densely covered with olfactory pores. If, in the case of a moth, even 10 per cent of the surface of the richly branched antenna offered receptor sites, this area wouId still be infinitesimal compared with the area over which the molecules would be dispersed within a few miles from a female. Only the results of a probability study, based on realistic data, could possibly predict the greatest distance over which a sensory response would be assured. Acknowledgements-I wish to thank especially Professor D. SCHNEIDER (Max-Planck Institut, Abt. Vergl. Neurophysiologie, Miinchen, Germany) for his careful review and criticism of the manuscript; also Professor F. HUBER (Institut Vergl. Tierphysiologie der
1584
BRUNHILD
STORCKOW
Universittit Kbln, Germany) for his constructive criticism, Dr. W. G. BODENSTEIN (Entomology Research Division, Agricultural Research Service, USDA, Beltsville, Maryland) for his assistance in preparing the manuscript and superimposing the EAG’s, and Dr. J. U. MCGUIRE (Biometrical Services, Agricultural Research Service, USDA, Beltsville, Maryland) for his audit of the analyses of variance. REFERENCES BLOCK B. C. (1960) Laboratory method for screening compounds as attractants to gypsy moth males. 3’. econ. Ent. 53, 172-173. BURKHARDT D. (1959) Die Erregungsvorglnge sensibler Gangienzellen in Abhlrngigkeit von der Temperatur. Biol. Zbl. 78, 22-62. EVANSD. R. and MELLON D. JR. (1962) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly. J. gen. Physiol. 45, 487-500. HODGSONE. S., LETTVIN J. Y., and ROEDERK. D. (19.55) The physiology of a primary chemoreceptor unit. Science 122, 417-418. JACOBSONM. (1962) Insect sex attractants. III. The optical resolution of DL-lO-aCetOxycis-7-hexadecen-l-01. J. org. Chem. 27, 2670-2671. JACOBSONM., BEROZAM., and JONESW. A. (1960) Isolation, identification, and synthesis of the sex attractant of gypsy moth. Science 132, 1011-1012. JACOBSONM., BEROZAM., and JONESW. A. (1961) Insect sex attractants. I. The isolation, identification, and synthesis of the sex attractant of the gypsy moth. J. Am. them. Sot. 83, 4819-4824. JACOBSONM. and JONESW. A. (1962) Insect sex attractants. II. The synthesis of a highly potent gypsy moth sex attractant and some related compounds. J. org. Chem. 27, 2523-2524. LINDAUER M. and MARTIN H. (1963) uber die Orientierung der Biene im Duftfeld. Naturwiss. 50, 509-514. MINNICH D. E. (1926) The organs of taste on the proboscis of the blowfly, Phmmia regina Meigen. Anat. Rec. 34, 126. OTTOSOND. (1956) Analysis of the electrical activity of the olfactory epithelium. Acta physiol. &and. 35, Suppl. 122, l-83. RICHTER S. (1962) Unmittelbarer Kontakt der Sinneszellen cuticularer Sinnesorgane mit der Aussenwelt. Z. Morph. okol. Tiere. 52, 171-196. SCHNEIDERD. (1957) Elektrophysiologische Untersuchungen von Chemo- und Mechanorezeptoren der Antenne des Seidenspinners Bombyx mori L. Z. oergl. Physiol. 40, 8-41. SCHNEIDERD. (1962) Electrophysiological investigation on the olfactory specificity of sexual attracting substances in different species of moths. J. Insect Physiol. 8, 1 S-30. SCHNEIDERD. (1963) Electrophysiological investigation of insect olfaction. Proc. 1st int. Symp. Olfaction and Taste, Stockholm 1962, 85-103. SCHNEIDERD. and HECKER E. (1956) Zur Elektrophysiologie der Antenne des Seidenspinners Bombyx mori bei Reizung mit angereicherten Extrakten des Sexuallockstoffes. Z. Nat&ouch. llb, 121-124. SCHWINCK I. (1955) Weitere Untersuchungen zur Frage der Geruchsorientierung der Nachtschmetterlinge : Partielle Fiihleramputation bei Spinnermiinnchen, insbesondere am Seidenspinner Bombyx mori L. Z. vergl. PhysioE. 37, 439458. STEINBRECHTR. A. (1964a) Feinstruktur und Histochemie der Sexualduftdriise des Seidenspinners Bombyx mori L. Z. Zellfovsch. 64, 227-261. STEINBRECHTR. A. (1964b) Die Abhgngigkeit der Lockwirkung des Sexualduftorgans weiblicher Seidenspinner (Bombyx mori) von Alter und Kopulation. Z. vergl. Physiol. 48, 341-356.
Printed in Great Britain
THE ELECTROANTENNOGRAM (EAG) AS AN ASSAY FOR THE RECEPTION OF ODOURS BY THE GYPSY MOTH BRUNHILD
STURCKOW
Entomology Research Division, Agricultural Research Service, USDA, Beltsville, Maryland (Received 14 June 1965) Abstract-The electroantennogram (EAG), an overall electrical response of the insect antenna to an adequate stimulus, has been used as a testing method for the reception of odours. Synthetic sex lures for the male gypsy moth Porthetriu dispar (IO-acetoxycis-7-hexadecen-l-01 and 12-acetoxy-cis-9-octadecen-l-01) were assayed electrophysiologically and behaviourally, and their effectiveness was compared with that of a natural sex pheromone extract derived from female gypsy moths, and with sexually neutral compounds. INTRODUCTION
THE RESPONSE of the male gypsy female was studied behaviourally by physiological (acetoxy-cis-hexadecenol) cannot be compared directly with obtained method. As
moth to the natural sex pheromone of the SCHNEIDER (1963). He also included electroisomers of a synthetic sex attractant JACOBSON However, results those from the lure since they were long as the knowledge of the relation between limited, stimulation-response compared directly obtained by the same the behavioural criteria used within each method should be comparable.
extended to assay both the of acetoxy-cis-hexadecenol gyplure (acetoxy-cis-octadecenol), another synthetic JACOBSONand JONES A few behavioural suitable technique could not be developed with the relatively
odours. antennae of are lacking, receptor potentials excited sense cells. nervous
of the receptor
Considered
activity exists, 1573
Since interneurons detail, it also contains contain the
1574
BIWNHILD STURCKOW
sum phenomenon of each type of potential derived from the central cells. However, the influence of the last two factors on the shape and kinetics of the EAG in general is negligible. MATERIALS
AND METHODS
From a total of 50 male pupae * of Porthetria dispar L. 16 moths, 3-4 days old, tests 31 moths were used for the electrophysiological tests. For the behavioural were used 2-5 days after emergence. All tests were made during the month of December. The air in the testing room was kept as clean as possible by ventilation. The temperature ranged from 22” to 25°C. The following materials were provided by Mr. Martin Jacobson (of this Division) as stock solution in hexane: 1. Crude natural pheromone (20 mg extract of 500,000 female abdominal tips, JACOBSONet al., 1961) 1 female unit = approximately 4 x 1O-2 pg. The stock solution contained 100 female units/O*5 ml hexane. 2. A synthetic sex lure (lo-acetoxy-cis-7-hexadecen-I-ol), D- and L-isomers and the racemic mixture (JACOBSON, 1962). Each stock solution contained 100 pg/O.5 ml hexane. The mol. wt. is 298. 3. A synthetic sex lure, gyplure (12-acetoxy-cis-9-octadecen-I-ol), n-isomer (JACOBSON and JONES, 1962). The stock solution contained 100 pgjO.5 ml hexane. The mol. wt. is 314. The stock solutions were diluted so that the same volume (5 ~1) could be used Cartridges loaded with hexane, for each test, except for each highest concentration. ammonia, and amyl acetate and an empty cartridge were used for comparison or as a control. In each test series, two compounds were tested alternately in 4 or 5 steps, each 10 times higher in concentration. In order to reduce the error by coupling effects, the sequence of the compounds in each concentration step was changed at random, for example: a b, b’ a’, b” a”, a”’ b”’ etc. Each substance was tested electrophysiologically in three or more series and behaviourally in one series as shown in Table 1. Electrophysiological
tests
Each moth was mounted on a piece of cardboard so that wing, head, and leg movements were prevented without inhibition of breathing. The antenna not used in the experiment was glued backward, the other was fixed forward between 2 electrodes. One of the electrodes (a capillary) was slipped over the tip of the antenna, the other (a needle) was inserted into its base. Experiments of l-2 hr duration were possible under these conditions. The stimulus, a sudden odorous puff, was directed into the basket-shaped antenna behind which an exhaust duct carried off the contaminated air. The movement of the plunger of a 50 ml syringe provided a measured volume of air which * The pupae were supplied by Mr. Thomas McIntyre, Methods Improvement Laboratory, Plant Pest Control Division, Agricultural Research Service, USDA, Falmouth, Mass.
THE EAG AS AN ASSAY FOR THE RECEPTION
OF ODOURS BY THE GYPSY MOTH
1575
was blown through a glass cartridge containing the test material on a piece of filter paper (Whatman No. 2). Since the same volume of solution was used for each test, the evaporating area on each filter paper was the same. The syringe was filled with air from outside of the stimulation system by means of a two-way cock. Three to seven EAG’s were made with each cartridge at intervals of 5.8 set _+0.3 standard error based on the measurement of 100 consecutive tests. Within this time the EAG potential had always returned to the base line. The cartridge was loaded outside of the room just before the test; loading and corking took 3 min. The use of ground-glass joints permitted rapid interchange of the cartridges. The platinum electrodes were connected to a DC amplifier (Grass, Model P67*. 4 junction potential, partly due to different electrodes (needle and capillary) and partly due to the structure of the antenna (as seen from measurements without antenna), was balanced out by the amplifier control. A light signal, indicating the release but not the duration of the stimulus, was activated by a foot control. The length of the puff from the hand-driven syringe was approximately 500 milliseconds. A Grass”* camera registered the results on paper film. The first of several EAG’s at each concentration were rephotographed and superimposed in printing with the aid of orientation marks. The number assigned to each antenna was carried over to the legend of the figures to make a comparison between EAG’s of the same antenna possible. The height of the on-effect (SCHNEIDER, 1962) of the first and second EAG in a series of tests with the same cartridge was used for the preparation of the stimulation-response curve expressed in arbitrary units.
Behavioural tests of at least 30 set Each moth was kept in an individual cage. Wing vibration was chosen as a positive criterium of sexual excitement. At about sunrise males, 2-5 days old, began to vibrate their wings, move about, and fly even without an odorous stimulus. They continued this activity interrupted by longer or shorter intervals throughout the day. Therefore, the tests were made from 4 to 8 a.m. in diffuse light when the moths were relatively quiet. The stimulus was applied to both antennae of an unrestrained moth from the same distance, with the same applicator, and in the same sequence as in the electrophysiological tests. The interval between tests was 20 min. Each filter paper was utilized for six puffs, the sequence of which was noted and altered in the next round. All moths were tested against all concentrations of all compounds (see Table 1). Sometimes the moths reacted to the mechanical component of the stimulating puff with tonic immobility which disappeared after l-5 min. The response to the * Mention of this proprietary product does not necessarily imply its endorsement by the U.S. Department of Agriculture.
BRUNHILDST~&CKOW
1.576
TABLE la-ELECTROPHY~IOLOGICALTESTS
Substance Natural lure extract
DL-Acetoxy-cis-hexadecenol
Antenna record number
Coupled with (all isomers refer to acetoxyhexadecenol) L-isomer L-isomer D-isomer D-isomer gyplure
2 4 3 4 5
L-isomer L-isomer gyplure amyl acetate iso-amyl acetate
8 9 9 11 11
D-isomer D-isomer gyplure and DL-aCetOXy-
L-Acetoxy-cis-hexadecenol
3 6 6 And see under natural lure extract hexadecenol
D-Acetoxy-cis-hexadecenol
See under natural lure extract and L-acetoxyhexadecenol
Gyplure
See under natural lure extract, DL-acetoxyhexadecenol, and L-acetoxyhexadecenol
iso-Amy1 acetate
6 amyl amyl 8 9 amyl 10 amyl And see under DL-acetoxyhexadecenol
Amy1 acetate
See under DL-acetoxyhexadecenol
acetate acetate acetate acetate
and iso-amyl acetate
TABLE 1b-BEH.4vIouR.4L TESTS L-Acetoxyhexadecenol coupled with D-acetoxyhexadecenol DL-Acetoxyhexadecenol coupled with gyplure Natural lure extract coupled with D-acetoxyhexadecenol iso-Amy1 acetate coupled with amyl acetate
odorous component of the puff was stored over this period as evidenced by differences between tests with air and with lures. For this reason the moths were observed up to 7 min after the application of the stimulus. RESULTS
Electrophysiological The first response in a series of tests with the same stimulus was usually stronger than the following ones, which remained equal for up to ten responses
FIG.
1.
Superimposition
of the first 4 EAG’s in a series of tests. (a) amyl acetate (11) 4 x 103. (b) gyplure (5) lo.* * NOTE FOR ALL FIGURES. The concentration of the stimulus compound is given in pg. The record number of the antenna is enclosed in parenthesis. Horizontal mark: 1 set; vertical mark: 2 mv (no dots), 4 mv (2 dots). The circle is a register The horizontal line above the EAG’s indicates mark used for the superimposition. the release of the stimulus.
FIG. 2. Different antennae excited by the same stimulus. (a) natural lure extract 5 x 10-2; I (S), 2 (2). (6) L-acetoxy-cis-hexadecenol 1; I (6), 2 (2), 3 (8). (c) iso-amyl acetate 4 x 102; I (6), 2 (ll), 3 (8). (d) amyl acetate 4 x 102; 1 (6), 2 (ll), 3 (8).
FIG. 3. Antenna hexadecenol; I to 4 (c) control with air. as in d. The voltage
8 stimulated in two series: a, b and d, e. (a) L-acetoxy-cis10d2 to 1O1. (b) nbacetoxy-cis-hexadecenol; I to 4 as in a. (d) iso-amyl acetate; I to 3 4 to 4 x 102. (e) amyl acetate; I to 3 mark equals in this case 4 mV.
.. -.
*“_I-
-_,_-_,_
.
-.L”
I ,-;:
*,.*
_/
.~l_“_x-__._
FIG. 4. Antenna 5 stimulated by the natural lure extract in comparison with a synthetic lure. (a) control with air. (b) natural lure extract; I to 4 5 x 10m4to 5 x 10-l. (c) gyplure; I to 4 lo-’ to 1O1. The voltage mark equals in this case 4 mV.
FIG. 5. Antenna 6 stimulated by the two optically active isomers of acetoxy-cishexadecenol. (a) control with air. (b) L-isomer; I to 5 10e3 to 10’. (c) D-isomer; I to 5 IO-3 to 101.
THE
1577
EAGAS AN ASSAYFORTHE RECEPTIONOF ODOURSBY THE GYPSYMOTH
depending on the intensity and quality of the stimulus (Fig. 1). After the group of the EAG’s dropped slowly. Sometimes the first relatively equal responses, response was not significantly higher than those following. In order to establish a reliable base for the evaluation of the tests, the first and second EAG of each series of tests were superimposed. Essential characteristics of the EAG were determined by the quality and intensity of the stimulus, the site of the electrodes, and the number and types of sensilla encountered by the stimulus (Figs 2 and 3). Therefore, a direct comparison between EAG’s from different antennae was approached with caution (see also SCHNEIDER, 1962). Different EAG’s were directly comparable if they were obtained from one antenna with the same placement of the electrodes, and with the same rise time, duration, and direction of the stimulating puff throughout the experiment, as in Figs 3 to 5. In order to compare the increase in the EAG with increasing concentrations between different antennae and different compounds, it was necessary to equate the height of the EAG’s derived from each antenna and each compound. This was done by adjusting the EAG’s derived from each highest concentration tested to 100 arbitrary units, and expressing their lower values in per cent. Two antennae were not stimulated by the highest concentration of amyl acetate (Table 2). In this case an adjustment to seventy-five arbitrary units was made. Fig. 6 and Table 2 show the stimulation-response curves obtained in this way. The tests with 2 rlO0
.
.
0
Y
CONTROL 10-S
-4
-3
-2
-1
1
lo+’
+!2
+a
MICRO(IRAM
FIG. 6. Means of the stimulation-response curves (electrophysiological tests). The response to air is taken as the control level. For the standard error and number in agreement. of replicates of all values see Table 2. 0 Means of the compounds Deviations from the means in agreement: 0 gyplure, o L-acetoxy-cis-hexadecenol, 0 iso-amyl acetate.
+4
1578
BRUNHILDST~~RCKOW
ammonia and hexane are not included because of their failure to produce an EAG, even at highest concentrations. The results were evaluated by the conventional analysis of variance with regard to the effectiveness of the synthetic lures and the natural lure extract. Since there were only three trials with gyplure on different antennae the samples used for each of the other compounds were reduced to three by the use of tables of random numbers. A correction and transformation of the values were indicated by the following characteristics : 1. The upper part of the S-shaped curves appeared to be slightly distorted by missing assays l-3 log units higher in concentration than those used. 2. The variance of the EAG showed a dependence on the mean (Fig. 6 and Table 2). TABLE ~-STANDARD ERRORANDNUMBEROF REPLICATES OF THE CURVESIN FIG. 6
Compound 1 Crude natural lure extract m-Acetoxy-cis-hexadecenol
+ 2.24 (5) * 1.59
f 5.48 (5) + 2.24
+ 2.74 (5) f 2.74
(‘5;
(is;
(5)
(5)
(5)
(5)
t 2.74 ;;
n-Acetoxy-cis-hexadecenol
&o-Amy1 acetate Amy1 acetate
5
*0 (5Y
L-Acetoxy-cis-hexadeceno1
n-Acetoxy-cis-octadecenol
Concentrations tested in increasing order (for actual amounts see Fig. 6) 2 3 4
F[ (3) *0 (5) ;p
(7) + 2.04 (4) * 3.54 (3) * 1.59 (5) f 1.94 (5)
f 2.99 (7) * 2.50 (4) f 7.64 (3) rf:5.00 (5) f 4.33 (5)
+ 3.45 (7) + 2.04 (4) _+2.89 (3) +_1.23 (5) + 6.02 (5)
f0 6; (‘4: ;; (13;
* Number of tests at each concentration in parenthesis.
3. Responses of a single antenna to different concentrations and compounds were treated in the analysis as responses of different antennae. However, these corrections were considered to be of minor importance and therefore were not applied. The comparison between the effectiveness of the 3 isomers of acetoxy-cishexadecenol and gyplure was of primary interest, see Table 3: 1. The analysis of variance indicates no difference between the means of the four synthetic lures -disregarding the differences in concentration, a significant difference between the effect of the four synthetic lures on the EAG is not demonstrable.
THE EAG AS AN ASSAY FOR THE RECEPTION OF ODOURS BY THE GYPSY MOTH TABLE ~-ANALYSIS Due to
1579
OF VARIANCE: EFFECTIVENESS OF THE FOUR SYNTHETIC LURES ss
DF
Compounds 38.33 Concentrations 74,227.09 Interaction (compound x concentration) 109.58 Sampling 896.67
3 4
12.77 18,556.77
12 40
9.15 22.41
Total
59
75,241.67
F
MS
1.39 2,028.06 -
P
in y. > 5 & 1 -
2. The differences between the means of the concentrations are significant-all four compounds show an influence by the concentration with respect to their effect on the EAG. Since there was no significant difference in the effectiveness of the synthetic lures, any of them could be compared with the natural lure extract; L-acetoxy-cishexadecenol was chosen for this comparison. The concentrations used in the calculation ranged from 10-4 to 1 pg. The corresponding values for the natural lure extract were taken from the stimulation-response curve of each antenna. The results are shown in Table 4:
TABLE 4-ANALYSIS
Due to
OF VARIANCE: EFFECTIVENESSOF L-ACETOXY-CZS-HEXADECENOLAND THE NATURAL LURE EXTRACT ss
DF
MS
F
P in y0 41
Compounds 7,680.OO Concentrations 28,628.33 Interaction (compound x concentration) 788.34 Sampling 650.00
1 4
7,680.OO 7,157.08
236.30 220.21
4 20
197.08 32.50
6.06 -
37,746.67
29
Total
1. The analysis of variance shows a significant difference between the means of the synthetic lure and natural lure extract-disregarding the differences in concentration, L-acetoxy-cis-hexadecenol and the natural lure extract influence the EAG differently. 2. The differences between the means of the concentrations are significantthe synthetic lure and natural lure extract show an influence by the concentration with respect to their effect on the EAG.
1.580
BRUNHILDST~RCKOW
3. A significant interaction exists between the means of the compounds and concentrations-the effective differences occurring with changes in the concentration depend on the compounds. n-Acetoxy-cis-hexadecenol had been determined to be the major attractive compound of the crude natural lure extract (JACOBSON et al., 1960, 1961). Therefore, a comparison between its effectiveness and that of the other lures on the basis of weight was justified. (The vapour pressure of all compounds could be expected to be approximately the same.) A comparison between the effectiveness of the lures and the amyl acetates was not feasible, since the vapour pressure of the amyl acetates appeared to be higher than that of the lures, as evidenced by cartridges which were used until exhausted. A legitimate comparison between the effectiveness of the different compounds may be based on the similar shape of the curves. The EAG’s incresased considerably for each compound within a relatively narrow range of increase in the stimulus, that is within 34 log units. Although the vapour pressure of both isomers of amyl acetate could not be ascertained, an approximate comparison between their effectiveness is possible based on their closely related boiling points (149°C for amyl acetate, 142°C for iso-amyl acetate). The stimulation-response curves of the two isomers were not significantly different. However, the shape and kinetics of the EAG’s differed distinctly (Fig. 2). Amy1 acetate evoked a characteristic on-effect and a regularly declining EAG, while the &o-isomer showed a slightly depressed and retarded on-effect, followed by a more or less steady level and a weak off-effect. Behavioural Males of the gypsy moth sexually excited by scent raise their antennae, Finally, they fly in occasionally clean them, vibrate their wings, and move about. the direction of the odorous source, which is in nature the fragrant female (SCHWINCK, 1955). When the male has found the female, he performs his display and copulation follows. BLOCK (1960) found that the male would begin his display from an artificially fixed position without the presence of a female and used this as a criterion for behavioural tests with sex lures in the laboratory. This display is a relatively high reaction level of copulatory behaviour which, however, could also be interpreted as an expedient behaviour because of the fixed position. In order to determine approximately the threshold at which flight and search begins, wing vibration of at least 30-set duration as a response to an odorous stimulus was chosen as the positive criterion for sexual excitement. Wing vibration of 30 set or longer was always connected with moving about. It generally lasted 1-5 min and would correspond to the fifth stage of sexual excitement noted by SCHWINCK (1955). Though the behavioural tests were of a preliminary nature, their results (Fig. 7) are about the same as those obtained electrophysiologically. The effect of the crude natural lure was stronger than the effect of the synthetic lures. Both forms of amyl acetate produced a few short wing flashes at their effective concentrations
THE
EAG
AS AN
ASSAY
FOR
THE
RECEPTION
OF
ODOURS
1581
BY THE GYPSY MOTH
(compare EAG’s); however, they failed to produce the typical wing fluttering of the sex lures. Ammonia and hexane caused no noticeable response even at much higher concentrations, as shown in Fig. 7.
dI
-4
-3
-2
-1
1
10+1
.2
+3
MICROGRAM
FIG. 7. Means of the stimulation-response curves (behavioural tests). Wing fluttering of 30 set or longer was considered as the positive criterion. The response to an air puff (9 trials with 31 moths, n = 279) is shown as control + standard error. The curves for the amyl acetates do not contain the short flashes of the wings mentioned in the text. DISCUSSION
Review of the method Excitement released by ascending and descending intensities of a stimulus results in the phenomenon of hysteresis. This is true for the activity of a single receptor cell (BURKHARDT, 1959) as well as for the activity of many receptor cells excited simultaneously (SCHNEIDER, 1963). Therefore, these tests were performed in ascending concentrations which show the least adaptation effect. The thresholds of the compounds had previously been determined with antennae which were not used for preparing the stimulation-response curves. A masking effect originating from the preceding test was excluded as far as possible by coupling one compound with different compounds in different test series and comparing their results (Table 1, and Figs 3-5), and by changing at random the succession of the compounds in each step. Although an adapting or masking effect could not be excluded completely in this type of assay, its influence was not pronounced as may best be seen by a comparison of tests with both amyl acetates (Fig. 2). These were made with the same concentration from 3 antennae: antenna No. 6 was stimulated first with normal- and then with iso-amyl acetate, while antennae Nos. 11 and 8 were stimulated inversely. There appeared to be no evidence that one compound significantly effected the other. The principal interest of the experiments was a comparison between the effectiveness of the natural lure extract and the synthetic lures. Studies with respect to the detailed relationship between stimulus and response were not possible
1582
BRUNHILDST~~RCKOW
Rise time, duration, and interval of the with the small number of moths available. stimulus were kept as uniform as possible because of their influence on the sensory response. The stimulation by an air puff from the hand-driven 50 ml syringe proved to be superior to the stimulation by an air flow from a pressure system. This superiority could have been due to the flow rate, which was about 6 I/min for the syringe and 0.36 to 3*6 l/min for the pressure system as measured with a flowmeter. The reliability of the stimulation method with the hand-driven syringe is evident from the uniform response to repeated puffs (Fig. 1). The electrophysiological tests with acetoxy-cis-hexadecenol were a repetition of tests made by SCHNEIDER (1963). The results of his tests and ours are in full agreement. Efiectiveness of the compounds The comparison between the effectiveness of the crude natural extract and the synthetic lures was based on the calculation that 1 female unit of the extract would have a weight of 4 x 10 -!z pg, since JACOBSONet al. (1961) obtained 20 mg of the crude attractive material from 500,000 females. The abdomens of 24-48 hr-old virgin females had been clipped and immediately dropped into the organic solvent (JACOBSON, personal communication). Although a loss of the active compound by enzymatic activity may be disregarded by this procedure, the accuracy of the calculation is questionable because of possible losses occurring in the separation and purification processes. In the female of BOW+ mori the amount of lure emitted equals approximately that of the stored lure (STEINBRECHT, 1964a, b). This result suggests that no storage of the active form of the lure takes place. Since STEINBRECHT does not mention a diurnal rhythm or other irregularities in the emission of the lure, an approximately equal content of lure for each clipped abdomen could also be expected in the case of the gypsy moth. If one assumes that the amount of lure per gypsy moth abdominal tip is 1 pg instead of the calculated 4 x 1O-2 pg, the significant difference between the effectiveness of the natural lure extract and the synthetic lures would not exist. This assumption would be a reasonable one in comparison with the amount of lure found in 24-48 hr-old virgin females of Bombyx mori (STEINBRECHT, 1964b). A biological assay to determine the effect of a single live female in comparison with that of a single female unit from an extract of insects proved to be active, appears to be the most practicable way to resolve the inconsistency between the effectiveness of the natural lure extract and the synthetic lures. Orientation by chemoreception It is generally accepted that a sensory the intensities of a stimulus in that range is found. In olfactory chemoreception
receptor is able to differentiate between in which its stimulation-response curve the differentiation between molecular
1583
densities is mainly used for orientation in the proximity of an odorous source (klinotaxis, tropotaxis, and finally osmotropotaxis, LINDAUERand MARTIN, 1963). Although it has been assumed that a male moth will be attracted by a female over a relatively great distance (6-12 miles), the principle of this attraction is not well understood. The extremely low thresholds of chemosensory reception in assays with the male of Bombyx mori (summarized by SCHNEIDER,1963) apparently explained this phenomenon by chemoreception. However, low thresholds could not be confirmed for either the synthetic lures or the natural lure extract of the gypsy moth. It is possible that orientation from afar requires only a stimulus of very low intensity which, when correlated by the insect with the direction of the wind, orients the insect without reference to the distance from the odorous source (anemotuxis, rhotaxis). This simultaneous evaluation of two different stimuli may explain attraction from a few hundred metres or even l-3 miles. The frequency of repeated stimuli may be more important in this case than the intensity. The continued production of the stimulus would be assured by the fragrant female. MINNICH (1926) was the first who stimulated a single labellar taste hair in the blowfly with a sugar solution and obtained a behavioural response. HODGSONet al. (1955) and EVANSand MELLON (1962) proved that in this case only two sense cells are excited, the sugar receptor and the water receptor. Although therefore, a sudden excitement of one or two single sense cells is capable of evoking a behavioural response, there must be, nevertheless, a limitation on the orientation curves the of insects by odour. At the threshold of the stimulation-response amount of material on the filter paper used in the experiments contained 1013 molecules for the synthetic lures and 5 x 1015 molecules for the amyl acetates. Only a part of these molecules were carried off with each puff, and only a part of those which were carried off impinged upon the adequate receptor sites on the antenna. Impingement on the receptor site is necessary for the excitation of chemoreceptive sense cells. OTTOSON(1956) obtained no response from the nasal mucosa of the frog after covering it with a plastic membrane, which impeded contact between the receptor sites and the stimulating molecules, but which transmitted infra-red radiation. studies of olfactory RICHTER (1962) calculated from electronmicroscopical organs of a fly larva that the total area of the olfactory pores in the cuticle measured only 5 per cent of the entire surface of the antenna1 tip, although this tip appeared to be densely covered with olfactory pores. If, in the case of a moth, even 10 per cent of the surface of the richly branched antenna offered receptor sites, this area wouId still be infinitesimal compared with the area over which the molecules would be dispersed within a few miles from a female. Only the results of a probability study, based on realistic data, could possibly predict the greatest distance over which a sensory response would be assured. Acknowledgements-I wish to thank especially Professor D. SCHNEIDER (Max-Planck Institut, Abt. Vergl. Neurophysiologie, Miinchen, Germany) for his careful review and criticism of the manuscript; also Professor F. HUBER (Institut Vergl. Tierphysiologie der
1584
BRUNHILD
STORCKOW
Universittit Kbln, Germany) for his constructive criticism, Dr. W. G. BODENSTEIN (Entomology Research Division, Agricultural Research Service, USDA, Beltsville, Maryland) for his assistance in preparing the manuscript and superimposing the EAG’s, and Dr. J. U. MCGUIRE (Biometrical Services, Agricultural Research Service, USDA, Beltsville, Maryland) for his audit of the analyses of variance. REFERENCES BLOCK B. C. (1960) Laboratory method for screening compounds as attractants to gypsy moth males. 3’. econ. Ent. 53, 172-173. BURKHARDT D. (1959) Die Erregungsvorglnge sensibler Gangienzellen in Abhlrngigkeit von der Temperatur. Biol. Zbl. 78, 22-62. EVANSD. R. and MELLON D. JR. (1962) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly. J. gen. Physiol. 45, 487-500. HODGSONE. S., LETTVIN J. Y., and ROEDERK. D. (19.55) The physiology of a primary chemoreceptor unit. Science 122, 417-418. JACOBSONM. (1962) Insect sex attractants. III. The optical resolution of DL-lO-aCetOxycis-7-hexadecen-l-01. J. org. Chem. 27, 2670-2671. JACOBSONM., BEROZAM., and JONESW. A. (1960) Isolation, identification, and synthesis of the sex attractant of gypsy moth. Science 132, 1011-1012. JACOBSONM., BEROZAM., and JONESW. A. (1961) Insect sex attractants. I. The isolation, identification, and synthesis of the sex attractant of the gypsy moth. J. Am. them. Sot. 83, 4819-4824. JACOBSONM. and JONESW. A. (1962) Insect sex attractants. II. The synthesis of a highly potent gypsy moth sex attractant and some related compounds. J. org. Chem. 27, 2523-2524. LINDAUER M. and MARTIN H. (1963) uber die Orientierung der Biene im Duftfeld. Naturwiss. 50, 509-514. MINNICH D. E. (1926) The organs of taste on the proboscis of the blowfly, Phmmia regina Meigen. Anat. Rec. 34, 126. OTTOSOND. (1956) Analysis of the electrical activity of the olfactory epithelium. Acta physiol. &and. 35, Suppl. 122, l-83. RICHTER S. (1962) Unmittelbarer Kontakt der Sinneszellen cuticularer Sinnesorgane mit der Aussenwelt. Z. Morph. okol. Tiere. 52, 171-196. SCHNEIDERD. (1957) Elektrophysiologische Untersuchungen von Chemo- und Mechanorezeptoren der Antenne des Seidenspinners Bombyx mori L. Z. oergl. Physiol. 40, 8-41. SCHNEIDERD. (1962) Electrophysiological investigation on the olfactory specificity of sexual attracting substances in different species of moths. J. Insect Physiol. 8, 1 S-30. SCHNEIDERD. (1963) Electrophysiological investigation of insect olfaction. Proc. 1st int. Symp. Olfaction and Taste, Stockholm 1962, 85-103. SCHNEIDERD. and HECKER E. (1956) Zur Elektrophysiologie der Antenne des Seidenspinners Bombyx mori bei Reizung mit angereicherten Extrakten des Sexuallockstoffes. Z. Nat&ouch. llb, 121-124. SCHWINCK I. (1955) Weitere Untersuchungen zur Frage der Geruchsorientierung der Nachtschmetterlinge : Partielle Fiihleramputation bei Spinnermiinnchen, insbesondere am Seidenspinner Bombyx mori L. Z. vergl. PhysioE. 37, 439458. STEINBRECHTR. A. (1964a) Feinstruktur und Histochemie der Sexualduftdriise des Seidenspinners Bombyx mori L. Z. Zellfovsch. 64, 227-261. STEINBRECHTR. A. (1964b) Die Abhgngigkeit der Lockwirkung des Sexualduftorgans weiblicher Seidenspinner (Bombyx mori) von Alter und Kopulation. Z. vergl. Physiol. 48, 341-356.