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Conditioning in Drosophila melanogaster
Anim . Behav ., 1967, 15, 259-262
CONDITIONING IN DROSOPHILA MELANOGASTER* BY
WAYNE A. HERSHBERGER & MAURICE P. SMITH
North. Ill. Univ ., Dept. Psychol., DeKalb, Ill. & Univ . Colo ., Dept. Psychol., Boulder, Colo .
Thorpe (1939) has found that Drosophila melanogaster reared on a food medium containing 0 .5 per cent peppermint essence show as imagoes a greater acceptance of this odour than do imagoes reared on a non-scented medium . He found this to be the case even when during the pupal stage the peppermint-reared insects were washed and transferred to a nonscented medium where they emerged and were tested as imagoes . He interpreted this process, which he termed olfactory conditioning, as involving some form of habituation (1943, a, b, c) . However, it may also be interpreted as involving a form of learning . A habituation interpretation supposes that the exposure to the scent is sufficient to condition an insect . A learning interpretation supposes further that conditioning depends upon the association of the scent with certain reinforcing stimuli, e.g., food . It is possible to test these two interpretations by confining insects to foodless chambers permeated with the scent of peppermint . If this confinement increases the acceptability of the peppermint scent, the habituation interpretation is substantiated . On the other hand, if it decreases, or at least fails to increase, the acceptability of the peppermint scent, olfactory conditioning in Drosophila melanogaster may be considered a form of learning. Such was the rationale of the present design .t Method A total of 1,074 insects served as subjects . A wild Oregon-Red-Eye strain of Drosophila melanogaster was used, with both sexes used indiscriminately . All subjects were reared in half-pint glass bottles containing a pre-cooked, artificial food medium composed of 300 ml water, 40 g yellow cornmeal, 3 g Agar-Agar, 55 ml Karo, and 2 ml Turtox mold inhibitor . The peppermint-scented medium included 0 .5
per cent, by volume, peppermint-essence (McKesson & Robbins) . The subjects' parents were removed from their respective bottles when the subjects began to pupate. The peppermintreared and normal-reared imagoes did not appear to differ in health and vigour . The olfactometer in which the scent preferences were tested is shown diagrammatically in Fig. 1 . The instrument was fixed in a horizontal plane . To bait the olfactometer, two drops of 5 per cent solution of peppermint essence in absolute alcohol were allowed to evaporate on 2 cm2 of filter paper which was then placed in one of the bait tubes (B), the other bait tube being left empty. One side was baited for half of the insects in each experimental group, and the other side for the other half. Currents of air were drawn through the two arms of the Y tube by a faucet aspirator attached to tube A, the flow being equalized by the adjustment of water level in the wash bottles (W) attached to the two inlet tubes (I) . A batch of ten to fifty insects to be tested was lured phototropically into the starting tube (S), which was then connected to the test apparatus . The room was darkened, light L ( 7 watt) was turned on, and clip C was removed . Proceeding toward light L the insects encountered the two air currents, chose either the baited or empty arm of the Y tube, and were subsequently trapped in the respective goal tubes (G) . They were transferred to a transparent tube for counting and then discarded . Prior to testing a batch of flies, the parts of the apparatus likely to be contaminated with the peppermint scent were always washed, and new silk caps were placed over the ends of the bait tubes . Results Table I lists and describes a total of twelve experimental groups, each treated differently prior to testing. Table I also shows the number of insects in each group and the percentage of each group selecting the baited arm of the olfactometer. Those percentages significantly different from the percentage of the control group (Group 1) are followed by an asterisk (P<0 .05, two-tailed, large-sample test of a difference between population proportions . Walker &
*This research was aided by United States Public Health Service Grant M-1963 . The authors are indebted to W. 0 . Evans and Elene Maginnes for earlier suggestions and work on this problem . tAlthough peppermint vapour may have irritant as well as olfactory properties, whether the effective conditioned stimulus is truly olfactory or not is an empirical question distinct from and incidental to the purpose of the present design . 259
ANIMAL BEHAVIOUR, 15, 2-3
260
C4
glau A
ilk filter bait
SIDE VIEW
W
Fig . 1 . Olfactometer
Table I. Twelve Experimental Groups Listed in Terms of the Cultures from which They Came, and the Intervening Conditions which They Experienced Prior to Testing
Intervening experience
Number of flies
Per cent choosing baited arm of olfactometer
Group
Culturet
1
Non-scented
None (control group)
2
Peppermint
None
66
53%*
3
Peppermint
1 day non-scented medium
46
46
4
Peppermint
2 days non-scented medium
76
33
5
Peppermint
4 days non-scented medium
64
34
6
Peppermint
6 days non-scented medium
64
25
7
Peppermint
Puparium washed and transferred to non-scented medium ; tested within 18 hr of emergence
104
44%*
8
Peppermint
Puparium washed and transferred to a bottle containing moist cotton ; tested within 18 hr of emergence
102
50%*
9
Non-scented
1 day empty scented bottle
102
21
10
Peppermint
1 day empty scented bottle
60
23
11
Non-scented
1 day empty non-scented bottle
94
42%*
12
Peppermint
1 day empty non-scented bottle
80
38%*
216
26 % (Control)
f Except for groups 7 and 8 all insects were selected from their respective cultures as imagoes .
'Significantly different (P<0 .05, two-tailed) from the percentage of control group .
HERSHBERGER & SMITH : CONDITIONING IN DROSOPHILA MELANOGASTER
Lev's (1953) formula 3 . 13 was used to compute the standardized normal deviate, Z .) The control group, Group 1, was reared exelusively on the non-scented medium and only 26 per cent of these insects chose the baited arm_ Groups 2 to 6. were reared to the imago stage on the peppermint-scented medium and then tested after zero, one, two, four, or six days interpolated experience with the normal medium respectively. These five groups . demonstrated conditioning and its reversal.' Group 2, reared exclusively on the scented medium, selected the baited arm twice as often as the controls : 53 per cent. The other peppermint-reared groups with progressively protracted experience with the normal medium found the peppermint scent progressively less acceptable, Group 6 being virtually identical to control Group 1 . The insects of Groups 7 and 8 were reared on the scented medium as larvae, but during the pupal stage the puparia were scrubbed in distilled water with a camel-hair brush and transferred to a bottle containing either the nonscented medium (Group 7) or moist cotton (Group 8) . They were tested within 18 hr of emergence . These groups, significantly different from the controls, had virtually no experience with the scented medium as adults, and, therefore, are said to evince pre-imaginal olfactory conditioning in their test performance . Groups 9 and 11, reared on the non-scented medium, and Groups 10 and 12, reared on the peppermint medium, were confined as imagoes for 24 hr in foodless containers just prior to testing . Groups 9 and 10 were confined to scented bottles . Groups 11 and. 12 were confined to non-scented bottles . The test performance of these four groups indicates that olfactory conditioning in Drosophila melanogaster is a form of learning rather than habituation or the like. Specifically, Groups 9 and 10, from nonscented and peppermint-reared cultures respectively, selected the baited arm of the olfactometer no more than the controls, even though both groups had been exposed to the peppermint scent for 24 hr just prior to testing . Conversely, Groups 11 and 12, from nonscented and peppermint-reared cultures respectively, selected the baited arm of the olfactometer significantly more than the controls even though neither group had been exposed to the peppermint scent during the 24 hr period immediately prior to testing. These results show that the conditioned acceptability of an olfactory
261
stimulus in Drosophila melanogaster depends upon the association of that conditioned stimulus with a reinforcing stimulus, in the present case food . Presenting the conditioned olfactory stimulus in the absence of food results in ex tinction . Apparently, such is the case in both Groups 10 and 11 . In Group 10 the conditioned acceptability of the peppermint scent in which the insects were reared is extinguished when the scent is presented in the absence of food . In Group 11 the conditioned acceptability of the neutral odour in which the insects were reared is extinguished when that neutral odour, or one similar to it, is presented in the absence of food, resulting in a subsequent increased selection of the baited arm of the olfactometer . Transferring peppermint-reared imagoes to the non-scented medium reduces the number of flies selecting the baited arm of the olfactometer, but this procedure resembles counterconditioning rather than simple extinction, as was implied by Thorpe . Discussion In general, the present data corroborate Thorpe's results without exception . They, nevertheless, clearly contradict his interpretation of those results . Olfactory conditioning in Drosophila melanogaster is not mere habituation, but appears to be a form of learning susceptible to both experimental extinction and counterconditioning . Such a learning interpretation also is in keeping with recent findings reported by Murphey & Katahn (unpublished) to the effect that Drosophila appear capable of learning operant responses 'reinforced by negative geotaxis . Drosophila melanogaster would appear to be an ideal subject for investigating the genetic correlates of learning . For example, breeding studies of the type employed by Hirsch & Boudreau (1958) might be used to determine the heritability of olfactory conditioning . On the one hand, there are available the techniques of describing and manipulating the genetic structure of Drosophila, and on the other hand, there are what appear to be fairly reliable measures of conditioning . Summary Imagoes reared on a peppermint-scented food medium subsequently selected the peppermintscented arm of an insect olfactometer more often than did controls reared on a non-scented medium . Further, confining peppermint-reared imagoes for 24 hr to peppermint-scented but
26 2
ANIMAL BEHAVIOUR, 15, 2-3
foodless containers extinguished the conditioned acceptability of that scent . REFERENCES Hirsch, J . & Boudreau, J. C. (1958) . Studies in experimental behavior genetics : I . The heritability of phototaxis in a population of Drosophila melanogaster . J. comp. physiol. Psychol., 51, 647-651 . Thorpe, W. H . (1939). Further studies on pre-imaginal olfactory conditioning in insects . Proc. Roy . Soc. B ., 127,424-433 .
Thorpe, W . H. (1943a) . Types of learning in insects and other arthropods, Part I. Brit. J. Psychol., 33, 220-234. Thorpe, W. H. (1943b) . Types of learning in insects and other arthropods, Part II . Brit . J. Psychol., 34, 20-31 . Thorpe, W. H. (1943c) . Types of learning in insects and other arthropods, Part III. Brit . J. Psychol., 34, 66-76. Walker, Helen M . & Lev, J. (1953) . Statistical Inference . New York : Holt . (Received 1 May 1964 ; revised 4 November 1966 ; Ms . number : A260)
CONDITIONING IN DROSOPHILA MELANOGASTER* BY
WAYNE A. HERSHBERGER & MAURICE P. SMITH
North. Ill. Univ ., Dept. Psychol., DeKalb, Ill. & Univ . Colo ., Dept. Psychol., Boulder, Colo .
Thorpe (1939) has found that Drosophila melanogaster reared on a food medium containing 0 .5 per cent peppermint essence show as imagoes a greater acceptance of this odour than do imagoes reared on a non-scented medium . He found this to be the case even when during the pupal stage the peppermint-reared insects were washed and transferred to a nonscented medium where they emerged and were tested as imagoes . He interpreted this process, which he termed olfactory conditioning, as involving some form of habituation (1943, a, b, c) . However, it may also be interpreted as involving a form of learning . A habituation interpretation supposes that the exposure to the scent is sufficient to condition an insect . A learning interpretation supposes further that conditioning depends upon the association of the scent with certain reinforcing stimuli, e.g., food . It is possible to test these two interpretations by confining insects to foodless chambers permeated with the scent of peppermint . If this confinement increases the acceptability of the peppermint scent, the habituation interpretation is substantiated . On the other hand, if it decreases, or at least fails to increase, the acceptability of the peppermint scent, olfactory conditioning in Drosophila melanogaster may be considered a form of learning. Such was the rationale of the present design .t Method A total of 1,074 insects served as subjects . A wild Oregon-Red-Eye strain of Drosophila melanogaster was used, with both sexes used indiscriminately . All subjects were reared in half-pint glass bottles containing a pre-cooked, artificial food medium composed of 300 ml water, 40 g yellow cornmeal, 3 g Agar-Agar, 55 ml Karo, and 2 ml Turtox mold inhibitor . The peppermint-scented medium included 0 .5
per cent, by volume, peppermint-essence (McKesson & Robbins) . The subjects' parents were removed from their respective bottles when the subjects began to pupate. The peppermintreared and normal-reared imagoes did not appear to differ in health and vigour . The olfactometer in which the scent preferences were tested is shown diagrammatically in Fig. 1 . The instrument was fixed in a horizontal plane . To bait the olfactometer, two drops of 5 per cent solution of peppermint essence in absolute alcohol were allowed to evaporate on 2 cm2 of filter paper which was then placed in one of the bait tubes (B), the other bait tube being left empty. One side was baited for half of the insects in each experimental group, and the other side for the other half. Currents of air were drawn through the two arms of the Y tube by a faucet aspirator attached to tube A, the flow being equalized by the adjustment of water level in the wash bottles (W) attached to the two inlet tubes (I) . A batch of ten to fifty insects to be tested was lured phototropically into the starting tube (S), which was then connected to the test apparatus . The room was darkened, light L ( 7 watt) was turned on, and clip C was removed . Proceeding toward light L the insects encountered the two air currents, chose either the baited or empty arm of the Y tube, and were subsequently trapped in the respective goal tubes (G) . They were transferred to a transparent tube for counting and then discarded . Prior to testing a batch of flies, the parts of the apparatus likely to be contaminated with the peppermint scent were always washed, and new silk caps were placed over the ends of the bait tubes . Results Table I lists and describes a total of twelve experimental groups, each treated differently prior to testing. Table I also shows the number of insects in each group and the percentage of each group selecting the baited arm of the olfactometer. Those percentages significantly different from the percentage of the control group (Group 1) are followed by an asterisk (P<0 .05, two-tailed, large-sample test of a difference between population proportions . Walker &
*This research was aided by United States Public Health Service Grant M-1963 . The authors are indebted to W. 0 . Evans and Elene Maginnes for earlier suggestions and work on this problem . tAlthough peppermint vapour may have irritant as well as olfactory properties, whether the effective conditioned stimulus is truly olfactory or not is an empirical question distinct from and incidental to the purpose of the present design . 259
ANIMAL BEHAVIOUR, 15, 2-3
260
C4
glau A
ilk filter bait
SIDE VIEW
W
Fig . 1 . Olfactometer
Table I. Twelve Experimental Groups Listed in Terms of the Cultures from which They Came, and the Intervening Conditions which They Experienced Prior to Testing
Intervening experience
Number of flies
Per cent choosing baited arm of olfactometer
Group
Culturet
1
Non-scented
None (control group)
2
Peppermint
None
66
53%*
3
Peppermint
1 day non-scented medium
46
46
4
Peppermint
2 days non-scented medium
76
33
5
Peppermint
4 days non-scented medium
64
34
6
Peppermint
6 days non-scented medium
64
25
7
Peppermint
Puparium washed and transferred to non-scented medium ; tested within 18 hr of emergence
104
44%*
8
Peppermint
Puparium washed and transferred to a bottle containing moist cotton ; tested within 18 hr of emergence
102
50%*
9
Non-scented
1 day empty scented bottle
102
21
10
Peppermint
1 day empty scented bottle
60
23
11
Non-scented
1 day empty non-scented bottle
94
42%*
12
Peppermint
1 day empty non-scented bottle
80
38%*
216
26 % (Control)
f Except for groups 7 and 8 all insects were selected from their respective cultures as imagoes .
'Significantly different (P<0 .05, two-tailed) from the percentage of control group .
HERSHBERGER & SMITH : CONDITIONING IN DROSOPHILA MELANOGASTER
Lev's (1953) formula 3 . 13 was used to compute the standardized normal deviate, Z .) The control group, Group 1, was reared exelusively on the non-scented medium and only 26 per cent of these insects chose the baited arm_ Groups 2 to 6. were reared to the imago stage on the peppermint-scented medium and then tested after zero, one, two, four, or six days interpolated experience with the normal medium respectively. These five groups . demonstrated conditioning and its reversal.' Group 2, reared exclusively on the scented medium, selected the baited arm twice as often as the controls : 53 per cent. The other peppermint-reared groups with progressively protracted experience with the normal medium found the peppermint scent progressively less acceptable, Group 6 being virtually identical to control Group 1 . The insects of Groups 7 and 8 were reared on the scented medium as larvae, but during the pupal stage the puparia were scrubbed in distilled water with a camel-hair brush and transferred to a bottle containing either the nonscented medium (Group 7) or moist cotton (Group 8) . They were tested within 18 hr of emergence . These groups, significantly different from the controls, had virtually no experience with the scented medium as adults, and, therefore, are said to evince pre-imaginal olfactory conditioning in their test performance . Groups 9 and 11, reared on the non-scented medium, and Groups 10 and 12, reared on the peppermint medium, were confined as imagoes for 24 hr in foodless containers just prior to testing . Groups 9 and 10 were confined to scented bottles . Groups 11 and. 12 were confined to non-scented bottles . The test performance of these four groups indicates that olfactory conditioning in Drosophila melanogaster is a form of learning rather than habituation or the like. Specifically, Groups 9 and 10, from nonscented and peppermint-reared cultures respectively, selected the baited arm of the olfactometer no more than the controls, even though both groups had been exposed to the peppermint scent for 24 hr just prior to testing . Conversely, Groups 11 and 12, from nonscented and peppermint-reared cultures respectively, selected the baited arm of the olfactometer significantly more than the controls even though neither group had been exposed to the peppermint scent during the 24 hr period immediately prior to testing. These results show that the conditioned acceptability of an olfactory
261
stimulus in Drosophila melanogaster depends upon the association of that conditioned stimulus with a reinforcing stimulus, in the present case food . Presenting the conditioned olfactory stimulus in the absence of food results in ex tinction . Apparently, such is the case in both Groups 10 and 11 . In Group 10 the conditioned acceptability of the peppermint scent in which the insects were reared is extinguished when the scent is presented in the absence of food . In Group 11 the conditioned acceptability of the neutral odour in which the insects were reared is extinguished when that neutral odour, or one similar to it, is presented in the absence of food, resulting in a subsequent increased selection of the baited arm of the olfactometer . Transferring peppermint-reared imagoes to the non-scented medium reduces the number of flies selecting the baited arm of the olfactometer, but this procedure resembles counterconditioning rather than simple extinction, as was implied by Thorpe . Discussion In general, the present data corroborate Thorpe's results without exception . They, nevertheless, clearly contradict his interpretation of those results . Olfactory conditioning in Drosophila melanogaster is not mere habituation, but appears to be a form of learning susceptible to both experimental extinction and counterconditioning . Such a learning interpretation also is in keeping with recent findings reported by Murphey & Katahn (unpublished) to the effect that Drosophila appear capable of learning operant responses 'reinforced by negative geotaxis . Drosophila melanogaster would appear to be an ideal subject for investigating the genetic correlates of learning . For example, breeding studies of the type employed by Hirsch & Boudreau (1958) might be used to determine the heritability of olfactory conditioning . On the one hand, there are available the techniques of describing and manipulating the genetic structure of Drosophila, and on the other hand, there are what appear to be fairly reliable measures of conditioning . Summary Imagoes reared on a peppermint-scented food medium subsequently selected the peppermintscented arm of an insect olfactometer more often than did controls reared on a non-scented medium . Further, confining peppermint-reared imagoes for 24 hr to peppermint-scented but
26 2
ANIMAL BEHAVIOUR, 15, 2-3
foodless containers extinguished the conditioned acceptability of that scent . REFERENCES Hirsch, J . & Boudreau, J. C. (1958) . Studies in experimental behavior genetics : I . The heritability of phototaxis in a population of Drosophila melanogaster . J. comp. physiol. Psychol., 51, 647-651 . Thorpe, W. H . (1939). Further studies on pre-imaginal olfactory conditioning in insects . Proc. Roy . Soc. B ., 127,424-433 .
Thorpe, W . H. (1943a) . Types of learning in insects and other arthropods, Part I. Brit. J. Psychol., 33, 220-234. Thorpe, W. H. (1943b) . Types of learning in insects and other arthropods, Part II . Brit . J. Psychol., 34, 20-31 . Thorpe, W. H. (1943c) . Types of learning in insects and other arthropods, Part III. Brit . J. Psychol., 34, 66-76. Walker, Helen M . & Lev, J. (1953) . Statistical Inference . New York : Holt . (Received 1 May 1964 ; revised 4 November 1966 ; Ms . number : A260)