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Species differences in the diurnal rhythmicity of courtship behaviour within the Melanogaster group of the genus Drosophila
.Anim. Behav., 1972,20, 170-174 SPECIES
DIFFERENCES
COURTSHIP
BEHAVIOUR
IN
THE
WITHIN
DIURNAL
RHYTHMICITY
THE MELANOGASTER
OF
GROUP
OF THE GENUS DROSOPHILA BY RI~DIGER HARDELAND L Zoologisches Institut, Universitiit Grttingen Abstract. Courtship activity of Drosophila males varies within the diurnal cycle. Sixteen species belonging to the melanogaster group were compared with respect to the diurnal rhythms of courting frequency. Several types of diurnal patterns, differing from each other in the phase of the rhythm or in the number of maxima, could be observed. Three species, D. melanogaster, D. yakuba, and D. kikkawai, showed maximal courtship activity before light-on. In the case of D. kikkawai, the use of flies which had lived isolated from the time of emergence led to marked alterations in the diurnal rhythmic pattern of courting frequency. A possible isolating mechanism based on temporal fixation of courtship be= haviour is discussed for the species of the melanogaster group. In the past, some attention has been paid to the influence of light and darkness on mating behaviour in Drosophila (Wallace & Dobzhansky 1947; Rendel 1951; Jacobs 1960, 1961; Spieth 1966; Grossfield 1966, 1970; Petit & Ehrman 1969). However, the possibility of a diurnal rhythm of courting frequency has not been considered in these papers. In some cases, this may have brought about misleading conclusions. Species differences in diurnal rhythmicity between members of the genus Drosophila have already been reported for locomotor activity (Pavan, Dobzhansky & Burla 1950; Lewis & Taylor 1964; Stange & Hardeland 1970), and puparium formation (Rensing & Hardeland 1967). If at all, these differences were mostly discussed under the ecological aspect of adaptation to arid climates. However, differences in temporal organization of related species could have some isolating function as well. Hence, a comparison between the diurnal courting rhythms of some closely related species might be of interest.
D. pulchrella: Kirishima, Japan; D. suzukii: Kirishima, Japan. Tests for frequency of courtship behaviour were carried out using 5- to 8-day-old males and females, which had lived together in one culture bottle, Only in one experiment were males and females of D. kikkawai kept individually in tubes with one fly in each. Experimental groups of flies, consisting of six males and six females, were placed in Petri dishes (10 cm in diameter) and supplied with sufficient food to be kept for several days under these conditions. Ageing of moist, uninfected food produced no detectable effect on the behaviour examined. Tests were performed on the flies' second or third day in the Petri dishes. The animals were subjected to a constant temperature of 25~ and to an artificial light-dark cycle of 12:12 hours. Light intensity was 0.3 lx produced by a dim red lamp during the dark-time and 60 lx throughout the light period. In one experiment, natural lighting conditions were used; however, it was necessary to maintain the dim red illumination during the dark period. Results obtained from flies which had been reared under experimental lighting and temperature conditions did not noticeably differ from those with flies which were adapted for several days to these conditions (data not shown). Courtship behaviour was recorded by observation during 10-min periods. The response of one male to one female was defined as a courtship event, irrespective of the reaction of the female. Circling around a female and following a decamping female were counted equally. If a male turned from one female to another one,
Methods
Drosophila stocks from the following localities were used: D. melanogaster: Princeton, New Jersey, U.S.A.; D. simulans: Lima, Peru; D. yakuba: Ivory Coast; D. ananassae: Brazil; D. bipectinata: Nepal; D. lutea: Sugadaira, Japan; D. takahashii: Nepal; D. pseudotakahashii: Australia; D. auraria: Hong Chow, China; D. nikananu: Ivory Coast; D. serrata: Heun Island, Australia; D. birchii: New Guinea; D. kikkawai: Malaya; D. szentivanii: Fiji; 170
HARDELAND: DIURNAL RHYTHMICITY OF DROSOPHILA COURTSHIP this was recorded as a second courtship event. Under the conditions described, courtship behaviour leads relatively seldom to copulation; thus, the high rates of courtship events observed within 10 rain can be understood. Each experimental group of flies was observed over a continuous 24-hr period. In order to ensure that the rhythmicity pattern recorded was typical for the experimental group tested, observations were often made over 48 hr or even more; however, these additional data were not evaluated. Means and three-fold standard errors were calculated from the data of five to seven experimental groups each. Results All species examined showed a diurnal rhythm of courtship behaviour (Fig. 1). This fact alone, however, could be trivial, since circadian variations in locomotor activity would produce rhythms of courtship frequency anyway. Therefore, it seems necessary to find out whether or not the rhythm of courtship activity is independent of the rate of locomotor activity. (Independency of the rate of locomotor activity does not necessarily imply independency of the locomotor activity rhythm. This would be rather a question of the circadian organization of the animal.) Although courtship activity appears to be controlled by general locomotor activity in the case of D. funebris (Hardeland 1971), courtship activity does not parallel locomotor activity in D. melanogaster; in the latter species, courtship activity is at its maximum when non-courting flies are normally not active (Hardeland & Stange 1971). Hence, in D. melanogaster courtship activity cannot depend on the rate of locomotor activity. In the reverse, the occurrence of courtship activity can alter the diurnal pattern of locomotion, as is also the case in D. melanogaster (Ohsawa et al. 1952; Hardeland & Stange 1971); but this aspect is not to be considered in this paper. Taking into account a clearly bimodal pattern of locomotor activity, with peaks at the beginning and at the end of the light period, which is typical for all species examined here except D. ananassae and D. bipectinata (Hardeland & Stange, in preparation), it can be seen from Fig. 1 th~/t the diurnal courting rhythm is independent of locomotor activity in several species. This might be true at least for D. melanogaster, D. yakuba, D. takahashii, D. pseudotakahashii, D. nikananu, and D. kikkawai. I n all these cases, maximum courtship activity is either associated with o n l y
171
one peak of locomotor activity, or does not coinside with one of these at all. With respect to the other species, especially D. ananassae, D. auraria, D. serrata, and D. pulchrella, one could argue that courting frequency might be correlated with general activity of the animals. It is worth noting that in all those species examined which do not court during the dark period of the diurnal cycle greatest courtship activity is observed in the morning hours (Fig. 1). This seems to be a peculiarity of the melanogaster group. In other species groups, in particular those belonging to the subgenus Drosophila, maximal courtship activity during the evening hours is not uncommon (unpublished data). Although it is known that mating behaviour of several Drosophila species is not affected by darkness (Petit & Ehrman 1969), it was somewhat surprising to me to find maximum courtship activity during the night hours in three cases: in D. melanogaster, D. yakuba, and D. kikkawai. D. melanogaster and D. yakuba are Very closely related; both are sibling species of another form, D. simulans. Among these three species, D. simulans courts almost exclusively during the light period of the diurnal cycle, D. melanogaster courts most frequently in the dark, while D. yakuba behaves somewhat intermediarily. As the observation for D. melanogaster seemed to be in contrast with earlier findings (Rendel 1951; Jacobs 1960), which suggested higher courtship activity during the light period, I performed one expenment under natural daylight conditions in order to exclude t h e possibility that my results were induced by the block Zeitgeber. This experiment revealed nocturnal courtship activity in this species (Fig. 2). D. auraria, the courting of which is blocked by darkness and which I had tested for control, showed the same phase relationship with respect to D. melanogaster as under artificial illumination. The diurnal courting pattern of the third species which showed significant mating activity in the night, D. kikkawai, differs markedly from those of all its nearest relatives, the members of the 'montium' subgroup, which have been tested. First, there is a maximum during the night, which coincides with that shown by D. melanogaster. Moreover, these flies exhibit a second peak during the day-time, though it is very variable and cannot be dearly observed in every run. Furthermore, D. kikkawai is characterized by much higher mating activity than the other related species.
172
ANIMAL
a
loo-4
9
9
I
BEHAVIOUR.,
Ib
lc
I
I
I
I
2 0 , :1
Id
9
I
BII
I--lOO
I
I
"6
3
9
15 21
3
9
15 21
3
9
15 21
3
9
15 21
I
Time of day Fig. 1. Diurnal rhythms of courtship activity in sixteen species of the melanogaster group. (a) D. melanogaster; (b) D. simulans; (c) D. yakuba; (d) D. ananassae; (e) D. bipectinata; (f) D. lutea; (g) D. takahashii; (h) D. pseudotakahashii; (i) D. auraria; (k) D. nikananu; (1) D. serrata; (m) D. birehii; (n) D. kikkawai; (o) D. szentivanii; (p) D. pulchrella; (q) D. suzukiL The number of courtship events Was recorded in an experimental population of six males and six females within 10 min. The horizontal black and white bars represent the light--dark cycle of 12.12 hours. 9 . 0 0 and 21 . 00 recordings were carried out during the light period (because of this, the black bars are drawn only from the 23.00 to the 7 . 0 recordings).
HARDELAND: DIURNAL RHYTHMICITY OF DROSOPHILA COURTSHIP I000
173
o
I00 IO
I 0. I
b 60
c
*,> 9
4=
2C
I
15
21
Time of
5
doy
I
9
15
I
Fig. 3. Courtship activity in the course of a light--dark cycle in specimens of D. kikkawai which had been kept isolated from the time of emergence. Males and females were kept singly, until they were put together on the 6th day of life; recordings were made from the 7th to the 8th day. Shaded columns: isolated animals; open columns: control animals. Black and white bar as in Fig. 1.
d z
4C
24
4
8 Time
12 of
16
20
doy
Fig. 2. Diurnal rhythms of courtship activity in D.
melanogaster and D. auraria under natural lighting conditions. (a) light intensity (ordinate: lux); (b) D. melanogaster, (c) D. auraria.
Courtship activity is greatly enhanced in specimens which have lived singly from the time of emergence. Flies subjected to this treatment showed strong alterations in the diurnal pattern of courtship frequency: they were much more active during the light than during the dark period (Fig. 3). Nonetheless, these animals were as active as the controls in the dark period when the latter were showing maximal courtship activity. In the light, however, the flies which had lived isolated courted far more frequently than observed in any other experiment; they were 'permanently' courting over several days. Discussion
As demonstrated by these experiments, Drosophila species exhibit strong diurnal variations in courting activity. On the other hand, court-
ship activity can be also controlled by nonrhythmic factors due to the pre-treatment of the animals. Hence, if experiments are performed concerning light dependency of courtship behaviour, the result largely depends on the time of day at which they are carried out, and on the material used, in particular whether or not the flies have been allowed to mate prior to the experiment. Such methodical differences m u s t be the reason for the findings of other workers (Rendel 1951; Jacobs 1960) that D. melanogaster preferably courts in the light, whereas I would like to call it a species which preferably courts in the dark. Of course one might obtain results different from those reported here, if one compared animals from constant light and constant dark conditions (which do not behave rhythmically!) or if animals which had lived under Zeitgeber conditions were carried from light to dark. Ill this context, it may be noted that courtship behaviour of D. melanogaster, which is shown during the second part of the night, can even be suppressed by light of intensities not higher than 60 lx (Hardeland, unpublished data). Thus, light or dark preferences with respect to courtship behaviour may depend on the time of day. Moreover, I think that my results on courtship activity in the dark period are supported by observations of Ohsawa et al.
174
ANIMAL
BEHAVIOUR,
(1952), who reported that nocturnal locomotor activity of D. melanogaster is greatly enhanced in mixed groups of males and females, compared with males and females alone. Nocturnal locomotor activity, however, is obviously related to nocturnal courtship activity in this species (cf. Hardeland & Stange 1971). The experimental conditions used for comparison of the sixteen species avoid some extremely::nnnatural situations such as great population densities and use of flies which have not been allowed to mate for a long time. Hence, one could speculate about the possible ecological value of differences in the diurnal pattern of courtship activity between related species. In the case of the three sibling species D. melanogaster, D. simulans and D. yakuba, the latter is geographically restricted to the African region, whereas both D. melanogaster and D. simulans are cosmopolitan species (cf. Patterson & Stone 1952). One could imagine that temporal fixation of courtship behaviour to different phases of the diurnal cycle may function as an isolating mechanism between these two cosmopolitans. O f course, this would not be the only isolating barrier, since other mechanisms, e.g. concerning the courtship song (Bennet-Clark & Ewing 1969), have already been worked out. Another isolating barrier due to the same mechanism could possibly exist between D. kikkawai and the other members of the 'montium' subgroup. Although not cosmopolitan, D. kikkawai is widely distributed, whereas the other members of the subgroup are restricted to smaller geographical regions. In this case, the maxima of the geographically isolated species coincide; in contrast, the only species with a wide geographical range, which cannot be isolated from its relatives simply by distribution, courts preferably at a time when the other flies usually do not show this behaviour. Even the second, variable maximum of the light period coincides with the minima of the other species. Whether or not the isolating mechanism supposed here is of ecological significance, cannot be decided from these experiments alone. In particular, the question should be answered whether the females' reactions to courting males vary within the diurnal cycle, and, if this were true, whether there are species differences. It should be t a k e n into consideration that diurnal rhythms in receptivity could be of great isolating effectiveness, especially if they would parallel the males' courting rhythms.
20,
1
Acknowledgment I wish to thank Mis M. A. Reveley, Austin, Texas for generous gifts of Drosophila stocks.
REFERENCES Bennet-Clark, H. C. & Ewing, A. W. (1969). Pulse interval as a critical parameter in the courtship song of Drosophila melanogaster. Anita. Behav.,. 17, 755-759. Grossfield, J. (1966). The influence of light on the mating behavior in Drosophila. Univ. of Texas Pubt., 6615, 147-176. Grossfield, J. (1970). Species differences in light-influenced mating behavior in Drosophila. Am. Natur., 104, 307-309. Hardeland, R. (1971). Lighting conditions and mating behavior in Drosophila. Am. Natur., 105, 198-200. Hardeland, R. & Stange, G. (1971). Einfltisse yon Geschlecht und Alter auf die lokomotorische Aktivit~tt yon Drosophila. J. lnsect. PhysioL, 17, 427-434. Jacobs, M. E. (1960). Influence of light on mating of Drosophila melanogaster, Ecology, 41, 182-188. Jacobs, M. E. (1961). The influence of light on gene frequency changes in laboratory populations of ebony and non-ebony Drosophila melanogaster. Genetics, 46, 1089-1095. Lewis, T. & Taylor, L. R. (1964). Diurnal periodicity of flight by insects. Trans. Roy. ent. Soc. Lond., 116, 393--469. Ohsawa, W., Matutani, K., Tukuda, H., Mori, S., Miyadi, D., Yanagisima, S. & Sato, Y. (1952). Sexual properties of the daily rhythmic activity in Drosophila melanogaster. Seiro-Seitai, 5, 26-30. Patterson, J. T. & Stone, W. S. (1952). Evolution in the Genus Drosophila. New York: Macmillan. Pavan, C., Dobzhansky, Th. & Burla, H. (1950). Diurnal behavior of some neotropical speciesof Drosophila. Ecology, 31, 36--43. Petit, C . & Ehrman, L. (1969). Sexual selection in Drosophila. Evolut. BioL, 2, 177-233. Rendel, J. M. (1951). Mating of ebony, vestigial, and wildtype Drosophila melanogaster in light and dark. Evolution, 5, 226-230. Rensing, L. & Hardeland, R. (1967). Zur Wirkung der circadianen Rhythmik auf die Entwicklung yon Drosophila. J. Insect PhysioL, 13, 1547-1568. Spieth, H. T. (1966). Drosophilid mating behavior: the behavior of decapitated females. Anim. Behav., 14, 226-235. Stange, G. & Hardeland, R. (1970). Eine Methode zur Registrierung der Laufaktivitat von kleinen Insecten. Oecologia, 5, 400--405. Wallace, B.& Dobzhansky, Th. (1947). Experiments on sexual isolation in Drosophila. VIII. Influence of light on the mating behavior of Drosophila subobscura, Drosophila persimilis and Drosophila pseudoobscura. Proc. nat. Acad. Sci., Wash., 32, 226-234. (Received 21 April 1971; revised 6 August 1971; MS. number: 1054)
DIFFERENCES
COURTSHIP
BEHAVIOUR
IN
THE
WITHIN
DIURNAL
RHYTHMICITY
THE MELANOGASTER
OF
GROUP
OF THE GENUS DROSOPHILA BY RI~DIGER HARDELAND L Zoologisches Institut, Universitiit Grttingen Abstract. Courtship activity of Drosophila males varies within the diurnal cycle. Sixteen species belonging to the melanogaster group were compared with respect to the diurnal rhythms of courting frequency. Several types of diurnal patterns, differing from each other in the phase of the rhythm or in the number of maxima, could be observed. Three species, D. melanogaster, D. yakuba, and D. kikkawai, showed maximal courtship activity before light-on. In the case of D. kikkawai, the use of flies which had lived isolated from the time of emergence led to marked alterations in the diurnal rhythmic pattern of courting frequency. A possible isolating mechanism based on temporal fixation of courtship be= haviour is discussed for the species of the melanogaster group. In the past, some attention has been paid to the influence of light and darkness on mating behaviour in Drosophila (Wallace & Dobzhansky 1947; Rendel 1951; Jacobs 1960, 1961; Spieth 1966; Grossfield 1966, 1970; Petit & Ehrman 1969). However, the possibility of a diurnal rhythm of courting frequency has not been considered in these papers. In some cases, this may have brought about misleading conclusions. Species differences in diurnal rhythmicity between members of the genus Drosophila have already been reported for locomotor activity (Pavan, Dobzhansky & Burla 1950; Lewis & Taylor 1964; Stange & Hardeland 1970), and puparium formation (Rensing & Hardeland 1967). If at all, these differences were mostly discussed under the ecological aspect of adaptation to arid climates. However, differences in temporal organization of related species could have some isolating function as well. Hence, a comparison between the diurnal courting rhythms of some closely related species might be of interest.
D. pulchrella: Kirishima, Japan; D. suzukii: Kirishima, Japan. Tests for frequency of courtship behaviour were carried out using 5- to 8-day-old males and females, which had lived together in one culture bottle, Only in one experiment were males and females of D. kikkawai kept individually in tubes with one fly in each. Experimental groups of flies, consisting of six males and six females, were placed in Petri dishes (10 cm in diameter) and supplied with sufficient food to be kept for several days under these conditions. Ageing of moist, uninfected food produced no detectable effect on the behaviour examined. Tests were performed on the flies' second or third day in the Petri dishes. The animals were subjected to a constant temperature of 25~ and to an artificial light-dark cycle of 12:12 hours. Light intensity was 0.3 lx produced by a dim red lamp during the dark-time and 60 lx throughout the light period. In one experiment, natural lighting conditions were used; however, it was necessary to maintain the dim red illumination during the dark period. Results obtained from flies which had been reared under experimental lighting and temperature conditions did not noticeably differ from those with flies which were adapted for several days to these conditions (data not shown). Courtship behaviour was recorded by observation during 10-min periods. The response of one male to one female was defined as a courtship event, irrespective of the reaction of the female. Circling around a female and following a decamping female were counted equally. If a male turned from one female to another one,
Methods
Drosophila stocks from the following localities were used: D. melanogaster: Princeton, New Jersey, U.S.A.; D. simulans: Lima, Peru; D. yakuba: Ivory Coast; D. ananassae: Brazil; D. bipectinata: Nepal; D. lutea: Sugadaira, Japan; D. takahashii: Nepal; D. pseudotakahashii: Australia; D. auraria: Hong Chow, China; D. nikananu: Ivory Coast; D. serrata: Heun Island, Australia; D. birchii: New Guinea; D. kikkawai: Malaya; D. szentivanii: Fiji; 170
HARDELAND: DIURNAL RHYTHMICITY OF DROSOPHILA COURTSHIP this was recorded as a second courtship event. Under the conditions described, courtship behaviour leads relatively seldom to copulation; thus, the high rates of courtship events observed within 10 rain can be understood. Each experimental group of flies was observed over a continuous 24-hr period. In order to ensure that the rhythmicity pattern recorded was typical for the experimental group tested, observations were often made over 48 hr or even more; however, these additional data were not evaluated. Means and three-fold standard errors were calculated from the data of five to seven experimental groups each. Results All species examined showed a diurnal rhythm of courtship behaviour (Fig. 1). This fact alone, however, could be trivial, since circadian variations in locomotor activity would produce rhythms of courtship frequency anyway. Therefore, it seems necessary to find out whether or not the rhythm of courtship activity is independent of the rate of locomotor activity. (Independency of the rate of locomotor activity does not necessarily imply independency of the locomotor activity rhythm. This would be rather a question of the circadian organization of the animal.) Although courtship activity appears to be controlled by general locomotor activity in the case of D. funebris (Hardeland 1971), courtship activity does not parallel locomotor activity in D. melanogaster; in the latter species, courtship activity is at its maximum when non-courting flies are normally not active (Hardeland & Stange 1971). Hence, in D. melanogaster courtship activity cannot depend on the rate of locomotor activity. In the reverse, the occurrence of courtship activity can alter the diurnal pattern of locomotion, as is also the case in D. melanogaster (Ohsawa et al. 1952; Hardeland & Stange 1971); but this aspect is not to be considered in this paper. Taking into account a clearly bimodal pattern of locomotor activity, with peaks at the beginning and at the end of the light period, which is typical for all species examined here except D. ananassae and D. bipectinata (Hardeland & Stange, in preparation), it can be seen from Fig. 1 th~/t the diurnal courting rhythm is independent of locomotor activity in several species. This might be true at least for D. melanogaster, D. yakuba, D. takahashii, D. pseudotakahashii, D. nikananu, and D. kikkawai. I n all these cases, maximum courtship activity is either associated with o n l y
171
one peak of locomotor activity, or does not coinside with one of these at all. With respect to the other species, especially D. ananassae, D. auraria, D. serrata, and D. pulchrella, one could argue that courting frequency might be correlated with general activity of the animals. It is worth noting that in all those species examined which do not court during the dark period of the diurnal cycle greatest courtship activity is observed in the morning hours (Fig. 1). This seems to be a peculiarity of the melanogaster group. In other species groups, in particular those belonging to the subgenus Drosophila, maximal courtship activity during the evening hours is not uncommon (unpublished data). Although it is known that mating behaviour of several Drosophila species is not affected by darkness (Petit & Ehrman 1969), it was somewhat surprising to me to find maximum courtship activity during the night hours in three cases: in D. melanogaster, D. yakuba, and D. kikkawai. D. melanogaster and D. yakuba are Very closely related; both are sibling species of another form, D. simulans. Among these three species, D. simulans courts almost exclusively during the light period of the diurnal cycle, D. melanogaster courts most frequently in the dark, while D. yakuba behaves somewhat intermediarily. As the observation for D. melanogaster seemed to be in contrast with earlier findings (Rendel 1951; Jacobs 1960), which suggested higher courtship activity during the light period, I performed one expenment under natural daylight conditions in order to exclude t h e possibility that my results were induced by the block Zeitgeber. This experiment revealed nocturnal courtship activity in this species (Fig. 2). D. auraria, the courting of which is blocked by darkness and which I had tested for control, showed the same phase relationship with respect to D. melanogaster as under artificial illumination. The diurnal courting pattern of the third species which showed significant mating activity in the night, D. kikkawai, differs markedly from those of all its nearest relatives, the members of the 'montium' subgroup, which have been tested. First, there is a maximum during the night, which coincides with that shown by D. melanogaster. Moreover, these flies exhibit a second peak during the day-time, though it is very variable and cannot be dearly observed in every run. Furthermore, D. kikkawai is characterized by much higher mating activity than the other related species.
172
ANIMAL
a
loo-4
9
9
I
BEHAVIOUR.,
Ib
lc
I
I
I
I
2 0 , :1
Id
9
I
BII
I--lOO
I
I
"6
3
9
15 21
3
9
15 21
3
9
15 21
3
9
15 21
I
Time of day Fig. 1. Diurnal rhythms of courtship activity in sixteen species of the melanogaster group. (a) D. melanogaster; (b) D. simulans; (c) D. yakuba; (d) D. ananassae; (e) D. bipectinata; (f) D. lutea; (g) D. takahashii; (h) D. pseudotakahashii; (i) D. auraria; (k) D. nikananu; (1) D. serrata; (m) D. birehii; (n) D. kikkawai; (o) D. szentivanii; (p) D. pulchrella; (q) D. suzukiL The number of courtship events Was recorded in an experimental population of six males and six females within 10 min. The horizontal black and white bars represent the light--dark cycle of 12.12 hours. 9 . 0 0 and 21 . 00 recordings were carried out during the light period (because of this, the black bars are drawn only from the 23.00 to the 7 . 0 recordings).
HARDELAND: DIURNAL RHYTHMICITY OF DROSOPHILA COURTSHIP I000
173
o
I00 IO
I 0. I
b 60
c
*,> 9
4=
2C
I
15
21
Time of
5
doy
I
9
15
I
Fig. 3. Courtship activity in the course of a light--dark cycle in specimens of D. kikkawai which had been kept isolated from the time of emergence. Males and females were kept singly, until they were put together on the 6th day of life; recordings were made from the 7th to the 8th day. Shaded columns: isolated animals; open columns: control animals. Black and white bar as in Fig. 1.
d z
4C
24
4
8 Time
12 of
16
20
doy
Fig. 2. Diurnal rhythms of courtship activity in D.
melanogaster and D. auraria under natural lighting conditions. (a) light intensity (ordinate: lux); (b) D. melanogaster, (c) D. auraria.
Courtship activity is greatly enhanced in specimens which have lived singly from the time of emergence. Flies subjected to this treatment showed strong alterations in the diurnal pattern of courtship frequency: they were much more active during the light than during the dark period (Fig. 3). Nonetheless, these animals were as active as the controls in the dark period when the latter were showing maximal courtship activity. In the light, however, the flies which had lived isolated courted far more frequently than observed in any other experiment; they were 'permanently' courting over several days. Discussion
As demonstrated by these experiments, Drosophila species exhibit strong diurnal variations in courting activity. On the other hand, court-
ship activity can be also controlled by nonrhythmic factors due to the pre-treatment of the animals. Hence, if experiments are performed concerning light dependency of courtship behaviour, the result largely depends on the time of day at which they are carried out, and on the material used, in particular whether or not the flies have been allowed to mate prior to the experiment. Such methodical differences m u s t be the reason for the findings of other workers (Rendel 1951; Jacobs 1960) that D. melanogaster preferably courts in the light, whereas I would like to call it a species which preferably courts in the dark. Of course one might obtain results different from those reported here, if one compared animals from constant light and constant dark conditions (which do not behave rhythmically!) or if animals which had lived under Zeitgeber conditions were carried from light to dark. Ill this context, it may be noted that courtship behaviour of D. melanogaster, which is shown during the second part of the night, can even be suppressed by light of intensities not higher than 60 lx (Hardeland, unpublished data). Thus, light or dark preferences with respect to courtship behaviour may depend on the time of day. Moreover, I think that my results on courtship activity in the dark period are supported by observations of Ohsawa et al.
174
ANIMAL
BEHAVIOUR,
(1952), who reported that nocturnal locomotor activity of D. melanogaster is greatly enhanced in mixed groups of males and females, compared with males and females alone. Nocturnal locomotor activity, however, is obviously related to nocturnal courtship activity in this species (cf. Hardeland & Stange 1971). The experimental conditions used for comparison of the sixteen species avoid some extremely::nnnatural situations such as great population densities and use of flies which have not been allowed to mate for a long time. Hence, one could speculate about the possible ecological value of differences in the diurnal pattern of courtship activity between related species. In the case of the three sibling species D. melanogaster, D. simulans and D. yakuba, the latter is geographically restricted to the African region, whereas both D. melanogaster and D. simulans are cosmopolitan species (cf. Patterson & Stone 1952). One could imagine that temporal fixation of courtship behaviour to different phases of the diurnal cycle may function as an isolating mechanism between these two cosmopolitans. O f course, this would not be the only isolating barrier, since other mechanisms, e.g. concerning the courtship song (Bennet-Clark & Ewing 1969), have already been worked out. Another isolating barrier due to the same mechanism could possibly exist between D. kikkawai and the other members of the 'montium' subgroup. Although not cosmopolitan, D. kikkawai is widely distributed, whereas the other members of the subgroup are restricted to smaller geographical regions. In this case, the maxima of the geographically isolated species coincide; in contrast, the only species with a wide geographical range, which cannot be isolated from its relatives simply by distribution, courts preferably at a time when the other flies usually do not show this behaviour. Even the second, variable maximum of the light period coincides with the minima of the other species. Whether or not the isolating mechanism supposed here is of ecological significance, cannot be decided from these experiments alone. In particular, the question should be answered whether the females' reactions to courting males vary within the diurnal cycle, and, if this were true, whether there are species differences. It should be t a k e n into consideration that diurnal rhythms in receptivity could be of great isolating effectiveness, especially if they would parallel the males' courting rhythms.
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1
Acknowledgment I wish to thank Mis M. A. Reveley, Austin, Texas for generous gifts of Drosophila stocks.
REFERENCES Bennet-Clark, H. C. & Ewing, A. W. (1969). Pulse interval as a critical parameter in the courtship song of Drosophila melanogaster. Anita. Behav.,. 17, 755-759. Grossfield, J. (1966). The influence of light on the mating behavior in Drosophila. Univ. of Texas Pubt., 6615, 147-176. Grossfield, J. (1970). Species differences in light-influenced mating behavior in Drosophila. Am. Natur., 104, 307-309. Hardeland, R. (1971). Lighting conditions and mating behavior in Drosophila. Am. Natur., 105, 198-200. Hardeland, R. & Stange, G. (1971). Einfltisse yon Geschlecht und Alter auf die lokomotorische Aktivit~tt yon Drosophila. J. lnsect. PhysioL, 17, 427-434. Jacobs, M. E. (1960). Influence of light on mating of Drosophila melanogaster, Ecology, 41, 182-188. Jacobs, M. E. (1961). The influence of light on gene frequency changes in laboratory populations of ebony and non-ebony Drosophila melanogaster. Genetics, 46, 1089-1095. Lewis, T. & Taylor, L. R. (1964). Diurnal periodicity of flight by insects. Trans. Roy. ent. Soc. Lond., 116, 393--469. Ohsawa, W., Matutani, K., Tukuda, H., Mori, S., Miyadi, D., Yanagisima, S. & Sato, Y. (1952). Sexual properties of the daily rhythmic activity in Drosophila melanogaster. Seiro-Seitai, 5, 26-30. Patterson, J. T. & Stone, W. S. (1952). Evolution in the Genus Drosophila. New York: Macmillan. Pavan, C., Dobzhansky, Th. & Burla, H. (1950). Diurnal behavior of some neotropical speciesof Drosophila. Ecology, 31, 36--43. Petit, C . & Ehrman, L. (1969). Sexual selection in Drosophila. Evolut. BioL, 2, 177-233. Rendel, J. M. (1951). Mating of ebony, vestigial, and wildtype Drosophila melanogaster in light and dark. Evolution, 5, 226-230. Rensing, L. & Hardeland, R. (1967). Zur Wirkung der circadianen Rhythmik auf die Entwicklung yon Drosophila. J. Insect PhysioL, 13, 1547-1568. Spieth, H. T. (1966). Drosophilid mating behavior: the behavior of decapitated females. Anim. Behav., 14, 226-235. Stange, G. & Hardeland, R. (1970). Eine Methode zur Registrierung der Laufaktivitat von kleinen Insecten. Oecologia, 5, 400--405. Wallace, B.& Dobzhansky, Th. (1947). Experiments on sexual isolation in Drosophila. VIII. Influence of light on the mating behavior of Drosophila subobscura, Drosophila persimilis and Drosophila pseudoobscura. Proc. nat. Acad. Sci., Wash., 32, 226-234. (Received 21 April 1971; revised 6 August 1971; MS. number: 1054)