A new behavioral bioassay for an analysis of sexual attraction and pheromones in insects

Moti Nissani

Source:  JOURNAL OF EXPERIMENTAL ZOOLOGY, 1975 May; 192 (2): 271-5.

[Author's note (2005):  As far as I'm aware, this ancient paper (1975)  was the first to suggest (1) the use of mosaics for the detection and localization of sex pheromones, (2) the existence of such pheromones in Drosophila melanogaster, (3) that most of female pheromones in Drosophila are produced in the integument, and, more specifically, in the abdominal tergites.]

Dmmale.gif (63176 bytes) Abstract: Drosophila melanogaster gynandromorphs were introduced into a mating chamber containing two wild type males. From the differential courtship responses of these males it could be concluded that the only important factor which enables a male to distinguish between conspecific males and females and to direct persistent courtship only toward females is tissue composition of females. This fact is interpreted in terms of a female-specific sex pheromone. Most of this pheromone is produced by cells whose precursors on the blastoderm surface are very close to, or identical with, the blastoderm precursors of the tergites. Some of this pheromone is probably produced by cells whose precursors are very close to, or identical with, the precursors of the sternites and thorax.

Key Words: Drosophila melanogaster; Sexual Attraction; Mating Behavior; Courtship Behavior; Insect Behavior; Pheromones; Animal Mosaicism; Gynandromorphs; Fate Mapping.

In a classical study, whiting ('32) demonstrated   the potential of gynandromorphs for behavioral genetics. He observed the behavior of gynandromorphs of the parasitic wasp, Habrobracon juglandis, and obtained evidence which suggested that the organ controlling sex-specific behavioral patterns in this wasp is the brain.  The experiment described below demonstrates that gynandromorphs may be also used in order to investigate what differences(s) between a male and a female cause a male to direct persistent courtship only toward a female and which anatomical sties(s) in the female are responsible for triggering and sustaining such a persistent courtship response by the male.

MATERIALS AND METHODS Wildtype Drosophila melanogaster males of the canton strain were lightly etherized 0-24 hours after eclosion and 10-20 males were placed in a fresh vial. All flies were reared at 25° C in half-pint milk bottles containing standard medium (Strickberger, ’62).

Two wildtype males, 6-12 days old, were introduced into a clean Pasteur pipette. The pointed tip of the pipette was removed so that with the aid of 2 glass rods of appropriate size a mating chamber of approximately 1.8 cm3 could be produced. The 2 males were left for 10 minutes in this chamber and their behavior was recorded. At the end of this 10-minute period, a single 4-12 days old gynandromorph was introduced. For each gynandromorph, 2 or more replicates were made. Lag time, persistency, and length of courtship were recorded. Gynandromorphs which were courted persistently and for more than 20 minutes were classified as sexually attractive. Gynandromorphs which evoked only sporadic and short bouts of courtship were classified as sexually intermediate. Gynandromorphs which were not courted during a 10-minute observation periods by the two wild type males present were classified as sexually unattractive. Gynandromorphs were assigned to either of the last two categories only if immediately after the observation period, when the gynandromorph was removed and a wildtype virgin female was introduced, one or both males started courting her persistently within 3 minutes.

Gynandromorphs were obtained by means of the unstable ring chromosome In(1)wvc (Hotta and Benzer, ’72). In order to rule out possible effects of specific recessive genes on the attractiveness of gynandromorphs, 4 differently marked rod X chromosomes were used: (1) y v f car (2) sn3 un4 (3) w sn3 (4) y sn3 un4 (Lindsley and Grell, ’68).

RESULTS AND DISCUSSION Out of 183 gynandromorphs observed, 117 were sexually attractive, 21 were sexually intermediate, and 45 were sexually unattractive. The gynandromorphs obtained had all possible combinations of male and female appearance, behavior, and tissue composition. It was thus possible to draw some conclusions regarding the relative importance of female appearance, behavior, and tissue composition in eliciting courtship in males. Since some gynandromorphs with typical male appearance and behavior were courted as persistently and as long as wildtype females, and since some gynandromorphs with female appearance and behavior were ignored , it could be concluded that female appearance and behavior do not play an important role in evoking persistent courtship of males toward wildtype conspecific females. The results also rule out the suggestion that males and females of this species may be equally attractive to conspecific males that that the male-specific rejection behavior has a strong inhibitory effect on a courting male (Bastock and Manning, ’55).

The results indicate that the only female-specific stimulus of major importance in evoking persistent courtship from males is associated with female composition of certain parts of the integument and that the effect of the various cuticular landmarks is not in proportion to their relative size. The clearest effect is that of the tergites since all gynandromorphs with 3 or more female half-tergites were courted persistently. Two sexually attractive and 7 sexually intermediate gynandromorphs had no female tergites. These facts suggest that female composition of 3 or more half-tergites is sufficient to evoke persistent male courtship, and that some other structure(s) in a female may also have an excitatory effect. Indeed, when the composition of the thorax and sternites is also considered, and if the assumption is made that the excitatory effect of one female tergite is roughly equivalent to the excitatory effect of 4 sternites or the whole thorax (=one tergite equivalent), the resultes are clearcut. All sexually unattractive gynandromorphs had less than one tergite equivalent, all 21 sexually intermediates had ½-1¾ tergite equivalents, and all 117 sexually attractive gynandromorphs had more than ¾ tergite equivalents; the means of the numbers of tergite equivalents in each of these 3 classes were 0.23, 1.12, and 4.45, respectively. In contrast to the effect of the tergites, thorax, and sternites, female composition of the head, wings, legs, and external genitalia appeared to have no excitatory effect.

It can be concluded that the only female-specific stimulus of major importance in eliciting persistent courtship of D. melanogaster is associated with integumental cells of female composition, and hence, that it is probably a sex pheromone(s) (Jacobson, ’72; Shorey and Bartell, ’70). Determination of the embryological origin of these cells (Hotta and Benzer,’72) was aided by the following considerations: (1) No flies with more than 2 female half-tergites were ignored, i.e., the mosaic boundary never passed between the precursors of these imaginal half-tergites and the precursors of the pheromone-producing cells. (2) All gynandromorphs whose thoracic and abdominal integument was male but whose external genitalia and/or head were female were ignored. This fact can be explained only by the assumption that the precursors of the pheromone-producing cells are very close to, or identical with, the precursors of the tergites, sternites, and thoracic integument. (3) As the number of tergite equivalents increased, gynandromorphs were courted more persistently and for longer periods of time. (4) All fore-and-aft gynandromorphs were courted unless the mosaic boundary passed between a female head and a male body, or passed too close to the posterior end of the fly.

Taken together, the data indicate that most of the pheromone-producing cells map to the tergites, and that some of these cells map to the sternites and to the thoracic integument. This conclusion, combined with embryological and anatomical observations (Demerec, ‘50), suggests that the pheromone-producing cells of adult D. melanogaster females are dispersed throughout, and are part of, the female tergites, sternites, and thorax, and that these cells are more numerous, or more active, in the tergites and sternites than in the thoracic integument.


Bastock, M., and A. Manning 1955 The courtship of Drosophila melanogaster. Behaviour, 8: 85-111.

Demerec, M (ed.) 1950 Biology of Drosophila John Wiley and Son, New York.

Hotta, Y, and S. Benzer 1972 Mapping of Behaviour in Drosophila mosaics. Nature, 240: 527-535.

Jacobson, M. 1972. Insect Sex Pheromones. Academic Press, new York.

Lindsey, D. L., and E. H. Gruel. 1968 Genetic Variations of Drosophila melanogaster. Carnegie Inst. of Was. Pub 627.

Sherry, H. H., and R. J. Barrel 1970 Role of a volatile female sex pheromone in stimulating male courtship behaviour in Drosophila melanogaster. Anim. Behav., 18: 159-164.

Strickberger, M. W. 1962 Experiments in Genetics with Drosophila. John Wiley and Son, New York.

Whiting, P.W. 1932 Reproductive reactions of sex mosaics of a parasitic wasp, Habrobracon juglandis. J. Comp. Psychol., 14: 345-363.

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