Sensory integration regulating male courtship behavior in Drosophila

Abstract

The courtship behavior of Drosophila melanogaster serves as an excellent model system to study how complex innate behaviors are controlled by the nervous system. To understand how the underlying neural network controls this behavior, it is not sufficient to unravel its architecture, but also crucial to decipher its logic. By systematic analysis of how variations in sensory inputs alter the courtship behavior of a naïve male in the single-choice courtship paradigm, we derive a model describing the logic of the network that integrates the various sensory stimuli and elicits this complex innate behavior. This approach and the model derived from it distinguish (i) between initiation and maintenance of courtship, (ii) between courtship in daylight and in the dark, where the male uses a scanning strategy to retrieve the decamping female, and (iii) between courtship towards receptive virgin females and mature males. The last distinction demonstrates that sexual orientation of the courting male, in the absence of discriminatory visual cues, depends on the integration of gustatory and behavioral feedback inputs, but not on olfactory signals from the courted animal. The model will complement studies on the connectivity and intrinsic properties of the neurons forming the circuitry that regulates male courtship behavior.

Figure 1. Poxn null allele and rescue transgenes used for manipulation of gustatory modality.

(A) Map of the Poxn gene , , the Poxn^ΔM22-B5 deletion , and the Poxn rescue constructs. The Poxn transgenes ΔPBs, ΔXBs, and SuperA, and the Poxn deficiency Df(2R)Poxn^ΔM22-B5 are shown with regard to a restriction map of the Poxn locus. Upstream region (green), 5′ leader and 3′ trailer (orange), coding region (black), introns (yellow), and downstream region (blue) are indicated. (B) Table of Poxn transgenes used to rescue development of antenna, leg, male genitalia, CNS, and of all or only very few gustatory bristles in Poxn^ΔM22-B5 null mutants. The Poxn-SuperA transgene (SuperA) rescues all mutant phenotypes of the Poxn gene . Since the ΔXBs transgene does not completely rescue the leg/antenna segmentation phenotype of Poxn^ΔM22-B5 null mutants, it was combined with one copy of the ΔPBs transgene. This combination, Poxn-pRes, rescued leg and antennal segmentation but also, in a random manner (data not shown), 2–4 of ∼50 taste bristles on the foreleg of a wild-type male. The genotypes Poxn-pRes and Poxn-SuperA are short for ΔXBs6; Poxn^ΔM22-B5/Poxn^ΔM22-B5 ΔPBs96.2 and Poxn^ΔM22-B5 SuperA-158, respectively. The SuperA transgene also rescued all courtship mutant phenotypes of Poxn-pRes males described in this paper (data not shown), when combined with the ΔXBs and ΔPBs transgenes (ΔXBs6; Poxn^ΔM22-B5 SuperA-158; ΔPBs69/+). This demonstrates that the insertions of the ΔXBs and ΔPBs transgenes do not interfere with the rescue of the Poxn mutant phenotype by the SuperA transgene. PK6 is a Poxn transgene that does not rescue any taste bristles . Poxn-PK6; Or83b² males showed no initiation of courtship in the dark (data not shown). However, we did not use these flies in our courtship assays because they lack the Poxn functions required for proper development of male genitalia as well as the Poxn ventral ganglion function, which all may not influence courtship initiation but interfere with copulation .

Figure 2. Light-dependent adaptation of male courtship strategy: visual tracking versus scanning.

(A) Average latency (in seconds) till courtship initiation, and (B) courtship vigor index were measured in single-choice courtship assays with mature males of indicated genotypes and receptive Ore-R virgin females in daylight (light colored columns) or under dim red light (dark colored columns). The fraction of males initiating courtship was 100% in all cases. In this and all other figures, the numbers below columns indicate the number of couples observed, unless indicated differently, and error bars always represent double standard errors of the mean. Red numbers above columns (B) denote copulation efficiencies. The copulation efficiencies of black-eyed Ore-R males are considerably reduced and hence not indicated. (C) Photograph of male (♂) in search of virgin (V) under infrared light. The dotted line indicates the zigzag course of the male scanning the mating chamber.

Figure 3. Chemosensory signals drive male courtship in the presence and absence of light.

Male courtship parameters, (A) the fraction of males initiating courtship, (B) the average latency (in seconds) till courtship initiation, and (C) the courtship vigor index, were measured in single-choice courtship assays with mature males of indicated genotypes and intact receptive Ore-R virgins in daylight (light colored columns) or under dim red light (dark colored columns).

Figure 4. Gustatory, but not olfactory, signals of the courted fly contribute to the heterosexual orientation of the courting male.

(A) The fraction of males initiating courtship, (B) the average latency (in seconds) till courtship initiation, and (C) the courtship vigor index were measured in single-choice courtship assays, performed under dim red light with courting males of indicated genotypes and decapitated receptive Ore-R virgins (V, filled columns) or decapitated mature Ore-R males (♂, hatched columns).

Figure 5. Integration of gustatory signals and feedback behavior of the courted fly enforce heterosexual orientation of males in the dark.

The courtship vigor index was measured in single-choice courtship assays performed under dim red light with courting males of indicated genotypes and decapitated, dewinged, or intact males. Below each column, the number of males that initiated courtship is shown. Measurements of cvi towards decapitated males were taken from Figure 4C.

Figure 6. Vision strongly supports the heterosexual orientation of males.

(A) Courtship vigor indices were measured in single-choice courtship assays, performed in the dark or in daylight with courting males of indicated genotypes and decapitated Ore-R virgins (V, filled columns) or decapitated Ore-R males (♂, hatched columns). The number of males that initiated courtship is shown below each column. (B) Fractions of males initiating courtship and (C) average latencies (in seconds) till courtship initiation correspond to the courtship assays in (A) of Ore-R and Poxn-pRes; Or83b² males courting decapitated flies in daylight.

Figure 7. Integration of feedback with visual signals inhibits homosexual orientation of males.

Courtship vigor indices were measured in single-choice courtship assays in daylight, with courting males of indicated genotypes and decapitated receptive Ore-R virgins (V, filled columns) or Ore-R males that were either decapitated (1), dewinged (2), or intact (3) (♂, hatched columns). The number of males that initiated courtship is shown below each column. Values of cvi of Ore-R and Poxn-pRes; Or83b² males courting decapitated flies in daylight were taken from Figure 6A.

Figure 8. Chaining behavior of taste-deficient males.

(A) Chain of four courting Poxn-pRes males. The picture was taken 5 minutes after eight mature, but sexually naïve, Poxn-pRes males were placed together into a small Petri dish. (B) In addition to the average chaining indices for groups of eight males of indicated genotypes, the number of groups for which chaining was observed over the total number of groups examined is shown for each genotype.

Figure 9. Model illustrating the regulation of Drosophila male courtship behavior through integration of sensory signals.

The model, derived from the results presented here, illustrates how courtship activity of a male is regulated by the various sensory inputs when the male (♂) faces a receptive virgin (V), shown on the left, or another male (♂), shown on the right. The sensory receptors of the male are responding to the presence (yellow sun) or absence of light through an unidentified light sensor (ls), and to olfactory (olf), gustatory (gust), visual (vis), or behavioral feedback (fb) signals. It is unclear which senses are stimulated by the fb signals. Tactile cues (tc) that are necessary but not sufficient to stimulate male courtship originate from the object animal and are always present in our courtship assays (see Discussion). Arrows and T-bars are stimulatory and inhibitory signals affecting a modality or behavior, but do not indicate differences in relative weights. However, qualitative information on relative weights where known is provided in the text. Red, green, and blue lines relay gustatory, olfactory, and visual information, respectively. Gray lines transport signals from the light sensor, while brown lines transmit the behavioral feedback of the object animal in response to being approached and courted (orange lines). Arrows converging on the same behavioral step, illustrated by a box (yellow: daylight; gray: dark; gray/yellow: daylight or dark), may be sufficient or necessary to trigger that behavioral step. Lines passing behind the boxes for tracking and scanning behavior indicate that the corresponding inputs maintain but do not trigger these behavioral steps. Numbers refer to those in parentheses behind the experimental evidence mentioned in the text, while their color refers to that of the corresponding sensory modality. Black numbers indicate where one modality is used to stimulate another. In cases where two colored numbers refer to the same arrow, the lower number refers to assays with intact, the other with decapitated females.