Joint control of Drosophila male courtship behavior by motion cues and activation of male-specific P1 neurons

Abstract

Sexual behaviors in animals are governed by inputs from multiple external sensory modalities. However, how these inputs are integrated to jointly control animal behavior is still poorly understood. Whereas visual information alone is not sufficient to induce courtship behavior in Drosophila melanogaster males, when a subset of male-specific fruitless (fru)- and doublesex (dsx)-expressing neurons that respond to chemosensory cues (P1 neurons) were artificially activated via a temperature-sensitive cation channel (dTRPA1), males followed and extended their wing toward moving objects (even a moving piece of rubber band) intensively. When stationary, these objects were not courted. Our results indicate that motion input and activation of P1 neurons are individually necessary, and under our assay conditions, jointly sufficient to elicit early courtship behaviors, and provide insights into how courtship decisions are made via sensory integration.

Fig. 1.

Activation of fru^M or dsx neurons promotes vision-based courtship behavior. (A) Diagram of two-layer chamber used for the assay of vision-based courtship behavior. A tester male fly was separated from two female targets by a thin transparent plastic barrier. (B) Courtship indices of males of the indicated genotypes toward two freely moving females in the vision-based courtship assay. Control males rarely court these females at either 22 °C or 27 °C (white or black). When all fru^M or dsx neurons were activated via dTRPA1 at 27 °C, these males showed consistently high levels of total courtship output (gray) and also significantly increased courtship toward female targets (fru^M drivers in red and blue, dsx drivers in yellow and cyan). (C) Courtship indices of males toward two immobile females (frozen overnight) in the vision-based courtship assay. n = 12–20 for each. Error bars indicate SEM.

Fig. 2.

Mutual enhancement of courtship behavior by R71G01 activation and motion input. (A) Courtship indices of males of indicated genotypes toward freely moving females in the vision-based courtship assay. Control UAS-dTrpA1/+; pBDPGAL4u/+ males (white) hardly courted female targets at either 22 °C or 30 °C. UAS-dTrpA1/+; R71G01-GAL4/+ males (gray) hardly courted female targets at 22 °C, but they followed the moving females and courted them intensively at 30 °C. (B) Courtship indices toward immobile females in the vision-based courtship assay. (C) Diagram of vision-based courtship assay with automatically rotating targets. The tester male fly was placed in a one-layer chamber, and two females or two pieces of rubber bands were rotated automatically by a rotator underneath the chamber in which the tester male was suspended. (D) Courtship indices toward constantly rotating females (frozen overnight). (E) Courtship indices toward constantly rotating rubber band pieces. (F) Courtship indices toward immobile rubber band pieces. n = 12–24 for each. Error bars indicate SEM.

Fig. 3.

Lateralized visual input determines the choice of unilateral wing extension. (A and B) Numbers of left wing extensions (white) and right wing extensions (gray) from UAS-dTrpA1/+; R71G01-GAL4/+ males to freely moving females (A) or counterclockwise rotating rubber band pieces (B). Males were separated from targets by a transparent barrier. (A) UAS-dTrpA1/+; R71G01-GAL4/+ males displayed approximately equal left or right wing extensions to moving females at 30 °C. When one eye (either left of right) was blinded (painted), these males displayed dominant contralateral wing extensions. While both eyes were blinded, these males rarely showed wing extension to targets. (B) UAS-dTrpA1/+; R71G01-GAL4/+ males displayed high level of left wing extensions and a lower level of right wing extensions to counterclockwise rotating rubber band pieces at 30 °C. While one eye was blinded, these males displayed dominant contralateral wing extensions. They hardly showed wing extension while both eyes were blinded. n = 12 for each. Error bars indicate SEM.

Fig. 4.

Male-specific R71G01 neurons (P1 neurons) mediate motion-based courtship behavior in males. (A and B) R71G01-GAL4 expression in the brains and VNCs of a male (A) and a female (B) visualized with pJFRC2-10XUAS-IVS-mCD8GFP. It targets a subset of male-specific neurons in the brain that project across the anterior dorsal commissure (arrow), as well as a few neurons common to both sexes. (C and D) R71G01-LexA expression in the brains and VNCs of a male (C) and a female (D) visualized with pJFRC19-13XLexAop2-IVS-myrGFP. It recapitulates R71G01-GAL4 expression, including the male-specific neurons in the brain (arrow). (E) Intersectional expression of R71G01-LexA and dsx^GAL4 in the male CNS [genotype: LexAop2-FlpL/R71G01-LexA; UAS > stop > myrGFP/dsx^GAL4(∆2)]. Male-specific P1 neurons (arrow) were targeted in the male CNS. (F) Expressing dTrpA1 specifically in the P1 neurons (arrow) in the male brain [genotype: UAS > stop > dTrpA1^myc/R71G01-LexA; LexAop2-FlpL/dsx^GAL4(∆2)]. The ectopic dTrpA1 expression was visualized with anti-myc antibody. (G) Box plots indicate courtship indices of males of the indicated genotypes to freely moving females or constantly rotating rubber band pieces in the vision-based courtship assay. Circles indicate mean values. The middle line represents the median and the ends of the vertical lines indicate the minimum and maximum. Upper and lower edges of the boxes represent the first and third quartiles. Data show that activating only the P1 neurons is sufficient to induce a significant level of motion-based courtship behavior in every individual. All genotypes were tested at 30 °C except for the one in yellow, which was tested at 32 °C. n = 12 for each.