Genetic feminization of the thoracic nervous system disrupts courtship song in male Drosophila melanogaster

Abstract

Despite the growing research investigating the sex-specific organization of courtship behavior in Drosophila melanogaster, much remains to be understood about the sex-specific organization of the motor circuit that drives this behavior. To investigate the sex-specification of a tightly patterned component of courtship behavior, courtship song, the authors used the GAL4/UAS targeted gene expression system to feminize the ventral ganglia in male Drosophila and analyzed the acoustic properties of courtship song. More specifically, the authors used the thoracic-specifying teashirt promoter (tsh(GAL4)) to express feminizing transgenes specifically in the ventral ganglia. When tsh(GAL4) drove expression of transformer (tra), males were unable to produce prolonged wing extensions. Transgenic expression of an RNAi construct directed against male-specific fruitless (fru(M)) transcripts resulted in normal wing extension, but highly defective courtship song, with 58% of males failing to generate detectable courtship song. Of those that did sing, widths of individual pulses were significantly broader than controls, suggesting thoracic fru(M) function serves to mediate proprioceptive-dependent wing vibration damping during pulse song. However, the most critical signal in the song, the interpulse interval, remained intact. The inability to phenocopy this effect by reducing fru(M) expression in motor neurons and proprioceptive neurons suggests thoracic interneurons require fru(M) for proper pulse song execution and patterning of pulse structure, but not for pulse timing. This provides evidence that genes establishing sex-specific activation of complex behaviors may also be used in establishing pattern-generating motor networks underlying these sex-specific behaviors.

Figure 1

Measurements of wing extension behavior. (A) No differences in courtship index (CI) were observed among genotypes (mean ± s.e.m.). (B) tsh^GAL4/UAS-tra flies had a significantly decreased median wing extension duration compared to tsh^GAL4/+ controls, while tsh^GAL4/UAS-fru^MIR males were no different than controls. (C) Mean of a fly’s median wing extension duration. tsh^GAL4/UAS-tra males display significantly shorter wing extensions than tsh^GAL4/+ controls. (D) Wing extension frequency, separating extensions shorter and longer than 0.5 s. tsh^GAL4/UAS-tra flies are not different in frequency of total wing extensions, but exhibit more frequent wing extensions shorter than 0.5 s and less frequent wing extensions longer than 0.5 s compared to tsh^GAL4/+ controls. There is no difference between tsh^GAL4/UAS-fru^MIR males and tsh^GAL4/+ control males. Sample size indicated within bars in (D). *: p < 0.05.

Figure 2

Expression of fru^MIR in tsh-specific pattern reduces amount of courtship song. Proportion of flies that produced audible output classified as pulse song (A). Only tsh^GAL4/UAS-fru^MIR males failed to produce courtship song. tsh^GAL4/UAS-fru^MIR also males exhibited fewer pulse trains per minute (B) and pulses per train (C) than tsh^GAL4/+ controls, while tsh^GAL4/UAS-fru^MIR; n-syb^GAL80 males were no different than controls. tsh^GAL4/UAS-fru^MIR; n-syb^GAL80 males rescued the decreased pulses per train in tsh^GAL4/UAS-fru^MIR males. *: p < 0.05, **: p < 0.01.

Figure 3

Representative traces of courtship song output. The left panel displays individual pulses within a pulse train aligned by midpoint of energy. Solid lines indicate amount of trace required to include 90% of the signal’s energy (pulse width). Right panel displays the whole trace. Arrowheads indicate sine song. (A) tsh^GAL4/+ controls, (B) tsh^GAL4/UAS-fru^MIR; n-syb^GAL80 rescue flies, (C) representative small amplitude and (D) polycyclic nature of tsh^GAL4/UAS-fru^MIR courtship song. Scale bars: Left panel, horizontal 5 ms, vertical 5 mm s⁻¹. Right panel, horizontal 25 ms, vertical 5 mm s⁻¹.

Figure 4

Intrapulse data. (A) Peak particle velocity within a pulse is reported here as a mean of medians in dB SPVL, using 50 nm s⁻¹ as a reference. There no significant differences were detected in dB SPVL, but there was a trend for a reduction in pulse amplitude for tsh^GAL4/UAS-fru^MIR males. (B) Peak frequency of sine songs (circle) and individual pulses (triangle). No significant differences were observed. (C) Pulse widths from tsh^GAL4/UAS-fru^MIR males were significantly broader compared to tsh^GAL4/+ controls. This broadened pulse width is rescued in tsh^GAL4/UAS-fru^MIR; n-syb^GAL80 males. (D) Mean interpusle interval (IPI) was unaffected by genotype. *: p < 0.05, **: p < 0.01, ‡: one-tailed, p < 0.05.

Figure 5

Immunocytochemistry of adult w; tsh^GAL4, UAS-mCD8-GFP/CyO CNS, visualizing endogenous, membrane bound GFP (green) and Fru^M immunoreactivity (magenta). (A) Dorsal-ventral view of adult ventral ganglia. Extensive labeling is visible in prothoracic (Pr), mesothoracic (Ms), metathoracic (Mt), and abdominal (Ab) segments. Anterior-posterior axis is indicated. (B–F) 3 – 5 µm representative sections of the five groups of fru^M neurons in the ventral ganglia, according to (Lee et al., 2000). Fru^M neural cluster 16 (B), 17 (C), 18 (D), 19 (E), and 20 (F). Arrowheads indicate examples of neurons coexpressing Fru^M and mCD8-GFP. In (C), a Fru^M-expressing cluster clearly coexpresses GFP (solid line), while an adjacent Fru^M cluster does not (dashed line). Scale bars (B–F) represent 5 µm.

Figure 6

Courtship song is unaffected by driving fru^MIR in motor neurons and sensory neurons. No significant effects of genotype were found on (A) pulse rate, (B) pulse and sine song peak frequency, or (C) pulse width compared to UAS-fru^MIR controls. The tsh^GAL4/UAS-fru^MIR mutant phenotype is replotted from Fig. 2 and Fig. 4 for comparison. n = 6 – 8. *: p < 0.005, **: p < 0.001.

Figure 7

Flight ability of tsh^GAL4/UAS-fru^MIR males. Males were placed in the center of a cylinder and allowed to freely fly. Landing sites were recorded as the bottom (white), side (gray), or out of the cylinder (black). There was a significant effect of genotype on flight performance (p < 0.001). Flight performance of control + / + males (n = 23) was not significantly different from tsh^GAL4 / UAS-fru^MIR males (n = 27), but both performed better than the known flight mutant, Mhc⁵ (n = 19) (p < 0.0005).