Mutations of the Drosophila myosin regulatory light chain affect courtship song and reduce reproductive success

Abstract

The Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2(Δ2-46) (Ext), an N-terminal extension truncation; (ii) Dmlc2(S66A,S67A) (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2(Δ2-46;S66A,S67A) (Dual), expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2 (+) rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight.

Figure 1. Courtship song oscillogram samples of control and mutant lines.

Courtship song samples from transgenic males (A) Control, (B) Ext (C) Phos, and (D) Dual. In all cases, male courtship song was induced by the presence of a wild type (Oregon R) virgin female D. melanogaster. All strains produce sine song and pulse song. Song recording was done at 22°C and 70% humidity in a dark room, with the only light source inside the song recording chamber , . The samples here were retrieved with Audacity software.

Figure 2. Dmlc2 mutations affect sine song frequency.

(A) Representative sine song oscillograms from Control, Ext, Phos and Dual males are shown (top to bottom panels, respectively). (B) Compared to Control (blue), sine song frequency is significantly higher in Ext (red) and Phos (green) mutants but similar in the Dual mutant (yellow). Also note that Ext mutant sings with a significantly higher sine song frequency compared to Phos and Dual mutants. (C) Sine song burst duration (SDUR) is similar for all the lines. See Materials and Methods and ref [76] for details and retrieval method of the sine song parameters. n = 7–8 males for each line. *(p<0.0001), § (p<0.0001) and Ω (p<0.0001) indicate significant difference from Control, Ext, and Phos respectively. Error bars indicate SEM.

Figure 3. Effect of Dmlc2 mutations on pulse song properties.

(A) Representative pulse song oscillograms from Control, Ext, Phos and Dual males (top to bottom panels, respectively). (B) Phos and Dual males sing with similar cycles per pulse (CPP) and (C) pulse length (PL) compared to Control males. Ext males produce songs with higher CPP and longer PL. (D) All the mutant males produce pulse song with normal intrapulse frequency (IPF), with only the Phos mutants’ IPF showing a slight reduction compared to Ext and Dual. (E) None of the mutations affect interpulse interval. (F) The Dual mutant has significantly reduced pulse duty cycle compared to Control, Ext, and Phos. (G) Amplitude ratio (AMP-RT) of consecutive sine to pulse song is significantly higher in individual (Ext, Phos) and Dual mutants compared to Control. n = 7–8 males for each line. *(p<0.05), ***(p<0.001), ****(p<0.0001) indicate significant differences from Control. § (p<0.05), Ω (p<0.05) and Δ (p<0.05) indicate significant difference from Ext, Phos and Dual mutants, respectively. Error bars indicate SEM.

Figure 4. Dmlc2 mutations affect courtship behavior.

Single pair mating assays, consisting of a wild-type (OR) female and a male of Control or mutant strain were performed to assess male courtship vigor. (A) Courtship index (CI) and (B) Wing extension index (WEI). Ext and Phos mutants had significantly reduced CI, and Dual mutant’s CI is marginally reduced compared to Control (p = 0.054). Ext, Phos, and Dual mutants had significantly reduced WEI compared to Control (B). The Ext mutant, in particular, had the greatest reduction in CI and WEI compared to Control and Dual. n = 4–6 for each mating competition group. *(p<0.05) indicate significant difference from Control. § (p<0.05) indicate significant differences from Ext mutant. Error bars indicate SEM.

Figure 5. Dmlc2 mutations affect male courtship vigor and female preference.

(A) Female preference index (FPI) is the relative advantage of a male of specific genotype over a male of a different genotype, i.e., the excess number of copulations with a male of specific genotype divided by the total number of copulations . Negative FPI indicate the Ext (red), Phos (green) and Dual (yellow) mutant males were out-competed by the Control male for female preference. Positive FPI indicate the Phos (green) and Dual (yellow) mutant males individually out-competed the Ext mutant males. There is no female preference for Phos or Dual males (FPI = 0). (B–C) Male courtship vigor in competitive mating situation was calculated via. courtship index (CI) and wing extension index (WEI). Ext and Dual mutants had significantly reduced CI and WEI but Phos mutant had only significantly reduced WEI compared to Control. In competition between mutants, Phos and Dual mutants have significantly higher CI and WEI compared to the Ext mutant. There is no difference between Phos and Dual. n = 20–30 for each mating competition group. *(p<0.05) indicate significant difference from Control. § (p<0.05) indicate significant differences from Ext mutant. No error bars in (A), (B–C) error bars indicate SEM.